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  • 99
    New England Biolabs epimark nucleosome assembly kit
    Epimark Nucleosome Assembly Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 97 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Zymo Research en nucleosome dna prep kit
    Nuclei of TGCs show decreased <t>nucleosomal</t> stability. ( a – c ) Nucleosome stability assay using a permeabilized individual cell in the microfluidic device. ( a ) Overview of the microfluidic device and experimental procedure for the nucleosome stability assay using a single cell. ( b ) Representative green fluorescence observed in H4-GFP-TSC and -dTSC at day 8 following exposure to NaCl buffer at the indicated concentrations in the micropockets. Bars = 5 μm. ( c) Measurement of fluorescence intensity of H4-GFP in TSCs and dTSCs at day 8. Fluorescence intensities were measured using the ImageJ software, and the nuclear position in each cell was determined using the genomic <t>DNA</t> image obtained after GelRed staining. Values indicate the mean ± S.D. (n = 5) and are relative to the value obtained after the 0.5 M NaCl treatment, which is arbitrarily set as 1. ( d ) Nucleosome stability assay. Each sample was extracted using a buffer containing 0.5 or 1 M NaCl. Extracted proteins equivalent to 0.5 μg of genomic DNA were subjected to 15% SDS-PAGE. ( e ) Proportion of each histone in the NaCl extract to total histone. Band intensity following western blotting of 1 M NaCl-extracted histone ( d ) was calculated and normalized to that of the corresponding band in Fig. S1 . The mean ± S.D. of three independent experiments are shown. * P
    En Nucleosome Dna Prep Kit, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Abcam dinucleosomes
    Fpr4 FKBP condenses <t>nucleosome</t> arrays. ( A ) The Fpr4 FKBP sensitizes nucleosome arrays to magnesium-dependent self-association. Recombinant Fpr4 FKBP was mixed with 25 mer nucleosome array in the presence of 0.25 mM, 0.5 mM and 1 mM MgCl 2 and centrifuged. Nucleosome arrays in the supernatant and pellet fraction were visualized by agarose gel electrophoresis with ethidium bromide staining. The supernatant and pellet fractions contain extended and condensed arrays, respectively. Full-length images of this cropped gel are available in the Supplementary Information File. ( B ) Chromatin fibre self-association is unique for Fpr4 but does not require peptidyl-prolyl isomerase activity. Recombinant Fpr4 FKBP, FK506-treated Fpr4 FKBP, Fpr4 FKBP F323Y, Fpr1 and Fpr4 NL were mixed with nucleosome arrays in the presence of 0.5 mM MgCl 2 and subjected to centrifugation to separate condensed and open fibres. Full-length images of this cropped gel are available in the Supplementary Information File. ( C ) The Fpr4 FKBP occupies linker regions in nucleosome arrays. Recombinant Fpr4 FKBP, FK506-treated Fpr4 FKBP, Fpr4 FKBP F323Y, Fpr1 and Fpr4 NL were mixed with 25 mer nucleosome array, and arrays digested with AvaI restriction enzyme for 15 and 30 minutes. DNA was purified and subjected to agarose gel electrophoresis and ethidium bromide staining. DNA from arrays with fully protected AvaI sites migrates at 5000 bp while digested arrays liberate fragments in 197 bp increments.
    Dinucleosomes, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore nucleosome
    Cofactor steady-state kinetics are affected by the mutation of ATXR5 PHD domain. Michaelis–Menten (M–M) plot of the initial velocity versus <t>nucleosome</t> concentration ( A ) and its Lineweaver–Burk (LB) double reciprocal plot ( B ) for full-length wild-type ATXR5, L39W mutant and ATXR5 ΔPHD. The kinetics for the cofactor were performed using 8 μM of recombinant nucleosome. The M-M plot of the initial velocity versus cofactor concentration ( C ) and its corresponding LB plot ( D ) for the constructs used in C. ( E ) Summary of the results obtained from the M-M plots for wild-type ATXR5, L39W mutant and ATXR5 ΔPHD.
    Nucleosome, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Roche nucleosomes
    A combined single molecule – bulk FRET approach to study nucleosome stability. A ) Theoretical diagram of nucleosome stability as a function of salt and nucleosome concentration (adapted from ref. [22] ). The solid line represents the amount of salt needed to destabilize <t>nucleosomes</t> at a given nucleosome concentration. Nucleosomes generally remain stable at higher concentrations and lower ionic strength, dissociation occurs at elevated ionic strength and nucleosome concentrations in the sub-nM range. The dashed line represents changes in nucleosome stability from altered nucleosome composition. B) DNA labeling for nucleosome FRET experiments. 170 bp long DNA fragments were labeled at positions -42 and +52 from the dyad axis. In the intact nucleosome both dyes are located ≈ 6 nm apart, allowing for FRET, while in a fully dissociated nucleosome or free DNA fragment both dyes are too far apart to undergo FRET. C ) (i) Schematic of confocal single molecule detection of nucleosomes in solution. A detailed description of the setup is given in Section S1 in File S1 . (ii) The passage of individual nucleosomes through the focus generates bursts of fluorescence. (iii) For each burst a proximity ratio is calculated and data binned for histogram analysis. The position of relevant subpopulations in the histogram is indicated. D ) (i) Schematic setup for microplate-scanning FRET (μpsFRET). Samples are loaded into a 384-well multiplate and imaged in three spectral channels using a commercial Typhoon™ multimode scanner with confocal optics (i). Grey scale images and intensity profiles of samples with different bulk FRET efficiencies (ii). Higher FRET leads to a decrease of signal in the donor channel and a corresponding increase of signal in the transfer channel. The signal in the acceptor channel remains unaffected. From these intensities P-values are calculated for each well. Abbreviations: DM: dichroic mirror, F: emission filter, APD: avalanche photodiode, PMT, photomultiplier tube, PH: pinhole.
    Nucleosomes, supplied by Roche, used in various techniques. Bioz Stars score: 95/100, based on 365 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    EpiCypher nucleosome
    Conditional knockout of PKM2 in myeloid cells protects septic mice. ( a ) Western blot analysis of expression of indicated proteins in BMDMs or lung isolated from myeloid cell-specific PKM2 -knockout mice ( PKM2 −/− ) and control WT mice ( PKM2 +/+ ). ( b ) Indicated mice ( n =10 mice per group) were pre-injected with LPS (2 mg kg − 1 , intraperitoneally) for 3 h and then challenged with NLRP3 activator ATP (200 mg kg − 1 , intraperitoneally) or AIM2 activator <t>nucleosome</t> (20 mg kg − 1 , intraperitoneally). Injection with LPS (2 mg kg − 1 , intraperitoneally) alone in these mice was used as a control ( n =10 mice per group). The Kaplan–Meyer method was used to compare differences in survival rates between groups (* P
    Nucleosome, supplied by EpiCypher, used in various techniques. Bioz Stars score: 94/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Seramun nucleosomes
    Conditional knockout of PKM2 in myeloid cells protects septic mice. ( a ) Western blot analysis of expression of indicated proteins in BMDMs or lung isolated from myeloid cell-specific PKM2 -knockout mice ( PKM2 −/− ) and control WT mice ( PKM2 +/+ ). ( b ) Indicated mice ( n =10 mice per group) were pre-injected with LPS (2 mg kg − 1 , intraperitoneally) for 3 h and then challenged with NLRP3 activator ATP (200 mg kg − 1 , intraperitoneally) or AIM2 activator <t>nucleosome</t> (20 mg kg − 1 , intraperitoneally). Injection with LPS (2 mg kg − 1 , intraperitoneally) alone in these mice was used as a control ( n =10 mice per group). The Kaplan–Meyer method was used to compare differences in survival rates between groups (* P
    Nucleosomes, supplied by Seramun, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Sutter Instrument nucleosomes
    CPD repair by photolyase in ARS1 of minichromosome YRpTRURAP. ( A and B ) Primer extension products of the bottom strand (short and long gel run, respectively). ( C ) Primer extension products of the top strand. Chromatin structure is illustrated according to F.Thoma (21), S.Tanaka and F.Thoma, unpublished results: positioned <t>nucleosomes</t> (ovals), presumed nucleosome position (dashed oval); ARS1 elements (boxes, according to Diffley and Cocker, 32): B3 (position 1912–1929), B2 (position 1959–1969), B1 (position 1996–2009), A (position 2018–2028). Indicated are: pyrimidine sites used for repair calculation (filled boxes, numbers refer to the 5′ nucleotide in the YRpTRURAP sequence), sites not quantified due to low signal intensity (open boxes). The lanes represent: dideoxy-sequencing reactions A, G, C and T (lanes 1–4); DNA damaged in vitro with 40 J/m 2 (lane 5); DNA of non-irradiated cells (lane 6); DNA of cells irradiated with 100 J/m 2 (chromatin, lanes 7–15), photoreactivated for 3–120 min (lanes 9–14) or incubated in the dark for 120 min (lane 15); damaged DNA (as in lane 8), but treated with E.coli photolyase to remove CPDs and to display 6-4PPs and other non-CPD lesions (lane 7).
    Nucleosomes, supplied by Sutter Instrument, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Reaction Biology Corporation mono dinucleosomes
    CPD repair by photolyase in ARS1 of minichromosome YRpTRURAP. ( A and B ) Primer extension products of the bottom strand (short and long gel run, respectively). ( C ) Primer extension products of the top strand. Chromatin structure is illustrated according to F.Thoma (21), S.Tanaka and F.Thoma, unpublished results: positioned <t>nucleosomes</t> (ovals), presumed nucleosome position (dashed oval); ARS1 elements (boxes, according to Diffley and Cocker, 32): B3 (position 1912–1929), B2 (position 1959–1969), B1 (position 1996–2009), A (position 2018–2028). Indicated are: pyrimidine sites used for repair calculation (filled boxes, numbers refer to the 5′ nucleotide in the YRpTRURAP sequence), sites not quantified due to low signal intensity (open boxes). The lanes represent: dideoxy-sequencing reactions A, G, C and T (lanes 1–4); DNA damaged in vitro with 40 J/m 2 (lane 5); DNA of non-irradiated cells (lane 6); DNA of cells irradiated with 100 J/m 2 (chromatin, lanes 7–15), photoreactivated for 3–120 min (lanes 9–14) or incubated in the dark for 120 min (lane 15); damaged DNA (as in lane 8), but treated with E.coli photolyase to remove CPDs and to display 6-4PPs and other non-CPD lesions (lane 7).
    Mono Dinucleosomes, supplied by Reaction Biology Corporation, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Roche nucleosome enrichment
    PL enhances APR-246-induced apoptosis and autophagy in HNSCC cells ( a ) UMSCC10A cells were treated with 10 μM PL and/or 25 μM APR-246 for 24 h. After the treatments, whole cell extracts were collected for the western blot analysis. Thirty μg proteins were loaded in each lane. GAPDH serves as a loading control. ( b ) UMSCC10A cells were treated with 10 μM PL and/or 25μM APR-246 in the presence or absence of 20 μM z-VAD-fmk for 72 h. After the treatment, cell apoptosis was quantified using a cell death ELISA kit (Roche Diagnostics) showing enrichment of <t>nucleosomes</t> in the cytoplasmic fraction of the cells. Values represent the mean ± S.D. * P
    Nucleosome Enrichment, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Illumina Inc nucleosome positions
    Different promoter types are differently packaged. ( A ) Cumulative distribution function (CDF) plots for two significant Kolmogorov-Smirnov (KS) enrichments. The gene set of 270 ribosomal genes is enriched for long NFRs ( left ), and close +1 to +3 <t>nucleosome</t> spacing ( right ). For example, 45% of ribosomal genes have 5′ nucleosome spacing of
    Nucleosome Positions, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 90/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa nucleosome preparation kit
    Inactivation of bptf or TGF-β signaling induces <t>nucleosome</t> repositioning within the wnt8 a promoter. A , MNase digestion of chromatin isolated from embryos at 75% epiboly stage. Digestion with 320 units per milliliters of MNase for 30 min was appropriate to produce mononucleosome-sized DNAs. B , C , The dynamic changes of nucleosomal positions at the wnt8a promoter in bptf morphants ( B ) or Δ kT β RII- overexpressing embryos ( C ). There were five positioned nucleosomes (N1, N2, N3, N4, and N5) within the −1449 to −416 region of the wnt8a promoter in cMO-injected embryos. Bptf (green) and Smad2 (red) binding motifs were located in the DNA sequences occupied by N3. A solid increase in DNA amount was detected at N3 positioning site in bptf morphants and Δ kT β RII- overexpressing embryos. NS, Nonsignificant. ** p
    Nucleosome Preparation Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs nucleosome control dna
    Mhrt inhibits chromatin targeting and gene regulation by Brg1 a, Gel electrophoresis and quantitation of nucleosomal 5SrDNA, Myh6 promoter and Neo <t>DNA.</t> Arrowheads: DNA-histone complex. Arrows: naked DNA. <t>Nucleosome</t> assembly efficiency is defined as the fraction of DNA bound to histones (arrowheads). P-value: Student’s t-test. Error bar: standard error of the mean (SEM). b-d, Quantification of amylose pull-down of MBP-D1D2 (D1D2) with nucleosomal and naked Myh6 promoter DNA ( b ), with nucleosomal Myh6 promoter, Neo , and 5SrDNA ( c ), or with nucleosomal Myh6 promoter in the presence of Mhrt779 ( d ). P-value: Student’s t-test. Error bar: SEM. e, Amylose pull-down of MBP-D1D2 and histone 3. Anti-histone 3 and anti-MBP antibodies were used for western blot analysis. f, ChIP analysis of Brg1 on chromatinized and naked Myh6 promoter in rat ventricular cardiomyocytes. GFP: green fluorescence protein control. P-value: Student’s t-test. Error bar: SEM. g, h, Luciferase reporter activity of Brg1 on naked Myh6 promoter ( g ) or of helicase-deficient Brg1 on chromatinized Myh6 promoter ( h ) in rat ventricular cardiomyocytes. ΔD1: Brg1 lacking amino acid 774–913; ΔD2: Brg1 lacking 1086–1246. GFP: green fluorescence protein control. ChIP: H-10 antibody recognizing N-terminus, non-disrupted region of Brg1. P-value: Student’s t-test. Error bar: SEM. i, j, ChIP analysis in SW13 cells of chromatinized Myh6 promoter in the presence of Mhrt779 ( i ) or helicase-deficient Brg1 ( j ). Vector: pAdd2 empty vector. Mhrt : pAdd2- Mhrt779 . P-value: Student’s t-test. Error bar: SEM. k, Schematic illustration and PCR of human MHRT. MHRT originates from MYH7 and is transcribed into MYH7. MYH7 e xons and introns are indicated. R1 and R2 are strand-specific PCR primers; F1 and R1 target MHRT and MYH7 ; F2 and R2 are specific for MHRT . l, Quantification of MHRT in human heart tissues. Ctrl: control. LVH: left ventricular hypertrophy. ICM: ischemic cardiomyopathy. IDCM: idiopathic dilated cardiomyopathy. P-value: Student’s t-test. Error bar: SEM. m, Working model of a Brg1- Mhrt negative feedback circuit in the heart. Brg1 represses Mhrt transcription, whereas Mhrt prevents Brg1 from recognizing its chromatin targets. Brg1 functions through two distinct promoter elements to bidirectionally repress Myh6 and Mhrt expression. n , Molecular model of how Brg1 binds to its genomic DNA targets. Brg1 helicase (D1D2) binds chromatinized DNA, C-terminal extension (CTE) binds histone 3 (H3), and bromodomain binds acetylated (Ac) histone 3 or 4 (H4).
    Nucleosome Control Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore nucleosome coated plates
    B-Cell-Intrinsic IFNαR 1 Is Required for ANA-Producing AFC Responses in B6. Sle1b Mice (A) Flow cytometric analysis of surface expression of IFNαR 1 on B220 + B cells in B6. Sle1b and B6. Sle1b .IFNαR 1 −/− chimeric mice 3 months after BM cell transfer. (B and C) The percentages of B220 + GL-7 hi Fas hi GC B cells (B) and CD4 + CXCR5 hi PD-1 hi Tfh cells (C) in total splenocytes of the chimeras. (D and E) Numbers of dsDNA-specific (D) and <t>nucleosome-specific</t> (E) splenic AFCs in chimeric mice described in (A)–(C). (F and G) Numbers of dsDNA-specific (F) and nucleosome-specific (G) long-lived bone marrow AFCs in chimeric mice described in (A)–(C). (H) Analysis of serum titers of total IgG2c antibodies in these mice. (I and J) Analysis of dsDNA-reactive (I) and nucleosome-reactive IgG2c (J) in the sera of these mice. These data represent one experiment of four or five mice of each genotype. Statistical significance was determined using an unpaired, nonparametric Mann-Whitney Student’s t test (NS, not significant, *p
    Nucleosome Coated Plates, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore nucleosome elisa
    Bcl-2 and Bcl-X L prevent the redistribution of cytochrome c in cells undergoing apoptosis. (A) Time course of cytochrome c release in Jurkat cells treated with TG. The release of cytochrome c in the cytosolic extract was determined by Western blot analysis and was quantified by densitometric scanning of the autoradiograph and plotted against time in hours after TG treatment. (B) Redistribution of cytochrome c in Bcl-2- and Bcl-X L -overexpressing Jurkat cells. JT/Neo, JT/Bcl-2, and JT/Bcl-X L cells were treated with 100 nM TG. Jurkat T cells were pretreated with the caspase inhibitor z-VAD-fmk (50 μM) for 1 h prior to addition of TG (right panel). After 3 h, the cells were mechanically lysed and separated into mitochondrial (M) and S100 (S) fractions. The amounts of cytochrome c and cytochrome oxidase (subunit IV) present in each fraction were determined by Western blot analysis. (C) Bcl-2 or Bcl-X L blocks TG-induced caspase-3 activation. Jurkat cells were treated with TG (100 nM) for various times. Caspase-3 activity was measured as specified by the manufacturer (see Materials and Methods). (D) The caspase inhibitors z-VAD-fmk and z-DEVD-fmk block TG- and CPA-induced apoptosis. Jurkat T cells were pretreated with the caspase inhibitor z-VAD-fmk (50 μM) or z-DEVD-fmk (50 μM) for 1 h and then treated with TG or CPA for an additional 36 h. Apoptosis was measured by a <t>nucleosome</t> <t>ELISA.</t>
    Nucleosome Elisa, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Active Motif recombinant nucleosomes
    ZRF1 facilitates the assembly of the UV – DDB – CUL4A E3 ligase complex. (A) ZRF1 displaces RING1B from chromatin during NER. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A ubiquitin and RING1B abundance was calculated. Values are given as mean ± SEM ( n = 3). (B) ZRF1 regulates chromatin association of CUL4A and CUL4B. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative CUL4B and CUL4A abundance was calculated. Values are given as mean ± SEM ( n = 3). (C) ZRF1 regulates CUL4A association with H2AX containing <t>nucleosomes.</t> Control cells and ZRF1 knockdown cells expressing FLAG H2AX were irradiated with UV. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (D) Knockdown of ZRF1 modulates CUL4A association with DDB2. Control cells and ZRF1 knockdown cells expressing FLAG DDB2 were irradiated with UV light. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (E) Assembly of the UV–DDB–CUL4A E3 ligase is facilitated by ZRF1. Control cells and ZRF1 knockdown HEK293T cells expressing HA RBX1 were irradiated with UV light. After immunoprecipitation with HA-specific antibodies the precipitated material was subjected to Western blotting, and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (F) ZRF1 competes with CUL4B and RING1B for DDB2 binding in vitro. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4B, and RING1B and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 over the other components (relative molarity ZRF1: DDB1–CUL4B–RING1B; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (G) ZRF1 does not compete with CUL4A and RBX1 for binding to DDB1–DDB2. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4A and RBX1 and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 (relative molarity ZRF1: DDB1–CUL4A–RBX1; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (H) ZRF1 mediates the formation of the UV-DDB-CUL4A complex in vitro. GFP and GFP-DDB2 were coupled to beads and incubated with CUL4B, DDB1 and RING1B. After washing, GFP and GFP-DDB2 (UV–RING1B complex) beads were incubated with an estimated fivefold excess of purified CUL4A and RBX1 (lanes 1–3) over the retained UV–RING1B complex. Simultaneously, ZRF1 (lanes 1 and 3) or GST (lane 2) was added to the incubations in equimolar amounts. The precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%.
    Recombinant Nucleosomes, supplied by Active Motif, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore bovine nucleosomes
    ZRF1 facilitates the assembly of the UV – DDB – CUL4A E3 ligase complex. (A) ZRF1 displaces RING1B from chromatin during NER. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A ubiquitin and RING1B abundance was calculated. Values are given as mean ± SEM ( n = 3). (B) ZRF1 regulates chromatin association of CUL4A and CUL4B. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative CUL4B and CUL4A abundance was calculated. Values are given as mean ± SEM ( n = 3). (C) ZRF1 regulates CUL4A association with H2AX containing <t>nucleosomes.</t> Control cells and ZRF1 knockdown cells expressing FLAG H2AX were irradiated with UV. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (D) Knockdown of ZRF1 modulates CUL4A association with DDB2. Control cells and ZRF1 knockdown cells expressing FLAG DDB2 were irradiated with UV light. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (E) Assembly of the UV–DDB–CUL4A E3 ligase is facilitated by ZRF1. Control cells and ZRF1 knockdown HEK293T cells expressing HA RBX1 were irradiated with UV light. After immunoprecipitation with HA-specific antibodies the precipitated material was subjected to Western blotting, and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (F) ZRF1 competes with CUL4B and RING1B for DDB2 binding in vitro. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4B, and RING1B and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 over the other components (relative molarity ZRF1: DDB1–CUL4B–RING1B; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (G) ZRF1 does not compete with CUL4A and RBX1 for binding to DDB1–DDB2. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4A and RBX1 and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 (relative molarity ZRF1: DDB1–CUL4A–RBX1; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (H) ZRF1 mediates the formation of the UV-DDB-CUL4A complex in vitro. GFP and GFP-DDB2 were coupled to beads and incubated with CUL4B, DDB1 and RING1B. After washing, GFP and GFP-DDB2 (UV–RING1B complex) beads were incubated with an estimated fivefold excess of purified CUL4A and RBX1 (lanes 1–3) over the retained UV–RING1B complex. Simultaneously, ZRF1 (lanes 1 and 3) or GST (lane 2) was added to the incubations in equimolar amounts. The precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%.
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    91
    Roche nucleosome detection kit
    ZRF1 facilitates the assembly of the UV – DDB – CUL4A E3 ligase complex. (A) ZRF1 displaces RING1B from chromatin during NER. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A ubiquitin and RING1B abundance was calculated. Values are given as mean ± SEM ( n = 3). (B) ZRF1 regulates chromatin association of CUL4A and CUL4B. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative CUL4B and CUL4A abundance was calculated. Values are given as mean ± SEM ( n = 3). (C) ZRF1 regulates CUL4A association with H2AX containing <t>nucleosomes.</t> Control cells and ZRF1 knockdown cells expressing FLAG H2AX were irradiated with UV. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (D) Knockdown of ZRF1 modulates CUL4A association with DDB2. Control cells and ZRF1 knockdown cells expressing FLAG DDB2 were irradiated with UV light. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (E) Assembly of the UV–DDB–CUL4A E3 ligase is facilitated by ZRF1. Control cells and ZRF1 knockdown HEK293T cells expressing HA RBX1 were irradiated with UV light. After immunoprecipitation with HA-specific antibodies the precipitated material was subjected to Western blotting, and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (F) ZRF1 competes with CUL4B and RING1B for DDB2 binding in vitro. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4B, and RING1B and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 over the other components (relative molarity ZRF1: DDB1–CUL4B–RING1B; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (G) ZRF1 does not compete with CUL4A and RBX1 for binding to DDB1–DDB2. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4A and RBX1 and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 (relative molarity ZRF1: DDB1–CUL4A–RBX1; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (H) ZRF1 mediates the formation of the UV-DDB-CUL4A complex in vitro. GFP and GFP-DDB2 were coupled to beads and incubated with CUL4B, DDB1 and RING1B. After washing, GFP and GFP-DDB2 (UV–RING1B complex) beads were incubated with an estimated fivefold excess of purified CUL4A and RBX1 (lanes 1–3) over the retained UV–RING1B complex. Simultaneously, ZRF1 (lanes 1 and 3) or GST (lane 2) was added to the incubations in equimolar amounts. The precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%.
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    97
    Bio-Rad nucleosomes
    The nucleosome containing histone H3.5 is unstable. a Sequence comparison between human H3.1, H3.2, H3.3, H3T, and H3.5. The amino acids in H3.5 that differ from those in H3.3 are indicated by black boxes with white characters. The epitope peptide sequence used to generate the H3.5 antibody is underlined. The α-helices and β-strands found in the crystal structures of the human <t>nucleosomes</t> are represented on the top of the panel. b 18 % SDS-PAGE analysis of purified histones H3.1, H3.3, H3T, and H3.5, stained with Coomassie Brilliant Blue (CBB). c Non-denaturing 6 % PAGE analysis of purified nucleosomes containing H3.1, H3.3, H3T, and H3.5, stained with ethidium bromide. Lane 1 represents the naked DNA used in the nucleosome reconstitution. Nucleosome core particles are denoted by NCPs. d Histone compositions of the purified nucleosomes containing H3.1, H3.3, H3T, and H3.5, analyzed by 18 % SDS-PAGE with Coomassie Brilliant Blue staining. e Salt resistance assays of the H3.1 and H3.3 nucleosomes and f the H3.3, H3T, and H3.5 nucleosomes. Bands corresponding to nucleosomes are indicated by NCPs. Asterisks represent bands corresponding to non-nucleosomal DNA-histone complexes [ 26 ]
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    Cell Signaling Technology Inc nucleosome
    Hierarchical looping at the gene level. Structural model of the GATA-4 gene locus in an open, active state ( left ) versus an epigenetically silent state ( right ). The GATA-4 gene has been shown to exhibit five distinct chromatin loops of ∼43, 61, 57, 157, and 109 <t>nucleosomes</t> each (colored in tan , blue , green , white , and purple , respectively); each loop is associated with enriched trimethylation of Lys27 of histone tail H3 (H3K27Me3) and PcG protein binding. The TSS, which resides between loop 3 and 4 (from 5′ to 3′), is enveloped by the chromatin loops when these contacts are enforced in our mesoscale model, suggesting a structural mechanism for epigenetic silencing of the GATA-4 gene. To see this figure in color, go online.
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    Double Helix nucleosomes
    ( A ) M601 DNA sequence used in the remodeling assays. GRP78-gene primer sequences flanking the DNA construct are underlined and CpG dinucleotides are highlighted in grey. The residues in bold denote the approximate position of the protection caused by the histone octamer on the nucleosome substrate. ( B ) Schematic representation of the procedure used to perform single molecule analyses of the remodeled products. <t>Nucleosomes</t> were assembled using the M601 DNA. After incubation with (or without) nucleosome remodeling factor, remodeled nucleosomes were methylated with the M.SssI CpG methyltransferase to create a footprint of DNA accessibility (the DNA methyltransferase only methylates cytosine residues that are not bound to the histones). After native electrophoresis, excision and elution from the gel, nucleosomes were deproteinized and the nucleosomal DNA molecules subjected to conventional bisulfite treatment to reveal their methylation pattern. Changes in these patterns were compared to the input nucleosome to assess alterations in histone–DNA contacts resulting from the action of the remodeling factors.
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    EUROIMMUN nucleosomes
    ( A ) M601 DNA sequence used in the remodeling assays. GRP78-gene primer sequences flanking the DNA construct are underlined and CpG dinucleotides are highlighted in grey. The residues in bold denote the approximate position of the protection caused by the histone octamer on the nucleosome substrate. ( B ) Schematic representation of the procedure used to perform single molecule analyses of the remodeled products. <t>Nucleosomes</t> were assembled using the M601 DNA. After incubation with (or without) nucleosome remodeling factor, remodeled nucleosomes were methylated with the M.SssI CpG methyltransferase to create a footprint of DNA accessibility (the DNA methyltransferase only methylates cytosine residues that are not bound to the histones). After native electrophoresis, excision and elution from the gel, nucleosomes were deproteinized and the nucleosomal DNA molecules subjected to conventional bisulfite treatment to reveal their methylation pattern. Changes in these patterns were compared to the input nucleosome to assess alterations in histone–DNA contacts resulting from the action of the remodeling factors.
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    93
    Active Motif nucleosomes
    ZRF1 facilitates the assembly of the UV – DDB – CUL4A E3 ligase complex. (A) ZRF1 displaces RING1B from chromatin during NER. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A ubiquitin and RING1B abundance was calculated. Values are given as mean ± SEM ( n = 3). (B) ZRF1 regulates chromatin association of CUL4A and CUL4B. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative CUL4B and CUL4A abundance was calculated. Values are given as mean ± SEM ( n = 3). (C) ZRF1 regulates CUL4A association with H2AX containing <t>nucleosomes.</t> Control cells and ZRF1 knockdown cells expressing FLAG H2AX were irradiated with UV. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (D) Knockdown of ZRF1 modulates CUL4A association with DDB2. Control cells and ZRF1 knockdown cells expressing FLAG DDB2 were irradiated with UV light. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (E) Assembly of the UV–DDB–CUL4A E3 ligase is facilitated by ZRF1. Control cells and ZRF1 knockdown HEK293T cells expressing HA RBX1 were irradiated with UV light. After immunoprecipitation with HA-specific antibodies the precipitated material was subjected to Western blotting, and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (F) ZRF1 competes with CUL4B and RING1B for DDB2 binding in vitro. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4B, and RING1B and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 over the other components (relative molarity ZRF1: DDB1–CUL4B–RING1B; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (G) ZRF1 does not compete with CUL4A and RBX1 for binding to DDB1–DDB2. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4A and RBX1 and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 (relative molarity ZRF1: DDB1–CUL4A–RBX1; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (H) ZRF1 mediates the formation of the UV-DDB-CUL4A complex in vitro. GFP and GFP-DDB2 were coupled to beads and incubated with CUL4B, DDB1 and RING1B. After washing, GFP and GFP-DDB2 (UV–RING1B complex) beads were incubated with an estimated fivefold excess of purified CUL4A and RBX1 (lanes 1–3) over the retained UV–RING1B complex. Simultaneously, ZRF1 (lanes 1 and 3) or GST (lane 2) was added to the incubations in equimolar amounts. The precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%.
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    Active Motif nucleosome purification kit
    ZRF1 facilitates the assembly of the UV – DDB – CUL4A E3 ligase complex. (A) ZRF1 displaces RING1B from chromatin during NER. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A ubiquitin and RING1B abundance was calculated. Values are given as mean ± SEM ( n = 3). (B) ZRF1 regulates chromatin association of CUL4A and CUL4B. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative CUL4B and CUL4A abundance was calculated. Values are given as mean ± SEM ( n = 3). (C) ZRF1 regulates CUL4A association with H2AX containing <t>nucleosomes.</t> Control cells and ZRF1 knockdown cells expressing FLAG H2AX were irradiated with UV. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (D) Knockdown of ZRF1 modulates CUL4A association with DDB2. Control cells and ZRF1 knockdown cells expressing FLAG DDB2 were irradiated with UV light. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (E) Assembly of the UV–DDB–CUL4A E3 ligase is facilitated by ZRF1. Control cells and ZRF1 knockdown HEK293T cells expressing HA RBX1 were irradiated with UV light. After immunoprecipitation with HA-specific antibodies the precipitated material was subjected to Western blotting, and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (F) ZRF1 competes with CUL4B and RING1B for DDB2 binding in vitro. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4B, and RING1B and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 over the other components (relative molarity ZRF1: DDB1–CUL4B–RING1B; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (G) ZRF1 does not compete with CUL4A and RBX1 for binding to DDB1–DDB2. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4A and RBX1 and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 (relative molarity ZRF1: DDB1–CUL4A–RBX1; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (H) ZRF1 mediates the formation of the UV-DDB-CUL4A complex in vitro. GFP and GFP-DDB2 were coupled to beads and incubated with CUL4B, DDB1 and RING1B. After washing, GFP and GFP-DDB2 (UV–RING1B complex) beads were incubated with an estimated fivefold excess of purified CUL4A and RBX1 (lanes 1–3) over the retained UV–RING1B complex. Simultaneously, ZRF1 (lanes 1 and 3) or GST (lane 2) was added to the incubations in equimolar amounts. The precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%.
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    Arotec Diagnostics nucleosome antigen
    Spontaneous humoral autoimmune response in Ly9 −/− (BALB/c.129) mice . (A) ANA titers in the serum of 3- to 12-month-old Ly9 +/+ (wt) and Ly9 −/− mice. (B) Representative immunofluorescence staining of permeabilized Hep-2 incubated with sera from 1-year-old wt as compared with 1-year-old Ly9 −/− mice (sera dilution 1:200). After washing, IgG was detected with anti-mouse IgG-Texas Red (red). Nucleus was stained with DAPI (blue). (C) Determination by ELISA of autoantibodies against double-stranded DNA (dsDNA) and (D) <t>nucleosome</t> in serum from 12-month-old wt and Ly9 −/− mice. Experiments were initially conducted with a total of n = 11 BALB/c (wt) and n = 15 Ly9 −/− (BALB/c.129) female mice. Small horizontal bars indicate the mean. Statistical significances are shown.
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    GraphPad Prism Inc nucleosome
    Specialization of ISWI remodellers for diverse <t>nucleosome</t> modifications a , Principal component analysis of library remodelling data. Nucleosomes, light blue; principal component (PC) weight values for remodellers, orange. Weights are scaled by a factor of 2 for visibility. b , ISWI remodelling data for selected nucleosome substrates in the library. Values capped at −4 and 4 for display purposes. All histones are unmodified unless otherwise specified. c , Single-site modifications mapped onto the nucleosome (PDB: 1KX5) and coloured according to whether they had consistently positive (green), consistently negative (red), or variable (purple) effects on nucleosome remodelling activity across all ISWI remodellers analysed.
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    Living Cell Technologies Inc nucleosomes
    Single amino acid substitutions in H2A affect the nucleosome surface and structure. ( A ) Threonine 16 of H2A is exposed on the surface of the nucleosome. A space-filling model of the canonical nucleosome was generated in PyMOL using the spheres feature. Threonine 16 on H2A is highlighted in purple. ( B ) Zoomed-in view of the region highlighted with a gray box in (A). ( C ) Zoomed-in molecular surface representations of <t>nucleosomes</t> containing threonine (C) or serine ( D ) at residue 16 of H2A (highlighted in purple) were generated in pymol using the mutagenesis and surface features. ( E ) Alanine 40 of H2A, highlighted in royal blue, interacts with isoleucine of H2B via hydrophobic side chain interactions. For all images, H3 is light blue, H4 is green, H2A is orange, and H2B is red. The crystal structure of the canonical nucleosome (PDB: 1AOI) was used for all images.
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    TaKaRa nucleosome
    Frequencies of occurrence of DNA dinucleotide steps in the +1 <t>nucleosomes</t> of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.
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    Applied DNA Sciences Inc nucleosomes
    Recombinant CHD3 and CHD4 exhibit distinct, sequence-specific nucleosome positioning behaviour . Recombinantly purified human CHD3/4 were titrated in increasing concentrations (A/C/ E : 25, 50, 75 and 100 nM; B/D: 50, 100 and 200 nM) to the indicated mono- and dinucleosomal templates containing different configurations of linker DNA (see also Materials and Methods). The reactions were started by adding ATP. <t>Nucleosomes</t> in the absence or presence of enzyme (without ATP) served as reference. To visualize the nucleosome movements, the reactions were loaded on PAA gels (ethidium bromide stain). Positions of mono- and dinucleosomes are indicated by ovals, according to ( 68 ). Filled triangles represent more intense bands, empty triangles less intense bands or no signal, comparing CHD3 and CHD4 remodeling patterns. Triangles with a dashed rim were added for better orientation in the intensity profiles (see below). Black asterisks indicate nucleosomes, which were probably pushed over the edge of the DNA strand. On the right side of each remodeling gel are intensity profiles of the indicated gel lanes, based on Multi Gauge software analysis. The triangles indicate the positions of the bands highlighted by the triangles in the respective lanes of the corresponding gel picture. All lanes shown for one remodeling template are from one gel. Number or replicates: ( A ) ≥3; ( B ) 3; ( C ) ≥3; ( D ) 3; ( E ) ≥3.
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    BPS Bioscience nucleosomes
    Reconstituted <t>nucleosome</t> from the purified histone octamer serves as a valid substrate to histone methyltransferases. (a) 4-12% native PAGE of the reconstituted nucleosome. (b) DOT1L HMTase assay demonstrating inhibition of DOT1L by SAH with recombinant
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    Bio Basic Canada nucleosomes
    Reconstituted <t>nucleosome</t> from the purified histone octamer serves as a valid substrate to histone methyltransferases. (a) 4-12% native PAGE of the reconstituted nucleosome. (b) DOT1L HMTase assay demonstrating inhibition of DOT1L by SAH with recombinant
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    Nuclei of TGCs show decreased nucleosomal stability. ( a – c ) Nucleosome stability assay using a permeabilized individual cell in the microfluidic device. ( a ) Overview of the microfluidic device and experimental procedure for the nucleosome stability assay using a single cell. ( b ) Representative green fluorescence observed in H4-GFP-TSC and -dTSC at day 8 following exposure to NaCl buffer at the indicated concentrations in the micropockets. Bars = 5 μm. ( c) Measurement of fluorescence intensity of H4-GFP in TSCs and dTSCs at day 8. Fluorescence intensities were measured using the ImageJ software, and the nuclear position in each cell was determined using the genomic DNA image obtained after GelRed staining. Values indicate the mean ± S.D. (n = 5) and are relative to the value obtained after the 0.5 M NaCl treatment, which is arbitrarily set as 1. ( d ) Nucleosome stability assay. Each sample was extracted using a buffer containing 0.5 or 1 M NaCl. Extracted proteins equivalent to 0.5 μg of genomic DNA were subjected to 15% SDS-PAGE. ( e ) Proportion of each histone in the NaCl extract to total histone. Band intensity following western blotting of 1 M NaCl-extracted histone ( d ) was calculated and normalized to that of the corresponding band in Fig. S1 . The mean ± S.D. of three independent experiments are shown. * P

    Journal: Scientific Reports

    Article Title: Nucleosomes of polyploid trophoblast giant cells mostly consist of histone variants and form a loose chromatin structure

    doi: 10.1038/s41598-018-23832-2

    Figure Lengend Snippet: Nuclei of TGCs show decreased nucleosomal stability. ( a – c ) Nucleosome stability assay using a permeabilized individual cell in the microfluidic device. ( a ) Overview of the microfluidic device and experimental procedure for the nucleosome stability assay using a single cell. ( b ) Representative green fluorescence observed in H4-GFP-TSC and -dTSC at day 8 following exposure to NaCl buffer at the indicated concentrations in the micropockets. Bars = 5 μm. ( c) Measurement of fluorescence intensity of H4-GFP in TSCs and dTSCs at day 8. Fluorescence intensities were measured using the ImageJ software, and the nuclear position in each cell was determined using the genomic DNA image obtained after GelRed staining. Values indicate the mean ± S.D. (n = 5) and are relative to the value obtained after the 0.5 M NaCl treatment, which is arbitrarily set as 1. ( d ) Nucleosome stability assay. Each sample was extracted using a buffer containing 0.5 or 1 M NaCl. Extracted proteins equivalent to 0.5 μg of genomic DNA were subjected to 15% SDS-PAGE. ( e ) Proportion of each histone in the NaCl extract to total histone. Band intensity following western blotting of 1 M NaCl-extracted histone ( d ) was calculated and normalized to that of the corresponding band in Fig. S1 . The mean ± S.D. of three independent experiments are shown. * P

    Article Snippet: MNase digestion analysis MNase digestion analysis for unfixed cells was performed using the EZ Nucleosomal DNA Prep kit (Zymo Research).

    Techniques: Stability Assay, Fluorescence, Software, Staining, SDS Page, Western Blot

    Fpr4 FKBP condenses nucleosome arrays. ( A ) The Fpr4 FKBP sensitizes nucleosome arrays to magnesium-dependent self-association. Recombinant Fpr4 FKBP was mixed with 25 mer nucleosome array in the presence of 0.25 mM, 0.5 mM and 1 mM MgCl 2 and centrifuged. Nucleosome arrays in the supernatant and pellet fraction were visualized by agarose gel electrophoresis with ethidium bromide staining. The supernatant and pellet fractions contain extended and condensed arrays, respectively. Full-length images of this cropped gel are available in the Supplementary Information File. ( B ) Chromatin fibre self-association is unique for Fpr4 but does not require peptidyl-prolyl isomerase activity. Recombinant Fpr4 FKBP, FK506-treated Fpr4 FKBP, Fpr4 FKBP F323Y, Fpr1 and Fpr4 NL were mixed with nucleosome arrays in the presence of 0.5 mM MgCl 2 and subjected to centrifugation to separate condensed and open fibres. Full-length images of this cropped gel are available in the Supplementary Information File. ( C ) The Fpr4 FKBP occupies linker regions in nucleosome arrays. Recombinant Fpr4 FKBP, FK506-treated Fpr4 FKBP, Fpr4 FKBP F323Y, Fpr1 and Fpr4 NL were mixed with 25 mer nucleosome array, and arrays digested with AvaI restriction enzyme for 15 and 30 minutes. DNA was purified and subjected to agarose gel electrophoresis and ethidium bromide staining. DNA from arrays with fully protected AvaI sites migrates at 5000 bp while digested arrays liberate fragments in 197 bp increments.

    Journal: Scientific Reports

    Article Title: Basic surface features of nuclear FKBPs facilitate chromatin binding

    doi: 10.1038/s41598-017-04194-7

    Figure Lengend Snippet: Fpr4 FKBP condenses nucleosome arrays. ( A ) The Fpr4 FKBP sensitizes nucleosome arrays to magnesium-dependent self-association. Recombinant Fpr4 FKBP was mixed with 25 mer nucleosome array in the presence of 0.25 mM, 0.5 mM and 1 mM MgCl 2 and centrifuged. Nucleosome arrays in the supernatant and pellet fraction were visualized by agarose gel electrophoresis with ethidium bromide staining. The supernatant and pellet fractions contain extended and condensed arrays, respectively. Full-length images of this cropped gel are available in the Supplementary Information File. ( B ) Chromatin fibre self-association is unique for Fpr4 but does not require peptidyl-prolyl isomerase activity. Recombinant Fpr4 FKBP, FK506-treated Fpr4 FKBP, Fpr4 FKBP F323Y, Fpr1 and Fpr4 NL were mixed with nucleosome arrays in the presence of 0.5 mM MgCl 2 and subjected to centrifugation to separate condensed and open fibres. Full-length images of this cropped gel are available in the Supplementary Information File. ( C ) The Fpr4 FKBP occupies linker regions in nucleosome arrays. Recombinant Fpr4 FKBP, FK506-treated Fpr4 FKBP, Fpr4 FKBP F323Y, Fpr1 and Fpr4 NL were mixed with 25 mer nucleosome array, and arrays digested with AvaI restriction enzyme for 15 and 30 minutes. DNA was purified and subjected to agarose gel electrophoresis and ethidium bromide staining. DNA from arrays with fully protected AvaI sites migrates at 5000 bp while digested arrays liberate fragments in 197 bp increments.

    Article Snippet: Resulting tailless dinucleosomes were confirmed by western blot analysis (anti-H3 Abcam #ab1791) and imaged using LiCor.

    Techniques: Recombinant, Agarose Gel Electrophoresis, Staining, Activity Assay, Centrifugation, Purification

    Fpr4 interacts with nucleosomes through the NPL and FKBP domains. ( A ) Predicted structure of Fpr4 NPM core domain. Fpr4 amino acids 1–169 are overlayed with crystal structure of human nucleoplasmin NPM1 (pink, PDB:1K5J). The location of the A1/B1 loop is coloured in red for the acidic portion and blue for the basic portion (amino acids 59–116), while domains D1 and D2 are coloured grey. ( B ) Domain architecture of Fpr4. Fpr4 NPL domain is comprised of a core homopentamerization motif encoded by regions D1 and D2, and is bifurcated by the A1/B1 loop. A second acidic (A2) and basic (B2) region follow before the Fpr4 FKBP prolyl isomerase domain. ( C ) The A2 region of Fpr4 is sufficient for histone binding. Histone pulldowns using calf thymus bulk histones and 6-His Fpr4 deletion constructs. Interactions were resolved by western blotting with anti-H3. Loading is shown by Ponceau staining of nitrocellulose membrane. Full-length images of this cropped blot are available in the Supplementary Information File. ( D ) The A2 region of Fpr4 is necessary for histone binding. Histone pulldowns using recombinant human histone H3 and either 6-His Fpr4 or indicated deletion mutants. Loading is shown by western blotting with anti-6-His. *Denotes non-specific band. Full-length images of this cropped blot are available in the Supplementary Information File. ( E ) The NPL and FKBP domains bind to nucleosomes. Electrophoretic mobility shift assay using either naked 601 template DNA, mononucleosomes or dinucleosomes. Recombinant histone H1, Fpr4 NL (1–280) or Fpr4 FKBP (280–392) were added to indicated substrates and binding inferred from mobility shifts visualized by agarose gel electrophoresis and ethidium bromide staining. ( F ) Histone H1 and Fpr4 FKBP do not co-occupy nucleosomes. Recombinant histone H1 was added to monucleosomes or dinucleosomes before (lanes 4, 10), after (lanes 5, 11), or simultaneously (lanes 6, 12) to the addition of Fpr4 FKBP. Gel shifts were visualized agarose gel electrophoresis and ethidium bromide staining.

    Journal: Scientific Reports

    Article Title: Basic surface features of nuclear FKBPs facilitate chromatin binding

    doi: 10.1038/s41598-017-04194-7

    Figure Lengend Snippet: Fpr4 interacts with nucleosomes through the NPL and FKBP domains. ( A ) Predicted structure of Fpr4 NPM core domain. Fpr4 amino acids 1–169 are overlayed with crystal structure of human nucleoplasmin NPM1 (pink, PDB:1K5J). The location of the A1/B1 loop is coloured in red for the acidic portion and blue for the basic portion (amino acids 59–116), while domains D1 and D2 are coloured grey. ( B ) Domain architecture of Fpr4. Fpr4 NPL domain is comprised of a core homopentamerization motif encoded by regions D1 and D2, and is bifurcated by the A1/B1 loop. A second acidic (A2) and basic (B2) region follow before the Fpr4 FKBP prolyl isomerase domain. ( C ) The A2 region of Fpr4 is sufficient for histone binding. Histone pulldowns using calf thymus bulk histones and 6-His Fpr4 deletion constructs. Interactions were resolved by western blotting with anti-H3. Loading is shown by Ponceau staining of nitrocellulose membrane. Full-length images of this cropped blot are available in the Supplementary Information File. ( D ) The A2 region of Fpr4 is necessary for histone binding. Histone pulldowns using recombinant human histone H3 and either 6-His Fpr4 or indicated deletion mutants. Loading is shown by western blotting with anti-6-His. *Denotes non-specific band. Full-length images of this cropped blot are available in the Supplementary Information File. ( E ) The NPL and FKBP domains bind to nucleosomes. Electrophoretic mobility shift assay using either naked 601 template DNA, mononucleosomes or dinucleosomes. Recombinant histone H1, Fpr4 NL (1–280) or Fpr4 FKBP (280–392) were added to indicated substrates and binding inferred from mobility shifts visualized by agarose gel electrophoresis and ethidium bromide staining. ( F ) Histone H1 and Fpr4 FKBP do not co-occupy nucleosomes. Recombinant histone H1 was added to monucleosomes or dinucleosomes before (lanes 4, 10), after (lanes 5, 11), or simultaneously (lanes 6, 12) to the addition of Fpr4 FKBP. Gel shifts were visualized agarose gel electrophoresis and ethidium bromide staining.

    Article Snippet: Resulting tailless dinucleosomes were confirmed by western blot analysis (anti-H3 Abcam #ab1791) and imaged using LiCor.

    Techniques: Binding Assay, Construct, Western Blot, Staining, Recombinant, Electrophoretic Mobility Shift Assay, Agarose Gel Electrophoresis

    The basic patches of the Fpr4 FKBP are required for chromatin interaction. ( A ) Basic patches are required for FKBP binding to dinucleosomes. Dinucleosomes were mixed with increasing concentrations of histone H1, Fpr4 FKBP, or basic patch mutants A-D proteins and subjected to EMSAs as in Figs 1 and 2 . ( B ) Basic patches are required for FKBP promotion of magnesium-dependent chromatin fibre self-association. Recombinant Fpr4 FKBP or basic patch mutants were mixed with 25mer nucleosome array in the presence of 0.25 and 0.5 mM MgCl 2 and centrifuged. Chromatin in the supernatant and pellet fraction were visualized by agarose gel electrophoresis with ethidium bromide staining. Full-length images of this cropped gel are available in the Supplementary Information File. ( C ) Basic surface patches are required for linker region interaction. Recombinant Fpr1, Fpr4 FKBP, or basic patch mutants were mixed with 25 mer nucleosome array, and arrays digested with AvaI restriction enzyme for 0 or 15 minutes. DNA was purified and subjected to agarose gel electrophoresis and ethidium bromide staining.

    Journal: Scientific Reports

    Article Title: Basic surface features of nuclear FKBPs facilitate chromatin binding

    doi: 10.1038/s41598-017-04194-7

    Figure Lengend Snippet: The basic patches of the Fpr4 FKBP are required for chromatin interaction. ( A ) Basic patches are required for FKBP binding to dinucleosomes. Dinucleosomes were mixed with increasing concentrations of histone H1, Fpr4 FKBP, or basic patch mutants A-D proteins and subjected to EMSAs as in Figs 1 and 2 . ( B ) Basic patches are required for FKBP promotion of magnesium-dependent chromatin fibre self-association. Recombinant Fpr4 FKBP or basic patch mutants were mixed with 25mer nucleosome array in the presence of 0.25 and 0.5 mM MgCl 2 and centrifuged. Chromatin in the supernatant and pellet fraction were visualized by agarose gel electrophoresis with ethidium bromide staining. Full-length images of this cropped gel are available in the Supplementary Information File. ( C ) Basic surface patches are required for linker region interaction. Recombinant Fpr1, Fpr4 FKBP, or basic patch mutants were mixed with 25 mer nucleosome array, and arrays digested with AvaI restriction enzyme for 0 or 15 minutes. DNA was purified and subjected to agarose gel electrophoresis and ethidium bromide staining.

    Article Snippet: Resulting tailless dinucleosomes were confirmed by western blot analysis (anti-H3 Abcam #ab1791) and imaged using LiCor.

    Techniques: Binding Assay, Recombinant, Agarose Gel Electrophoresis, Staining, Purification

    Fpr4 FKBP-nucleosome interaction requires linker DNA and histone tails. ( A ) Linker DNA is required for FKBP interaction with mononucleosomes. Nucleosomes assembled with 197 bp and 147 bp Widom 601 nucleosome positioning sequence DNA were incubated with no protein, histone H1 or the Fpr4 FKBP domain and subjected to EMSA as described in the Methods. ( B ) Anti-H3 Western blot analysis of trypsinized dinucleosomes. Recombinant human histone H3 was used as a positive control. Full-length images of this cropped blot are available in the Supplementary Information File. ( C ) FKBP interaction with nucleosomes is influenced by histone tails. Recombinant histone H1 or two concentrations of Fpr4 FKBP were mixed with intact or trypsinized dinucleosomes and visualized by agarose gel electrophoresis with ethidium bromide staining.

    Journal: Scientific Reports

    Article Title: Basic surface features of nuclear FKBPs facilitate chromatin binding

    doi: 10.1038/s41598-017-04194-7

    Figure Lengend Snippet: Fpr4 FKBP-nucleosome interaction requires linker DNA and histone tails. ( A ) Linker DNA is required for FKBP interaction with mononucleosomes. Nucleosomes assembled with 197 bp and 147 bp Widom 601 nucleosome positioning sequence DNA were incubated with no protein, histone H1 or the Fpr4 FKBP domain and subjected to EMSA as described in the Methods. ( B ) Anti-H3 Western blot analysis of trypsinized dinucleosomes. Recombinant human histone H3 was used as a positive control. Full-length images of this cropped blot are available in the Supplementary Information File. ( C ) FKBP interaction with nucleosomes is influenced by histone tails. Recombinant histone H1 or two concentrations of Fpr4 FKBP were mixed with intact or trypsinized dinucleosomes and visualized by agarose gel electrophoresis with ethidium bromide staining.

    Article Snippet: Resulting tailless dinucleosomes were confirmed by western blot analysis (anti-H3 Abcam #ab1791) and imaged using LiCor.

    Techniques: Sequencing, Incubation, Western Blot, Recombinant, Positive Control, Agarose Gel Electrophoresis, Staining

    Cofactor steady-state kinetics are affected by the mutation of ATXR5 PHD domain. Michaelis–Menten (M–M) plot of the initial velocity versus nucleosome concentration ( A ) and its Lineweaver–Burk (LB) double reciprocal plot ( B ) for full-length wild-type ATXR5, L39W mutant and ATXR5 ΔPHD. The kinetics for the cofactor were performed using 8 μM of recombinant nucleosome. The M-M plot of the initial velocity versus cofactor concentration ( C ) and its corresponding LB plot ( D ) for the constructs used in C. ( E ) Summary of the results obtained from the M-M plots for wild-type ATXR5, L39W mutant and ATXR5 ΔPHD.

    Journal: Nucleic Acids Research

    Article Title: Molecular basis for the methylation specificity of ATXR5 for histone H3

    doi: 10.1093/nar/gkx224

    Figure Lengend Snippet: Cofactor steady-state kinetics are affected by the mutation of ATXR5 PHD domain. Michaelis–Menten (M–M) plot of the initial velocity versus nucleosome concentration ( A ) and its Lineweaver–Burk (LB) double reciprocal plot ( B ) for full-length wild-type ATXR5, L39W mutant and ATXR5 ΔPHD. The kinetics for the cofactor were performed using 8 μM of recombinant nucleosome. The M-M plot of the initial velocity versus cofactor concentration ( C ) and its corresponding LB plot ( D ) for the constructs used in C. ( E ) Summary of the results obtained from the M-M plots for wild-type ATXR5, L39W mutant and ATXR5 ΔPHD.

    Article Snippet: Tritiated S -adenosyl-l -methionine ([3 H-methyl ]-AdoMet; PerkinElmer Life Sciences; catalog no. NET155V250UC) was used as a methyl donor and methylated nucleosome was captured and quantified on filter plate (Millipore; catalog no. MSFBN6B50).

    Techniques: Mutagenesis, Concentration Assay, Recombinant, Construct

    Increasing cofactor concentration bypasses the inability of ATXR5 mutant to bind the nucleosome. EMSA experiments were performed by incubating increasing amounts of ATXR5 WT in presence of 10X ( A ) or 1000X ( C ) of sinefugin (SFG) and fixed amounts of fluorescently-labeled NCP. Similar EMSAs were performed with ATXR5 L39W in 10X ( B ) or 1000X ( D ). Reactions were resolved on non-denaturing polyacrylamide gels. The * indicates the ATXR5–NCP complexes and the protein concentrations are indicated on the top of each gel. ( E ) GST pull-down assays showing the interaction between ATXR5 PHD domain with untagged ATXR5 SET domain in presence of sinefungin. Proteins were resolved on a denaturating SDS-PAGE gel and stained with Coomassie Brilliant Blue. ( F ) Model of the regulatory elements controlling the activity of ATXR5. The SET and PHD domains of ATXR5 are colored in green and blue respectively and are shown in two different conformations on active (top) or silent (bottom) chromatin and the cofactor is represented by a yellow circle. Histone tails of H3.3 and H3.1 are shown as black lines and the residues with their respective PTMs are indicated.

    Journal: Nucleic Acids Research

    Article Title: Molecular basis for the methylation specificity of ATXR5 for histone H3

    doi: 10.1093/nar/gkx224

    Figure Lengend Snippet: Increasing cofactor concentration bypasses the inability of ATXR5 mutant to bind the nucleosome. EMSA experiments were performed by incubating increasing amounts of ATXR5 WT in presence of 10X ( A ) or 1000X ( C ) of sinefugin (SFG) and fixed amounts of fluorescently-labeled NCP. Similar EMSAs were performed with ATXR5 L39W in 10X ( B ) or 1000X ( D ). Reactions were resolved on non-denaturing polyacrylamide gels. The * indicates the ATXR5–NCP complexes and the protein concentrations are indicated on the top of each gel. ( E ) GST pull-down assays showing the interaction between ATXR5 PHD domain with untagged ATXR5 SET domain in presence of sinefungin. Proteins were resolved on a denaturating SDS-PAGE gel and stained with Coomassie Brilliant Blue. ( F ) Model of the regulatory elements controlling the activity of ATXR5. The SET and PHD domains of ATXR5 are colored in green and blue respectively and are shown in two different conformations on active (top) or silent (bottom) chromatin and the cofactor is represented by a yellow circle. Histone tails of H3.3 and H3.1 are shown as black lines and the residues with their respective PTMs are indicated.

    Article Snippet: Tritiated S -adenosyl-l -methionine ([3 H-methyl ]-AdoMet; PerkinElmer Life Sciences; catalog no. NET155V250UC) was used as a methyl donor and methylated nucleosome was captured and quantified on filter plate (Millipore; catalog no. MSFBN6B50).

    Techniques: Concentration Assay, Mutagenesis, Labeling, SDS Page, Staining, Activity Assay

    A combined single molecule – bulk FRET approach to study nucleosome stability. A ) Theoretical diagram of nucleosome stability as a function of salt and nucleosome concentration (adapted from ref. [22] ). The solid line represents the amount of salt needed to destabilize nucleosomes at a given nucleosome concentration. Nucleosomes generally remain stable at higher concentrations and lower ionic strength, dissociation occurs at elevated ionic strength and nucleosome concentrations in the sub-nM range. The dashed line represents changes in nucleosome stability from altered nucleosome composition. B) DNA labeling for nucleosome FRET experiments. 170 bp long DNA fragments were labeled at positions -42 and +52 from the dyad axis. In the intact nucleosome both dyes are located ≈ 6 nm apart, allowing for FRET, while in a fully dissociated nucleosome or free DNA fragment both dyes are too far apart to undergo FRET. C ) (i) Schematic of confocal single molecule detection of nucleosomes in solution. A detailed description of the setup is given in Section S1 in File S1 . (ii) The passage of individual nucleosomes through the focus generates bursts of fluorescence. (iii) For each burst a proximity ratio is calculated and data binned for histogram analysis. The position of relevant subpopulations in the histogram is indicated. D ) (i) Schematic setup for microplate-scanning FRET (μpsFRET). Samples are loaded into a 384-well multiplate and imaged in three spectral channels using a commercial Typhoon™ multimode scanner with confocal optics (i). Grey scale images and intensity profiles of samples with different bulk FRET efficiencies (ii). Higher FRET leads to a decrease of signal in the donor channel and a corresponding increase of signal in the transfer channel. The signal in the acceptor channel remains unaffected. From these intensities P-values are calculated for each well. Abbreviations: DM: dichroic mirror, F: emission filter, APD: avalanche photodiode, PMT, photomultiplier tube, PH: pinhole.

    Journal: PLoS ONE

    Article Title: Closing the Gap between Single Molecule and Bulk FRET Analysis of Nucleosomes

    doi: 10.1371/journal.pone.0057018

    Figure Lengend Snippet: A combined single molecule – bulk FRET approach to study nucleosome stability. A ) Theoretical diagram of nucleosome stability as a function of salt and nucleosome concentration (adapted from ref. [22] ). The solid line represents the amount of salt needed to destabilize nucleosomes at a given nucleosome concentration. Nucleosomes generally remain stable at higher concentrations and lower ionic strength, dissociation occurs at elevated ionic strength and nucleosome concentrations in the sub-nM range. The dashed line represents changes in nucleosome stability from altered nucleosome composition. B) DNA labeling for nucleosome FRET experiments. 170 bp long DNA fragments were labeled at positions -42 and +52 from the dyad axis. In the intact nucleosome both dyes are located ≈ 6 nm apart, allowing for FRET, while in a fully dissociated nucleosome or free DNA fragment both dyes are too far apart to undergo FRET. C ) (i) Schematic of confocal single molecule detection of nucleosomes in solution. A detailed description of the setup is given in Section S1 in File S1 . (ii) The passage of individual nucleosomes through the focus generates bursts of fluorescence. (iii) For each burst a proximity ratio is calculated and data binned for histogram analysis. The position of relevant subpopulations in the histogram is indicated. D ) (i) Schematic setup for microplate-scanning FRET (μpsFRET). Samples are loaded into a 384-well multiplate and imaged in three spectral channels using a commercial Typhoon™ multimode scanner with confocal optics (i). Grey scale images and intensity profiles of samples with different bulk FRET efficiencies (ii). Higher FRET leads to a decrease of signal in the donor channel and a corresponding increase of signal in the transfer channel. The signal in the acceptor channel remains unaffected. From these intensities P-values are calculated for each well. Abbreviations: DM: dichroic mirror, F: emission filter, APD: avalanche photodiode, PMT, photomultiplier tube, PH: pinhole.

    Article Snippet: b) Confocal single molecule experiments For smFRET experiments, nucleosomes were freshly diluted into the experimental buffer; TE buffer, pH 7.5, supplemented with 0.01% Nonidet P40 (Roche Diagnostics), 0.5 mM ascorbic acid to minimize photobleaching, and NaCl as noted.

    Techniques: Concentration Assay, DNA Labeling, Labeling, Fluorescence

    smFRET results on nucleosome stability are consistent to μpsFRET data. ( A ) Average proximity ratio calculated from all photons from double-labeled nucleosomes as a function of salt concentration. Photons from the donor and transfer channel were summed for all detected molecules, except donor-only and acceptor-only species. ( B ) Salt dependence of the fraction of intact nucleosomes in smFRET histograms from Fig. 5. For each histogram, the donor-only and acceptor-only population was excluded from the analysis. The relative fraction of FRET-active molecules (0.25

    Journal: PLoS ONE

    Article Title: Closing the Gap between Single Molecule and Bulk FRET Analysis of Nucleosomes

    doi: 10.1371/journal.pone.0057018

    Figure Lengend Snippet: smFRET results on nucleosome stability are consistent to μpsFRET data. ( A ) Average proximity ratio calculated from all photons from double-labeled nucleosomes as a function of salt concentration. Photons from the donor and transfer channel were summed for all detected molecules, except donor-only and acceptor-only species. ( B ) Salt dependence of the fraction of intact nucleosomes in smFRET histograms from Fig. 5. For each histogram, the donor-only and acceptor-only population was excluded from the analysis. The relative fraction of FRET-active molecules (0.25

    Article Snippet: b) Confocal single molecule experiments For smFRET experiments, nucleosomes were freshly diluted into the experimental buffer; TE buffer, pH 7.5, supplemented with 0.01% Nonidet P40 (Roche Diagnostics), 0.5 mM ascorbic acid to minimize photobleaching, and NaCl as noted.

    Techniques: Labeling, Concentration Assay

    Characterization of microplate-scanning FRET spectroscopy (μpsFRET). A) μpsFRET grey scale images of a nucleosome sample (nuc) and a DNA fragment (DNA) at different sample concentrations (donor channel: excitation at 488 nm, detection at 500–540 nm; transfer channel: excitation at 488 nm, detection at 595–625 nm; acceptor channel: excitation at 532 nm, detection at 595–625 nm). Due to the absence of FRET, the DNA sample has a lower signal in the transfer channel. Concentrations are (from left to right): 2.5 nM, 1.7 nM, 1.1 nM, 600 pM, 350 pM, 180 pM, 120 pM, 70 p M, 40 pM, 20 pM, The last row to the right contained pure buffer solution. B ) A plot showing the integrated fluorescence signal (donor channel + transfer channel) as a function of sample concentration. The measured intensity is linear throughout the dilution series. Concentrations below 50 pM can still be distinguished from background. C ) A plot showing calculated P-values of nucleosomes and DNA as a function of sample concentration. For both samples P-values were consistent at larger concentrations, while for DNA P deviated at concentrations lower than 200 pM. Nucleosomal P-values were consistent to slightly lower concentrations (100 pM). D ) Noise analysis of P-values from a donor-only sample under sub-nanomolar concentrations. Black circles are experimental standard deviations from 25 wells, white circles show estimated shot noise values. The low signal to noise level at lowest concentrations results in a large well-to-well variation in P. Shot noise accounts for

    Journal: PLoS ONE

    Article Title: Closing the Gap between Single Molecule and Bulk FRET Analysis of Nucleosomes

    doi: 10.1371/journal.pone.0057018

    Figure Lengend Snippet: Characterization of microplate-scanning FRET spectroscopy (μpsFRET). A) μpsFRET grey scale images of a nucleosome sample (nuc) and a DNA fragment (DNA) at different sample concentrations (donor channel: excitation at 488 nm, detection at 500–540 nm; transfer channel: excitation at 488 nm, detection at 595–625 nm; acceptor channel: excitation at 532 nm, detection at 595–625 nm). Due to the absence of FRET, the DNA sample has a lower signal in the transfer channel. Concentrations are (from left to right): 2.5 nM, 1.7 nM, 1.1 nM, 600 pM, 350 pM, 180 pM, 120 pM, 70 p M, 40 pM, 20 pM, The last row to the right contained pure buffer solution. B ) A plot showing the integrated fluorescence signal (donor channel + transfer channel) as a function of sample concentration. The measured intensity is linear throughout the dilution series. Concentrations below 50 pM can still be distinguished from background. C ) A plot showing calculated P-values of nucleosomes and DNA as a function of sample concentration. For both samples P-values were consistent at larger concentrations, while for DNA P deviated at concentrations lower than 200 pM. Nucleosomal P-values were consistent to slightly lower concentrations (100 pM). D ) Noise analysis of P-values from a donor-only sample under sub-nanomolar concentrations. Black circles are experimental standard deviations from 25 wells, white circles show estimated shot noise values. The low signal to noise level at lowest concentrations results in a large well-to-well variation in P. Shot noise accounts for

    Article Snippet: b) Confocal single molecule experiments For smFRET experiments, nucleosomes were freshly diluted into the experimental buffer; TE buffer, pH 7.5, supplemented with 0.01% Nonidet P40 (Roche Diagnostics), 0.5 mM ascorbic acid to minimize photobleaching, and NaCl as noted.

    Techniques: Spectroscopy, Fluorescence, Concentration Assay

    smFRET analysis reveals a conformational transition prior to nucleosome unwrapping. A,B ) smFRET histograms of non-acetylated and H3-acetylated nucleosomes at various salt concentrations and 300 pM total nucleosome concentration. Above 300 mM NaCl, a fraction of H3-acetylated nucleosomes populates a second conformation with slightly increased proximity ratio compared to non-acetylated nucleosomes, which appear to retain their initial structure. C, D ) Overlay of histograms for salt concentrations between 150 mM and 600 mM NaCl for non-acetylated (C) and H3-acetylated nucleosomes (D). Data were smoothed once to better visualize the gradual transition of nucleosomes into the high FRET state.

    Journal: PLoS ONE

    Article Title: Closing the Gap between Single Molecule and Bulk FRET Analysis of Nucleosomes

    doi: 10.1371/journal.pone.0057018

    Figure Lengend Snippet: smFRET analysis reveals a conformational transition prior to nucleosome unwrapping. A,B ) smFRET histograms of non-acetylated and H3-acetylated nucleosomes at various salt concentrations and 300 pM total nucleosome concentration. Above 300 mM NaCl, a fraction of H3-acetylated nucleosomes populates a second conformation with slightly increased proximity ratio compared to non-acetylated nucleosomes, which appear to retain their initial structure. C, D ) Overlay of histograms for salt concentrations between 150 mM and 600 mM NaCl for non-acetylated (C) and H3-acetylated nucleosomes (D). Data were smoothed once to better visualize the gradual transition of nucleosomes into the high FRET state.

    Article Snippet: b) Confocal single molecule experiments For smFRET experiments, nucleosomes were freshly diluted into the experimental buffer; TE buffer, pH 7.5, supplemented with 0.01% Nonidet P40 (Roche Diagnostics), 0.5 mM ascorbic acid to minimize photobleaching, and NaCl as noted.

    Techniques: Concentration Assay

    Working range of conventional and quasi-bulk single particle FRET. A – C ) smFRET histograms and burst size to burst duration distributions for a binary DNA mixture (noFRET and FRET-active) at 60 pM (A), 150 pM (B), and 330 pM (C) sample concentrations. While at 60 pM both subpopulations are clearly separated, coincident detection of both species occurs at 150 pM and above. The presence of multi-particle events is evident from the burst size to burst duration distribution. While at 50 pM burst duration and burst size strongly correlate, additional populations appear outside the ellipsoidal point cloud at higher sample concentrations. D, E ) Principle of quasi-bulk smFRET of nucleosomes. Nucleosomes were reconstituted on 5S rDNA (D) or the high affinity Widom 601 sequence (E). Histograms are shown for 5 mM or 150 mM salt concentrations and in the presence or absence of 10 nM unlabeled nucleosomes. At 5 mM NaCl (left panels) most nucleosomes were intact as expected from Figure 1A . At 150 mM NaCl (right panels) and in the absence of unlabeled nucleosomes, diluted nucleosomes dissociated, whereas under quasi-bulk conditions, nucleosomes on both 5S and 601 DNA remained intact.

    Journal: PLoS ONE

    Article Title: Closing the Gap between Single Molecule and Bulk FRET Analysis of Nucleosomes

    doi: 10.1371/journal.pone.0057018

    Figure Lengend Snippet: Working range of conventional and quasi-bulk single particle FRET. A – C ) smFRET histograms and burst size to burst duration distributions for a binary DNA mixture (noFRET and FRET-active) at 60 pM (A), 150 pM (B), and 330 pM (C) sample concentrations. While at 60 pM both subpopulations are clearly separated, coincident detection of both species occurs at 150 pM and above. The presence of multi-particle events is evident from the burst size to burst duration distribution. While at 50 pM burst duration and burst size strongly correlate, additional populations appear outside the ellipsoidal point cloud at higher sample concentrations. D, E ) Principle of quasi-bulk smFRET of nucleosomes. Nucleosomes were reconstituted on 5S rDNA (D) or the high affinity Widom 601 sequence (E). Histograms are shown for 5 mM or 150 mM salt concentrations and in the presence or absence of 10 nM unlabeled nucleosomes. At 5 mM NaCl (left panels) most nucleosomes were intact as expected from Figure 1A . At 150 mM NaCl (right panels) and in the absence of unlabeled nucleosomes, diluted nucleosomes dissociated, whereas under quasi-bulk conditions, nucleosomes on both 5S and 601 DNA remained intact.

    Article Snippet: b) Confocal single molecule experiments For smFRET experiments, nucleosomes were freshly diluted into the experimental buffer; TE buffer, pH 7.5, supplemented with 0.01% Nonidet P40 (Roche Diagnostics), 0.5 mM ascorbic acid to minimize photobleaching, and NaCl as noted.

    Techniques: Sequencing

    Acetylation of histone H3 decreases nucleosome stability. Salt-dependent proximity ratio at 1.5 nM and 300 pM nucleosome concentration measured with μpsFRET. A loss in P is interpreted as nucleosome dissociation. Salt titration curves were approximated by a sigmoidal function and nucleosome stability was quantified in terms of the c 1/2 value, the salt concentration at which P is half the maximum observed around 500–600 mM NaCl. Measured c 1/2 values were (995±20) mM and (980±15) mM for 1.5 nM and 300 pM non-acetylated nucleosomes, while measured c 1/2 -values were 120−130 mM lower for H3-acetylated nucleosomes ((875±10) mM and (850±20) mM for 1.5 nM and 300 pM).

    Journal: PLoS ONE

    Article Title: Closing the Gap between Single Molecule and Bulk FRET Analysis of Nucleosomes

    doi: 10.1371/journal.pone.0057018

    Figure Lengend Snippet: Acetylation of histone H3 decreases nucleosome stability. Salt-dependent proximity ratio at 1.5 nM and 300 pM nucleosome concentration measured with μpsFRET. A loss in P is interpreted as nucleosome dissociation. Salt titration curves were approximated by a sigmoidal function and nucleosome stability was quantified in terms of the c 1/2 value, the salt concentration at which P is half the maximum observed around 500–600 mM NaCl. Measured c 1/2 values were (995±20) mM and (980±15) mM for 1.5 nM and 300 pM non-acetylated nucleosomes, while measured c 1/2 -values were 120−130 mM lower for H3-acetylated nucleosomes ((875±10) mM and (850±20) mM for 1.5 nM and 300 pM).

    Article Snippet: b) Confocal single molecule experiments For smFRET experiments, nucleosomes were freshly diluted into the experimental buffer; TE buffer, pH 7.5, supplemented with 0.01% Nonidet P40 (Roche Diagnostics), 0.5 mM ascorbic acid to minimize photobleaching, and NaCl as noted.

    Techniques: Concentration Assay, Titration

    Nucleosomes positioned in IFN-γ promoter chromatin in CD4 T cells. (a and b) Naive CD4 T cells were cross-linked with formaldehyde, and their chromatin was treated with MNase ( 5 , 7 .5, and 10 enzyme units). DNAs were then purified, digested with the indicated RE (or left undigested; 0), and analyzed by Southern blots using a probe indicated in panel c (directly adjacent to the RE site for HincII-cut DNAs [a] or EcoRI digests [b]). (c) Diagram of RE sites and inferred nucleosome positions at the IFN-γ promoter. The fragment used as a probe in Southern blotting and primers used for LM-PCR are shown above the gene; the transcription start site (+1) is indicated by an arrow. (d) LM-PCR mapping of nucleosome boundaries in the IFN-γ promoter. DNA purified from MNase (2.5 and 1 enzyme unit)-cleaved chromatin of naive or Th1 (6-d culture) CD4 T cells was analyzed by LM-PCR using primer x (nucleosome 1) or y (nucleosome 2) and a linker primer and was analyzed by Southern blot probed with an internal oligonucleotide (1 and 2 for nucleosomes 1 and 2). As a control for MNase cleavage preferences in DNA, pure cellular DNA was analyzed using the same preparation of MNase, linker ligation, and PCR (naked DNA). Shown is an autoradiograph representative of five independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: T helper type 1-specific Brg1 recruitment and remodeling of nucleosomes positioned at the IFN-? promoter are Stat4 dependent

    doi: 10.1084/jem.20060066

    Figure Lengend Snippet: Nucleosomes positioned in IFN-γ promoter chromatin in CD4 T cells. (a and b) Naive CD4 T cells were cross-linked with formaldehyde, and their chromatin was treated with MNase ( 5 , 7 .5, and 10 enzyme units). DNAs were then purified, digested with the indicated RE (or left undigested; 0), and analyzed by Southern blots using a probe indicated in panel c (directly adjacent to the RE site for HincII-cut DNAs [a] or EcoRI digests [b]). (c) Diagram of RE sites and inferred nucleosome positions at the IFN-γ promoter. The fragment used as a probe in Southern blotting and primers used for LM-PCR are shown above the gene; the transcription start site (+1) is indicated by an arrow. (d) LM-PCR mapping of nucleosome boundaries in the IFN-γ promoter. DNA purified from MNase (2.5 and 1 enzyme unit)-cleaved chromatin of naive or Th1 (6-d culture) CD4 T cells was analyzed by LM-PCR using primer x (nucleosome 1) or y (nucleosome 2) and a linker primer and was analyzed by Southern blot probed with an internal oligonucleotide (1 and 2 for nucleosomes 1 and 2). As a control for MNase cleavage preferences in DNA, pure cellular DNA was analyzed using the same preparation of MNase, linker ligation, and PCR (naked DNA). Shown is an autoradiograph representative of five independent experiments.

    Article Snippet: For ChIP assays, T cells were briefly fixed with formaldehyde, rinsed, and lysed as for nucleosome mapping with MNase.

    Techniques: Purification, Southern Blot, Polymerase Chain Reaction, Ligation, Autoradiography

    Chromatin remodeling at the IFN-γ promoter. (a) Diagram of the IFN-γ promoter with nucleosome positions identified in Fig. 2 . Sites analyzed in the conserved IFN-γ promoter region are indicated by black bars; the positions of nucleosomes are indicated by open ovals; and the transcription start site is indicate by an arrow. Below the line, an expanded view of nucleosome 1 indicates the position of binding sites that comprise the proximal conserved element ( 46 , 47 ). Further below, nucleotide sequences in pertinent sites are indicated, with lines designating binding motifs; papers identifying these sites ( 7 , 45 , 44 ) are noted. (b) Naive CD4 T cells were analyzed directly or activated (αCD3 + αCD28), differentiated under Th1 conditions for 6 d, and rested or restimulated as described previously ( 18 ). Nuclei from each cell population were digested with MseI ( 10 , 25 , and 50 enzyme units), HaeIII ( 10 , 25 , and 50 enzyme units), or no enzyme (0 in the right lanes) as indicated. Purified genomic DNAs were then cut to completion with HincII and analyzed using Southern blots. Black dots note the positions of bands generated from an upstream MseI site (nucleotide −510). Arrows point to the bands that are indicative of accessibility to the restriction endonuclease being tested. P, parental band.

    Journal: The Journal of Experimental Medicine

    Article Title: T helper type 1-specific Brg1 recruitment and remodeling of nucleosomes positioned at the IFN-? promoter are Stat4 dependent

    doi: 10.1084/jem.20060066

    Figure Lengend Snippet: Chromatin remodeling at the IFN-γ promoter. (a) Diagram of the IFN-γ promoter with nucleosome positions identified in Fig. 2 . Sites analyzed in the conserved IFN-γ promoter region are indicated by black bars; the positions of nucleosomes are indicated by open ovals; and the transcription start site is indicate by an arrow. Below the line, an expanded view of nucleosome 1 indicates the position of binding sites that comprise the proximal conserved element ( 46 , 47 ). Further below, nucleotide sequences in pertinent sites are indicated, with lines designating binding motifs; papers identifying these sites ( 7 , 45 , 44 ) are noted. (b) Naive CD4 T cells were analyzed directly or activated (αCD3 + αCD28), differentiated under Th1 conditions for 6 d, and rested or restimulated as described previously ( 18 ). Nuclei from each cell population were digested with MseI ( 10 , 25 , and 50 enzyme units), HaeIII ( 10 , 25 , and 50 enzyme units), or no enzyme (0 in the right lanes) as indicated. Purified genomic DNAs were then cut to completion with HincII and analyzed using Southern blots. Black dots note the positions of bands generated from an upstream MseI site (nucleotide −510). Arrows point to the bands that are indicative of accessibility to the restriction endonuclease being tested. P, parental band.

    Article Snippet: For ChIP assays, T cells were briefly fixed with formaldehyde, rinsed, and lysed as for nucleosome mapping with MNase.

    Techniques: Binding Assay, Purification, Generated

    Conditional knockout of PKM2 in myeloid cells protects septic mice. ( a ) Western blot analysis of expression of indicated proteins in BMDMs or lung isolated from myeloid cell-specific PKM2 -knockout mice ( PKM2 −/− ) and control WT mice ( PKM2 +/+ ). ( b ) Indicated mice ( n =10 mice per group) were pre-injected with LPS (2 mg kg − 1 , intraperitoneally) for 3 h and then challenged with NLRP3 activator ATP (200 mg kg − 1 , intraperitoneally) or AIM2 activator nucleosome (20 mg kg − 1 , intraperitoneally). Injection with LPS (2 mg kg − 1 , intraperitoneally) alone in these mice was used as a control ( n =10 mice per group). The Kaplan–Meyer method was used to compare differences in survival rates between groups (* P

    Journal: Nature Communications

    Article Title: PKM2-dependent glycolysis promotes NLRP3 and AIM2 inflammasome activation

    doi: 10.1038/ncomms13280

    Figure Lengend Snippet: Conditional knockout of PKM2 in myeloid cells protects septic mice. ( a ) Western blot analysis of expression of indicated proteins in BMDMs or lung isolated from myeloid cell-specific PKM2 -knockout mice ( PKM2 −/− ) and control WT mice ( PKM2 +/+ ). ( b ) Indicated mice ( n =10 mice per group) were pre-injected with LPS (2 mg kg − 1 , intraperitoneally) for 3 h and then challenged with NLRP3 activator ATP (200 mg kg − 1 , intraperitoneally) or AIM2 activator nucleosome (20 mg kg − 1 , intraperitoneally). Injection with LPS (2 mg kg − 1 , intraperitoneally) alone in these mice was used as a control ( n =10 mice per group). The Kaplan–Meyer method was used to compare differences in survival rates between groups (* P

    Article Snippet: Purified nucleosome (#16-0002) was obtained from EpiCypher (Research Triangle Park, NC, USA)

    Techniques: Knock-Out, Mouse Assay, Western Blot, Expressing, Isolation, Injection

    CPD repair by photolyase in ARS1 of minichromosome YRpTRURAP. ( A and B ) Primer extension products of the bottom strand (short and long gel run, respectively). ( C ) Primer extension products of the top strand. Chromatin structure is illustrated according to F.Thoma (21), S.Tanaka and F.Thoma, unpublished results: positioned nucleosomes (ovals), presumed nucleosome position (dashed oval); ARS1 elements (boxes, according to Diffley and Cocker, 32): B3 (position 1912–1929), B2 (position 1959–1969), B1 (position 1996–2009), A (position 2018–2028). Indicated are: pyrimidine sites used for repair calculation (filled boxes, numbers refer to the 5′ nucleotide in the YRpTRURAP sequence), sites not quantified due to low signal intensity (open boxes). The lanes represent: dideoxy-sequencing reactions A, G, C and T (lanes 1–4); DNA damaged in vitro with 40 J/m 2 (lane 5); DNA of non-irradiated cells (lane 6); DNA of cells irradiated with 100 J/m 2 (chromatin, lanes 7–15), photoreactivated for 3–120 min (lanes 9–14) or incubated in the dark for 120 min (lane 15); damaged DNA (as in lane 8), but treated with E.coli photolyase to remove CPDs and to display 6-4PPs and other non-CPD lesions (lane 7).

    Journal: Nucleic Acids Research

    Article Title: DNA repair in a yeast origin of replication: contributions of photolyase and nucleotide excision repair

    doi:

    Figure Lengend Snippet: CPD repair by photolyase in ARS1 of minichromosome YRpTRURAP. ( A and B ) Primer extension products of the bottom strand (short and long gel run, respectively). ( C ) Primer extension products of the top strand. Chromatin structure is illustrated according to F.Thoma (21), S.Tanaka and F.Thoma, unpublished results: positioned nucleosomes (ovals), presumed nucleosome position (dashed oval); ARS1 elements (boxes, according to Diffley and Cocker, 32): B3 (position 1912–1929), B2 (position 1959–1969), B1 (position 1996–2009), A (position 2018–2028). Indicated are: pyrimidine sites used for repair calculation (filled boxes, numbers refer to the 5′ nucleotide in the YRpTRURAP sequence), sites not quantified due to low signal intensity (open boxes). The lanes represent: dideoxy-sequencing reactions A, G, C and T (lanes 1–4); DNA damaged in vitro with 40 J/m 2 (lane 5); DNA of non-irradiated cells (lane 6); DNA of cells irradiated with 100 J/m 2 (chromatin, lanes 7–15), photoreactivated for 3–120 min (lanes 9–14) or incubated in the dark for 120 min (lane 15); damaged DNA (as in lane 8), but treated with E.coli photolyase to remove CPDs and to display 6-4PPs and other non-CPD lesions (lane 7).

    Article Snippet: This result is consistent with repair in other nucleosomes (B.Suter and F.Thoma, unpublished results) and suggests a modulation of PR by nucleosomes.

    Techniques: Sequencing, In Vitro, Irradiation, Incubation

    Comparison of DNA repair in ARS1 of YRpTRURAP with chromatin structure. ( A ) PR; ( B ) NER. Indicated are a nucleosome positions (ovals), presumed nucleosome positions (dashed ovals) and functional elements of ARS1 (boxes B3, B2, B1 and A). Bars show the time (min) used to remove 50% of the lesions ( T 50% ). T 50% values were calculated from repair curves (Figs 2 and 4). Asterisks indicate data points which could be quantified in one panel only (A or B).

    Journal: Nucleic Acids Research

    Article Title: DNA repair in a yeast origin of replication: contributions of photolyase and nucleotide excision repair

    doi:

    Figure Lengend Snippet: Comparison of DNA repair in ARS1 of YRpTRURAP with chromatin structure. ( A ) PR; ( B ) NER. Indicated are a nucleosome positions (ovals), presumed nucleosome positions (dashed ovals) and functional elements of ARS1 (boxes B3, B2, B1 and A). Bars show the time (min) used to remove 50% of the lesions ( T 50% ). T 50% values were calculated from repair curves (Figs 2 and 4). Asterisks indicate data points which could be quantified in one panel only (A or B).

    Article Snippet: This result is consistent with repair in other nucleosomes (B.Suter and F.Thoma, unpublished results) and suggests a modulation of PR by nucleosomes.

    Techniques: Functional Assay

    Composition of polyY tract affects gene expression and nucleosome positioning Luciferase assays were performed after insertion of a FLUC reporter construct containing the short GT‐rich promoter and either the endogenous GPEET polyY tract (green), a long T‐rich polyY tract (light blue), or no polyY tract (dark blue). To account for technical variations, values were normalized to rRNA promoter‐driven Fluc activity. Data are presented as mean ± SD. Error bars indicate standard deviation between two replicates. Nucleosome occupancy was determined for the three cell lines described in (A). The maps are aligned to the respective splice acceptor site (position 0). The maps were generated from histone H3 MNase‐ChIP‐seq data processed with bowtie 1.1.1 ( Dataset EV2 ) and default nucwave settings (Quintales et al , 2015 ). The location of the endogenous GPEET polyY tract is highlighted in gray.

    Journal: The EMBO Journal

    Article Title: GT‐rich promoters can drive RNA pol II transcription and deposition of H2A.Z in African trypanosomes

    doi: 10.15252/embj.201695323

    Figure Lengend Snippet: Composition of polyY tract affects gene expression and nucleosome positioning Luciferase assays were performed after insertion of a FLUC reporter construct containing the short GT‐rich promoter and either the endogenous GPEET polyY tract (green), a long T‐rich polyY tract (light blue), or no polyY tract (dark blue). To account for technical variations, values were normalized to rRNA promoter‐driven Fluc activity. Data are presented as mean ± SD. Error bars indicate standard deviation between two replicates. Nucleosome occupancy was determined for the three cell lines described in (A). The maps are aligned to the respective splice acceptor site (position 0). The maps were generated from histone H3 MNase‐ChIP‐seq data processed with bowtie 1.1.1 ( Dataset EV2 ) and default nucwave settings (Quintales et al , 2015 ). The location of the endogenous GPEET polyY tract is highlighted in gray.

    Article Snippet: In addition, at the DNA level, homopolymeric sequences such as polyY tracts are intrinsically rigid and are thus strongly inhibitory to nucleosome formation (Suter et al , ).

    Techniques: Expressing, Luciferase, Construct, Activity Assay, Standard Deviation, Generated, Chromatin Immunoprecipitation

    Establishment of a high‐resolution MNase‐ChIP‐seq protocol for Trypanosoma brucei Outline of MNase‐ChIP‐seq. T. brucei cells were formaldehyde‐cross‐linked and permeabilized, and chromatin was digested into mononucleosomes using MNase. Nucleosomes containing histone H3 were isolated via affinity purification using rabbit H3 antiserum. After reversing cross‐links, the nucleosomal DNA was purified and paired‐end‐sequenced using Illumina HiSeq 2500. The sequencing reads were joined to fragments and assembled according to their midpoints. 2% agarose gel with 100 ng of mononucleosomal DNA after an MNase digest. Fragment size distribution after sequencing and joining of paired sequencing reads. Dashed lines indicate the fragment sizes 100, 137, 147, and 157 bp. Relative frequencies of AA/AT/TA/TT and CC/CG/GC/GG dinucleotides throughout 147 bp of nucleosomal DNA for each bp relative to the nucleosome dyad. Dashed lines indicate distance of 10 bp from position −74 bp.

    Journal: The EMBO Journal

    Article Title: GT‐rich promoters can drive RNA pol II transcription and deposition of H2A.Z in African trypanosomes

    doi: 10.15252/embj.201695323

    Figure Lengend Snippet: Establishment of a high‐resolution MNase‐ChIP‐seq protocol for Trypanosoma brucei Outline of MNase‐ChIP‐seq. T. brucei cells were formaldehyde‐cross‐linked and permeabilized, and chromatin was digested into mononucleosomes using MNase. Nucleosomes containing histone H3 were isolated via affinity purification using rabbit H3 antiserum. After reversing cross‐links, the nucleosomal DNA was purified and paired‐end‐sequenced using Illumina HiSeq 2500. The sequencing reads were joined to fragments and assembled according to their midpoints. 2% agarose gel with 100 ng of mononucleosomal DNA after an MNase digest. Fragment size distribution after sequencing and joining of paired sequencing reads. Dashed lines indicate the fragment sizes 100, 137, 147, and 157 bp. Relative frequencies of AA/AT/TA/TT and CC/CG/GC/GG dinucleotides throughout 147 bp of nucleosomal DNA for each bp relative to the nucleosome dyad. Dashed lines indicate distance of 10 bp from position −74 bp.

    Article Snippet: In addition, at the DNA level, homopolymeric sequences such as polyY tracts are intrinsically rigid and are thus strongly inhibitory to nucleosome formation (Suter et al , ).

    Techniques: Chromatin Immunoprecipitation, Isolation, Affinity Purification, Purification, Sequencing, Agarose Gel Electrophoresis

    Nucleosome depletion correlates with the level of gene expression Schematic display of a PTU. Nucleosome occupancy is plotted relative to the start codon (ATG) of the first gene of a PTU and averaged across all PTUs ( n = 184). The definition of the first gene of a PTU is based on a previous study (Kolev et al , 2010 ) and genome version Tb927v24. Total nucleosome occupancy is plotted relative to the ATG and averaged across all genes except the first gene of a PTU ( n = 12,220). Nucleosome occupancy is plotted relative to the splice acceptor sites and averaged across the 25% of genes containing the highest RNA levels (left panel, n = 690), the 25% of genes containing intermediate RNA levels (middle panel, n = 690), and the 25% of genes containing the lowest RNA levels (lower panel, n = 690). RNA levels were determined previously (Fadda et al , 2014 ).

    Journal: The EMBO Journal

    Article Title: GT‐rich promoters can drive RNA pol II transcription and deposition of H2A.Z in African trypanosomes

    doi: 10.15252/embj.201695323

    Figure Lengend Snippet: Nucleosome depletion correlates with the level of gene expression Schematic display of a PTU. Nucleosome occupancy is plotted relative to the start codon (ATG) of the first gene of a PTU and averaged across all PTUs ( n = 184). The definition of the first gene of a PTU is based on a previous study (Kolev et al , 2010 ) and genome version Tb927v24. Total nucleosome occupancy is plotted relative to the ATG and averaged across all genes except the first gene of a PTU ( n = 12,220). Nucleosome occupancy is plotted relative to the splice acceptor sites and averaged across the 25% of genes containing the highest RNA levels (left panel, n = 690), the 25% of genes containing intermediate RNA levels (middle panel, n = 690), and the 25% of genes containing the lowest RNA levels (lower panel, n = 690). RNA levels were determined previously (Fadda et al , 2014 ).

    Article Snippet: In addition, at the DNA level, homopolymeric sequences such as polyY tracts are intrinsically rigid and are thus strongly inhibitory to nucleosome formation (Suter et al , ).

    Techniques: Expressing

    In vivo reporter assay reveals promoter activity of distinct sequence elements Outline of the genome organization. Boundaries of PTUs are marked by nucleosomes containing different types of histone variants. H2A.Z and H2B.V (cyan nucleosomes) are located at divergent (dTSR) and non‐divergent transcription start regions (ndTSR). H3.V and H4.V (green nucleosomes) are located at transcription termination regions (TTRs). Orange arrows indicate the direction of transcription. Outline of reporter assay. A region enriched in H2A.Z (H2A.Z MNase‐ChIP‐seq data are shown as counts per billion reads, CPB; cyan), which we defined as TSR, was cloned upstream of a firefly luciferase gene ( FLUC ). The reporter construct was targeted to a non‐transcribed locus between a dTSR of chr. 1 (mRNA levels are shown in gray and were determined previously, Vasquez et al , 2014 ), containing low levels of H3.V (H3.V levels are shown in green and were determined previously, Siegel et al , 2009 ). The luciferase gene cassette consists, from 5′ to 3′, of trans ‐splicing motifs and 5′ UTR from a GPEET gene, the luciferase CDS, and the 3′ UTR of aldolase including a polyadenylation site. Gray boxes represent regions of homology. Luciferase assays were performed after insertion of complete or partial TSR DNA sequences. Two different TSR DNA sequences were analyzed (regA, blue; regB, pink). Striped bars represent the respective fragment inserted as reverse complement (regA2rc, regB1rc). To account for differences in cell number, Fluc activity was normalized to ectopically expressed Renilla luciferase activity. To account for technical variations, values were normalized to rRNA promoter‐driven Fluc activity. Data are presented as mean ± SD. Error bars indicate standard deviation between two replicates.

    Journal: The EMBO Journal

    Article Title: GT‐rich promoters can drive RNA pol II transcription and deposition of H2A.Z in African trypanosomes

    doi: 10.15252/embj.201695323

    Figure Lengend Snippet: In vivo reporter assay reveals promoter activity of distinct sequence elements Outline of the genome organization. Boundaries of PTUs are marked by nucleosomes containing different types of histone variants. H2A.Z and H2B.V (cyan nucleosomes) are located at divergent (dTSR) and non‐divergent transcription start regions (ndTSR). H3.V and H4.V (green nucleosomes) are located at transcription termination regions (TTRs). Orange arrows indicate the direction of transcription. Outline of reporter assay. A region enriched in H2A.Z (H2A.Z MNase‐ChIP‐seq data are shown as counts per billion reads, CPB; cyan), which we defined as TSR, was cloned upstream of a firefly luciferase gene ( FLUC ). The reporter construct was targeted to a non‐transcribed locus between a dTSR of chr. 1 (mRNA levels are shown in gray and were determined previously, Vasquez et al , 2014 ), containing low levels of H3.V (H3.V levels are shown in green and were determined previously, Siegel et al , 2009 ). The luciferase gene cassette consists, from 5′ to 3′, of trans ‐splicing motifs and 5′ UTR from a GPEET gene, the luciferase CDS, and the 3′ UTR of aldolase including a polyadenylation site. Gray boxes represent regions of homology. Luciferase assays were performed after insertion of complete or partial TSR DNA sequences. Two different TSR DNA sequences were analyzed (regA, blue; regB, pink). Striped bars represent the respective fragment inserted as reverse complement (regA2rc, regB1rc). To account for differences in cell number, Fluc activity was normalized to ectopically expressed Renilla luciferase activity. To account for technical variations, values were normalized to rRNA promoter‐driven Fluc activity. Data are presented as mean ± SD. Error bars indicate standard deviation between two replicates.

    Article Snippet: In addition, at the DNA level, homopolymeric sequences such as polyY tracts are intrinsically rigid and are thus strongly inhibitory to nucleosome formation (Suter et al , ).

    Techniques: In Vivo, Reporter Assay, Activity Assay, Sequencing, Chromatin Immunoprecipitation, Clone Assay, Luciferase, Construct, Standard Deviation

    TSR s exhibit increased MN ase sensitivity MNase‐ChIP‐seq data of H2A.Z‐containing mononucleosomes and total mononucleosomes (nucleosome occupancy) grouped based on size of digestion products (for outline, see Fig EV5 ). Black boxes represent open reading frames. Orange arrows indicate the direction of transcription. Shown is a representative TSR of chr. 10. The enrichment of H2A.Z and total nucleosome occupancy averaged across all divergent TSRs (left panel) and non‐divergent TSRs (right panel) are plotted relative to the midpoint of the region between the TSRs and the TSR center, respectively. Dashed lines mark the respective TSR centers.

    Journal: The EMBO Journal

    Article Title: GT‐rich promoters can drive RNA pol II transcription and deposition of H2A.Z in African trypanosomes

    doi: 10.15252/embj.201695323

    Figure Lengend Snippet: TSR s exhibit increased MN ase sensitivity MNase‐ChIP‐seq data of H2A.Z‐containing mononucleosomes and total mononucleosomes (nucleosome occupancy) grouped based on size of digestion products (for outline, see Fig EV5 ). Black boxes represent open reading frames. Orange arrows indicate the direction of transcription. Shown is a representative TSR of chr. 10. The enrichment of H2A.Z and total nucleosome occupancy averaged across all divergent TSRs (left panel) and non‐divergent TSRs (right panel) are plotted relative to the midpoint of the region between the TSRs and the TSR center, respectively. Dashed lines mark the respective TSR centers.

    Article Snippet: In addition, at the DNA level, homopolymeric sequences such as polyY tracts are intrinsically rigid and are thus strongly inhibitory to nucleosome formation (Suter et al , ).

    Techniques: Chromatin Immunoprecipitation

    PL enhances APR-246-induced apoptosis and autophagy in HNSCC cells ( a ) UMSCC10A cells were treated with 10 μM PL and/or 25 μM APR-246 for 24 h. After the treatments, whole cell extracts were collected for the western blot analysis. Thirty μg proteins were loaded in each lane. GAPDH serves as a loading control. ( b ) UMSCC10A cells were treated with 10 μM PL and/or 25μM APR-246 in the presence or absence of 20 μM z-VAD-fmk for 72 h. After the treatment, cell apoptosis was quantified using a cell death ELISA kit (Roche Diagnostics) showing enrichment of nucleosomes in the cytoplasmic fraction of the cells. Values represent the mean ± S.D. * P

    Journal: Oncogene

    Article Title: Piperlongumine and p53-Reactivator APR-246 Selectively Induce Cell Death in HNSCC by Targeting GSTP1

    doi: 10.1038/s41388-017-0110-2

    Figure Lengend Snippet: PL enhances APR-246-induced apoptosis and autophagy in HNSCC cells ( a ) UMSCC10A cells were treated with 10 μM PL and/or 25 μM APR-246 for 24 h. After the treatments, whole cell extracts were collected for the western blot analysis. Thirty μg proteins were loaded in each lane. GAPDH serves as a loading control. ( b ) UMSCC10A cells were treated with 10 μM PL and/or 25μM APR-246 in the presence or absence of 20 μM z-VAD-fmk for 72 h. After the treatment, cell apoptosis was quantified using a cell death ELISA kit (Roche Diagnostics) showing enrichment of nucleosomes in the cytoplasmic fraction of the cells. Values represent the mean ± S.D. * P

    Article Snippet: Apoptosis in cells exposed to different treatments was measured with a kit from Roche Diagnostics quantifying nucleosome enrichment in cytoplasm.

    Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

    Different promoter types are differently packaged. ( A ) Cumulative distribution function (CDF) plots for two significant Kolmogorov-Smirnov (KS) enrichments. The gene set of 270 ribosomal genes is enriched for long NFRs ( left ), and close +1 to +3 nucleosome spacing ( right ). For example, 45% of ribosomal genes have 5′ nucleosome spacing of

    Journal: Genome Research

    Article Title: High-resolution nucleosome mapping reveals transcription-dependent promoter packaging

    doi: 10.1101/gr.098509.109

    Figure Lengend Snippet: Different promoter types are differently packaged. ( A ) Cumulative distribution function (CDF) plots for two significant Kolmogorov-Smirnov (KS) enrichments. The gene set of 270 ribosomal genes is enriched for long NFRs ( left ), and close +1 to +3 nucleosome spacing ( right ). For example, 45% of ribosomal genes have 5′ nucleosome spacing of

    Article Snippet: Comparing nucleosome positions and properties before and after Pol II inactivation, we confirm the role of RNA polymerase in nucleosome eviction at promoters, and find a surprising role in retrograde movement of nucleosomes over genes.

    Techniques:

    Nucleosomes relax toward in vitro preferred locations after Pol II loss. ( A ) Three examples of promoters where data from Pol II inactivation matches in vitro nucleosome assembly data better than data from before Pol II inactivation. Shown are extended read coverage along 1000 bp centered on TSS. Numbers shown in the inset are correlations between in vitro coverage and t = 0 (blue) and t = 120 (red) in vivo coverage. ( B ). Histograms show a global shift of promoters toward the in vitro nucleosome pattern. ( C ) Normalized occupancy of −1 nucleosome better matches in vitro data after polymerase loss. For all −1 nucleosomes (called at t = 0 or at t = 120), the difference between in vivo normalized occupancy and in vitro normalized occupancy at the center of the in vivo nucleosome were calculated and presented as a histogram. ( D ) As in C , but for all +1 nucleosomes.

    Journal: Genome Research

    Article Title: High-resolution nucleosome mapping reveals transcription-dependent promoter packaging

    doi: 10.1101/gr.098509.109

    Figure Lengend Snippet: Nucleosomes relax toward in vitro preferred locations after Pol II loss. ( A ) Three examples of promoters where data from Pol II inactivation matches in vitro nucleosome assembly data better than data from before Pol II inactivation. Shown are extended read coverage along 1000 bp centered on TSS. Numbers shown in the inset are correlations between in vitro coverage and t = 0 (blue) and t = 120 (red) in vivo coverage. ( B ). Histograms show a global shift of promoters toward the in vitro nucleosome pattern. ( C ) Normalized occupancy of −1 nucleosome better matches in vitro data after polymerase loss. For all −1 nucleosomes (called at t = 0 or at t = 120), the difference between in vivo normalized occupancy and in vitro normalized occupancy at the center of the in vivo nucleosome were calculated and presented as a histogram. ( D ) As in C , but for all +1 nucleosomes.

    Article Snippet: Comparing nucleosome positions and properties before and after Pol II inactivation, we confirm the role of RNA polymerase in nucleosome eviction at promoters, and find a surprising role in retrograde movement of nucleosomes over genes.

    Techniques: In Vitro, In Vivo

    Template filtering overview. ( A ) Deep sequencing data for a typical stretch of the yeast genome. Coverage by forward-strand sequencing reads are shown as red peaks, whereas coverage by reverse-strand sequencing reads are shown as inverted green peaks. ( B ) Templates. Forward and reverse-strand read distributions are cross-correlated with each of the seven templates shown. ( C ) Correlation coefficient heat map of template 1 for forward and reverse templates at varying center positions ( x -axis) and distances ( y -axis). ( D ) Examples of templates spaced too far apart ( top ), at the optimal distance ( middle ), or too close together ( bottom ). Dotted lines indicate template outlines being compared with the underlying data. ( E ) Read distributions explained by the optimal template matches are shown as dotted lines for the region in A . ( F ) Schematic of nucleosome calls and underlying gene annotations.

    Journal: Genome Research

    Article Title: High-resolution nucleosome mapping reveals transcription-dependent promoter packaging

    doi: 10.1101/gr.098509.109

    Figure Lengend Snippet: Template filtering overview. ( A ) Deep sequencing data for a typical stretch of the yeast genome. Coverage by forward-strand sequencing reads are shown as red peaks, whereas coverage by reverse-strand sequencing reads are shown as inverted green peaks. ( B ) Templates. Forward and reverse-strand read distributions are cross-correlated with each of the seven templates shown. ( C ) Correlation coefficient heat map of template 1 for forward and reverse templates at varying center positions ( x -axis) and distances ( y -axis). ( D ) Examples of templates spaced too far apart ( top ), at the optimal distance ( middle ), or too close together ( bottom ). Dotted lines indicate template outlines being compared with the underlying data. ( E ) Read distributions explained by the optimal template matches are shown as dotted lines for the region in A . ( F ) Schematic of nucleosome calls and underlying gene annotations.

    Article Snippet: Comparing nucleosome positions and properties before and after Pol II inactivation, we confirm the role of RNA polymerase in nucleosome eviction at promoters, and find a surprising role in retrograde movement of nucleosomes over genes.

    Techniques: Sequencing

    Effects of MNase level on chromatin structure. ( A ) Mononucleosomal DNA was isolated from ladders from three different MNase titration levels, and sequenced by Illumina sequencing. ( B ) Data from titration series was subjected to template filtering to generate nucleosome calls. Width distributions for nucleosomes from the three titration steps are plotted. Green, yellow, and red correspond to under-, mid-, and overdigested chromatin, respectively. ( C ) Data for under- (green), mid- (yellow), and over- (red) digested chromatin is shown in cluster view. Genes are aligned using BY10 +1 nucleosome center (indicated); all three clusters have genes ordered by clustering for BY10 data. Red bar indicates genes with wide NFRs in mid- and overdigested chromatin (largely highly expressed genes such as ribosomal genes), which are partially filled in underdigested chromatin. ( D ) TSS-aligned nucleosome occupancy data for all genes. ( E ) Stop-codon-aligned nucleosome occupancy for all genes. ( F ) As in D , but only for genes with Pol II ChIP occupancy > 1, top 7% of genes.

    Journal: Genome Research

    Article Title: High-resolution nucleosome mapping reveals transcription-dependent promoter packaging

    doi: 10.1101/gr.098509.109

    Figure Lengend Snippet: Effects of MNase level on chromatin structure. ( A ) Mononucleosomal DNA was isolated from ladders from three different MNase titration levels, and sequenced by Illumina sequencing. ( B ) Data from titration series was subjected to template filtering to generate nucleosome calls. Width distributions for nucleosomes from the three titration steps are plotted. Green, yellow, and red correspond to under-, mid-, and overdigested chromatin, respectively. ( C ) Data for under- (green), mid- (yellow), and over- (red) digested chromatin is shown in cluster view. Genes are aligned using BY10 +1 nucleosome center (indicated); all three clusters have genes ordered by clustering for BY10 data. Red bar indicates genes with wide NFRs in mid- and overdigested chromatin (largely highly expressed genes such as ribosomal genes), which are partially filled in underdigested chromatin. ( D ) TSS-aligned nucleosome occupancy data for all genes. ( E ) Stop-codon-aligned nucleosome occupancy for all genes. ( F ) As in D , but only for genes with Pol II ChIP occupancy > 1, top 7% of genes.

    Article Snippet: Comparing nucleosome positions and properties before and after Pol II inactivation, we confirm the role of RNA polymerase in nucleosome eviction at promoters, and find a surprising role in retrograde movement of nucleosomes over genes.

    Techniques: Isolation, Titration, Sequencing, Chromatin Immunoprecipitation

    Effects of RNA polymerase on chromatin structure. ( A ) Nucleosomes were isolated from rpb1-1 yeast grown at 25°C, and shifted to 37°C for 20 or 120 min. Data are presented in TSS-aligned average. ( B ) As in A , but for highly expressed genes. ( C ) Nucleosomes over genes shift downstream upon Pol II loss. For each indicated nucleosome type (−1, +1, +2, +3) we plot the distribution of center-to-center distances between the nucleosome calls at 0 and 120 min after Pol II inactivation. We find that 43% of −1 nucleosomes, 59% of +1 nucleosomes, 61% of +2 nucleosomes, and 60% of +3 nucleosomes shift away from the NFR. ( D ) Global view of +1 nucleosome shifts during Pol II inactivation. Nucleosome calls for all promoters with a downstream +1 nucleosome shift are shown as a heatmap, aligned by the center of the +1 nucleosome (yellow) before Pol II inactivation (red). After 2 h of Pol II inactivation (green), downstream shifts of these 59% of +1 nucleosomes are apparent.

    Journal: Genome Research

    Article Title: High-resolution nucleosome mapping reveals transcription-dependent promoter packaging

    doi: 10.1101/gr.098509.109

    Figure Lengend Snippet: Effects of RNA polymerase on chromatin structure. ( A ) Nucleosomes were isolated from rpb1-1 yeast grown at 25°C, and shifted to 37°C for 20 or 120 min. Data are presented in TSS-aligned average. ( B ) As in A , but for highly expressed genes. ( C ) Nucleosomes over genes shift downstream upon Pol II loss. For each indicated nucleosome type (−1, +1, +2, +3) we plot the distribution of center-to-center distances between the nucleosome calls at 0 and 120 min after Pol II inactivation. We find that 43% of −1 nucleosomes, 59% of +1 nucleosomes, 61% of +2 nucleosomes, and 60% of +3 nucleosomes shift away from the NFR. ( D ) Global view of +1 nucleosome shifts during Pol II inactivation. Nucleosome calls for all promoters with a downstream +1 nucleosome shift are shown as a heatmap, aligned by the center of the +1 nucleosome (yellow) before Pol II inactivation (red). After 2 h of Pol II inactivation (green), downstream shifts of these 59% of +1 nucleosomes are apparent.

    Article Snippet: Comparing nucleosome positions and properties before and after Pol II inactivation, we confirm the role of RNA polymerase in nucleosome eviction at promoters, and find a surprising role in retrograde movement of nucleosomes over genes.

    Techniques: Isolation

    Change in Accessibility of Functional Transcription Factor Binding Sites Transcription factors were classified into three groups based on the change in accessibility of their functional binding sites because of nucleosome repositioning after heat shock. Graphs of accessibility changes in arbitrary units (see Materials and Methods ) are plotted for transcription factor binding sites that (A) showed an increase in accessibility, (B) showed no significant change in accessibility, and (C) showed a decrease in accessibility upon heat shock. The right of each graph shows a schematic of the relationship between nucleosomes and transcription factor binding sites.

    Journal: PLoS Biology

    Article Title: Dynamic Remodeling of Individual Nucleosomes Across a Eukaryotic Genome in Response to Transcriptional Perturbation

    doi: 10.1371/journal.pbio.0060065

    Figure Lengend Snippet: Change in Accessibility of Functional Transcription Factor Binding Sites Transcription factors were classified into three groups based on the change in accessibility of their functional binding sites because of nucleosome repositioning after heat shock. Graphs of accessibility changes in arbitrary units (see Materials and Methods ) are plotted for transcription factor binding sites that (A) showed an increase in accessibility, (B) showed no significant change in accessibility, and (C) showed a decrease in accessibility upon heat shock. The right of each graph shows a schematic of the relationship between nucleosomes and transcription factor binding sites.

    Article Snippet: To map the location of individual nucleosomes on a genomic scale and at high resolution, we used ultra-high–throughput sequencing methodology (Solexa/Illumina) to sequence the ends of nucleosome-associated DNA.

    Techniques: Functional Assay, Binding Assay

    Nucleosome Positioning over Coding Regions Depends on Transcription Rate and Sequence Characteristics (A) Genes were aligned to the first nucleosome downstream of the TSS and sorted by their nucleosome positioning periodicity (NPP) score (see Materials and Methods ). Genes were sorted by their NPP scores in normally growing cells, and the data from heat-shocked cells are shown in the same order. The unaligned TSS is indicated by the approximate curve. (B) The transcription rate of genes with high NPP scores (well-positioned nucleosomes) is significantly lower than that of genes with low NPP scores (poorly positioned). In these box plots, the red line indicates the median, the upper and lower bounds of the box indicate the interquartile range, the horizontal lines that are connected to the box by a dashed line indicate the upper and lower bounds of nonoutlier values, and the open circles indicate outliers. (C) Genes were sorted in descending order according to their transcription rates, and the average nucleosome profiles over the coding regions for top 500 genes (orange) and the bottom 500 genes (black) are plotted. (D) Frequency of AA/TT dinucleotide at each position in the DNA sequence associated with the most strongly positioned first nucleosomes. The frequency profiles for the dinucleotides AA and TT for the first nucleosome shown in (A) were summed and smoothed using a 3-bp moving average. The same analysis was also performed for a comparable set of randomly chosen DNA sequences from the yeast genome. (E) Correlation coefficients of the AA/TT profiles for the DNA sequences underlying each of the indicated coding nucleosome positions from (A), with the positioning profile derived earlier. Each of the correlation values was significantly higher than background.

    Journal: PLoS Biology

    Article Title: Dynamic Remodeling of Individual Nucleosomes Across a Eukaryotic Genome in Response to Transcriptional Perturbation

    doi: 10.1371/journal.pbio.0060065

    Figure Lengend Snippet: Nucleosome Positioning over Coding Regions Depends on Transcription Rate and Sequence Characteristics (A) Genes were aligned to the first nucleosome downstream of the TSS and sorted by their nucleosome positioning periodicity (NPP) score (see Materials and Methods ). Genes were sorted by their NPP scores in normally growing cells, and the data from heat-shocked cells are shown in the same order. The unaligned TSS is indicated by the approximate curve. (B) The transcription rate of genes with high NPP scores (well-positioned nucleosomes) is significantly lower than that of genes with low NPP scores (poorly positioned). In these box plots, the red line indicates the median, the upper and lower bounds of the box indicate the interquartile range, the horizontal lines that are connected to the box by a dashed line indicate the upper and lower bounds of nonoutlier values, and the open circles indicate outliers. (C) Genes were sorted in descending order according to their transcription rates, and the average nucleosome profiles over the coding regions for top 500 genes (orange) and the bottom 500 genes (black) are plotted. (D) Frequency of AA/TT dinucleotide at each position in the DNA sequence associated with the most strongly positioned first nucleosomes. The frequency profiles for the dinucleotides AA and TT for the first nucleosome shown in (A) were summed and smoothed using a 3-bp moving average. The same analysis was also performed for a comparable set of randomly chosen DNA sequences from the yeast genome. (E) Correlation coefficients of the AA/TT profiles for the DNA sequences underlying each of the indicated coding nucleosome positions from (A), with the positioning profile derived earlier. Each of the correlation values was significantly higher than background.

    Article Snippet: To map the location of individual nucleosomes on a genomic scale and at high resolution, we used ultra-high–throughput sequencing methodology (Solexa/Illumina) to sequence the ends of nucleosome-associated DNA.

    Techniques: Sequencing, Derivative Assay

    Dynamic Nucleosome Remodeling Affects the Accessibility of Transcription Factor Binding Sites and the TSS (A) Example of nucleosome eviction at the heat-shock–activated UBC4 promoter (blue line). Nucleosomes defined by our sequencing data are indicated by ovals, colored according to their stability score. The positions of transcription factor binding sites are from [ 17 ] and are shaded according to their confidence. Binding sites for other transcription factors are also affected by remodeling (unpublished data), but these are not known to be related to heat shock. (B) Example of nucleosome appearance at the heat-shock–repressed RPL17B promoter (red line).

    Journal: PLoS Biology

    Article Title: Dynamic Remodeling of Individual Nucleosomes Across a Eukaryotic Genome in Response to Transcriptional Perturbation

    doi: 10.1371/journal.pbio.0060065

    Figure Lengend Snippet: Dynamic Nucleosome Remodeling Affects the Accessibility of Transcription Factor Binding Sites and the TSS (A) Example of nucleosome eviction at the heat-shock–activated UBC4 promoter (blue line). Nucleosomes defined by our sequencing data are indicated by ovals, colored according to their stability score. The positions of transcription factor binding sites are from [ 17 ] and are shaded according to their confidence. Binding sites for other transcription factors are also affected by remodeling (unpublished data), but these are not known to be related to heat shock. (B) Example of nucleosome appearance at the heat-shock–repressed RPL17B promoter (red line).

    Article Snippet: To map the location of individual nucleosomes on a genomic scale and at high resolution, we used ultra-high–throughput sequencing methodology (Solexa/Illumina) to sequence the ends of nucleosome-associated DNA.

    Techniques: Binding Assay, Sequencing

    Patterns of Chromatin Organization in the Yeast Genome (A) Average nucleosome profiles of all genes in the yeast genome from −600 bp to +1,000 bp with respect to the transcription start site (TSS). Nucleosome positions are shown as gray ovals below the profile. The intensity of the filled oval reflects the average probability score of the nucleosomes (see Figure 1 for the color scale), and the dotted oval marks the spread of that nucleosome across all genes. (B) The 3′ end of genes is marked by a strongly positioned nucleosome, followed by a relatively nucleosome-free region. The inset shows the 3′ end of convergently transcribed genes in which the 3′ end is not followed by another promoter. (C) Distinct classes of nucleosome profiles revealed by k -means clustering of all promoters in the yeast genome. Each row in the clusters shows the position of a nucleosome at an individual promoter. Nucleosomes are colored according to their probability using the shown color scale. Clusters 1–3 showed a significant enrichment for genes with lower transcription rates and for TATA-less genes ( p ≤ 10 −10 ). Cluster 6 showed a significant enrichment for genes with high transcription rates and for TATA-containing genes ( p

    Journal: PLoS Biology

    Article Title: Dynamic Remodeling of Individual Nucleosomes Across a Eukaryotic Genome in Response to Transcriptional Perturbation

    doi: 10.1371/journal.pbio.0060065

    Figure Lengend Snippet: Patterns of Chromatin Organization in the Yeast Genome (A) Average nucleosome profiles of all genes in the yeast genome from −600 bp to +1,000 bp with respect to the transcription start site (TSS). Nucleosome positions are shown as gray ovals below the profile. The intensity of the filled oval reflects the average probability score of the nucleosomes (see Figure 1 for the color scale), and the dotted oval marks the spread of that nucleosome across all genes. (B) The 3′ end of genes is marked by a strongly positioned nucleosome, followed by a relatively nucleosome-free region. The inset shows the 3′ end of convergently transcribed genes in which the 3′ end is not followed by another promoter. (C) Distinct classes of nucleosome profiles revealed by k -means clustering of all promoters in the yeast genome. Each row in the clusters shows the position of a nucleosome at an individual promoter. Nucleosomes are colored according to their probability using the shown color scale. Clusters 1–3 showed a significant enrichment for genes with lower transcription rates and for TATA-less genes ( p ≤ 10 −10 ). Cluster 6 showed a significant enrichment for genes with high transcription rates and for TATA-containing genes ( p

    Article Snippet: To map the location of individual nucleosomes on a genomic scale and at high resolution, we used ultra-high–throughput sequencing methodology (Solexa/Illumina) to sequence the ends of nucleosome-associated DNA.

    Techniques:

    Classification of Promoter Nucleosome Remodeling Profiles All profiles are aligned with respect to the TSS. (A) Remodeling profiles of genes activated greater than 2-fold after heat shock and (B), genes repressed greater than 2-fold by heat shock. Nucleosomes present during normal growth but evicted by heat shock are indicated in yellow, and nucleosomes that appeared after heat shock are shown in blue. The average profiles of nucleosomes in each group before and after heat shock are shown on the right. The k -means clustering for (A) and (B) was done based on data from −200 to TSS, but data are shown for −300 to +100. (C) A remodeling score for eviction and for appearance was separately calculated for activated genes and repressed genes (Materials and Methods), and the data were plotted using box plots similar to Figure 3 B. Activated genes showed significantly higher eviction scores than appearance scores, whereas repressed genes showed significantly higher appearance scores than eviction scores. (D) Nucleosome positions at the promoters of ribosomal protein genes during normal growth and after heat shock, clustered on data from −200 to +100 bp. (E) Remodeling profiles of genes whose expression changed by less than 1.2-fold after heat shock, clustered based on data from −300 to +100 bp.

    Journal: PLoS Biology

    Article Title: Dynamic Remodeling of Individual Nucleosomes Across a Eukaryotic Genome in Response to Transcriptional Perturbation

    doi: 10.1371/journal.pbio.0060065

    Figure Lengend Snippet: Classification of Promoter Nucleosome Remodeling Profiles All profiles are aligned with respect to the TSS. (A) Remodeling profiles of genes activated greater than 2-fold after heat shock and (B), genes repressed greater than 2-fold by heat shock. Nucleosomes present during normal growth but evicted by heat shock are indicated in yellow, and nucleosomes that appeared after heat shock are shown in blue. The average profiles of nucleosomes in each group before and after heat shock are shown on the right. The k -means clustering for (A) and (B) was done based on data from −200 to TSS, but data are shown for −300 to +100. (C) A remodeling score for eviction and for appearance was separately calculated for activated genes and repressed genes (Materials and Methods), and the data were plotted using box plots similar to Figure 3 B. Activated genes showed significantly higher eviction scores than appearance scores, whereas repressed genes showed significantly higher appearance scores than eviction scores. (D) Nucleosome positions at the promoters of ribosomal protein genes during normal growth and after heat shock, clustered on data from −200 to +100 bp. (E) Remodeling profiles of genes whose expression changed by less than 1.2-fold after heat shock, clustered based on data from −300 to +100 bp.

    Article Snippet: To map the location of individual nucleosomes on a genomic scale and at high resolution, we used ultra-high–throughput sequencing methodology (Solexa/Illumina) to sequence the ends of nucleosome-associated DNA.

    Techniques: Expressing

    Ultra-High–Throughput Sequencing Recapitulates In Vivo Nucleosome Positions (A) Detailed view of the PHO5 locus showing the raw sequence reads (brown and red profiles). The nucleosome positions calculated using our analysis algorithm are shown as ovals, shaded according to their nucleosome score as indicated. The positions of the amplicons used for qPCR analysis are marked as red (peaks) and green (troughs) lines below. The black arrows indicate the positions of genes in that region. (B) qPCR verification of the three nucleosome peaks and three troughs identified by sequencing confirm that their positions remain the same before and after heat shock. (C) The heat-shock–induced SSA4 gene and flanking regions, showing that nucleosomes are displaced specifically at the SSA4 promoter and coding region after heat shock (thick purple arrow). (D) The heat-shock–repressed ribosomal protein gene RPL17B and flanking regions, showing that a single positioned nucleosome appears after heat shock specifically at the RPL17B promoter (thin purple arrow). The nucleosome positions calculated using our analysis algorithm are indicated as in (A).

    Journal: PLoS Biology

    Article Title: Dynamic Remodeling of Individual Nucleosomes Across a Eukaryotic Genome in Response to Transcriptional Perturbation

    doi: 10.1371/journal.pbio.0060065

    Figure Lengend Snippet: Ultra-High–Throughput Sequencing Recapitulates In Vivo Nucleosome Positions (A) Detailed view of the PHO5 locus showing the raw sequence reads (brown and red profiles). The nucleosome positions calculated using our analysis algorithm are shown as ovals, shaded according to their nucleosome score as indicated. The positions of the amplicons used for qPCR analysis are marked as red (peaks) and green (troughs) lines below. The black arrows indicate the positions of genes in that region. (B) qPCR verification of the three nucleosome peaks and three troughs identified by sequencing confirm that their positions remain the same before and after heat shock. (C) The heat-shock–induced SSA4 gene and flanking regions, showing that nucleosomes are displaced specifically at the SSA4 promoter and coding region after heat shock (thick purple arrow). (D) The heat-shock–repressed ribosomal protein gene RPL17B and flanking regions, showing that a single positioned nucleosome appears after heat shock specifically at the RPL17B promoter (thin purple arrow). The nucleosome positions calculated using our analysis algorithm are indicated as in (A).

    Article Snippet: To map the location of individual nucleosomes on a genomic scale and at high resolution, we used ultra-high–throughput sequencing methodology (Solexa/Illumina) to sequence the ends of nucleosome-associated DNA.

    Techniques: Next-Generation Sequencing, In Vivo, Sequencing, Real-time Polymerase Chain Reaction

    Predicted and observed nucleosome occupancy around ChIP–seq peaks. A. Mean MNase midpoint depth around ChIP-seq peak summits, aggregated across 5 transcription factors (CTCF, NF-kB, Irf4, C-fos and GABP). Regions are aligned such that the estimated locations of the +1 nucleosome, the −1 nucleosome and the midpoint between the nucleosomes are at the same position. Segments that have data from less than 50% of the ChIP-seq peaks (because of the variable spacing between nucleosomes) are omitted. Regions are stratified into ChIP-seq read depth quintiles, (higher quintiles indicate higher transcription factor occupancy). B. Predicted nucleosome occupancy from an in vitro sequence model [33] . Each region is normalized by the mean predicted occupancy of the entire region. As in A, regions are aligned on putative nucleosome positions and are stratified into ChIP-seq read depth quintiles and segments with data from less than 50% of the ChIP-seq peaks are omitted. The inset shows Spearman's rank correlation ( ρ ) between predicted and observed nucleosome occupancy for these regions and for 1000 randomly sampled genomic regions.

    Journal: PLoS Genetics

    Article Title: Controls of Nucleosome Positioning in the Human Genome

    doi: 10.1371/journal.pgen.1003036

    Figure Lengend Snippet: Predicted and observed nucleosome occupancy around ChIP–seq peaks. A. Mean MNase midpoint depth around ChIP-seq peak summits, aggregated across 5 transcription factors (CTCF, NF-kB, Irf4, C-fos and GABP). Regions are aligned such that the estimated locations of the +1 nucleosome, the −1 nucleosome and the midpoint between the nucleosomes are at the same position. Segments that have data from less than 50% of the ChIP-seq peaks (because of the variable spacing between nucleosomes) are omitted. Regions are stratified into ChIP-seq read depth quintiles, (higher quintiles indicate higher transcription factor occupancy). B. Predicted nucleosome occupancy from an in vitro sequence model [33] . Each region is normalized by the mean predicted occupancy of the entire region. As in A, regions are aligned on putative nucleosome positions and are stratified into ChIP-seq read depth quintiles and segments with data from less than 50% of the ChIP-seq peaks are omitted. The inset shows Spearman's rank correlation ( ρ ) between predicted and observed nucleosome occupancy for these regions and for 1000 randomly sampled genomic regions.

    Article Snippet: Sequencing and read mapping Our seven libraries of nucleosome fragments were prepared for paired-end sequencing using the standard Illumina protocol.

    Techniques: Chromatin Immunoprecipitation, In Vitro, Sequencing

    Quantifying translational nucleosome positioning in the human genome. A. Distribution of nucleosome positioning scores from a random sample of one million 200 bp regions (smoothed using a Gaussian kernel with bandwidth 0.01). Scores were also calculated in the same regions using midpoints from non-duplicate read pairs and from simulated read pairs. B. Distribution of nucleosome array log likelihood ratios (LLRs) for 23,763 randomly sampled 1 kb regions (smoothed using a Gaussian kernel with bandwidth 1.0). LLRs were also calculated using midpoints from simulated reads and using permuted versions of the same regions. C. Heatmap of MNase midpoints in the randomly sampled regions from B, prior to their alignment. D. Heatmap of MNase midpoints from panel D, after their alignment. Regions were aligned according to the most likely position of the central nucleosome. E. Heatmap of aligned MNase midpoints for permuted regions. Heatmaps in C, D, and E are ordered by the LLR of the observed midpoints.

    Journal: PLoS Genetics

    Article Title: Controls of Nucleosome Positioning in the Human Genome

    doi: 10.1371/journal.pgen.1003036

    Figure Lengend Snippet: Quantifying translational nucleosome positioning in the human genome. A. Distribution of nucleosome positioning scores from a random sample of one million 200 bp regions (smoothed using a Gaussian kernel with bandwidth 0.01). Scores were also calculated in the same regions using midpoints from non-duplicate read pairs and from simulated read pairs. B. Distribution of nucleosome array log likelihood ratios (LLRs) for 23,763 randomly sampled 1 kb regions (smoothed using a Gaussian kernel with bandwidth 1.0). LLRs were also calculated using midpoints from simulated reads and using permuted versions of the same regions. C. Heatmap of MNase midpoints in the randomly sampled regions from B, prior to their alignment. D. Heatmap of MNase midpoints from panel D, after their alignment. Regions were aligned according to the most likely position of the central nucleosome. E. Heatmap of aligned MNase midpoints for permuted regions. Heatmaps in C, D, and E are ordered by the LLR of the observed midpoints.

    Article Snippet: Sequencing and read mapping Our seven libraries of nucleosome fragments were prepared for paired-end sequencing using the standard Illumina protocol.

    Techniques:

    Arrays of positioned nucleosomes flanking transcription factor (TF) binding sites. A. Heatmaps of MNase midpoints (columns 1–2) and DNase I cuts (column 3) surrounding 1000 randomly sampled ChIP-seq peaks for CTCF, NF-kB, Irf4, GABP and C-fos. Heatmap rows are ordered from top to bottom by the nucleosome array log likelihood ratio (LLR). Columns 2 and 3 are aligned according to the most likely location of the upstream and downstream arrays of positioned nucleosomes. B. Aggregate MNase midpoint and DNase I cutsite depths across all regions and for the subset of regions with LLR > 500.

    Journal: PLoS Genetics

    Article Title: Controls of Nucleosome Positioning in the Human Genome

    doi: 10.1371/journal.pgen.1003036

    Figure Lengend Snippet: Arrays of positioned nucleosomes flanking transcription factor (TF) binding sites. A. Heatmaps of MNase midpoints (columns 1–2) and DNase I cuts (column 3) surrounding 1000 randomly sampled ChIP-seq peaks for CTCF, NF-kB, Irf4, GABP and C-fos. Heatmap rows are ordered from top to bottom by the nucleosome array log likelihood ratio (LLR). Columns 2 and 3 are aligned according to the most likely location of the upstream and downstream arrays of positioned nucleosomes. B. Aggregate MNase midpoint and DNase I cutsite depths across all regions and for the subset of regions with LLR > 500.

    Article Snippet: Sequencing and read mapping Our seven libraries of nucleosome fragments were prepared for paired-end sequencing using the standard Illumina protocol.

    Techniques: Binding Assay, Chromatin Immunoprecipitation

    Fine scale characteristics of nucleosome sequences. A. Frequencies of AA/AT/TA/TT and CC/CG/GC/GG dinucleotides across nucleosome sequences normalized by expected dinucleotide frequencies (log2 ratio). Expected frequencies were taken from a set of simulated fragments, which were sampled such that they had the same MNase cutting bias as the observed fragments. B. DNase I cut rates across nucleosome sequences normalized by the expected cut rates (log2 ratio). Expected DNase I cut frequencies were estimated from the composition of all observed DNase I cut sites in the human genome. C. MNase-seq fragment midpoints from 3 cell lines. Expected midpoint frequencies were estimated from the same simulated fragments used in A.

    Journal: PLoS Genetics

    Article Title: Controls of Nucleosome Positioning in the Human Genome

    doi: 10.1371/journal.pgen.1003036

    Figure Lengend Snippet: Fine scale characteristics of nucleosome sequences. A. Frequencies of AA/AT/TA/TT and CC/CG/GC/GG dinucleotides across nucleosome sequences normalized by expected dinucleotide frequencies (log2 ratio). Expected frequencies were taken from a set of simulated fragments, which were sampled such that they had the same MNase cutting bias as the observed fragments. B. DNase I cut rates across nucleosome sequences normalized by the expected cut rates (log2 ratio). Expected DNase I cut frequencies were estimated from the composition of all observed DNase I cut sites in the human genome. C. MNase-seq fragment midpoints from 3 cell lines. Expected midpoint frequencies were estimated from the same simulated fragments used in A.

    Article Snippet: Sequencing and read mapping Our seven libraries of nucleosome fragments were prepared for paired-end sequencing using the standard Illumina protocol.

    Techniques:

    Nucleosome organization in regions with an association between DNase I sensitivity and genotype (dsQTLs). Data are aggregated across dsQTLs and are scaled by the total number of sequenced reads. The DNase-seq data are from 70 individuals and the MNase-seq data are from 7 individuals. This plot was created using a subset of dsQTLs (n = 1101) that have a narrow region of DNase I sensitivity (below the median) and a large difference in sensitivity between genotypes (above the median). The complete set of filtered dsQTLs shows the same trend ( Figure S16 ). A. The density of DNase I nicks for different dsQTL genotypes. B. The density of MNase midpoints for different dsQTL genotypes.

    Journal: PLoS Genetics

    Article Title: Controls of Nucleosome Positioning in the Human Genome

    doi: 10.1371/journal.pgen.1003036

    Figure Lengend Snippet: Nucleosome organization in regions with an association between DNase I sensitivity and genotype (dsQTLs). Data are aggregated across dsQTLs and are scaled by the total number of sequenced reads. The DNase-seq data are from 70 individuals and the MNase-seq data are from 7 individuals. This plot was created using a subset of dsQTLs (n = 1101) that have a narrow region of DNase I sensitivity (below the median) and a large difference in sensitivity between genotypes (above the median). The complete set of filtered dsQTLs shows the same trend ( Figure S16 ). A. The density of DNase I nicks for different dsQTL genotypes. B. The density of MNase midpoints for different dsQTL genotypes.

    Article Snippet: Sequencing and read mapping Our seven libraries of nucleosome fragments were prepared for paired-end sequencing using the standard Illumina protocol.

    Techniques:

    Examples of nucleosome arrays. A. MNase midpoint density (smoothed using a 30 bp sliding window) across a 76 kb region near the chromosome 12 centromere. This region contains an array of ∼400 nucleosomes with regular, consistent positioning. B. A small 10 kb subsection of the larger nucleosome array. Predicted nucleosome occupancy from the in vitro sequence model of Kaplan et al. [33] corresponds very well with MNase midpoint density. Kaplan scores predict the affinity of nucleosomes for the sequence but, unlike predicted occupancies, do not incorporate steric exclusion. DNase I nick density (smoothed with a 10 bp sliding window) indicates the location of DNase I sensitive regions (there are none in this region). The density of simulated MNase midpoints and Yoruba DNA sequencing read depth (aggregated across individuals from the 1000 genomes project) are not strongly correlated with MNase midpoint density, which shows that the array is not an artifact of sequencing or mapping bias. C. MNase midpoint density around the gene NPM3 . In this region there is consistent, regular spacing of nucleosomes, but their positions are not well predicted by the Kaplan model, particularly in the DNase I hypersensitive sites, which are depleted of nucleosomes.

    Journal: PLoS Genetics

    Article Title: Controls of Nucleosome Positioning in the Human Genome

    doi: 10.1371/journal.pgen.1003036

    Figure Lengend Snippet: Examples of nucleosome arrays. A. MNase midpoint density (smoothed using a 30 bp sliding window) across a 76 kb region near the chromosome 12 centromere. This region contains an array of ∼400 nucleosomes with regular, consistent positioning. B. A small 10 kb subsection of the larger nucleosome array. Predicted nucleosome occupancy from the in vitro sequence model of Kaplan et al. [33] corresponds very well with MNase midpoint density. Kaplan scores predict the affinity of nucleosomes for the sequence but, unlike predicted occupancies, do not incorporate steric exclusion. DNase I nick density (smoothed with a 10 bp sliding window) indicates the location of DNase I sensitive regions (there are none in this region). The density of simulated MNase midpoints and Yoruba DNA sequencing read depth (aggregated across individuals from the 1000 genomes project) are not strongly correlated with MNase midpoint density, which shows that the array is not an artifact of sequencing or mapping bias. C. MNase midpoint density around the gene NPM3 . In this region there is consistent, regular spacing of nucleosomes, but their positions are not well predicted by the Kaplan model, particularly in the DNase I hypersensitive sites, which are depleted of nucleosomes.

    Article Snippet: Sequencing and read mapping Our seven libraries of nucleosome fragments were prepared for paired-end sequencing using the standard Illumina protocol.

    Techniques: In Vitro, Sequencing, DNA Sequencing

    Inactivation of bptf or TGF-β signaling induces nucleosome repositioning within the wnt8 a promoter. A , MNase digestion of chromatin isolated from embryos at 75% epiboly stage. Digestion with 320 units per milliliters of MNase for 30 min was appropriate to produce mononucleosome-sized DNAs. B , C , The dynamic changes of nucleosomal positions at the wnt8a promoter in bptf morphants ( B ) or Δ kT β RII- overexpressing embryos ( C ). There were five positioned nucleosomes (N1, N2, N3, N4, and N5) within the −1449 to −416 region of the wnt8a promoter in cMO-injected embryos. Bptf (green) and Smad2 (red) binding motifs were located in the DNA sequences occupied by N3. A solid increase in DNA amount was detected at N3 positioning site in bptf morphants and Δ kT β RII- overexpressing embryos. NS, Nonsignificant. ** p

    Journal: The Journal of Neuroscience

    Article Title: The Chromatin Remodeling Protein Bptf Promotes Posterior Neuroectodermal Fate by Enhancing Smad2-Activated wnt8a Expression

    doi: 10.1523/JNEUROSCI.0377-15.2015

    Figure Lengend Snippet: Inactivation of bptf or TGF-β signaling induces nucleosome repositioning within the wnt8 a promoter. A , MNase digestion of chromatin isolated from embryos at 75% epiboly stage. Digestion with 320 units per milliliters of MNase for 30 min was appropriate to produce mononucleosome-sized DNAs. B , C , The dynamic changes of nucleosomal positions at the wnt8a promoter in bptf morphants ( B ) or Δ kT β RII- overexpressing embryos ( C ). There were five positioned nucleosomes (N1, N2, N3, N4, and N5) within the −1449 to −416 region of the wnt8a promoter in cMO-injected embryos. Bptf (green) and Smad2 (red) binding motifs were located in the DNA sequences occupied by N3. A solid increase in DNA amount was detected at N3 positioning site in bptf morphants and Δ kT β RII- overexpressing embryos. NS, Nonsignificant. ** p

    Article Snippet: Wild-type and bptf MOs or Δ kT β RII mRNA injected embryos were harvested at 75% epiboly stage, and mononucleosomes were prepared using Nucleosome Preparation Kit (5333, TaKaRa) according to the manufacturer's instructions.

    Techniques: Isolation, Injection, Binding Assay

    Mhrt inhibits chromatin targeting and gene regulation by Brg1 a, Gel electrophoresis and quantitation of nucleosomal 5SrDNA, Myh6 promoter and Neo DNA. Arrowheads: DNA-histone complex. Arrows: naked DNA. Nucleosome assembly efficiency is defined as the fraction of DNA bound to histones (arrowheads). P-value: Student’s t-test. Error bar: standard error of the mean (SEM). b-d, Quantification of amylose pull-down of MBP-D1D2 (D1D2) with nucleosomal and naked Myh6 promoter DNA ( b ), with nucleosomal Myh6 promoter, Neo , and 5SrDNA ( c ), or with nucleosomal Myh6 promoter in the presence of Mhrt779 ( d ). P-value: Student’s t-test. Error bar: SEM. e, Amylose pull-down of MBP-D1D2 and histone 3. Anti-histone 3 and anti-MBP antibodies were used for western blot analysis. f, ChIP analysis of Brg1 on chromatinized and naked Myh6 promoter in rat ventricular cardiomyocytes. GFP: green fluorescence protein control. P-value: Student’s t-test. Error bar: SEM. g, h, Luciferase reporter activity of Brg1 on naked Myh6 promoter ( g ) or of helicase-deficient Brg1 on chromatinized Myh6 promoter ( h ) in rat ventricular cardiomyocytes. ΔD1: Brg1 lacking amino acid 774–913; ΔD2: Brg1 lacking 1086–1246. GFP: green fluorescence protein control. ChIP: H-10 antibody recognizing N-terminus, non-disrupted region of Brg1. P-value: Student’s t-test. Error bar: SEM. i, j, ChIP analysis in SW13 cells of chromatinized Myh6 promoter in the presence of Mhrt779 ( i ) or helicase-deficient Brg1 ( j ). Vector: pAdd2 empty vector. Mhrt : pAdd2- Mhrt779 . P-value: Student’s t-test. Error bar: SEM. k, Schematic illustration and PCR of human MHRT. MHRT originates from MYH7 and is transcribed into MYH7. MYH7 e xons and introns are indicated. R1 and R2 are strand-specific PCR primers; F1 and R1 target MHRT and MYH7 ; F2 and R2 are specific for MHRT . l, Quantification of MHRT in human heart tissues. Ctrl: control. LVH: left ventricular hypertrophy. ICM: ischemic cardiomyopathy. IDCM: idiopathic dilated cardiomyopathy. P-value: Student’s t-test. Error bar: SEM. m, Working model of a Brg1- Mhrt negative feedback circuit in the heart. Brg1 represses Mhrt transcription, whereas Mhrt prevents Brg1 from recognizing its chromatin targets. Brg1 functions through two distinct promoter elements to bidirectionally repress Myh6 and Mhrt expression. n , Molecular model of how Brg1 binds to its genomic DNA targets. Brg1 helicase (D1D2) binds chromatinized DNA, C-terminal extension (CTE) binds histone 3 (H3), and bromodomain binds acetylated (Ac) histone 3 or 4 (H4).

    Journal: Nature

    Article Title: A long non-coding RNA protects the heart from pathological hypertrophy

    doi: 10.1038/nature13596

    Figure Lengend Snippet: Mhrt inhibits chromatin targeting and gene regulation by Brg1 a, Gel electrophoresis and quantitation of nucleosomal 5SrDNA, Myh6 promoter and Neo DNA. Arrowheads: DNA-histone complex. Arrows: naked DNA. Nucleosome assembly efficiency is defined as the fraction of DNA bound to histones (arrowheads). P-value: Student’s t-test. Error bar: standard error of the mean (SEM). b-d, Quantification of amylose pull-down of MBP-D1D2 (D1D2) with nucleosomal and naked Myh6 promoter DNA ( b ), with nucleosomal Myh6 promoter, Neo , and 5SrDNA ( c ), or with nucleosomal Myh6 promoter in the presence of Mhrt779 ( d ). P-value: Student’s t-test. Error bar: SEM. e, Amylose pull-down of MBP-D1D2 and histone 3. Anti-histone 3 and anti-MBP antibodies were used for western blot analysis. f, ChIP analysis of Brg1 on chromatinized and naked Myh6 promoter in rat ventricular cardiomyocytes. GFP: green fluorescence protein control. P-value: Student’s t-test. Error bar: SEM. g, h, Luciferase reporter activity of Brg1 on naked Myh6 promoter ( g ) or of helicase-deficient Brg1 on chromatinized Myh6 promoter ( h ) in rat ventricular cardiomyocytes. ΔD1: Brg1 lacking amino acid 774–913; ΔD2: Brg1 lacking 1086–1246. GFP: green fluorescence protein control. ChIP: H-10 antibody recognizing N-terminus, non-disrupted region of Brg1. P-value: Student’s t-test. Error bar: SEM. i, j, ChIP analysis in SW13 cells of chromatinized Myh6 promoter in the presence of Mhrt779 ( i ) or helicase-deficient Brg1 ( j ). Vector: pAdd2 empty vector. Mhrt : pAdd2- Mhrt779 . P-value: Student’s t-test. Error bar: SEM. k, Schematic illustration and PCR of human MHRT. MHRT originates from MYH7 and is transcribed into MYH7. MYH7 e xons and introns are indicated. R1 and R2 are strand-specific PCR primers; F1 and R1 target MHRT and MYH7 ; F2 and R2 are specific for MHRT . l, Quantification of MHRT in human heart tissues. Ctrl: control. LVH: left ventricular hypertrophy. ICM: ischemic cardiomyopathy. IDCM: idiopathic dilated cardiomyopathy. P-value: Student’s t-test. Error bar: SEM. m, Working model of a Brg1- Mhrt negative feedback circuit in the heart. Brg1 represses Mhrt transcription, whereas Mhrt prevents Brg1 from recognizing its chromatin targets. Brg1 functions through two distinct promoter elements to bidirectionally repress Myh6 and Mhrt expression. n , Molecular model of how Brg1 binds to its genomic DNA targets. Brg1 helicase (D1D2) binds chromatinized DNA, C-terminal extension (CTE) binds histone 3 (H3), and bromodomain binds acetylated (Ac) histone 3 or 4 (H4).

    Article Snippet: In brief, recombinant human core histone octamer, which consist of the 2:1 mix of histone H2A/H2B dimer and histone H3.1/H4 tetramer, were mixed with purified 5SrDNA (208bp, N1202S, NEB), Neo (512bp, amplified from pST18-Neo , 1175025, Roche), Myh6 core promoter (596bp, −426 to +170) and Mhrt core promoter (a3a4, 596bp, −2290 to −1775) DNA at 2 M NaCl.

    Techniques: Nucleic Acid Electrophoresis, Quantitation Assay, Western Blot, Chromatin Immunoprecipitation, Fluorescence, Luciferase, Activity Assay, Plasmid Preparation, Polymerase Chain Reaction, Expressing

    B-Cell-Intrinsic IFNαR 1 Is Required for ANA-Producing AFC Responses in B6. Sle1b Mice (A) Flow cytometric analysis of surface expression of IFNαR 1 on B220 + B cells in B6. Sle1b and B6. Sle1b .IFNαR 1 −/− chimeric mice 3 months after BM cell transfer. (B and C) The percentages of B220 + GL-7 hi Fas hi GC B cells (B) and CD4 + CXCR5 hi PD-1 hi Tfh cells (C) in total splenocytes of the chimeras. (D and E) Numbers of dsDNA-specific (D) and nucleosome-specific (E) splenic AFCs in chimeric mice described in (A)–(C). (F and G) Numbers of dsDNA-specific (F) and nucleosome-specific (G) long-lived bone marrow AFCs in chimeric mice described in (A)–(C). (H) Analysis of serum titers of total IgG2c antibodies in these mice. (I and J) Analysis of dsDNA-reactive (I) and nucleosome-reactive IgG2c (J) in the sera of these mice. These data represent one experiment of four or five mice of each genotype. Statistical significance was determined using an unpaired, nonparametric Mann-Whitney Student’s t test (NS, not significant, *p

    Journal: Cell reports

    Article Title: B-Cell-Intrinsic Type 1 Interferon Signaling Is Crucial for Loss of Tolerance and the Development of Autoreactive B Cells

    doi: 10.1016/j.celrep.2018.06.046

    Figure Lengend Snippet: B-Cell-Intrinsic IFNαR 1 Is Required for ANA-Producing AFC Responses in B6. Sle1b Mice (A) Flow cytometric analysis of surface expression of IFNαR 1 on B220 + B cells in B6. Sle1b and B6. Sle1b .IFNαR 1 −/− chimeric mice 3 months after BM cell transfer. (B and C) The percentages of B220 + GL-7 hi Fas hi GC B cells (B) and CD4 + CXCR5 hi PD-1 hi Tfh cells (C) in total splenocytes of the chimeras. (D and E) Numbers of dsDNA-specific (D) and nucleosome-specific (E) splenic AFCs in chimeric mice described in (A)–(C). (F and G) Numbers of dsDNA-specific (F) and nucleosome-specific (G) long-lived bone marrow AFCs in chimeric mice described in (A)–(C). (H) Analysis of serum titers of total IgG2c antibodies in these mice. (I and J) Analysis of dsDNA-reactive (I) and nucleosome-reactive IgG2c (J) in the sera of these mice. These data represent one experiment of four or five mice of each genotype. Statistical significance was determined using an unpaired, nonparametric Mann-Whitney Student’s t test (NS, not significant, *p

    Article Snippet: Briefly, splenocytes in RPMI containing 10% fetal bovine serum were plated at a concentration of 1 × 105 cells/well onto anti-IgM-, anti-IgG-, salmon sperm dsDNA- (Invitrogen, Grand Island, NY), calf thymus histone- (Sigma Aldrich, St. Louis, MO), or nucleosome-coated plates (Millipore, Bedford, MA).

    Techniques: Mouse Assay, Flow Cytometry, Expressing, MANN-WHITNEY

    IFNαR 1 Is Required for ANA Production in B6. Sle1b Mice (A) ANA detection from sera of 6-month-old B6, B6.IFNαR 1 −/− , B6. Sle1b , and B6. Sle1b .IFNαR 1 −/− female mice by fluorescent Hep-2 assay. Representative images are shown with pie charts that quantitate the distribution of the serum samples as negative staining (non-Hep-2 reactive), reactive to cytoplasmic antigens (cytoplasmic), reactive to nuclear and cytoplasmic antigens (nuclear and cytoplasmic), and reactive to nuclear and/or nucleolar antigens (nucleolar/nuclear). The scale bars represent 75 μm. (B-D) Quantification of dsDNA-specific (B), histone-specific (C), and nucleosome-specific (D) AFCs in total splenocytes from 6-month-old mice of the indicated genotypes. (E-G) Analysis of serum titers of dsDNA-reactive (E), histone-reactive (F), and nucleosome-reactive (G) ANAs in the sera of 6-month-old female mice of the indicated genotypes (key at bottom of figure) by ELISA. These data are representative of two independent experiments, and each symbol represents a mouse. Statistical significance was determined by one-way ANOVA with a follow-up Tukey multiple-comparison test (**p

    Journal: Cell reports

    Article Title: B-Cell-Intrinsic Type 1 Interferon Signaling Is Crucial for Loss of Tolerance and the Development of Autoreactive B Cells

    doi: 10.1016/j.celrep.2018.06.046

    Figure Lengend Snippet: IFNαR 1 Is Required for ANA Production in B6. Sle1b Mice (A) ANA detection from sera of 6-month-old B6, B6.IFNαR 1 −/− , B6. Sle1b , and B6. Sle1b .IFNαR 1 −/− female mice by fluorescent Hep-2 assay. Representative images are shown with pie charts that quantitate the distribution of the serum samples as negative staining (non-Hep-2 reactive), reactive to cytoplasmic antigens (cytoplasmic), reactive to nuclear and cytoplasmic antigens (nuclear and cytoplasmic), and reactive to nuclear and/or nucleolar antigens (nucleolar/nuclear). The scale bars represent 75 μm. (B-D) Quantification of dsDNA-specific (B), histone-specific (C), and nucleosome-specific (D) AFCs in total splenocytes from 6-month-old mice of the indicated genotypes. (E-G) Analysis of serum titers of dsDNA-reactive (E), histone-reactive (F), and nucleosome-reactive (G) ANAs in the sera of 6-month-old female mice of the indicated genotypes (key at bottom of figure) by ELISA. These data are representative of two independent experiments, and each symbol represents a mouse. Statistical significance was determined by one-way ANOVA with a follow-up Tukey multiple-comparison test (**p

    Article Snippet: Briefly, splenocytes in RPMI containing 10% fetal bovine serum were plated at a concentration of 1 × 105 cells/well onto anti-IgM-, anti-IgG-, salmon sperm dsDNA- (Invitrogen, Grand Island, NY), calf thymus histone- (Sigma Aldrich, St. Louis, MO), or nucleosome-coated plates (Millipore, Bedford, MA).

    Techniques: Mouse Assay, Negative Staining, Enzyme-linked Immunosorbent Assay

    IFNαR 1 Signaling in GC B Cells Is Required for GC Stability and IgG2c ANA Production in B6. Sle1b Mice (A) Flow cytometric analysis of surface expression of IFNαR 1 on B220 + GL-7 hi Fas hi GC B cells in 6-month-old B6. Sle1b .IFNαR 1 fl/fl (white) and B6. Sle1b .IFNαR 1 fl/fl GC Cre/+ (orange) female mice. (B and C) The percentages of B220 + GL-7 hi Fas hi GC B cells (B) and CD4 + CXCR5 hi PD-1 hi Tfh cells (C) in total splenocytes of 6-month-old B6. Sle1b .IFNαR 1 fl/fl (white) and B6. Sle1b .IFNαR 1 fl/fl GC Cre/+ (orange) mice. Each symbol represents a mouse, and horizontal lines indicate mean values. (D) Spleen sections from 5 mice per group were stained with anti-CD4 (red), GL-7 (green), and anti-IgD (blue). Representative images are shown in the left panels. GC areas were measured for 10 GCs per spleen section (right panel). The scale bars represent 100 μm. (E and F) Total IgG (E) and IgG2c (F) serum titers in these mice. (G and H) Serum titers of dsDNA-specific (G) and nucleosome-specific (H) IgG2c Abs in these mice. (I) Serum IgG2c ANA reactivity was measured by Hep-2 slides. The scale bars represent 250 μm. These data are obtained from two independent experiments, and each symbol represents a mouse. Statistical significance was determined using an unpaired, nonparametric Mann-Whitney Student’s t test (NS, not significant, *p

    Journal: Cell reports

    Article Title: B-Cell-Intrinsic Type 1 Interferon Signaling Is Crucial for Loss of Tolerance and the Development of Autoreactive B Cells

    doi: 10.1016/j.celrep.2018.06.046

    Figure Lengend Snippet: IFNαR 1 Signaling in GC B Cells Is Required for GC Stability and IgG2c ANA Production in B6. Sle1b Mice (A) Flow cytometric analysis of surface expression of IFNαR 1 on B220 + GL-7 hi Fas hi GC B cells in 6-month-old B6. Sle1b .IFNαR 1 fl/fl (white) and B6. Sle1b .IFNαR 1 fl/fl GC Cre/+ (orange) female mice. (B and C) The percentages of B220 + GL-7 hi Fas hi GC B cells (B) and CD4 + CXCR5 hi PD-1 hi Tfh cells (C) in total splenocytes of 6-month-old B6. Sle1b .IFNαR 1 fl/fl (white) and B6. Sle1b .IFNαR 1 fl/fl GC Cre/+ (orange) mice. Each symbol represents a mouse, and horizontal lines indicate mean values. (D) Spleen sections from 5 mice per group were stained with anti-CD4 (red), GL-7 (green), and anti-IgD (blue). Representative images are shown in the left panels. GC areas were measured for 10 GCs per spleen section (right panel). The scale bars represent 100 μm. (E and F) Total IgG (E) and IgG2c (F) serum titers in these mice. (G and H) Serum titers of dsDNA-specific (G) and nucleosome-specific (H) IgG2c Abs in these mice. (I) Serum IgG2c ANA reactivity was measured by Hep-2 slides. The scale bars represent 250 μm. These data are obtained from two independent experiments, and each symbol represents a mouse. Statistical significance was determined using an unpaired, nonparametric Mann-Whitney Student’s t test (NS, not significant, *p

    Article Snippet: Briefly, splenocytes in RPMI containing 10% fetal bovine serum were plated at a concentration of 1 × 105 cells/well onto anti-IgM-, anti-IgG-, salmon sperm dsDNA- (Invitrogen, Grand Island, NY), calf thymus histone- (Sigma Aldrich, St. Louis, MO), or nucleosome-coated plates (Millipore, Bedford, MA).

    Techniques: Mouse Assay, Flow Cytometry, Expressing, Staining, MANN-WHITNEY

    Bcl-2 and Bcl-X L prevent the redistribution of cytochrome c in cells undergoing apoptosis. (A) Time course of cytochrome c release in Jurkat cells treated with TG. The release of cytochrome c in the cytosolic extract was determined by Western blot analysis and was quantified by densitometric scanning of the autoradiograph and plotted against time in hours after TG treatment. (B) Redistribution of cytochrome c in Bcl-2- and Bcl-X L -overexpressing Jurkat cells. JT/Neo, JT/Bcl-2, and JT/Bcl-X L cells were treated with 100 nM TG. Jurkat T cells were pretreated with the caspase inhibitor z-VAD-fmk (50 μM) for 1 h prior to addition of TG (right panel). After 3 h, the cells were mechanically lysed and separated into mitochondrial (M) and S100 (S) fractions. The amounts of cytochrome c and cytochrome oxidase (subunit IV) present in each fraction were determined by Western blot analysis. (C) Bcl-2 or Bcl-X L blocks TG-induced caspase-3 activation. Jurkat cells were treated with TG (100 nM) for various times. Caspase-3 activity was measured as specified by the manufacturer (see Materials and Methods). (D) The caspase inhibitors z-VAD-fmk and z-DEVD-fmk block TG- and CPA-induced apoptosis. Jurkat T cells were pretreated with the caspase inhibitor z-VAD-fmk (50 μM) or z-DEVD-fmk (50 μM) for 1 h and then treated with TG or CPA for an additional 36 h. Apoptosis was measured by a nucleosome ELISA.

    Journal: Molecular and Cellular Biology

    Article Title: Bcl-2 and Bcl-XL Block Thapsigargin-Induced Nitric Oxide Generation, c-Jun NH2-Terminal Kinase Activity, and Apoptosis

    doi:

    Figure Lengend Snippet: Bcl-2 and Bcl-X L prevent the redistribution of cytochrome c in cells undergoing apoptosis. (A) Time course of cytochrome c release in Jurkat cells treated with TG. The release of cytochrome c in the cytosolic extract was determined by Western blot analysis and was quantified by densitometric scanning of the autoradiograph and plotted against time in hours after TG treatment. (B) Redistribution of cytochrome c in Bcl-2- and Bcl-X L -overexpressing Jurkat cells. JT/Neo, JT/Bcl-2, and JT/Bcl-X L cells were treated with 100 nM TG. Jurkat T cells were pretreated with the caspase inhibitor z-VAD-fmk (50 μM) for 1 h prior to addition of TG (right panel). After 3 h, the cells were mechanically lysed and separated into mitochondrial (M) and S100 (S) fractions. The amounts of cytochrome c and cytochrome oxidase (subunit IV) present in each fraction were determined by Western blot analysis. (C) Bcl-2 or Bcl-X L blocks TG-induced caspase-3 activation. Jurkat cells were treated with TG (100 nM) for various times. Caspase-3 activity was measured as specified by the manufacturer (see Materials and Methods). (D) The caspase inhibitors z-VAD-fmk and z-DEVD-fmk block TG- and CPA-induced apoptosis. Jurkat T cells were pretreated with the caspase inhibitor z-VAD-fmk (50 μM) or z-DEVD-fmk (50 μM) for 1 h and then treated with TG or CPA for an additional 36 h. Apoptosis was measured by a nucleosome ELISA.

    Article Snippet: They were harvested for the nucleosome ELISA as specified by the manufacturer (Oncogene Research Products, Cambridge, Mass.).

    Techniques: Western Blot, Autoradiography, Activation Assay, Activity Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay

    Inhibition of TG- or CPA-induced apoptosis by chelating intracellular calcium or overexpressing Bcl-2 and Bcl-X L . (A) Time course of TG-induced apoptosis in JT/Neo, JT/Bcl-2, and JT/Bcl-X L cells. Cells were treated with TG (50 nM) for 4, 6, 18, 24, 36, and 48 h. (B and C) Chelation of intracellular calcium by BAPTA-AM blocks TG- or CPA-induced apoptosis. Cells were pretreated with BAPTA-AM (10 μM) for 45 min, washed, reseeded and treated with TG (10, 50, or 100 nM), or CPA (0.1, 1, or 10 μM) for 36 h. (D and E) Overexpression of Bcl-2 or Bcl-X L blocks TG- or CPA-induced apoptosis. Cells were treated with TG (10, 50, or 100 nM) or CPA (0.1, 1, or 10 μM) for 36 h. A nucleosome ELISA was used to measure apoptosis.

    Journal: Molecular and Cellular Biology

    Article Title: Bcl-2 and Bcl-XL Block Thapsigargin-Induced Nitric Oxide Generation, c-Jun NH2-Terminal Kinase Activity, and Apoptosis

    doi:

    Figure Lengend Snippet: Inhibition of TG- or CPA-induced apoptosis by chelating intracellular calcium or overexpressing Bcl-2 and Bcl-X L . (A) Time course of TG-induced apoptosis in JT/Neo, JT/Bcl-2, and JT/Bcl-X L cells. Cells were treated with TG (50 nM) for 4, 6, 18, 24, 36, and 48 h. (B and C) Chelation of intracellular calcium by BAPTA-AM blocks TG- or CPA-induced apoptosis. Cells were pretreated with BAPTA-AM (10 μM) for 45 min, washed, reseeded and treated with TG (10, 50, or 100 nM), or CPA (0.1, 1, or 10 μM) for 36 h. (D and E) Overexpression of Bcl-2 or Bcl-X L blocks TG- or CPA-induced apoptosis. Cells were treated with TG (10, 50, or 100 nM) or CPA (0.1, 1, or 10 μM) for 36 h. A nucleosome ELISA was used to measure apoptosis.

    Article Snippet: They were harvested for the nucleosome ELISA as specified by the manufacturer (Oncogene Research Products, Cambridge, Mass.).

    Techniques: Inhibition, Over Expression, Enzyme-linked Immunosorbent Assay

    ZRF1 facilitates the assembly of the UV – DDB – CUL4A E3 ligase complex. (A) ZRF1 displaces RING1B from chromatin during NER. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A ubiquitin and RING1B abundance was calculated. Values are given as mean ± SEM ( n = 3). (B) ZRF1 regulates chromatin association of CUL4A and CUL4B. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative CUL4B and CUL4A abundance was calculated. Values are given as mean ± SEM ( n = 3). (C) ZRF1 regulates CUL4A association with H2AX containing nucleosomes. Control cells and ZRF1 knockdown cells expressing FLAG H2AX were irradiated with UV. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (D) Knockdown of ZRF1 modulates CUL4A association with DDB2. Control cells and ZRF1 knockdown cells expressing FLAG DDB2 were irradiated with UV light. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (E) Assembly of the UV–DDB–CUL4A E3 ligase is facilitated by ZRF1. Control cells and ZRF1 knockdown HEK293T cells expressing HA RBX1 were irradiated with UV light. After immunoprecipitation with HA-specific antibodies the precipitated material was subjected to Western blotting, and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (F) ZRF1 competes with CUL4B and RING1B for DDB2 binding in vitro. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4B, and RING1B and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 over the other components (relative molarity ZRF1: DDB1–CUL4B–RING1B; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (G) ZRF1 does not compete with CUL4A and RBX1 for binding to DDB1–DDB2. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4A and RBX1 and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 (relative molarity ZRF1: DDB1–CUL4A–RBX1; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (H) ZRF1 mediates the formation of the UV-DDB-CUL4A complex in vitro. GFP and GFP-DDB2 were coupled to beads and incubated with CUL4B, DDB1 and RING1B. After washing, GFP and GFP-DDB2 (UV–RING1B complex) beads were incubated with an estimated fivefold excess of purified CUL4A and RBX1 (lanes 1–3) over the retained UV–RING1B complex. Simultaneously, ZRF1 (lanes 1 and 3) or GST (lane 2) was added to the incubations in equimolar amounts. The precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%.

    Journal: The Journal of Cell Biology

    Article Title: ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair

    doi: 10.1083/jcb.201506099

    Figure Lengend Snippet: ZRF1 facilitates the assembly of the UV – DDB – CUL4A E3 ligase complex. (A) ZRF1 displaces RING1B from chromatin during NER. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A ubiquitin and RING1B abundance was calculated. Values are given as mean ± SEM ( n = 3). (B) ZRF1 regulates chromatin association of CUL4A and CUL4B. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative CUL4B and CUL4A abundance was calculated. Values are given as mean ± SEM ( n = 3). (C) ZRF1 regulates CUL4A association with H2AX containing nucleosomes. Control cells and ZRF1 knockdown cells expressing FLAG H2AX were irradiated with UV. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (D) Knockdown of ZRF1 modulates CUL4A association with DDB2. Control cells and ZRF1 knockdown cells expressing FLAG DDB2 were irradiated with UV light. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (E) Assembly of the UV–DDB–CUL4A E3 ligase is facilitated by ZRF1. Control cells and ZRF1 knockdown HEK293T cells expressing HA RBX1 were irradiated with UV light. After immunoprecipitation with HA-specific antibodies the precipitated material was subjected to Western blotting, and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (F) ZRF1 competes with CUL4B and RING1B for DDB2 binding in vitro. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4B, and RING1B and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 over the other components (relative molarity ZRF1: DDB1–CUL4B–RING1B; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (G) ZRF1 does not compete with CUL4A and RBX1 for binding to DDB1–DDB2. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4A and RBX1 and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 (relative molarity ZRF1: DDB1–CUL4A–RBX1; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (H) ZRF1 mediates the formation of the UV-DDB-CUL4A complex in vitro. GFP and GFP-DDB2 were coupled to beads and incubated with CUL4B, DDB1 and RING1B. After washing, GFP and GFP-DDB2 (UV–RING1B complex) beads were incubated with an estimated fivefold excess of purified CUL4A and RBX1 (lanes 1–3) over the retained UV–RING1B complex. Simultaneously, ZRF1 (lanes 1 and 3) or GST (lane 2) was added to the incubations in equimolar amounts. The precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%.

    Article Snippet: In vitro ubiquitylation assays In vitro ubiquitylation reactions were performed with 3 µg purified histone H2A (New England Biolabs, Inc.) or 5 µg recombinant nucleosomes (Active Motif), 200 ng purified HIS-UBA1 (E1), 20 ng purified GST-UBC5H (E2), 150 ng purified UV-RING1B (E3), or 150 ng GST (control) in UBAB buffer (25 mM Tris/HCl, pH 7.5, 50 mM NaCl, and 10 mM MgCl2 ) supplemented with 20 mM ATP, 1.5 mg/ml ubiquitin, 10 mM DTT, and 1 U creatine phosphokinase.

    Techniques: Irradiation, Western Blot, Expressing, Immunoprecipitation, Purification, Incubation, Binding Assay, In Vitro

    H2A ubiquitylation after UV irradiation is performed by the UV–RING1B complex. (A) Protein interaction partners of RING1B and DDB2. Mass spectrometry analysis after sequential immunoprecipitations with FLAG and RING1B antibodies revealed DDB1 and CUL4B as main interaction partners of DDB2 and RING1B. A comprehensive list of the identified unique peptides after RING1B and control immunoprecipitations (with or without UV irradiation) is provided in Table S5 . (B) Assembly of the UV–RING1B complex. Plasmids expressing FLAG DDB1, FLAG DDB2, and FLAG RING1B were cotransfected in combination with either control plasmid or a plasmid encoding FLAG-STREP CUL4B. After immunoprecipitation with STREP-Tactin beads, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (C) Visualization of the UV–RING1B complex. Purified UV–RING1B complex was subjected to SDS gel electrophoresis and colloidal Coomassie staining. Mass spectrometry analysis revealed the presence of all four subunits (bold). A comprehensive list of unique peptides is provided in Table S6 . (D) The UV–RING1B complex catalyzes ubiquitylation of H2A in vitro. Ubiquitylation assays were performed with recombinant H2A, E1 (UBA1), E2 (UBCH5), and either GST (control) or the UV–RING1B complex. Reactions were performed at 37°C, and samples were taken at the indicated time points. Material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. (E) The UV–RING1B complex catalyzes monoubiquitylation of nucleosomal H2A. Ubiquitylation assays were performed with recombinant nucleosomes, E1 (UBA1), E2 (UBCH5), and either GST (control) or UV-RING1B complex. Reactions lacking E1 (−E1) were performed as additional controls. The ubiquitylation assays were performed at 37°C for 5 h, and samples or pure substrate (Substrate) were subjected to Western blotting and probed with H2A antibodies.

    Journal: The Journal of Cell Biology

    Article Title: ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair

    doi: 10.1083/jcb.201506099

    Figure Lengend Snippet: H2A ubiquitylation after UV irradiation is performed by the UV–RING1B complex. (A) Protein interaction partners of RING1B and DDB2. Mass spectrometry analysis after sequential immunoprecipitations with FLAG and RING1B antibodies revealed DDB1 and CUL4B as main interaction partners of DDB2 and RING1B. A comprehensive list of the identified unique peptides after RING1B and control immunoprecipitations (with or without UV irradiation) is provided in Table S5 . (B) Assembly of the UV–RING1B complex. Plasmids expressing FLAG DDB1, FLAG DDB2, and FLAG RING1B were cotransfected in combination with either control plasmid or a plasmid encoding FLAG-STREP CUL4B. After immunoprecipitation with STREP-Tactin beads, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (C) Visualization of the UV–RING1B complex. Purified UV–RING1B complex was subjected to SDS gel electrophoresis and colloidal Coomassie staining. Mass spectrometry analysis revealed the presence of all four subunits (bold). A comprehensive list of unique peptides is provided in Table S6 . (D) The UV–RING1B complex catalyzes ubiquitylation of H2A in vitro. Ubiquitylation assays were performed with recombinant H2A, E1 (UBA1), E2 (UBCH5), and either GST (control) or the UV–RING1B complex. Reactions were performed at 37°C, and samples were taken at the indicated time points. Material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. (E) The UV–RING1B complex catalyzes monoubiquitylation of nucleosomal H2A. Ubiquitylation assays were performed with recombinant nucleosomes, E1 (UBA1), E2 (UBCH5), and either GST (control) or UV-RING1B complex. Reactions lacking E1 (−E1) were performed as additional controls. The ubiquitylation assays were performed at 37°C for 5 h, and samples or pure substrate (Substrate) were subjected to Western blotting and probed with H2A antibodies.

    Article Snippet: In vitro ubiquitylation assays In vitro ubiquitylation reactions were performed with 3 µg purified histone H2A (New England Biolabs, Inc.) or 5 µg recombinant nucleosomes (Active Motif), 200 ng purified HIS-UBA1 (E1), 20 ng purified GST-UBC5H (E2), 150 ng purified UV-RING1B (E3), or 150 ng GST (control) in UBAB buffer (25 mM Tris/HCl, pH 7.5, 50 mM NaCl, and 10 mM MgCl2 ) supplemented with 20 mM ATP, 1.5 mg/ml ubiquitin, 10 mM DTT, and 1 U creatine phosphokinase.

    Techniques: Irradiation, Mass Spectrometry, Expressing, Plasmid Preparation, Immunoprecipitation, Purification, Western Blot, Incubation, SDS-Gel, Electrophoresis, Staining, In Vitro, Recombinant

    The nucleosome containing histone H3.5 is unstable. a Sequence comparison between human H3.1, H3.2, H3.3, H3T, and H3.5. The amino acids in H3.5 that differ from those in H3.3 are indicated by black boxes with white characters. The epitope peptide sequence used to generate the H3.5 antibody is underlined. The α-helices and β-strands found in the crystal structures of the human nucleosomes are represented on the top of the panel. b 18 % SDS-PAGE analysis of purified histones H3.1, H3.3, H3T, and H3.5, stained with Coomassie Brilliant Blue (CBB). c Non-denaturing 6 % PAGE analysis of purified nucleosomes containing H3.1, H3.3, H3T, and H3.5, stained with ethidium bromide. Lane 1 represents the naked DNA used in the nucleosome reconstitution. Nucleosome core particles are denoted by NCPs. d Histone compositions of the purified nucleosomes containing H3.1, H3.3, H3T, and H3.5, analyzed by 18 % SDS-PAGE with Coomassie Brilliant Blue staining. e Salt resistance assays of the H3.1 and H3.3 nucleosomes and f the H3.3, H3T, and H3.5 nucleosomes. Bands corresponding to nucleosomes are indicated by NCPs. Asterisks represent bands corresponding to non-nucleosomal DNA-histone complexes [ 26 ]

    Journal: Epigenetics & Chromatin

    Article Title: Histone H3.5 forms an unstable nucleosome and accumulates around transcription start sites in human testis

    doi: 10.1186/s13072-016-0051-y

    Figure Lengend Snippet: The nucleosome containing histone H3.5 is unstable. a Sequence comparison between human H3.1, H3.2, H3.3, H3T, and H3.5. The amino acids in H3.5 that differ from those in H3.3 are indicated by black boxes with white characters. The epitope peptide sequence used to generate the H3.5 antibody is underlined. The α-helices and β-strands found in the crystal structures of the human nucleosomes are represented on the top of the panel. b 18 % SDS-PAGE analysis of purified histones H3.1, H3.3, H3T, and H3.5, stained with Coomassie Brilliant Blue (CBB). c Non-denaturing 6 % PAGE analysis of purified nucleosomes containing H3.1, H3.3, H3T, and H3.5, stained with ethidium bromide. Lane 1 represents the naked DNA used in the nucleosome reconstitution. Nucleosome core particles are denoted by NCPs. d Histone compositions of the purified nucleosomes containing H3.1, H3.3, H3T, and H3.5, analyzed by 18 % SDS-PAGE with Coomassie Brilliant Blue staining. e Salt resistance assays of the H3.1 and H3.3 nucleosomes and f the H3.3, H3T, and H3.5 nucleosomes. Bands corresponding to nucleosomes are indicated by NCPs. Asterisks represent bands corresponding to non-nucleosomal DNA-histone complexes [ 26 ]

    Article Snippet: The nucleosomes were reconstituted by the salt-dialysis method, heated at 55 °C for 2 h, and further purified from the free DNA and histones by non-denaturing polyacrylamide gel electrophoresis (Prep Cell, Bio-Rad).

    Techniques: Sequencing, SDS Page, Purification, Staining, Polyacrylamide Gel Electrophoresis

    Crystal structure of the H3.5 nucleosome. a Overall structure of the H3.5 nucleosome. The H3.5, H4, H2A, H2B, and DNA molecules are colored sky blue , light orange , pale green , pale yellow , and gray , respectively. The H3.5-specific Leu103 residues are colored red , and their side chains are represented. b Stereo view of the H3.5 ( sky blue ) and H4 ( light orange ) region around the H3.5 Leu103 residue ( red ). The 2mFo-DFc electron density map around the H3.5 Leu103 residue is shown as a blue mesh, contoured at 1.5σ. The van der Waals surfaces of the H3.5 Leu103 side chain atoms, and the H4 Ile34, Ile50, and Thr54 side chain atoms, are represented. c Stereo view of the H3.3 ( deep purple ) and H4 ( light orange ) region around the H3.3 Phe104 residue in the H3.3 nucleosome structure [PDB:3AV2] [ 27 ]. The 2mFo-DFc electron density map around the H3.3 Phe104 residue is shown as a blue mesh, contoured at 1.5σ. The van der Waals surfaces of the H3.3 Phe104 side chain atoms, and the H4 Ile34, Ile50, and Thr54 side chain atoms, are represented

    Journal: Epigenetics & Chromatin

    Article Title: Histone H3.5 forms an unstable nucleosome and accumulates around transcription start sites in human testis

    doi: 10.1186/s13072-016-0051-y

    Figure Lengend Snippet: Crystal structure of the H3.5 nucleosome. a Overall structure of the H3.5 nucleosome. The H3.5, H4, H2A, H2B, and DNA molecules are colored sky blue , light orange , pale green , pale yellow , and gray , respectively. The H3.5-specific Leu103 residues are colored red , and their side chains are represented. b Stereo view of the H3.5 ( sky blue ) and H4 ( light orange ) region around the H3.5 Leu103 residue ( red ). The 2mFo-DFc electron density map around the H3.5 Leu103 residue is shown as a blue mesh, contoured at 1.5σ. The van der Waals surfaces of the H3.5 Leu103 side chain atoms, and the H4 Ile34, Ile50, and Thr54 side chain atoms, are represented. c Stereo view of the H3.3 ( deep purple ) and H4 ( light orange ) region around the H3.3 Phe104 residue in the H3.3 nucleosome structure [PDB:3AV2] [ 27 ]. The 2mFo-DFc electron density map around the H3.3 Phe104 residue is shown as a blue mesh, contoured at 1.5σ. The van der Waals surfaces of the H3.3 Phe104 side chain atoms, and the H4 Ile34, Ile50, and Thr54 side chain atoms, are represented

    Article Snippet: The nucleosomes were reconstituted by the salt-dialysis method, heated at 55 °C for 2 h, and further purified from the free DNA and histones by non-denaturing polyacrylamide gel electrophoresis (Prep Cell, Bio-Rad).

    Techniques:

    Mutational analysis of H3.5 and H3.3. a Non-denaturing 6 % PAGE analysis of the purified nucleosomes containing H3.5 and H3.3 mutants, stained with ethidium bromide. Lane 1 represents the naked DNA used in the nucleosome reconstitution. Nucleosome core particles are denoted by NCPs. b Histone compositions of the purified nucleosomes containing H3.3 and H3.5 mutants, analyzed by 18 % SDS-PAGE with Coomassie Brilliant Blue staining. c Salt resistance assays of the H3.5 mutant nucleosomes, and d the H3.3 mutant nucleosomes. Bands corresponding to nucleosomes are indicated as NCPs. Asterisks represent bands corresponding to non-nucleosomal DNA-histone complexes [ 26 ]

    Journal: Epigenetics & Chromatin

    Article Title: Histone H3.5 forms an unstable nucleosome and accumulates around transcription start sites in human testis

    doi: 10.1186/s13072-016-0051-y

    Figure Lengend Snippet: Mutational analysis of H3.5 and H3.3. a Non-denaturing 6 % PAGE analysis of the purified nucleosomes containing H3.5 and H3.3 mutants, stained with ethidium bromide. Lane 1 represents the naked DNA used in the nucleosome reconstitution. Nucleosome core particles are denoted by NCPs. b Histone compositions of the purified nucleosomes containing H3.3 and H3.5 mutants, analyzed by 18 % SDS-PAGE with Coomassie Brilliant Blue staining. c Salt resistance assays of the H3.5 mutant nucleosomes, and d the H3.3 mutant nucleosomes. Bands corresponding to nucleosomes are indicated as NCPs. Asterisks represent bands corresponding to non-nucleosomal DNA-histone complexes [ 26 ]

    Article Snippet: The nucleosomes were reconstituted by the salt-dialysis method, heated at 55 °C for 2 h, and further purified from the free DNA and histones by non-denaturing polyacrylamide gel electrophoresis (Prep Cell, Bio-Rad).

    Techniques: Polyacrylamide Gel Electrophoresis, Purification, Staining, SDS Page, Mutagenesis

    Hierarchical looping at the gene level. Structural model of the GATA-4 gene locus in an open, active state ( left ) versus an epigenetically silent state ( right ). The GATA-4 gene has been shown to exhibit five distinct chromatin loops of ∼43, 61, 57, 157, and 109 nucleosomes each (colored in tan , blue , green , white , and purple , respectively); each loop is associated with enriched trimethylation of Lys27 of histone tail H3 (H3K27Me3) and PcG protein binding. The TSS, which resides between loop 3 and 4 (from 5′ to 3′), is enveloped by the chromatin loops when these contacts are enforced in our mesoscale model, suggesting a structural mechanism for epigenetic silencing of the GATA-4 gene. To see this figure in color, go online.

    Journal: Biophysical Journal

    Article Title: Linking Chromatin Fibers to Gene Folding by Hierarchical Looping

    doi: 10.1016/j.bpj.2017.01.003

    Figure Lengend Snippet: Hierarchical looping at the gene level. Structural model of the GATA-4 gene locus in an open, active state ( left ) versus an epigenetically silent state ( right ). The GATA-4 gene has been shown to exhibit five distinct chromatin loops of ∼43, 61, 57, 157, and 109 nucleosomes each (colored in tan , blue , green , white , and purple , respectively); each loop is associated with enriched trimethylation of Lys27 of histone tail H3 (H3K27Me3) and PcG protein binding. The TSS, which resides between loop 3 and 4 (from 5′ to 3′), is enveloped by the chromatin loops when these contacts are enforced in our mesoscale model, suggesting a structural mechanism for epigenetic silencing of the GATA-4 gene. To see this figure in color, go online.

    Article Snippet: Role of DNA sequence in nucleosome stability and dynamics.

    Techniques: Protein Binding

    A schematic view of the levels and structures involved as the chromatin fiber packs genomic DNA in the cell nucleus. Several levels of structural and functional states are known but not well understood. Double-stranded DNA binds to histone proteins to form complexes, known as nucleosomes, the building blocks of chromatin. Each nucleosome is composed of ∼147 bp of DNA coiled around eight histone proteins, two dimers each of H2A, H2B, H3, and H4. Many nucleosomes have been resolved at atomic resolution. Nucleosomes connect to one another to form the chromatin fiber, where an open state forms at low salt without linker histones called beads-on-a-string. A more condensed state forms at high salt with linker histones like H1 or H5. The long-assumed compact 30-nm fiber may adopt more variable forms in vivo, as shown for the subsaturated LH fibers (one LH per two nucleosomes, or ½LH) and LH-deficient (−LH) fibers shown in the bottom left. Relatively straight +LH fibers show little self-association, whereas ½LH fibers show moderate self-association, and −LH fibers show high levels of self-association. All three types show variable widths. Additionally, these self-associating fibers may involve hierarchical looping or flaking, as used in mountain climbing and rappelling, which is more pronounced in ½LH and −LH fibers. Flaking, in this context, refers to the lateral compaction of a long polymer due to loops of various size undergoing further folding in space, while avoiding tangles or knots. In the context of rope folding, flaking refers to packaging flexible elongated rope into neatly stacked loops (by winding the loops back and forth) so that the rope can be easily unraveled. A simple scheme for such rope stacking is shown at the bottom left. At bottom right, we show another possible variation of such compact networks of stacked loops. Active gene elements form large loose loops that bridge gene promoters to gene-transcribing regions. In cooperation with other epigenetic marks and scaffolding proteins, these gene elements organize into chromosomes. In interphase chromosomes, individual chromosomes are separated into different chromosomal territories. In the metaphase cell, individual chromosomes can be distinguished, as shown by different colors. Interphase chromosomes are also known to exist in different levels of condensation. The more loosely packed genomic state is known as “euchromatin”, while the more densely packed genomic material is denoted as “heterochromatin”. A possible internal organization for these two related states involves tight versus open networks of chromatin loops, which build upon the proposed flaking or hierarchical looping motif. To see this figure in color, go online.

    Journal: Biophysical Journal

    Article Title: Linking Chromatin Fibers to Gene Folding by Hierarchical Looping

    doi: 10.1016/j.bpj.2017.01.003

    Figure Lengend Snippet: A schematic view of the levels and structures involved as the chromatin fiber packs genomic DNA in the cell nucleus. Several levels of structural and functional states are known but not well understood. Double-stranded DNA binds to histone proteins to form complexes, known as nucleosomes, the building blocks of chromatin. Each nucleosome is composed of ∼147 bp of DNA coiled around eight histone proteins, two dimers each of H2A, H2B, H3, and H4. Many nucleosomes have been resolved at atomic resolution. Nucleosomes connect to one another to form the chromatin fiber, where an open state forms at low salt without linker histones called beads-on-a-string. A more condensed state forms at high salt with linker histones like H1 or H5. The long-assumed compact 30-nm fiber may adopt more variable forms in vivo, as shown for the subsaturated LH fibers (one LH per two nucleosomes, or ½LH) and LH-deficient (−LH) fibers shown in the bottom left. Relatively straight +LH fibers show little self-association, whereas ½LH fibers show moderate self-association, and −LH fibers show high levels of self-association. All three types show variable widths. Additionally, these self-associating fibers may involve hierarchical looping or flaking, as used in mountain climbing and rappelling, which is more pronounced in ½LH and −LH fibers. Flaking, in this context, refers to the lateral compaction of a long polymer due to loops of various size undergoing further folding in space, while avoiding tangles or knots. In the context of rope folding, flaking refers to packaging flexible elongated rope into neatly stacked loops (by winding the loops back and forth) so that the rope can be easily unraveled. A simple scheme for such rope stacking is shown at the bottom left. At bottom right, we show another possible variation of such compact networks of stacked loops. Active gene elements form large loose loops that bridge gene promoters to gene-transcribing regions. In cooperation with other epigenetic marks and scaffolding proteins, these gene elements organize into chromosomes. In interphase chromosomes, individual chromosomes are separated into different chromosomal territories. In the metaphase cell, individual chromosomes can be distinguished, as shown by different colors. Interphase chromosomes are also known to exist in different levels of condensation. The more loosely packed genomic state is known as “euchromatin”, while the more densely packed genomic material is denoted as “heterochromatin”. A possible internal organization for these two related states involves tight versus open networks of chromatin loops, which build upon the proposed flaking or hierarchical looping motif. To see this figure in color, go online.

    Article Snippet: Role of DNA sequence in nucleosome stability and dynamics.

    Techniques: Functional Assay, In Vivo, Scaffolding

    Hierarchical looping at the fiber level. ( a ), so that the electrostatic field is reasonably reproduced as a function of the monovalent salt concentration. These DNA, LH, and histone tail beads are combined with the nucleosome to form our mesoscale chromatin model. ( b – d ) Sample 96-nucleosome configurations are shown for LH-deficient (−LH) ( b ), partially LH-saturated (one LH per two nucleosomes, or ½LH) ( c ), and LH-saturated (one LH per nucleosome, or +LH) ( d ) fibers. Internucleosome contact matrices are calculated from each mesoscale configuration by measuring the distance between any two core, tail, or linker DNA beads belonging to separate nucleosomes, where any distance measured

    Journal: Biophysical Journal

    Article Title: Linking Chromatin Fibers to Gene Folding by Hierarchical Looping

    doi: 10.1016/j.bpj.2017.01.003

    Figure Lengend Snippet: Hierarchical looping at the fiber level. ( a ), so that the electrostatic field is reasonably reproduced as a function of the monovalent salt concentration. These DNA, LH, and histone tail beads are combined with the nucleosome to form our mesoscale chromatin model. ( b – d ) Sample 96-nucleosome configurations are shown for LH-deficient (−LH) ( b ), partially LH-saturated (one LH per two nucleosomes, or ½LH) ( c ), and LH-saturated (one LH per nucleosome, or +LH) ( d ) fibers. Internucleosome contact matrices are calculated from each mesoscale configuration by measuring the distance between any two core, tail, or linker DNA beads belonging to separate nucleosomes, where any distance measured

    Article Snippet: Role of DNA sequence in nucleosome stability and dynamics.

    Techniques: Concentration Assay

    ( A ) M601 DNA sequence used in the remodeling assays. GRP78-gene primer sequences flanking the DNA construct are underlined and CpG dinucleotides are highlighted in grey. The residues in bold denote the approximate position of the protection caused by the histone octamer on the nucleosome substrate. ( B ) Schematic representation of the procedure used to perform single molecule analyses of the remodeled products. Nucleosomes were assembled using the M601 DNA. After incubation with (or without) nucleosome remodeling factor, remodeled nucleosomes were methylated with the M.SssI CpG methyltransferase to create a footprint of DNA accessibility (the DNA methyltransferase only methylates cytosine residues that are not bound to the histones). After native electrophoresis, excision and elution from the gel, nucleosomes were deproteinized and the nucleosomal DNA molecules subjected to conventional bisulfite treatment to reveal their methylation pattern. Changes in these patterns were compared to the input nucleosome to assess alterations in histone–DNA contacts resulting from the action of the remodeling factors.

    Journal: Nucleic Acids Research

    Article Title: Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF

    doi: 10.1093/nar/gkp524

    Figure Lengend Snippet: ( A ) M601 DNA sequence used in the remodeling assays. GRP78-gene primer sequences flanking the DNA construct are underlined and CpG dinucleotides are highlighted in grey. The residues in bold denote the approximate position of the protection caused by the histone octamer on the nucleosome substrate. ( B ) Schematic representation of the procedure used to perform single molecule analyses of the remodeled products. Nucleosomes were assembled using the M601 DNA. After incubation with (or without) nucleosome remodeling factor, remodeled nucleosomes were methylated with the M.SssI CpG methyltransferase to create a footprint of DNA accessibility (the DNA methyltransferase only methylates cytosine residues that are not bound to the histones). After native electrophoresis, excision and elution from the gel, nucleosomes were deproteinized and the nucleosomal DNA molecules subjected to conventional bisulfite treatment to reveal their methylation pattern. Changes in these patterns were compared to the input nucleosome to assess alterations in histone–DNA contacts resulting from the action of the remodeling factors.

    Article Snippet: In another study, Wang and colleagues probed histone-DNA contacts on single nucleosomes that were remodeled by ySWI/SNF by unzipping their DNA double helix and compared their ‘disruption signature’ to that of the nucleosome substrate.

    Techniques: Sequencing, Construct, Incubation, Methylation, Electrophoresis

    ( A ) Native electrophoresis of hACF remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of hACF complex (lane 6: 8 nM; lane 7: 24 nM; lanes 8 and 9: 72 nM) in the presence (lanes 6–8) or absence (lane 9) of ATP (1 mM) as indicated on top. Reactions were handled identically and in parallel to samples in Figure 3 . ( B–E ) Schematic representation of individual DNA molecules remodeled by hACF. Bisulfite-converted DNAs from gel slices (black frames, lanes 6–9) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 3 . ( F ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel E (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–D (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.

    Journal: Nucleic Acids Research

    Article Title: Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF

    doi: 10.1093/nar/gkp524

    Figure Lengend Snippet: ( A ) Native electrophoresis of hACF remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of hACF complex (lane 6: 8 nM; lane 7: 24 nM; lanes 8 and 9: 72 nM) in the presence (lanes 6–8) or absence (lane 9) of ATP (1 mM) as indicated on top. Reactions were handled identically and in parallel to samples in Figure 3 . ( B–E ) Schematic representation of individual DNA molecules remodeled by hACF. Bisulfite-converted DNAs from gel slices (black frames, lanes 6–9) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 3 . ( F ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel E (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–D (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.

    Article Snippet: In another study, Wang and colleagues probed histone-DNA contacts on single nucleosomes that were remodeled by ySWI/SNF by unzipping their DNA double helix and compared their ‘disruption signature’ to that of the nucleosome substrate.

    Techniques: Electrophoresis, Incubation, Amplification, Polymerase Chain Reaction, Clone Assay, Methylation

    ( A ) Native electrophoresis of BRG1 remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of BRG1 (lane 10: 55 nM; lane 11: 170 nM; lanes 12 and 13: 500 nM) in the presence (lanes 10–12) or absence (lane 13) of ATP (1 mM) as indicated on top. Reactions were handled identically and in parallel to samples in Figure 3 . ( B–F ) Schematic representation of individual DNA molecules remodeled by BRG1. Bisulfite-converted DNAs from gel slices (black frames, lanes 10–13) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 3 . ( G ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel F (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–E (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.

    Journal: Nucleic Acids Research

    Article Title: Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF

    doi: 10.1093/nar/gkp524

    Figure Lengend Snippet: ( A ) Native electrophoresis of BRG1 remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of BRG1 (lane 10: 55 nM; lane 11: 170 nM; lanes 12 and 13: 500 nM) in the presence (lanes 10–12) or absence (lane 13) of ATP (1 mM) as indicated on top. Reactions were handled identically and in parallel to samples in Figure 3 . ( B–F ) Schematic representation of individual DNA molecules remodeled by BRG1. Bisulfite-converted DNAs from gel slices (black frames, lanes 10–13) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 3 . ( G ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel F (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–E (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.

    Article Snippet: In another study, Wang and colleagues probed histone-DNA contacts on single nucleosomes that were remodeled by ySWI/SNF by unzipping their DNA double helix and compared their ‘disruption signature’ to that of the nucleosome substrate.

    Techniques: Electrophoresis, Incubation, Amplification, Polymerase Chain Reaction, Clone Assay, Methylation

    ( A ) Native electrophoresis of SNF2H remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of SNF2H (lane 2: 6 nM; lane 3: 19 nM; lanes 4 and 5: 57 nM) in the presence (lanes 2–4) or absence (lane 5) of ATP (1 mM) as indicated on top, for 1 h at 30°C. Reactions were stopped by addition of ADP (10 mM) and incubation on ice for 10 min. Methylation of the remodeled products were performed as follow. The reactions were incubated for 15 min at 37°C after addition of M.SssI (5 U) and S-adenosylmethionine (160 μM). The remodeling reactions were separated by native gel electrophoresis after addition of competitor plasmid DNA and visualized by ethidium bromide staining. Gel areas excised and used for analysis are delimited by a black frame. Back frames are connected by dashed lines when gel slices were combined. ( B–F ) Schematic representation of individual DNA molecules remodeled by SNF2H. Bisulfite-converted DNAs from gel slices (black frames, lanes 3–5) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 2 D. The number of remodeled molecules shown is proportional to the average intensity of the bands generated after remodeling at enzyme concentration allowing maximal remodeling in three independent experiments. ( G ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel F (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–E (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.

    Journal: Nucleic Acids Research

    Article Title: Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF

    doi: 10.1093/nar/gkp524

    Figure Lengend Snippet: ( A ) Native electrophoresis of SNF2H remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of SNF2H (lane 2: 6 nM; lane 3: 19 nM; lanes 4 and 5: 57 nM) in the presence (lanes 2–4) or absence (lane 5) of ATP (1 mM) as indicated on top, for 1 h at 30°C. Reactions were stopped by addition of ADP (10 mM) and incubation on ice for 10 min. Methylation of the remodeled products were performed as follow. The reactions were incubated for 15 min at 37°C after addition of M.SssI (5 U) and S-adenosylmethionine (160 μM). The remodeling reactions were separated by native gel electrophoresis after addition of competitor plasmid DNA and visualized by ethidium bromide staining. Gel areas excised and used for analysis are delimited by a black frame. Back frames are connected by dashed lines when gel slices were combined. ( B–F ) Schematic representation of individual DNA molecules remodeled by SNF2H. Bisulfite-converted DNAs from gel slices (black frames, lanes 3–5) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 2 D. The number of remodeled molecules shown is proportional to the average intensity of the bands generated after remodeling at enzyme concentration allowing maximal remodeling in three independent experiments. ( G ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel F (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–E (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.

    Article Snippet: In another study, Wang and colleagues probed histone-DNA contacts on single nucleosomes that were remodeled by ySWI/SNF by unzipping their DNA double helix and compared their ‘disruption signature’ to that of the nucleosome substrate.

    Techniques: Electrophoresis, Incubation, Methylation, Nucleic Acid Electrophoresis, Plasmid Preparation, Staining, Amplification, Polymerase Chain Reaction, Clone Assay, Generated, Concentration Assay

    ( A ) Proteins used in the nucleosome remodeling assays were separated by SDS-PAGE and visualized by Coomassie Blue staining. Molecular masses are indicated on the left and enzymes indicated on top. ( B ) Analysis of remodeling activities in the presence of M.SssI. The restriction enzyme-accessibility (REA) assays measured the ability of the remodeling factors to expose an MfeI restriction site at bp position 108 (31-bp away from the first protected site) in the absence or presence of M.SssI. M601 nucleosomes (50 nM) were incubated with MfeI (25 U) in the absence (blue curves) or the presence (2.5 U, purple curves; 5 U, red curves) of M.SssI. Reactions in the absence or presence of remodeling enzyme are specified by diamond or circle plots, respectively (as indicated on top of each panel). Reactions in the presence of remodeler but absence of ATP are plotted in black. Enzymes were used at the following concentrations: SNF2H: 530 nM; hACF: 160 nM; BRG1: 690 nM and hSWI/SNF 68 nM. Curve fits of the data (obtained from averaging at least two independent experiments) were achieved using first-order exponential decay using an apparent endpoint of the reactions with the KaleidaGraph software. k obs for SNF2H = 0.18 ± 0.01 min −1 without M.SssI, 0.19 ± 0.01 min −1 + 2.5 U M.SssI and 0.15 ± 0.01 min −1 + 5 U M.SssI. k obs for hACF= 0.08 ± 0.01 min −1 without M.SssI, 0.09 ± 0.01 min −1 + 2.5 U M.SssI and 0.09 ± 0.01 min −1 + 5 U M.SssI. k obs for BRG1 = 0.02 ± 0.001 min −1 without M.SssI, 0.019 ± 0.001 min −1 + 2.5 U M.SssI and 0.02 ± 0.001 min −1 + 5 U M.SssI. k obs for hSWI/SNF = 0.13 ± 0.01 min −1 without M.SssI, 0.11 ± 0.01 min −1 + 2.5 U M.SssI and 0.1 ± 0.005 min −1 + 5 U M.SssI. ( C ) Nucleosomes (50 nM) were incubated as in Figure 3 , but addition of remodeler was omitted. Nucleosomes were then methylated with of M.SssI (5 U), separated by native gel electrophoresis and visualized by ethidium bromide staining. The gel area excised and used for analysis is delimited by a black frame. ( D ) Schematic representation of individual DNA molecules. Bisulfite-converted DNA from the excised gel slice (black frame, lane 1) was amplified by PCR, cloned and sequenced. Each line represents the sequence of individual DNA clones and the circles represent CpG dinucleotides. Methylated and unmethylated CpGs are indicated by filled (black) and open circles, respectively. ( E ) The frequency of methylation was determined at given CpG sites by averaging methylation for all the DNA molecules showed in panel D and expressed as a percentage. The position of the CpGs relative to the DNA sequence is indicated on the X -axis and clone numbers are indicated on the Y -axis.

    Journal: Nucleic Acids Research

    Article Title: Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF

    doi: 10.1093/nar/gkp524

    Figure Lengend Snippet: ( A ) Proteins used in the nucleosome remodeling assays were separated by SDS-PAGE and visualized by Coomassie Blue staining. Molecular masses are indicated on the left and enzymes indicated on top. ( B ) Analysis of remodeling activities in the presence of M.SssI. The restriction enzyme-accessibility (REA) assays measured the ability of the remodeling factors to expose an MfeI restriction site at bp position 108 (31-bp away from the first protected site) in the absence or presence of M.SssI. M601 nucleosomes (50 nM) were incubated with MfeI (25 U) in the absence (blue curves) or the presence (2.5 U, purple curves; 5 U, red curves) of M.SssI. Reactions in the absence or presence of remodeling enzyme are specified by diamond or circle plots, respectively (as indicated on top of each panel). Reactions in the presence of remodeler but absence of ATP are plotted in black. Enzymes were used at the following concentrations: SNF2H: 530 nM; hACF: 160 nM; BRG1: 690 nM and hSWI/SNF 68 nM. Curve fits of the data (obtained from averaging at least two independent experiments) were achieved using first-order exponential decay using an apparent endpoint of the reactions with the KaleidaGraph software. k obs for SNF2H = 0.18 ± 0.01 min −1 without M.SssI, 0.19 ± 0.01 min −1 + 2.5 U M.SssI and 0.15 ± 0.01 min −1 + 5 U M.SssI. k obs for hACF= 0.08 ± 0.01 min −1 without M.SssI, 0.09 ± 0.01 min −1 + 2.5 U M.SssI and 0.09 ± 0.01 min −1 + 5 U M.SssI. k obs for BRG1 = 0.02 ± 0.001 min −1 without M.SssI, 0.019 ± 0.001 min −1 + 2.5 U M.SssI and 0.02 ± 0.001 min −1 + 5 U M.SssI. k obs for hSWI/SNF = 0.13 ± 0.01 min −1 without M.SssI, 0.11 ± 0.01 min −1 + 2.5 U M.SssI and 0.1 ± 0.005 min −1 + 5 U M.SssI. ( C ) Nucleosomes (50 nM) were incubated as in Figure 3 , but addition of remodeler was omitted. Nucleosomes were then methylated with of M.SssI (5 U), separated by native gel electrophoresis and visualized by ethidium bromide staining. The gel area excised and used for analysis is delimited by a black frame. ( D ) Schematic representation of individual DNA molecules. Bisulfite-converted DNA from the excised gel slice (black frame, lane 1) was amplified by PCR, cloned and sequenced. Each line represents the sequence of individual DNA clones and the circles represent CpG dinucleotides. Methylated and unmethylated CpGs are indicated by filled (black) and open circles, respectively. ( E ) The frequency of methylation was determined at given CpG sites by averaging methylation for all the DNA molecules showed in panel D and expressed as a percentage. The position of the CpGs relative to the DNA sequence is indicated on the X -axis and clone numbers are indicated on the Y -axis.

    Article Snippet: In another study, Wang and colleagues probed histone-DNA contacts on single nucleosomes that were remodeled by ySWI/SNF by unzipping their DNA double helix and compared their ‘disruption signature’ to that of the nucleosome substrate.

    Techniques: SDS Page, Staining, Incubation, Software, Methylation, Nucleic Acid Electrophoresis, Amplification, Polymerase Chain Reaction, Clone Assay, Sequencing

    ( A ) Size distribution of the protections observed after incubation without (Nuc) or with (as indicated on the X -axis) remodeling factor. Bars indicate the percentage of sequences showing a protection in the following size ranges: between 0–45, 46–70, 71–95, 96–120, 121–145, 146–170, 171–195, 196–220 and 221–245-bp, as indicated by the color code on the right. ( B ) Direct comparison of average DNA accessibility generated by nucleosome remodeling factors (as indicated by the color code on the right). Methylation averages at given CpGs obtained for nucleosome ( Figure 2 E), SNF2H ( Figure 3 G, lower panel), hACF ( Figure 4 F, lower panel), BRG1 ( Figure 5 G, lower panel) and hSWI/SNF ( Figure 6 F, lower panel) are displayed as curves for clarity.

    Journal: Nucleic Acids Research

    Article Title: Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF

    doi: 10.1093/nar/gkp524

    Figure Lengend Snippet: ( A ) Size distribution of the protections observed after incubation without (Nuc) or with (as indicated on the X -axis) remodeling factor. Bars indicate the percentage of sequences showing a protection in the following size ranges: between 0–45, 46–70, 71–95, 96–120, 121–145, 146–170, 171–195, 196–220 and 221–245-bp, as indicated by the color code on the right. ( B ) Direct comparison of average DNA accessibility generated by nucleosome remodeling factors (as indicated by the color code on the right). Methylation averages at given CpGs obtained for nucleosome ( Figure 2 E), SNF2H ( Figure 3 G, lower panel), hACF ( Figure 4 F, lower panel), BRG1 ( Figure 5 G, lower panel) and hSWI/SNF ( Figure 6 F, lower panel) are displayed as curves for clarity.

    Article Snippet: In another study, Wang and colleagues probed histone-DNA contacts on single nucleosomes that were remodeled by ySWI/SNF by unzipping their DNA double helix and compared their ‘disruption signature’ to that of the nucleosome substrate.

    Techniques: Incubation, Generated, Methylation

    ( A ) Native electrophoresis of hSWI/SNF remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of hSWI/SNF complex (lane 14: 6 nM; lane 15: 19 nM; lanes 16 and 17: 56 nM) in the presence (lanes 14–16) or absence (lane 17) of ATP (1 mM) as indicated on top. Reactions were handled identically and in parallel to samples in Figure 3 . ( B–E ) Schematic representation of individual DNA molecules remodeled by hSWI/SNF. Bisulfite-converted DNAs from gel slices (black frames, lanes 14–17) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 3 . ( F ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel E (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–D (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.

    Journal: Nucleic Acids Research

    Article Title: Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF

    doi: 10.1093/nar/gkp524

    Figure Lengend Snippet: ( A ) Native electrophoresis of hSWI/SNF remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of hSWI/SNF complex (lane 14: 6 nM; lane 15: 19 nM; lanes 16 and 17: 56 nM) in the presence (lanes 14–16) or absence (lane 17) of ATP (1 mM) as indicated on top. Reactions were handled identically and in parallel to samples in Figure 3 . ( B–E ) Schematic representation of individual DNA molecules remodeled by hSWI/SNF. Bisulfite-converted DNAs from gel slices (black frames, lanes 14–17) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 3 . ( F ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel E (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–D (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.

    Article Snippet: In another study, Wang and colleagues probed histone-DNA contacts on single nucleosomes that were remodeled by ySWI/SNF by unzipping their DNA double helix and compared their ‘disruption signature’ to that of the nucleosome substrate.

    Techniques: Electrophoresis, Incubation, Amplification, Polymerase Chain Reaction, Clone Assay, Methylation

    X-ray structure of the nucleosome core particle. These views of NCP147, at Å resolution, show the two strands of the double-helix in purple and green, with the protein core in grey. (A) shows the curvature of DNA around the histone core, with the dyad at the top, center; (B) represents a rotation of the particle, showing the adjacent segments of DNA, opposite the dyad; and (C) represents a rotation in the opposite direction, showing the DNA crossing over the dyad. As indicated by the coordinate system axes, in (A) the y-axis is pointing out of the page, in (B) the z-axis is pointing into the page, and in (C) the z-axis is pointing out of the page.

    Journal: PLoS Computational Biology

    Article Title: Learning a Weighted Sequence Model of the Nucleosome Core and Linker Yields More Accurate Predictions in Saccharomyces cerevisiae and Homo sapiens

    doi: 10.1371/journal.pcbi.1000834

    Figure Lengend Snippet: X-ray structure of the nucleosome core particle. These views of NCP147, at Å resolution, show the two strands of the double-helix in purple and green, with the protein core in grey. (A) shows the curvature of DNA around the histone core, with the dyad at the top, center; (B) represents a rotation of the particle, showing the adjacent segments of DNA, opposite the dyad; and (C) represents a rotation in the opposite direction, showing the DNA crossing over the dyad. As indicated by the coordinate system axes, in (A) the y-axis is pointing out of the page, in (B) the z-axis is pointing into the page, and in (C) the z-axis is pointing out of the page.

    Article Snippet: The dominant hypothesis regarding DNA sequence preference of nucleosome formation is related to the curvature required to wrap the double helix tightly around the histone core .

    Techniques: Polyacrylamide Gel Electrophoresis

    Distribution of distances between successive nucleosome dyad positions. The distributions shown here were derived from Field et al. [4] S. cerevisiae data (red), and from genome-wide model predictions in S. cerevisiae (green), and in H. sapiens (dark blue). The predicted dyad positions in H. sapiens are also shown partitioned according to the fraction of the neighboring 200 bases that are marked as repetitive ( 25% repeat in pink, and 75% repeat in aqua). For the purposes of this analysis, a predicted dyad position is a local maximum in the dyad score trace. The grey line shows the geometric distribution resulting from random positions with an average spacing of 165 bp.

    Journal: PLoS Computational Biology

    Article Title: Learning a Weighted Sequence Model of the Nucleosome Core and Linker Yields More Accurate Predictions in Saccharomyces cerevisiae and Homo sapiens

    doi: 10.1371/journal.pcbi.1000834

    Figure Lengend Snippet: Distribution of distances between successive nucleosome dyad positions. The distributions shown here were derived from Field et al. [4] S. cerevisiae data (red), and from genome-wide model predictions in S. cerevisiae (green), and in H. sapiens (dark blue). The predicted dyad positions in H. sapiens are also shown partitioned according to the fraction of the neighboring 200 bases that are marked as repetitive ( 25% repeat in pink, and 75% repeat in aqua). For the purposes of this analysis, a predicted dyad position is a local maximum in the dyad score trace. The grey line shows the geometric distribution resulting from random positions with an average spacing of 165 bp.

    Article Snippet: The dominant hypothesis regarding DNA sequence preference of nucleosome formation is related to the curvature required to wrap the double helix tightly around the histone core .

    Techniques: Derivative Assay, Genome Wide

    Classification performance comparisons. (A) Comparison in S. cerevisiae between our model and the models of Field et al. [4] and Kaplan et al. [10] . These two previously published models each produce three types of scores at each nucleotide: a raw binding score, a probability that a nucleosome starts at that position, and a nucleosome-occupancy probability. The S. cerevisiae dataset used in this evaluation contains the top-scoring 6,355 positions or approximately 1/8 of the entire dataset. (Top-scoring means most well-positioned based on experimental data, not highest pattern-correlation scores.) (B) Similar comparison in H. sapiens between our model and the models of Field et al. and Kaplan et al. The raw binding scores and the occupancy probabilities were downloaded from the Segal lab website. The H. sapiens dataset used in this evaluation contains 200,000 dyad positions.

    Journal: PLoS Computational Biology

    Article Title: Learning a Weighted Sequence Model of the Nucleosome Core and Linker Yields More Accurate Predictions in Saccharomyces cerevisiae and Homo sapiens

    doi: 10.1371/journal.pcbi.1000834

    Figure Lengend Snippet: Classification performance comparisons. (A) Comparison in S. cerevisiae between our model and the models of Field et al. [4] and Kaplan et al. [10] . These two previously published models each produce three types of scores at each nucleotide: a raw binding score, a probability that a nucleosome starts at that position, and a nucleosome-occupancy probability. The S. cerevisiae dataset used in this evaluation contains the top-scoring 6,355 positions or approximately 1/8 of the entire dataset. (Top-scoring means most well-positioned based on experimental data, not highest pattern-correlation scores.) (B) Similar comparison in H. sapiens between our model and the models of Field et al. and Kaplan et al. The raw binding scores and the occupancy probabilities were downloaded from the Segal lab website. The H. sapiens dataset used in this evaluation contains 200,000 dyad positions.

    Article Snippet: The dominant hypothesis regarding DNA sequence preference of nucleosome formation is related to the curvature required to wrap the double helix tightly around the histone core .

    Techniques: Binding Assay

    Mono-nucleotide patterns in H. sapiens . These patterns were derived by aligning DNA sequences at experimentally determined nucleosome dyads, and computing the resulting position specific frequency matrix. The correlation between the corresponding mono-nucleotide patterns derived from the Barski nucleosome positions (top) and the Schones nucleosome positions (bottom) is .

    Journal: PLoS Computational Biology

    Article Title: Learning a Weighted Sequence Model of the Nucleosome Core and Linker Yields More Accurate Predictions in Saccharomyces cerevisiae and Homo sapiens

    doi: 10.1371/journal.pcbi.1000834

    Figure Lengend Snippet: Mono-nucleotide patterns in H. sapiens . These patterns were derived by aligning DNA sequences at experimentally determined nucleosome dyads, and computing the resulting position specific frequency matrix. The correlation between the corresponding mono-nucleotide patterns derived from the Barski nucleosome positions (top) and the Schones nucleosome positions (bottom) is .

    Article Snippet: The dominant hypothesis regarding DNA sequence preference of nucleosome formation is related to the curvature required to wrap the double helix tightly around the histone core .

    Techniques: Derivative Assay

    Estimated proportions from the 3-component Gaussian mixture model of the cell-free fragment lengths separated by chromosome. For both samples I1_M_plasma and G1_M_plasma, these estimates approximate the proportion of mono-, di- and tri-nucleosome lengths in each chromosome. All other mixture model parameters are reported in Additional file 4 . The solid lines depict the average value in each component while the dashed lines demarcate +/- 3 standard deviations from the mean

    Journal: BMC Medical Genomics

    Article Title: High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA

    doi: 10.1186/s12920-015-0107-z

    Figure Lengend Snippet: Estimated proportions from the 3-component Gaussian mixture model of the cell-free fragment lengths separated by chromosome. For both samples I1_M_plasma and G1_M_plasma, these estimates approximate the proportion of mono-, di- and tri-nucleosome lengths in each chromosome. All other mixture model parameters are reported in Additional file 4 . The solid lines depict the average value in each component while the dashed lines demarcate +/- 3 standard deviations from the mean

    Article Snippet: This further adds evidence for the hypothesis that it is the nucleosome packaging and the approximately 10 bp 360° turn of the double helix that are key determinants for the fragmentation of autosomal cell free DNA.

    Techniques:

    Different genomic codes are subject to selective pressures. Shown is a hypothetical genomic region (top) with a gene (black rectangle), nucleosomes (red ellipses), and various transcription factors (colored circles). Gray rectangles (middle) represent genomic regions in which the primary order of the DNA nucleotide sequence encodes functional information. Pink rectangles (bottom) represent regions where genomic information is encoded in DNA shape. These various encodings can be dispersed, overlapping, redundant, and, importantly, can represent different types of biological function. Considering the molecular structure of DNA in addition to the primary nucleotide sequence can help in understanding how biological function is encoded in genomes. Note that symbols and spacing are not drawn to scale.

    Journal: Current opinion in structural biology

    Article Title: DNA shape, genetic codes, and evolution

    doi: 10.1016/j.sbi.2011.03.002

    Figure Lengend Snippet: Different genomic codes are subject to selective pressures. Shown is a hypothetical genomic region (top) with a gene (black rectangle), nucleosomes (red ellipses), and various transcription factors (colored circles). Gray rectangles (middle) represent genomic regions in which the primary order of the DNA nucleotide sequence encodes functional information. Pink rectangles (bottom) represent regions where genomic information is encoded in DNA shape. These various encodings can be dispersed, overlapping, redundant, and, importantly, can represent different types of biological function. Considering the molecular structure of DNA in addition to the primary nucleotide sequence can help in understanding how biological function is encoded in genomes. Note that symbols and spacing are not drawn to scale.

    Article Snippet: Consistent with this finding is extensive evidence that long A-tracts—stretches of consecutive deoxyadenosine nucleotides on one strand of the double helix—strongly influence nucleosome organization [ ].

    Techniques: Sequencing, Functional Assay

    ZRF1 facilitates the assembly of the UV – DDB – CUL4A E3 ligase complex. (A) ZRF1 displaces RING1B from chromatin during NER. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A ubiquitin and RING1B abundance was calculated. Values are given as mean ± SEM ( n = 3). (B) ZRF1 regulates chromatin association of CUL4A and CUL4B. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative CUL4B and CUL4A abundance was calculated. Values are given as mean ± SEM ( n = 3). (C) ZRF1 regulates CUL4A association with H2AX containing nucleosomes. Control cells and ZRF1 knockdown cells expressing FLAG H2AX were irradiated with UV. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (D) Knockdown of ZRF1 modulates CUL4A association with DDB2. Control cells and ZRF1 knockdown cells expressing FLAG DDB2 were irradiated with UV light. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (E) Assembly of the UV–DDB–CUL4A E3 ligase is facilitated by ZRF1. Control cells and ZRF1 knockdown HEK293T cells expressing HA RBX1 were irradiated with UV light. After immunoprecipitation with HA-specific antibodies the precipitated material was subjected to Western blotting, and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (F) ZRF1 competes with CUL4B and RING1B for DDB2 binding in vitro. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4B, and RING1B and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 over the other components (relative molarity ZRF1: DDB1–CUL4B–RING1B; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (G) ZRF1 does not compete with CUL4A and RBX1 for binding to DDB1–DDB2. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4A and RBX1 and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 (relative molarity ZRF1: DDB1–CUL4A–RBX1; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (H) ZRF1 mediates the formation of the UV-DDB-CUL4A complex in vitro. GFP and GFP-DDB2 were coupled to beads and incubated with CUL4B, DDB1 and RING1B. After washing, GFP and GFP-DDB2 (UV–RING1B complex) beads were incubated with an estimated fivefold excess of purified CUL4A and RBX1 (lanes 1–3) over the retained UV–RING1B complex. Simultaneously, ZRF1 (lanes 1 and 3) or GST (lane 2) was added to the incubations in equimolar amounts. The precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%.

    Journal: The Journal of Cell Biology

    Article Title: ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair

    doi: 10.1083/jcb.201506099

    Figure Lengend Snippet: ZRF1 facilitates the assembly of the UV – DDB – CUL4A E3 ligase complex. (A) ZRF1 displaces RING1B from chromatin during NER. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative H2A ubiquitin and RING1B abundance was calculated. Values are given as mean ± SEM ( n = 3). (B) ZRF1 regulates chromatin association of CUL4A and CUL4B. Chromatin association assays of control and ZRF1 knockdown HEK293T cell lines after UV irradiation. De–cross-linked material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. The relative CUL4B and CUL4A abundance was calculated. Values are given as mean ± SEM ( n = 3). (C) ZRF1 regulates CUL4A association with H2AX containing nucleosomes. Control cells and ZRF1 knockdown cells expressing FLAG H2AX were irradiated with UV. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (D) Knockdown of ZRF1 modulates CUL4A association with DDB2. Control cells and ZRF1 knockdown cells expressing FLAG DDB2 were irradiated with UV light. After immunoprecipitation with FLAG-M2-agarose, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 3%. (E) Assembly of the UV–DDB–CUL4A E3 ligase is facilitated by ZRF1. Control cells and ZRF1 knockdown HEK293T cells expressing HA RBX1 were irradiated with UV light. After immunoprecipitation with HA-specific antibodies the precipitated material was subjected to Western blotting, and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (F) ZRF1 competes with CUL4B and RING1B for DDB2 binding in vitro. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4B, and RING1B and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 over the other components (relative molarity ZRF1: DDB1–CUL4B–RING1B; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (G) ZRF1 does not compete with CUL4A and RBX1 for binding to DDB1–DDB2. GFP and GFP-DDB2 immobilized on beads were incubated with equimolar amounts of purified DDB1, CUL4A and RBX1 and increasing amounts of ZRF1. ZRF1 levels were doubled stepwise reaching an eightfold molar excess of ZRF1 (relative molarity ZRF1: DDB1–CUL4A–RBX1; lane 3, 1:1; lane 4, 2:1; lane 5, 4:1; lane 6, 8:1). Precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 10%. (H) ZRF1 mediates the formation of the UV-DDB-CUL4A complex in vitro. GFP and GFP-DDB2 were coupled to beads and incubated with CUL4B, DDB1 and RING1B. After washing, GFP and GFP-DDB2 (UV–RING1B complex) beads were incubated with an estimated fivefold excess of purified CUL4A and RBX1 (lanes 1–3) over the retained UV–RING1B complex. Simultaneously, ZRF1 (lanes 1 and 3) or GST (lane 2) was added to the incubations in equimolar amounts. The precipitated material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%.

    Article Snippet: Purification of recombinant proteins Proteins were purified as suggested by GE Healthcare (GST-tagged proteins) or QIAGEN (His-tagged proteins) after inducing BL21 bacterial strains transformed with the respective plasmids at an OD = 0.5 with 0.2 mM isopropyl-β-D-thiogalactoside for 4 h at 37°C or at 20°C for 14 h. The following recombinant proteins were purchased: H2A (New England Biolabs), Ubiquitin (Boston Biochem), nucleosomes (Active Motif), GST-RBX1 (Novus Biologicals), and RAD23A (Abcam).

    Techniques: Irradiation, Western Blot, Expressing, Immunoprecipitation, Purification, Incubation, Binding Assay, In Vitro

    H2A ubiquitylation after UV irradiation is performed by the UV–RING1B complex. (A) Protein interaction partners of RING1B and DDB2. Mass spectrometry analysis after sequential immunoprecipitations with FLAG and RING1B antibodies revealed DDB1 and CUL4B as main interaction partners of DDB2 and RING1B. A comprehensive list of the identified unique peptides after RING1B and control immunoprecipitations (with or without UV irradiation) is provided in Table S5 . (B) Assembly of the UV–RING1B complex. Plasmids expressing FLAG DDB1, FLAG DDB2, and FLAG RING1B were cotransfected in combination with either control plasmid or a plasmid encoding FLAG-STREP CUL4B. After immunoprecipitation with STREP-Tactin beads, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (C) Visualization of the UV–RING1B complex. Purified UV–RING1B complex was subjected to SDS gel electrophoresis and colloidal Coomassie staining. Mass spectrometry analysis revealed the presence of all four subunits (bold). A comprehensive list of unique peptides is provided in Table S6 . (D) The UV–RING1B complex catalyzes ubiquitylation of H2A in vitro. Ubiquitylation assays were performed with recombinant H2A, E1 (UBA1), E2 (UBCH5), and either GST (control) or the UV–RING1B complex. Reactions were performed at 37°C, and samples were taken at the indicated time points. Material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. (E) The UV–RING1B complex catalyzes monoubiquitylation of nucleosomal H2A. Ubiquitylation assays were performed with recombinant nucleosomes, E1 (UBA1), E2 (UBCH5), and either GST (control) or UV-RING1B complex. Reactions lacking E1 (−E1) were performed as additional controls. The ubiquitylation assays were performed at 37°C for 5 h, and samples or pure substrate (Substrate) were subjected to Western blotting and probed with H2A antibodies.

    Journal: The Journal of Cell Biology

    Article Title: ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair

    doi: 10.1083/jcb.201506099

    Figure Lengend Snippet: H2A ubiquitylation after UV irradiation is performed by the UV–RING1B complex. (A) Protein interaction partners of RING1B and DDB2. Mass spectrometry analysis after sequential immunoprecipitations with FLAG and RING1B antibodies revealed DDB1 and CUL4B as main interaction partners of DDB2 and RING1B. A comprehensive list of the identified unique peptides after RING1B and control immunoprecipitations (with or without UV irradiation) is provided in Table S5 . (B) Assembly of the UV–RING1B complex. Plasmids expressing FLAG DDB1, FLAG DDB2, and FLAG RING1B were cotransfected in combination with either control plasmid or a plasmid encoding FLAG-STREP CUL4B. After immunoprecipitation with STREP-Tactin beads, the purified material was subjected to Western blotting and blots were incubated with the indicated antibodies. Inputs correspond to 5%. (C) Visualization of the UV–RING1B complex. Purified UV–RING1B complex was subjected to SDS gel electrophoresis and colloidal Coomassie staining. Mass spectrometry analysis revealed the presence of all four subunits (bold). A comprehensive list of unique peptides is provided in Table S6 . (D) The UV–RING1B complex catalyzes ubiquitylation of H2A in vitro. Ubiquitylation assays were performed with recombinant H2A, E1 (UBA1), E2 (UBCH5), and either GST (control) or the UV–RING1B complex. Reactions were performed at 37°C, and samples were taken at the indicated time points. Material of the respective time points was subjected to Western blotting and probed with the indicated antibodies. (E) The UV–RING1B complex catalyzes monoubiquitylation of nucleosomal H2A. Ubiquitylation assays were performed with recombinant nucleosomes, E1 (UBA1), E2 (UBCH5), and either GST (control) or UV-RING1B complex. Reactions lacking E1 (−E1) were performed as additional controls. The ubiquitylation assays were performed at 37°C for 5 h, and samples or pure substrate (Substrate) were subjected to Western blotting and probed with H2A antibodies.

    Article Snippet: Purification of recombinant proteins Proteins were purified as suggested by GE Healthcare (GST-tagged proteins) or QIAGEN (His-tagged proteins) after inducing BL21 bacterial strains transformed with the respective plasmids at an OD = 0.5 with 0.2 mM isopropyl-β-D-thiogalactoside for 4 h at 37°C or at 20°C for 14 h. The following recombinant proteins were purchased: H2A (New England Biolabs), Ubiquitin (Boston Biochem), nucleosomes (Active Motif), GST-RBX1 (Novus Biologicals), and RAD23A (Abcam).

    Techniques: Irradiation, Mass Spectrometry, Expressing, Plasmid Preparation, Immunoprecipitation, Purification, Western Blot, Incubation, SDS-Gel, Electrophoresis, Staining, In Vitro, Recombinant

    Spontaneous humoral autoimmune response in Ly9 −/− (BALB/c.129) mice . (A) ANA titers in the serum of 3- to 12-month-old Ly9 +/+ (wt) and Ly9 −/− mice. (B) Representative immunofluorescence staining of permeabilized Hep-2 incubated with sera from 1-year-old wt as compared with 1-year-old Ly9 −/− mice (sera dilution 1:200). After washing, IgG was detected with anti-mouse IgG-Texas Red (red). Nucleus was stained with DAPI (blue). (C) Determination by ELISA of autoantibodies against double-stranded DNA (dsDNA) and (D) nucleosome in serum from 12-month-old wt and Ly9 −/− mice. Experiments were initially conducted with a total of n = 11 BALB/c (wt) and n = 15 Ly9 −/− (BALB/c.129) female mice. Small horizontal bars indicate the mean. Statistical significances are shown.

    Journal: Frontiers in Immunology

    Article Title: Ly9 (CD229) Cell-Surface Receptor is Crucial for the Development of Spontaneous Autoantibody Production to Nuclear Antigens

    doi: 10.3389/fimmu.2013.00225

    Figure Lengend Snippet: Spontaneous humoral autoimmune response in Ly9 −/− (BALB/c.129) mice . (A) ANA titers in the serum of 3- to 12-month-old Ly9 +/+ (wt) and Ly9 −/− mice. (B) Representative immunofluorescence staining of permeabilized Hep-2 incubated with sera from 1-year-old wt as compared with 1-year-old Ly9 −/− mice (sera dilution 1:200). After washing, IgG was detected with anti-mouse IgG-Texas Red (red). Nucleus was stained with DAPI (blue). (C) Determination by ELISA of autoantibodies against double-stranded DNA (dsDNA) and (D) nucleosome in serum from 12-month-old wt and Ly9 −/− mice. Experiments were initially conducted with a total of n = 11 BALB/c (wt) and n = 15 Ly9 −/− (BALB/c.129) female mice. Small horizontal bars indicate the mean. Statistical significances are shown.

    Article Snippet: Anti-chromatin autoantibodies were detected using nucleosome antigen (Arotec Diagnostics Limited, Wellington, New Zealand).

    Techniques: Mouse Assay, Immunofluorescence, Staining, Incubation, Enzyme-linked Immunosorbent Assay

    Autoantibody development in Ly9 −/− (B6.129) mice . (A) Determination of anti-nuclear autoantibody (ANA) titers in the serum from Ly9 +/+ (wt) and Ly9 −/− mice obtained at the indicated time points, and as described in Section “Materials and Methods.” (B) Representative immunofluorescence image of permeabilized Hep-2 incubated with sera from 1-year-old wt as compared with 1-year-old Ly9 −/− mice (sera dilution 1:600). After washing, IgG was detected with anti-mouse IgG-Texas Red (red). Nucleus was stained with DAPI (blue). (C) ELISA was performed to assess autoantibodies against double-stranded DNA (dsDNA) and (D) nucleosome in serum from 12-month-old wt and Ly9 −/− mice (see Materials and Methods ). Experiments were initially conducted with a total of n = 11 B6 (wt) and n = 11 Ly9 −/− (B6.129) female mice. Small horizontal bars indicate the mean. Statistical significances are shown.

    Journal: Frontiers in Immunology

    Article Title: Ly9 (CD229) Cell-Surface Receptor is Crucial for the Development of Spontaneous Autoantibody Production to Nuclear Antigens

    doi: 10.3389/fimmu.2013.00225

    Figure Lengend Snippet: Autoantibody development in Ly9 −/− (B6.129) mice . (A) Determination of anti-nuclear autoantibody (ANA) titers in the serum from Ly9 +/+ (wt) and Ly9 −/− mice obtained at the indicated time points, and as described in Section “Materials and Methods.” (B) Representative immunofluorescence image of permeabilized Hep-2 incubated with sera from 1-year-old wt as compared with 1-year-old Ly9 −/− mice (sera dilution 1:600). After washing, IgG was detected with anti-mouse IgG-Texas Red (red). Nucleus was stained with DAPI (blue). (C) ELISA was performed to assess autoantibodies against double-stranded DNA (dsDNA) and (D) nucleosome in serum from 12-month-old wt and Ly9 −/− mice (see Materials and Methods ). Experiments were initially conducted with a total of n = 11 B6 (wt) and n = 11 Ly9 −/− (B6.129) female mice. Small horizontal bars indicate the mean. Statistical significances are shown.

    Article Snippet: Anti-chromatin autoantibodies were detected using nucleosome antigen (Arotec Diagnostics Limited, Wellington, New Zealand).

    Techniques: Mouse Assay, Immunofluorescence, Incubation, Staining, Enzyme-linked Immunosorbent Assay

    Specialization of ISWI remodellers for diverse nucleosome modifications a , Principal component analysis of library remodelling data. Nucleosomes, light blue; principal component (PC) weight values for remodellers, orange. Weights are scaled by a factor of 2 for visibility. b , ISWI remodelling data for selected nucleosome substrates in the library. Values capped at −4 and 4 for display purposes. All histones are unmodified unless otherwise specified. c , Single-site modifications mapped onto the nucleosome (PDB: 1KX5) and coloured according to whether they had consistently positive (green), consistently negative (red), or variable (purple) effects on nucleosome remodelling activity across all ISWI remodellers analysed.

    Journal: Nature

    Article Title: ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference

    doi: 10.1038/nature23671

    Figure Lengend Snippet: Specialization of ISWI remodellers for diverse nucleosome modifications a , Principal component analysis of library remodelling data. Nucleosomes, light blue; principal component (PC) weight values for remodellers, orange. Weights are scaled by a factor of 2 for visibility. b , ISWI remodelling data for selected nucleosome substrates in the library. Values capped at −4 and 4 for display purposes. All histones are unmodified unless otherwise specified. c , Single-site modifications mapped onto the nucleosome (PDB: 1KX5) and coloured according to whether they had consistently positive (green), consistently negative (red), or variable (purple) effects on nucleosome remodelling activity across all ISWI remodellers analysed.

    Article Snippet: For each enzyme, remodelling rate constants per nucleosome were calculated for experimental triplicates in GraphPad Prism.

    Techniques: Activity Assay

    High-throughput chromatin remodelling and binding data a , Heat-map displaying ISWI remodelling data (as in ) against the nucleosome library with CHD4 data for comparison. Rows were sorted on the basis of values for SNF2h (low to high). b , Heat map displaying binding of chromatin factors RCC1 and Sir3 against the nucleosome library relative to unmodified nucleosomes. Values were capped at − 4 and 4 for display purposes. All data are represented as the mean of experimental replicates ( n = 3). Fig. 2b

    Journal: Nature

    Article Title: ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference

    doi: 10.1038/nature23671

    Figure Lengend Snippet: High-throughput chromatin remodelling and binding data a , Heat-map displaying ISWI remodelling data (as in ) against the nucleosome library with CHD4 data for comparison. Rows were sorted on the basis of values for SNF2h (low to high). b , Heat map displaying binding of chromatin factors RCC1 and Sir3 against the nucleosome library relative to unmodified nucleosomes. Values were capped at − 4 and 4 for display purposes. All data are represented as the mean of experimental replicates ( n = 3). Fig. 2b

    Article Snippet: For each enzyme, remodelling rate constants per nucleosome were calculated for experimental triplicates in GraphPad Prism.

    Techniques: High Throughput Screening Assay, Binding Assay

    Characterization of barcoded 601 (BC-601) DNA a , BC-601 DNA prepared for all 115 nucleosome library members as described in Methods (Barcoded 601 (BC-601) DNA preparation). Ligation products are 192 bp in size and were visualized by polyacrylamide gel electrophoresis (5% acrylamide, 0.5× TBE, 200 V, 40 min) and staining with SYBR Safe DNA gel stain. A faint band corresponding to unligated 601 DNA (601) is slightly visible in certain cases. b .

    Journal: Nature

    Article Title: ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference

    doi: 10.1038/nature23671

    Figure Lengend Snippet: Characterization of barcoded 601 (BC-601) DNA a , BC-601 DNA prepared for all 115 nucleosome library members as described in Methods (Barcoded 601 (BC-601) DNA preparation). Ligation products are 192 bp in size and were visualized by polyacrylamide gel electrophoresis (5% acrylamide, 0.5× TBE, 200 V, 40 min) and staining with SYBR Safe DNA gel stain. A faint band corresponding to unligated 601 DNA (601) is slightly visible in certain cases. b .

    Article Snippet: For each enzyme, remodelling rate constants per nucleosome were calculated for experimental triplicates in GraphPad Prism.

    Techniques: Ligation, Polyacrylamide Gel Electrophoresis, Staining

    Principal component (PC) analysis of library remodelling data Percentages show the fractions of the variance accounted for by each PC. Individual nucleosomes are shown in light blue, and PC weight values for each remodeller are shown in either orange or black. Weights are scaled by a factor of 2 for visibility. a , PC1 vs. PC2 and PC1 vs. PC3 are plotted. b ). Fig. 3a

    Journal: Nature

    Article Title: ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference

    doi: 10.1038/nature23671

    Figure Lengend Snippet: Principal component (PC) analysis of library remodelling data Percentages show the fractions of the variance accounted for by each PC. Individual nucleosomes are shown in light blue, and PC weight values for each remodeller are shown in either orange or black. Weights are scaled by a factor of 2 for visibility. a , PC1 vs. PC2 and PC1 vs. PC3 are plotted. b ). Fig. 3a

    Article Snippet: For each enzyme, remodelling rate constants per nucleosome were calculated for experimental triplicates in GraphPad Prism.

    Techniques:

    Remodelling of nucleosomes containing modifications preferred by histone recognition domains Library remodelling data generated by the NoRC ( a ), WICH ( b ), and NURF ( c ) complexes for nucleosomes containing residues known to interact with histone binding modules in accessory subunits of each complex (NoRC: TIP5; WICH: WSTF; NURF: BPTF). Literature binding specificities are displayed in corresponding tables on the right. Bar graphs display log 2 values of the rate of remodelling of individual nucleosome library members ( k MN ) relative to unmodified nucleosomes ( k unmod. ). Data are represented as the ratio of the mean of experimental replicates ± s.e.m. ( n ). All histones are unmodified unless otherwise specified. BRD, bromodomain; PHD, PHD-finger; PHD–BRD, tandem PHD-finger–bromodomain module; ND, not determined.

    Journal: Nature

    Article Title: ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference

    doi: 10.1038/nature23671

    Figure Lengend Snippet: Remodelling of nucleosomes containing modifications preferred by histone recognition domains Library remodelling data generated by the NoRC ( a ), WICH ( b ), and NURF ( c ) complexes for nucleosomes containing residues known to interact with histone binding modules in accessory subunits of each complex (NoRC: TIP5; WICH: WSTF; NURF: BPTF). Literature binding specificities are displayed in corresponding tables on the right. Bar graphs display log 2 values of the rate of remodelling of individual nucleosome library members ( k MN ) relative to unmodified nucleosomes ( k unmod. ). Data are represented as the ratio of the mean of experimental replicates ± s.e.m. ( n ). All histones are unmodified unless otherwise specified. BRD, bromodomain; PHD, PHD-finger; PHD–BRD, tandem PHD-finger–bromodomain module; ND, not determined.

    Article Snippet: For each enzyme, remodelling rate constants per nucleosome were calculated for experimental triplicates in GraphPad Prism.

    Techniques: Generated, Binding Assay

    A high-throughput nucleosome remodelling assay for ISWI family chromatin remodellers a , Schematic of the restriction enzyme accessibility assay used with the DNA-barcoded library. Individual remodelling rates are calculated from unique DNA sequencing reads. b , Heat map displaying ISWI remodelling data against the nucleosome library. Rows were sorted on the basis of values for SNF2h (low to high). k MN , nucleosome remodelling rate; k unmod ., unmodified nucleosome remodelling rate. Values were capped at −4 and 4 for display purposes. c , Example decay curves depicting individual rates ( k MN ) as in b. d , Rank-ordered remodelling rates for the ACF complex ( k MN ) against the library. Dashed red line, k unmod . e , Relative remodelling rates as in b for select library members. All data are represented as the mean of experimental replicates ( n = 3). Error bars represent s.e.m. All histones are unmodified unless otherwise specified.

    Journal: Nature

    Article Title: ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference

    doi: 10.1038/nature23671

    Figure Lengend Snippet: A high-throughput nucleosome remodelling assay for ISWI family chromatin remodellers a , Schematic of the restriction enzyme accessibility assay used with the DNA-barcoded library. Individual remodelling rates are calculated from unique DNA sequencing reads. b , Heat map displaying ISWI remodelling data against the nucleosome library. Rows were sorted on the basis of values for SNF2h (low to high). k MN , nucleosome remodelling rate; k unmod ., unmodified nucleosome remodelling rate. Values were capped at −4 and 4 for display purposes. c , Example decay curves depicting individual rates ( k MN ) as in b. d , Rank-ordered remodelling rates for the ACF complex ( k MN ) against the library. Dashed red line, k unmod . e , Relative remodelling rates as in b for select library members. All data are represented as the mean of experimental replicates ( n = 3). Error bars represent s.e.m. All histones are unmodified unless otherwise specified.

    Article Snippet: For each enzyme, remodelling rate constants per nucleosome were calculated for experimental triplicates in GraphPad Prism.

    Techniques: High Throughput Screening Assay, DNA Sequencing

    Remodelling assays carried out on individual nucleosomes measured via standard gel-based read-out to validate library data a , Activity of the NURF complex towards H3K4me3+H4K16ac relative to unmodified nucleosomes as measured in the context of the nucleosome library (library) or individual assays (individual). b , Activity of the ACF complex on unmodified and acidic patch mutant nucleosomes. c , Remodelling of unmodified nucleosomes is inhibited by the presence of the LANA peptide when compared to a LANA peptide with key binding residues mutated (LRS to AAA). d , Activity of the ACF complex towards nucleosomes modified near the acidic patch (H2BK108ac and H2BS112GlcNac) relative to unmodified nucleosomes as measured in the context of the nucleosome library (library) or individual assays (individual). Gel images of example replicates used to generate densitometry measurements in each subpanel are shown above respective graphs. a, c , and d use a restriction enzyme accessibility assay. b uses a nucleosome repositioning electrophoretic mobility shift assay. All histones are unmodified unless otherwise specified. All data are represented as the mean of experimental replicates ± s.e.m. ( n .

    Journal: Nature

    Article Title: ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference

    doi: 10.1038/nature23671

    Figure Lengend Snippet: Remodelling assays carried out on individual nucleosomes measured via standard gel-based read-out to validate library data a , Activity of the NURF complex towards H3K4me3+H4K16ac relative to unmodified nucleosomes as measured in the context of the nucleosome library (library) or individual assays (individual). b , Activity of the ACF complex on unmodified and acidic patch mutant nucleosomes. c , Remodelling of unmodified nucleosomes is inhibited by the presence of the LANA peptide when compared to a LANA peptide with key binding residues mutated (LRS to AAA). d , Activity of the ACF complex towards nucleosomes modified near the acidic patch (H2BK108ac and H2BS112GlcNac) relative to unmodified nucleosomes as measured in the context of the nucleosome library (library) or individual assays (individual). Gel images of example replicates used to generate densitometry measurements in each subpanel are shown above respective graphs. a, c , and d use a restriction enzyme accessibility assay. b uses a nucleosome repositioning electrophoretic mobility shift assay. All histones are unmodified unless otherwise specified. All data are represented as the mean of experimental replicates ± s.e.m. ( n .

    Article Snippet: For each enzyme, remodelling rate constants per nucleosome were calculated for experimental triplicates in GraphPad Prism.

    Techniques: Activity Assay, Mutagenesis, Binding Assay, Modification, Electrophoretic Mobility Shift Assay

    Alteration of histone–DNA contacts affects remodelling activity a , Modified histone residues in the nucleosome library that lie under the DNA (tan) are highlighted on the nucleosome (PDB: 1KX5) in red. PTMs are numbered and labelled on the nucleosome structure. Values were capped at −2 and 2 for display purposes. b , Histone mutants present in the nucleosome library that lie under the DNA (tan) are highlighted on the nucleosome (PDB: 1KX5) in red. The heatmap is displayed as in a. Locations of each mutation are individually labelled on the nucleosome structure. Values were capped at −3 and 3 for display purposes. All histones are unmodified unless otherwise specified.

    Journal: Nature

    Article Title: ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference

    doi: 10.1038/nature23671

    Figure Lengend Snippet: Alteration of histone–DNA contacts affects remodelling activity a , Modified histone residues in the nucleosome library that lie under the DNA (tan) are highlighted on the nucleosome (PDB: 1KX5) in red. PTMs are numbered and labelled on the nucleosome structure. Values were capped at −2 and 2 for display purposes. b , Histone mutants present in the nucleosome library that lie under the DNA (tan) are highlighted on the nucleosome (PDB: 1KX5) in red. The heatmap is displayed as in a. Locations of each mutation are individually labelled on the nucleosome structure. Values were capped at −3 and 3 for display purposes. All histones are unmodified unless otherwise specified.

    Article Snippet: For each enzyme, remodelling rate constants per nucleosome were calculated for experimental triplicates in GraphPad Prism.

    Techniques: Activity Assay, Modification, Mutagenesis

    Nucleosome remodelling activity is negligible in the absence of ATP Bar graphs show individual DNA cleavage rates ( k MN ) from library remodelling experiments for each member of the library in the presence of the indicated chromatin remodeller with and without ATP. Rate values were rank ordered and are displayed from low to high. The dashed red line represents the rate of remodelling of unmodified nucleosomes. The related graphs for the ACF complex can be found in . Data are represented as the mean of experimental replicates ± s.e.m. ( n = 3). Fig. 2d

    Journal: Nature

    Article Title: ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference

    doi: 10.1038/nature23671

    Figure Lengend Snippet: Nucleosome remodelling activity is negligible in the absence of ATP Bar graphs show individual DNA cleavage rates ( k MN ) from library remodelling experiments for each member of the library in the presence of the indicated chromatin remodeller with and without ATP. Rate values were rank ordered and are displayed from low to high. The dashed red line represents the rate of remodelling of unmodified nucleosomes. The related graphs for the ACF complex can be found in . Data are represented as the mean of experimental replicates ± s.e.m. ( n = 3). Fig. 2d

    Article Snippet: For each enzyme, remodelling rate constants per nucleosome were calculated for experimental triplicates in GraphPad Prism.

    Techniques: Activity Assay

    Analysis of the quality and integrity of the nucleosome library a , b , Analysis of individual nucleosome preparations ( a ) and the final library after pooling of nucleosomes ( b ) by native gel electrophoresis and staining with ethidium bromide. c .

    Journal: Nature

    Article Title: ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference

    doi: 10.1038/nature23671

    Figure Lengend Snippet: Analysis of the quality and integrity of the nucleosome library a , b , Analysis of individual nucleosome preparations ( a ) and the final library after pooling of nucleosomes ( b ) by native gel electrophoresis and staining with ethidium bromide. c .

    Article Snippet: For each enzyme, remodelling rate constants per nucleosome were calculated for experimental triplicates in GraphPad Prism.

    Techniques: Nucleic Acid Electrophoresis, Staining

    A diverse library of modified nucleosomes Diagram depicting all histone modifications, mutants, and variants present in the 115-member nucleosome library used in this study. Residues modified or mutated were mapped on to the nucleosome (PDB: 1KX5) in black using UCSF Chimera. H2A (light yellow), H2B (light red), H3 (light blue) and H4 (light green) modification and mutation locations are indicated by boxes and lines. For clarity, connections are shown to only a single copy of each histone protein.

    Journal: Nature

    Article Title: ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference

    doi: 10.1038/nature23671

    Figure Lengend Snippet: A diverse library of modified nucleosomes Diagram depicting all histone modifications, mutants, and variants present in the 115-member nucleosome library used in this study. Residues modified or mutated were mapped on to the nucleosome (PDB: 1KX5) in black using UCSF Chimera. H2A (light yellow), H2B (light red), H3 (light blue) and H4 (light green) modification and mutation locations are indicated by boxes and lines. For clarity, connections are shown to only a single copy of each histone protein.

    Article Snippet: For each enzyme, remodelling rate constants per nucleosome were calculated for experimental triplicates in GraphPad Prism.

    Techniques: Modification, Mutagenesis

    The p12 CTD directly binds recombinant nucleosomal arrays. Direct interactions between p12 CTD peptides and nucleosomal histone proteins was tested using biolayer interferometry (BIL). Streptavidin sensor probes were coated with biotinylated p12 CTD or h CBS (positive control) peptides to equivalent levels and then immersed in analyte solutions containing recombinant poly-nucleosomes to measure binding. (A) Representative sensorgrams showing binding of peptides corresponding to the CTD of Mo-MLV p12_M63I, phos p12-M63I (S61-phosphorylated), p12_M63I/R66A or h CBS to recombinant poly-nucleosomes (~250 nM). Black arrows indicate the beginning and end of the association phase. (B-D) Affinity measurements were derived by probing peptides corresponding to h CBS (B-D) or the CTD of Mo-MLV p12_M63I, phos p12-M63I (B), GaLV p12, FeLV p12 (C) or Mo-MLV p12_WT (D) against a range of serially-diluted nucleosomes. Equilibrium binding data from two technical replicates were pooled for the analysis. The plotted data are fractional saturation of binding as a function of nucleosome concentration. (E) Competition assay inhibiting nucleosome binding to immobilised biotin-tagged h CBS with soluble non-biotinylated h CBS or Mo-MLV p12 CTD peptides as indicated in key (100 μM). Left panel shows representative sensorgrams with black arrows indicating the beginning and end of the association phase. The bar chart shows equilibrium binding of h CBS in the presence and absence of competing peptides as mean ± SD from three technical replicates. (F) Representative sensorgrams showing competition assay inhibiting nucleosome binding to immobilised biotin-tagged p12 CTD peptides or h CBS, as indicated in graph title, with soluble non-biotinylated h CBS (100 μM, red lines). Black arrows indicate the beginning and end of the association phase. See methods for peptide sequences.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: The p12 CTD directly binds recombinant nucleosomal arrays. Direct interactions between p12 CTD peptides and nucleosomal histone proteins was tested using biolayer interferometry (BIL). Streptavidin sensor probes were coated with biotinylated p12 CTD or h CBS (positive control) peptides to equivalent levels and then immersed in analyte solutions containing recombinant poly-nucleosomes to measure binding. (A) Representative sensorgrams showing binding of peptides corresponding to the CTD of Mo-MLV p12_M63I, phos p12-M63I (S61-phosphorylated), p12_M63I/R66A or h CBS to recombinant poly-nucleosomes (~250 nM). Black arrows indicate the beginning and end of the association phase. (B-D) Affinity measurements were derived by probing peptides corresponding to h CBS (B-D) or the CTD of Mo-MLV p12_M63I, phos p12-M63I (B), GaLV p12, FeLV p12 (C) or Mo-MLV p12_WT (D) against a range of serially-diluted nucleosomes. Equilibrium binding data from two technical replicates were pooled for the analysis. The plotted data are fractional saturation of binding as a function of nucleosome concentration. (E) Competition assay inhibiting nucleosome binding to immobilised biotin-tagged h CBS with soluble non-biotinylated h CBS or Mo-MLV p12 CTD peptides as indicated in key (100 μM). Left panel shows representative sensorgrams with black arrows indicating the beginning and end of the association phase. The bar chart shows equilibrium binding of h CBS in the presence and absence of competing peptides as mean ± SD from three technical replicates. (F) Representative sensorgrams showing competition assay inhibiting nucleosome binding to immobilised biotin-tagged p12 CTD peptides or h CBS, as indicated in graph title, with soluble non-biotinylated h CBS (100 μM, red lines). Black arrows indicate the beginning and end of the association phase. See methods for peptide sequences.

    Article Snippet: The estimated binding amplitudes were then plotted against approximate nucleosome concentrations on GraphPad Prism 7.

    Techniques: Recombinant, Positive Control, Binding Assay, Derivative Assay, Concentration Assay, Competitive Binding Assay

    Models for p12-chromatin binding. (A) Proposed model for the different functions of p12. The p12 region of Gag and p12 protein in the viral PIC differ in their affinity for cellular proteins and chromatin. We propose that as part of Gag, or when expressed as a recombinant GST-fusion protein, p12 exists in an unstructured conformation with low affinity for nucleosomes but relatively high affinity for host proteins such as clathrin and NEDD4-like E3 ligases which facilitate late replication events. Following Gag cleavage, the binding of the p12 NTD to the CA lattice promotes a change in the conformation of p12 which increases the affinity of the p12 CTD for nucleosomes. During mitosis, the breakdown of the nuclear envelope allows the p12/CA-containing PIC to access chromatin. The PIC is targeted to nucleosomes on mitotic chromatin by CA-bound, phosphorylated p12. Exit from mitosis promotes the de-phosphorylation of p12 and the dissociation of p12 and CA from chromatin. BET proteins can then bind IN and direct the viral cDNA to gene promoter regions where integration occurs. (B) Proposed relationship between virus infectivity and affinity of p12 for chromatin. We suggest that the affinity of p12 for chromatin is fine-tuned for optimal infectivity with deviations incurring a fitness cost. Mutations in p12 that increase or decrease chromatin binding (measured, in blue, or extrapolated, in red) alter viral infectivity as shown on the left. Only interactions above an arbitrary threshold can be detected by GST-pull down assays.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: Models for p12-chromatin binding. (A) Proposed model for the different functions of p12. The p12 region of Gag and p12 protein in the viral PIC differ in their affinity for cellular proteins and chromatin. We propose that as part of Gag, or when expressed as a recombinant GST-fusion protein, p12 exists in an unstructured conformation with low affinity for nucleosomes but relatively high affinity for host proteins such as clathrin and NEDD4-like E3 ligases which facilitate late replication events. Following Gag cleavage, the binding of the p12 NTD to the CA lattice promotes a change in the conformation of p12 which increases the affinity of the p12 CTD for nucleosomes. During mitosis, the breakdown of the nuclear envelope allows the p12/CA-containing PIC to access chromatin. The PIC is targeted to nucleosomes on mitotic chromatin by CA-bound, phosphorylated p12. Exit from mitosis promotes the de-phosphorylation of p12 and the dissociation of p12 and CA from chromatin. BET proteins can then bind IN and direct the viral cDNA to gene promoter regions where integration occurs. (B) Proposed relationship between virus infectivity and affinity of p12 for chromatin. We suggest that the affinity of p12 for chromatin is fine-tuned for optimal infectivity with deviations incurring a fitness cost. Mutations in p12 that increase or decrease chromatin binding (measured, in blue, or extrapolated, in red) alter viral infectivity as shown on the left. Only interactions above an arbitrary threshold can be detected by GST-pull down assays.

    Article Snippet: The estimated binding amplitudes were then plotted against approximate nucleosome concentrations on GraphPad Prism 7.

    Techniques: Binding Assay, Recombinant, De-Phosphorylation Assay, Infection

    Single amino acid substitutions in H2A affect the nucleosome surface and structure. ( A ) Threonine 16 of H2A is exposed on the surface of the nucleosome. A space-filling model of the canonical nucleosome was generated in PyMOL using the spheres feature. Threonine 16 on H2A is highlighted in purple. ( B ) Zoomed-in view of the region highlighted with a gray box in (A). ( C ) Zoomed-in molecular surface representations of nucleosomes containing threonine (C) or serine ( D ) at residue 16 of H2A (highlighted in purple) were generated in pymol using the mutagenesis and surface features. ( E ) Alanine 40 of H2A, highlighted in royal blue, interacts with isoleucine of H2B via hydrophobic side chain interactions. For all images, H3 is light blue, H4 is green, H2A is orange, and H2B is red. The crystal structure of the canonical nucleosome (PDB: 1AOI) was used for all images.

    Journal: Nucleic Acids Research

    Article Title: Replication-dependent histone isoforms: a new source of complexity in chromatin structure and function

    doi: 10.1093/nar/gky768

    Figure Lengend Snippet: Single amino acid substitutions in H2A affect the nucleosome surface and structure. ( A ) Threonine 16 of H2A is exposed on the surface of the nucleosome. A space-filling model of the canonical nucleosome was generated in PyMOL using the spheres feature. Threonine 16 on H2A is highlighted in purple. ( B ) Zoomed-in view of the region highlighted with a gray box in (A). ( C ) Zoomed-in molecular surface representations of nucleosomes containing threonine (C) or serine ( D ) at residue 16 of H2A (highlighted in purple) were generated in pymol using the mutagenesis and surface features. ( E ) Alanine 40 of H2A, highlighted in royal blue, interacts with isoleucine of H2B via hydrophobic side chain interactions. For all images, H3 is light blue, H4 is green, H2A is orange, and H2B is red. The crystal structure of the canonical nucleosome (PDB: 1AOI) was used for all images.

    Article Snippet: All these modifications of H3.1T confer distinct properties to this isoform, including its in vitro and in vivo instability, weaker association to H2A/H2B dimer, defective incorporation into the nucleosome by Nap1, and more rapid exchange in the nucleosomes of living cell ( , ).

    Techniques: Generated, Mutagenesis

    Single amino acid substitutions in H2B affect nucleosome structure. ( A ) Threonine 33 of the H2B N-terminal tail (colored in cyan) is positioned between the DNA gyres of the nucleosome and hydrogen bonds to one strand of DNA. ( B ) Zoomed-in view of the region highlighted with a gray box in (A). Dashed yellow lines indicate hydrogen bonds identified by the PyMol polar contacts tool. ( C ) Serine 76 of H2B (shown in white) makes intra-chain interactions with the adjacent arginine. The hydrogen bond network thus formed between adjacent residues is shown with yellow dashed lines. Glycine 76 was replaced with serine using the PyMol mutagenesis tool. For all images, H3 is light blue, H4 is green, H2A is orange, and H2B is red. The crystal structure of the canonical nucleosome (PDB: 1AOI) was used for all images.

    Journal: Nucleic Acids Research

    Article Title: Replication-dependent histone isoforms: a new source of complexity in chromatin structure and function

    doi: 10.1093/nar/gky768

    Figure Lengend Snippet: Single amino acid substitutions in H2B affect nucleosome structure. ( A ) Threonine 33 of the H2B N-terminal tail (colored in cyan) is positioned between the DNA gyres of the nucleosome and hydrogen bonds to one strand of DNA. ( B ) Zoomed-in view of the region highlighted with a gray box in (A). Dashed yellow lines indicate hydrogen bonds identified by the PyMol polar contacts tool. ( C ) Serine 76 of H2B (shown in white) makes intra-chain interactions with the adjacent arginine. The hydrogen bond network thus formed between adjacent residues is shown with yellow dashed lines. Glycine 76 was replaced with serine using the PyMol mutagenesis tool. For all images, H3 is light blue, H4 is green, H2A is orange, and H2B is red. The crystal structure of the canonical nucleosome (PDB: 1AOI) was used for all images.

    Article Snippet: All these modifications of H3.1T confer distinct properties to this isoform, including its in vitro and in vivo instability, weaker association to H2A/H2B dimer, defective incorporation into the nucleosome by Nap1, and more rapid exchange in the nucleosomes of living cell ( , ).

    Techniques: Mutagenesis

    Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.

    Journal: Nucleic Acids Research

    Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

    doi: 10.1093/nar/gky502

    Figure Lengend Snippet: Frequencies of occurrence of DNA dinucleotide steps in the +1 nucleosomes of yeast and the sketch of MNase-seq experiments. ( A ) Frequencies of occurrence of dinucleotide steps at each position in the +1 nucleosomes of yeast were plotted. The DNA sequences were aligned from the DNA entry to exit site. It is shown that the frequencies of AA/TT dinucleotide step are distinctively higher than those of the other dinucleotide steps at all positions and that the DNA entry site of +1 nucleosomes in yeast is AA/TT-rich. ( B ) Schematic illustration of MNase-seq experiments carried out in this study is shown.

    Article Snippet: For the MNase digestion experiments with nuc19 and its mutants , each nucleosome (94 nM) was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as described above.

    Techniques:

    MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.

    Journal: Nucleic Acids Research

    Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

    doi: 10.1093/nar/gky502

    Figure Lengend Snippet: MNase digestions on TA- and AA-repeated regions. ( A ) Read frequencies of TA-repeated regions from the sense/+ strand of nuc01, nuc02 and nuc10 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that although TAs are favourably cleaved in free DNA, they are generally well wrapped on histones and cleavages on nucleosomal TAs are suspended by the upstream. Therefore, MNase cleaves TAs in nucleosomes from the 5′ end of DNA as an exonuclease. ( B ) Read frequencies of AA-repeated regions from the antisense/− strand of nuc01, nuc03 and nuc07 are shown as a function of incubation time and DNA position. The digestions of nucleosomal DNAs (left panel) are compared with the digestions of free DNAs (right panel). It shows that at the inward sites of nucleosomes, digestions of AAs are allowed via nucleosome site exposures. The evenly distributed read frequencies in AA-repeated regions suggest that MNase cleaves AAs as an endonuclease in the early stage of digestion.

    Article Snippet: For the MNase digestion experiments with nuc19 and its mutants , each nucleosome (94 nM) was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as described above.

    Techniques: Incubation, Atomic Absorption Spectroscopy

    Read frequency as a function of read start position and overall digestion profile as a function of time. Read frequencies of the 20 +1 nucleosomes as a function of read start position for the 1-min and 3-min assays are shown in ( A ) and ( B ), respectively. The 5′ terminal nucleotides of both DNA strands are indexed from ‘0’. Read frequencies of the DNA sense/+ and antisense/− strands are shown in the upper and lower panels in the figures, respectively. DNA base compositions for each read start position are also indicated. ( C ) Cumulative read frequency as a function of read start position at different incubation times. Cumulative read frequencies were calculated from position 1 (instead of 0), averaged over the 40 strands of the 20 +1 nucleosomes. ( D ) Differentials of cumulative read frequencies between incubation times are shown.

    Journal: Nucleic Acids Research

    Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

    doi: 10.1093/nar/gky502

    Figure Lengend Snippet: Read frequency as a function of read start position and overall digestion profile as a function of time. Read frequencies of the 20 +1 nucleosomes as a function of read start position for the 1-min and 3-min assays are shown in ( A ) and ( B ), respectively. The 5′ terminal nucleotides of both DNA strands are indexed from ‘0’. Read frequencies of the DNA sense/+ and antisense/− strands are shown in the upper and lower panels in the figures, respectively. DNA base compositions for each read start position are also indicated. ( C ) Cumulative read frequency as a function of read start position at different incubation times. Cumulative read frequencies were calculated from position 1 (instead of 0), averaged over the 40 strands of the 20 +1 nucleosomes. ( D ) Differentials of cumulative read frequencies between incubation times are shown.

    Article Snippet: For the MNase digestion experiments with nuc19 and its mutants , each nucleosome (94 nM) was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as described above.

    Techniques: Incubation

    Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).

    Journal: Nucleic Acids Research

    Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

    doi: 10.1093/nar/gky502

    Figure Lengend Snippet: Sequence-dependent site exposure in nucleosome. ( A ) MNase digestion on preferential sequence. When the preferential sequence (e.g. TATA) is at the end region where MNase can access from the 5′ end of DNA, TATA would be favourably cleaved. However, if it is at the internal region where TATA is tightly bound on histones, cleavages are prohibited. ( B ) MNase digestion on site-exposure sequence. When the site-exposure sequence (e.g. AAAA) is at the end region, because MNase can access from the 5′ end of DNA and sequence-dependent site exposure occurs, cleavages on AAAA are allowed, though less favourably than TATA. When it is at the internal site, due to site exposure, cleavages will also occur. ( C ) When multiple sites composed of the site-exposure sequence are assembled at one end of nucleosome (i.e. DNA entry site), the overall affinities between DNA and histones will dwindle to assist nucleosome unwrapping with the presence of transcription factors or chromatin remodellers (shown in green ellipse).

    Article Snippet: For the MNase digestion experiments with nuc19 and its mutants , each nucleosome (94 nM) was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as described above.

    Techniques: Sequencing

    Correlation between MNase digestions and contents of site-exposure sequence by comparing the two ends of a nucleosome. Correlations between MNase digestions and AA/TT contents in the 1- and 3-min assays are shown on the left and right panels of ( A ), respectively. Similarly, ( B ) shows the correlations of MNase digestion versus AAAA/TTTT content from the 1- and 3-min assays. The shaded rectangle regions in red indicate that the entry site of a nucleosome with more AA/TTs or AAAA/TTTTs gets more digested; the regions in blue indicate that the exit site with more AA/TTs or AAAA/TTTTs gets more digested. The 20 +1 nucleosomes are divided into two groups based on the numbers of s ite e xposure s equence e lements (SESEs, defined as discrete AAAA or TTTT segments) in their sequences. Specifically, nucleosomes with SESEs (coloured in black) are those with three or more SESEs on either strand of the nucleosomes, including nuc01, nuc02, nuc03, nuc05, nuc07, nuc10 and nuc20. Oppositely, nucleosomes with no or fewer SESEs (coloured in orange) consisting of the rest of the +1 nucleosomes, are those with two or fewer SESEs on each strand. Correlation coefficients for each class of nucleosomes under each incubation time are also indicated.

    Journal: Nucleic Acids Research

    Article Title: MNase, as a probe to study the sequence-dependent site exposures in the +1 nucleosomes of yeast

    doi: 10.1093/nar/gky502

    Figure Lengend Snippet: Correlation between MNase digestions and contents of site-exposure sequence by comparing the two ends of a nucleosome. Correlations between MNase digestions and AA/TT contents in the 1- and 3-min assays are shown on the left and right panels of ( A ), respectively. Similarly, ( B ) shows the correlations of MNase digestion versus AAAA/TTTT content from the 1- and 3-min assays. The shaded rectangle regions in red indicate that the entry site of a nucleosome with more AA/TTs or AAAA/TTTTs gets more digested; the regions in blue indicate that the exit site with more AA/TTs or AAAA/TTTTs gets more digested. The 20 +1 nucleosomes are divided into two groups based on the numbers of s ite e xposure s equence e lements (SESEs, defined as discrete AAAA or TTTT segments) in their sequences. Specifically, nucleosomes with SESEs (coloured in black) are those with three or more SESEs on either strand of the nucleosomes, including nuc01, nuc02, nuc03, nuc05, nuc07, nuc10 and nuc20. Oppositely, nucleosomes with no or fewer SESEs (coloured in orange) consisting of the rest of the +1 nucleosomes, are those with two or fewer SESEs on each strand. Correlation coefficients for each class of nucleosomes under each incubation time are also indicated.

    Article Snippet: For the MNase digestion experiments with nuc19 and its mutants , each nucleosome (94 nM) was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1 and 3 min under the same conditions as described above.

    Techniques: Sequencing, Incubation

    Recombinant CHD3 and CHD4 exhibit distinct, sequence-specific nucleosome positioning behaviour . Recombinantly purified human CHD3/4 were titrated in increasing concentrations (A/C/ E : 25, 50, 75 and 100 nM; B/D: 50, 100 and 200 nM) to the indicated mono- and dinucleosomal templates containing different configurations of linker DNA (see also Materials and Methods). The reactions were started by adding ATP. Nucleosomes in the absence or presence of enzyme (without ATP) served as reference. To visualize the nucleosome movements, the reactions were loaded on PAA gels (ethidium bromide stain). Positions of mono- and dinucleosomes are indicated by ovals, according to ( 68 ). Filled triangles represent more intense bands, empty triangles less intense bands or no signal, comparing CHD3 and CHD4 remodeling patterns. Triangles with a dashed rim were added for better orientation in the intensity profiles (see below). Black asterisks indicate nucleosomes, which were probably pushed over the edge of the DNA strand. On the right side of each remodeling gel are intensity profiles of the indicated gel lanes, based on Multi Gauge software analysis. The triangles indicate the positions of the bands highlighted by the triangles in the respective lanes of the corresponding gel picture. All lanes shown for one remodeling template are from one gel. Number or replicates: ( A ) ≥3; ( B ) 3; ( C ) ≥3; ( D ) 3; ( E ) ≥3.

    Journal: Nucleic Acids Research

    Article Title: CHD3 and CHD4 form distinct NuRD complexes with different yet overlapping functionality

    doi: 10.1093/nar/gkx711

    Figure Lengend Snippet: Recombinant CHD3 and CHD4 exhibit distinct, sequence-specific nucleosome positioning behaviour . Recombinantly purified human CHD3/4 were titrated in increasing concentrations (A/C/ E : 25, 50, 75 and 100 nM; B/D: 50, 100 and 200 nM) to the indicated mono- and dinucleosomal templates containing different configurations of linker DNA (see also Materials and Methods). The reactions were started by adding ATP. Nucleosomes in the absence or presence of enzyme (without ATP) served as reference. To visualize the nucleosome movements, the reactions were loaded on PAA gels (ethidium bromide stain). Positions of mono- and dinucleosomes are indicated by ovals, according to ( 68 ). Filled triangles represent more intense bands, empty triangles less intense bands or no signal, comparing CHD3 and CHD4 remodeling patterns. Triangles with a dashed rim were added for better orientation in the intensity profiles (see below). Black asterisks indicate nucleosomes, which were probably pushed over the edge of the DNA strand. On the right side of each remodeling gel are intensity profiles of the indicated gel lanes, based on Multi Gauge software analysis. The triangles indicate the positions of the bands highlighted by the triangles in the respective lanes of the corresponding gel picture. All lanes shown for one remodeling template are from one gel. Number or replicates: ( A ) ≥3; ( B ) 3; ( C ) ≥3; ( D ) 3; ( E ) ≥3.

    Article Snippet: The success of an assembly reaction was checked by loading 200–500 ng of the nucleosomes (concentration determined via the applied DNA amount per reaction) on a PAA-gel, using non-assembled DNA as a control.

    Techniques: Recombinant, Sequencing, Purification, Staining, Software

    CHD3 and CHD4 form distinct NuRD complexes with different yet overlapping functionality . Our experiments propose that CHD3 and CHD4 form isoform-specific NuRD complexes in living cells, harboring one single copy of the respective remodeling enzyme. Beside well known ‘shared’ core subunits like HDAC1/2 or MTA2/3 proteins, we found Brg1, Snf2h and HP1 to be associated with both CHD3- and CHD4-containing NuRD complexes. DNA damage experiments, performed in this study, suggest that both CHD3- and CHD4-containing NuRD complexes exert a role in the DNA damage response. Nevertheless, an individual role of mouse CHD3 in DSB repair was proposed by the CHD3 specific binding protein KAP-1 ( 11 , 81 ). In addition to interaction partners that might act as functional NuRD regulators, we propose intrinsic remodeling properties as another possibility to influence the physiological role of NuRD complexes in vivo . Nucleosome remodeling assays with recombinant, single CHD3 and CHD4 proteins revealed that both enzymes move nucleosomes to distinct, sequence-specific positions, supporting the idea of CHD remodeling enzymes acting as chromatin specific organizers. In line with this, we observe mainly distinct nuclear localization patterns for CHD3- and CHD4-NuRD complexes in living cells, arguing for the existence of structurally and spatially separated complexes acting independently from each other in different genomic regions with a putative effect on gene activity. Indeed, our RNA-seq and qPCR experiments showed that CHD3 and CHD4 mainly regulate distinct genes. Taken together our data suggest that CHD3 and CHD4 form distinct NuRD complexes with different yet overlapping functionality (see also Results and Discussion).

    Journal: Nucleic Acids Research

    Article Title: CHD3 and CHD4 form distinct NuRD complexes with different yet overlapping functionality

    doi: 10.1093/nar/gkx711

    Figure Lengend Snippet: CHD3 and CHD4 form distinct NuRD complexes with different yet overlapping functionality . Our experiments propose that CHD3 and CHD4 form isoform-specific NuRD complexes in living cells, harboring one single copy of the respective remodeling enzyme. Beside well known ‘shared’ core subunits like HDAC1/2 or MTA2/3 proteins, we found Brg1, Snf2h and HP1 to be associated with both CHD3- and CHD4-containing NuRD complexes. DNA damage experiments, performed in this study, suggest that both CHD3- and CHD4-containing NuRD complexes exert a role in the DNA damage response. Nevertheless, an individual role of mouse CHD3 in DSB repair was proposed by the CHD3 specific binding protein KAP-1 ( 11 , 81 ). In addition to interaction partners that might act as functional NuRD regulators, we propose intrinsic remodeling properties as another possibility to influence the physiological role of NuRD complexes in vivo . Nucleosome remodeling assays with recombinant, single CHD3 and CHD4 proteins revealed that both enzymes move nucleosomes to distinct, sequence-specific positions, supporting the idea of CHD remodeling enzymes acting as chromatin specific organizers. In line with this, we observe mainly distinct nuclear localization patterns for CHD3- and CHD4-NuRD complexes in living cells, arguing for the existence of structurally and spatially separated complexes acting independently from each other in different genomic regions with a putative effect on gene activity. Indeed, our RNA-seq and qPCR experiments showed that CHD3 and CHD4 mainly regulate distinct genes. Taken together our data suggest that CHD3 and CHD4 form distinct NuRD complexes with different yet overlapping functionality (see also Results and Discussion).

    Article Snippet: The success of an assembly reaction was checked by loading 200–500 ng of the nucleosomes (concentration determined via the applied DNA amount per reaction) on a PAA-gel, using non-assembled DNA as a control.

    Techniques: Binding Assay, Activated Clotting Time Assay, Functional Assay, In Vivo, Recombinant, Sequencing, Activity Assay, RNA Sequencing Assay, Real-time Polymerase Chain Reaction

    Reconstituted nucleosome from the purified histone octamer serves as a valid substrate to histone methyltransferases. (a) 4-12% native PAGE of the reconstituted nucleosome. (b) DOT1L HMTase assay demonstrating inhibition of DOT1L by SAH with recombinant

    Journal: Protein expression and purification

    Article Title: One-pot refolding of core histones from bacterial inclusion bodies allows rapid reconstitution of histone octamer

    doi: 10.1016/j.pep.2015.02.007

    Figure Lengend Snippet: Reconstituted nucleosome from the purified histone octamer serves as a valid substrate to histone methyltransferases. (a) 4-12% native PAGE of the reconstituted nucleosome. (b) DOT1L HMTase assay demonstrating inhibition of DOT1L by SAH with recombinant

    Article Snippet: The HMTase reaction for DOT1L was carried out by incubation of 125 nM GST-DOT1L with 0.7 μg of either recombinant nucleosomes or HeLa extracted nucleosomes (52015; BPS Bioscience) and 125 nM (0.28 μCi) 3 H-S-adenosyl methionine (NET155250UC; Perkin Elmer).

    Techniques: Purification, Clear Native PAGE, Inhibition, Recombinant