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  • 93
    New England Biolabs epimark nucleosome assembly kit
    Epimark Nucleosome Assembly Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 153 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Roche nucleosomes
    Effects of p38 mitogen-activated protein kinases (MAPK) inhibitor and nuclear factor kappa-light-chain-enhancer of activated B cells ( NF-κB ) siRNA on <t>nucleosome</t> release. The apoptosis of retinal tissues was evaluated by nucleosome release ELISA. Eyes injected with SB203580 or NF-κB p65 siRNA had higher nucleosome release than the ischemia/reperfusion (I/R) control groups. Control: normal retinas, the other five groups were individually as I/R, I/R+vehicle (0.1% DMSO), I/R+control of siRNA in vehicle (Lipofectamine 2000 [Invitrogen] and DMEM/F12: no serum and no antibiotics), I/R+SB203580 and I/R+ NF-κB siRNA. *Statistically significant compared to the I/R control: p
    Nucleosomes, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 405 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    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: 91/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Reaction Biology Corporation mono dinucleosomes
    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.
    Mono Dinucleosomes, supplied by Reaction Biology Corporation, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    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: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    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: 90/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    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: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    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: 89/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    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: 92/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    New England Biolabs nucleosomes
    Changes in the probability of YpY dimer formation in DNA during <t>nucleosome</t> assembly and disassembly experiments. PhAST signals are presented in terms of log 2 of the intensity ratios (IR) along the 601 sequence expressed in SHLs; they are given for decreasing (top panel) or increasing (bottom panel) ionic strengths, as indicated by the green and black arrow respectively. The IR quantities are the ratios calculated between the normalised peak heights of nucleosomal and free DNA at each YpY position (see the text and Materials and Methods); they represent changes in the probability of YpY dimer formation. Red and blue bars correspond to DNA residues involved in the interface with H3-H4 and H2A-H2B, respectively. The black bars correspond to dinucleotides contacted by both H3-H4 and H2A-H2B. Minor-groove inward facing regions observed in the nucleosome structures are represented by grey boxes; they approximatively correspond to the SHL centres. Error bars are standard errors (n≥3, see Material and Methods for details).
    Nucleosomes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Active Motif nucleosome purification kit
    Changes in the probability of YpY dimer formation in DNA during <t>nucleosome</t> assembly and disassembly experiments. PhAST signals are presented in terms of log 2 of the intensity ratios (IR) along the 601 sequence expressed in SHLs; they are given for decreasing (top panel) or increasing (bottom panel) ionic strengths, as indicated by the green and black arrow respectively. The IR quantities are the ratios calculated between the normalised peak heights of nucleosomal and free DNA at each YpY position (see the text and Materials and Methods); they represent changes in the probability of YpY dimer formation. Red and blue bars correspond to DNA residues involved in the interface with H3-H4 and H2A-H2B, respectively. The black bars correspond to dinucleotides contacted by both H3-H4 and H2A-H2B. Minor-groove inward facing regions observed in the nucleosome structures are represented by grey boxes; they approximatively correspond to the SHL centres. Error bars are standard errors (n≥3, see Material and Methods for details).
    Nucleosome Purification Kit, supplied by Active Motif, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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: 93/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    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.
    Nucleosome Antigen, supplied by Arotec Diagnostics, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    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: 88/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    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: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    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: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    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%.
    Nucleosome Detection Kit, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Bio-Rad nucleosomes
    The HS4 insulator is enriched with ubiquitinated histones. A) Sucrose gradient fractionation of native MNase-digested <t>nucleosomes.</t> Fractions containing di- and tri- nucleosomes (e.g., 5–7) are pooled for ChIP analysis. B) SDS-PAGE analysis of histone purity in di/tri-nucleosome preparations from 10 day chick embryo red cells (R) and whole brain (B). C) Western blot analysis of mono-ubiquitinated H2B present in immunoprecipitates from 6C2 cell di/tri-nucleosomes. D, E) Native ChIP of histone ubiquitination at sites across the chicken β-globin gene neighborhood in 10 day chick embryo red cells (D) and whole brain (E). The enrichment of each sequence is normalized to the background observed at the downstream inactive OR51M1 ( COR3′ ) gene. Significant ChIP enrichments are represented by asterisks (⋆ = p
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    92
    Double Helix nucleosomes
    Basic structure and folding of the nucleosomal filament. Panel I: ( A ) A nucleosomal filament with five <t>nucleosomes.</t> ( B ) Core particle. ( C ) Histone H1, ( D ) A nucleosomal repeat length (NRL) of 200 bp consisting of 146 bp of nucleosomal DNA (red) plus 54 bp of linker DNA (black). ( E ) The angle (α) between the entering and exiting linkers, the change in direction of the linkers (δ = 180°−α) and the dyad axis (a d ). ( F ) The rotational angle (β) between the flat faces of the nucleosomes. ( G ) The slope of the DNA (γ) and the angle (η 1 ) between the projection of the linkers into a plane through the axis of symmetry of the nucleosome (a s ) and the dyad axis, bisecting α. ( H ) The projection (α 0 ) of α in a chromatin fiber into a plane perpendicular to the fiber axis. Panel II: Unfolding the nucleosomal filament from α = 0° to α = 180°. Panel III: The effect of β on the direction and coiling of the filament. Rotation of the terminal nucleosomes of a trinucleosome of a polygon fiber ( A ) and a star fiber ( B ). The trinucleosomes are oriented with the dyad axis of the central nucleosome (yellow) in the viewing direction. Arrows indicate the direction of the filament from nucleosome No. 1→3. ( C ) Side-on view of a dinucleosomes showing the pitch ( p ) of the dinucleosomes, as defined by the height difference between the entry and exit sites of the terminal linkers in a plane perpendicular to the interconnecting linker. ( D ) Dinucleosomes oriented with the interconnecting linker in the viewing direction and nucleosome no. 1 in front. Clockwise and counter clockwise rotations are indicated by (+) and (−) and the pitch of the dinucleosomes is indicated by arrows. White and black asterisks indicate the entry and exit sites of the terminal linkers. Panel IV: Possible effects of binding of histone H1 on the geometry of the dinucleosome, as shown for NRL = 200 bp. ( A and B ) The entry and exit sites of the linkers on the nucleosomes approach each other, corresponding to two full coils of DNA around the core particles, while the size of α is decreased below 180° by bending of the linkers close to their entry/exit sites, causing the nucleosomes to rotate away from each other (arrows). ( C ) Rotation of the nucleosomes until α = 0°, forming a stem conformation, which occupies 25–30 bp of the linker. ( D – F) Bending of the linker may introduce a twist angle ( τ ) between the flat faces of the nucleosomes. 146 bp nucleosomal DNA (gray); 54 bp connecting linker DNA (blue). The angle (75°) between the terminals of the 146 bp nucleosomal DNA is shown by the white sectors on the core particles.
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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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    Image Search Results


    Effects of p38 mitogen-activated protein kinases (MAPK) inhibitor and nuclear factor kappa-light-chain-enhancer of activated B cells ( NF-κB ) siRNA on nucleosome release. The apoptosis of retinal tissues was evaluated by nucleosome release ELISA. Eyes injected with SB203580 or NF-κB p65 siRNA had higher nucleosome release than the ischemia/reperfusion (I/R) control groups. Control: normal retinas, the other five groups were individually as I/R, I/R+vehicle (0.1% DMSO), I/R+control of siRNA in vehicle (Lipofectamine 2000 [Invitrogen] and DMEM/F12: no serum and no antibiotics), I/R+SB203580 and I/R+ NF-κB siRNA. *Statistically significant compared to the I/R control: p

    Journal: Molecular Vision

    Article Title: p38 mitogen-activated protein kinase-induced nuclear factor kappa-light-chain-enhancer of activated B cell activity is required for neuroprotection in retinal ischemia/reperfusion injury

    doi:

    Figure Lengend Snippet: Effects of p38 mitogen-activated protein kinases (MAPK) inhibitor and nuclear factor kappa-light-chain-enhancer of activated B cells ( NF-κB ) siRNA on nucleosome release. The apoptosis of retinal tissues was evaluated by nucleosome release ELISA. Eyes injected with SB203580 or NF-κB p65 siRNA had higher nucleosome release than the ischemia/reperfusion (I/R) control groups. Control: normal retinas, the other five groups were individually as I/R, I/R+vehicle (0.1% DMSO), I/R+control of siRNA in vehicle (Lipofectamine 2000 [Invitrogen] and DMEM/F12: no serum and no antibiotics), I/R+SB203580 and I/R+ NF-κB siRNA. *Statistically significant compared to the I/R control: p

    Article Snippet: The presence of nucleosomes from apoptosis can be quantified by using a Cell Death Detection enzyme-linked immunosorbent assay (ELISA; Roche Applied Science, Indianapolis, IN) with antibodies directed against DNA and histones.

    Techniques: Enzyme-linked Immunosorbent Assay, Injection

    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

    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

    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

    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

    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: Published methods for calling nucleosome positions from Illumina data typically involve extending each single-end short sequenced read to the expected segment length of ∼140 bp and then examining the coverage of different genomic loci by the accumulated extended segments.

    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: Published methods for calling nucleosome positions from Illumina data typically involve extending each single-end short sequenced read to the expected segment length of ∼140 bp and then examining the coverage of different genomic loci by the accumulated extended segments.

    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: Published methods for calling nucleosome positions from Illumina data typically involve extending each single-end short sequenced read to the expected segment length of ∼140 bp and then examining the coverage of different genomic loci by the accumulated extended segments.

    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: Published methods for calling nucleosome positions from Illumina data typically involve extending each single-end short sequenced read to the expected segment length of ∼140 bp and then examining the coverage of different genomic loci by the accumulated extended segments.

    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: Published methods for calling nucleosome positions from Illumina data typically involve extending each single-end short sequenced read to the expected segment length of ∼140 bp and then examining the coverage of different genomic loci by the accumulated extended segments.

    Techniques: Isolation

    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

    Changes in the probability of YpY dimer formation in DNA during nucleosome assembly and disassembly experiments. PhAST signals are presented in terms of log 2 of the intensity ratios (IR) along the 601 sequence expressed in SHLs; they are given for decreasing (top panel) or increasing (bottom panel) ionic strengths, as indicated by the green and black arrow respectively. The IR quantities are the ratios calculated between the normalised peak heights of nucleosomal and free DNA at each YpY position (see the text and Materials and Methods); they represent changes in the probability of YpY dimer formation. Red and blue bars correspond to DNA residues involved in the interface with H3-H4 and H2A-H2B, respectively. The black bars correspond to dinucleotides contacted by both H3-H4 and H2A-H2B. Minor-groove inward facing regions observed in the nucleosome structures are represented by grey boxes; they approximatively correspond to the SHL centres. Error bars are standard errors (n≥3, see Material and Methods for details).

    Journal: bioRxiv

    Article Title: Nucleosome assembly and disassembly pathways

    doi: 10.1101/2020.04.01.020263

    Figure Lengend Snippet: Changes in the probability of YpY dimer formation in DNA during nucleosome assembly and disassembly experiments. PhAST signals are presented in terms of log 2 of the intensity ratios (IR) along the 601 sequence expressed in SHLs; they are given for decreasing (top panel) or increasing (bottom panel) ionic strengths, as indicated by the green and black arrow respectively. The IR quantities are the ratios calculated between the normalised peak heights of nucleosomal and free DNA at each YpY position (see the text and Materials and Methods); they represent changes in the probability of YpY dimer formation. Red and blue bars correspond to DNA residues involved in the interface with H3-H4 and H2A-H2B, respectively. The black bars correspond to dinucleotides contacted by both H3-H4 and H2A-H2B. Minor-groove inward facing regions observed in the nucleosome structures are represented by grey boxes; they approximatively correspond to the SHL centres. Error bars are standard errors (n≥3, see Material and Methods for details).

    Article Snippet: Nucleosomes were reconstituted with a salt dilution method according to manufacturer’s instructions (New England BioLabs) with a slight modification.

    Techniques: Sequencing

    Representation of asymmetries in the DNA/histone interactions on nucleosome models according to PhAST experiments. In the panels A and B, the DNA double helix was coloured with a gradient of grey, from light to dark grey for low and high R log2(IR) values (see Figure 5 ). Low and high R log2(IR) values were interpreted as weak and strong DNA/histone interactions. The double helix in yellow corresponds to the DNA region devoid of YpY steps. The nucleosome DNA was split into two parts containing either the 5’ or 3’ half of the 601 sequence. Histones are represented by semi-transparent models (H3-H4 in red and H2A-H2B in blue) to reflect their location in the intact nucleosome. For clarity, the histone tails are not represented. Comparison of the PhAST derived data R log2(IR) along t he 5’ (left) and 3’ (right) DNA halves A : at 1.0 M NaCl in the experiments of nucleosome dissociation and B : at 1.5 M NaCl in the experiments of nucleosome association.

    Journal: bioRxiv

    Article Title: Nucleosome assembly and disassembly pathways

    doi: 10.1101/2020.04.01.020263

    Figure Lengend Snippet: Representation of asymmetries in the DNA/histone interactions on nucleosome models according to PhAST experiments. In the panels A and B, the DNA double helix was coloured with a gradient of grey, from light to dark grey for low and high R log2(IR) values (see Figure 5 ). Low and high R log2(IR) values were interpreted as weak and strong DNA/histone interactions. The double helix in yellow corresponds to the DNA region devoid of YpY steps. The nucleosome DNA was split into two parts containing either the 5’ or 3’ half of the 601 sequence. Histones are represented by semi-transparent models (H3-H4 in red and H2A-H2B in blue) to reflect their location in the intact nucleosome. For clarity, the histone tails are not represented. Comparison of the PhAST derived data R log2(IR) along t he 5’ (left) and 3’ (right) DNA halves A : at 1.0 M NaCl in the experiments of nucleosome dissociation and B : at 1.5 M NaCl in the experiments of nucleosome association.

    Article Snippet: Nucleosomes were reconstituted with a salt dilution method according to manufacturer’s instructions (New England BioLabs) with a slight modification.

    Techniques: Sequencing, Derivative Assay

    Outline of the PhAST experiments monitoring successive DNA structural changes induced during nucleosome assembly and disassembly by using stepwise decrease and increase of NaCl concentration.

    Journal: bioRxiv

    Article Title: Nucleosome assembly and disassembly pathways

    doi: 10.1101/2020.04.01.020263

    Figure Lengend Snippet: Outline of the PhAST experiments monitoring successive DNA structural changes induced during nucleosome assembly and disassembly by using stepwise decrease and increase of NaCl concentration.

    Article Snippet: Nucleosomes were reconstituted with a salt dilution method according to manufacturer’s instructions (New England BioLabs) with a slight modification.

    Techniques: Concentration Assay

    The DNA/histone interface. In both A and B panels, the DNA/histone interface data were extracted and analysed from NCP simulations [ 33 ]. A: Schematic representation of DNA regions involved in the DNA/histone interface. The nucleotides interacting with the histone structured and unstructured domains are positioned along the 601 sequence, specifying the contacts with the different chains (A, B, C, etc…) of each histone type (H3 and H4 in red, H2A and H2B in blue). The lower banner summarises those regions contacted by H3-H4, H2A-H2B, both H3-H4 and H2A-H2B (black) or none of them (grey). The DNA sequence is labeled by SHL (Super Helical Location, defined in Material and Methods). The dotted lines correspond to intervals of 1 SHL. In the upper banner, the location of YY steps along the two strands of the 601 sequence is pictured by black bars. B: Quantification of the DNA/histone interactions. The average contact areas (CA av ) quantified the DNA/histone interactions at the base pair level of DNA regions, and uses the same color code as in panel A. The vertical error bars are standard deviations over the simulation trajectory. The data presented in panels A and B were extracted from the VLDM analysis of structures from a 1.2 μs simulation of a 601-nucleosome.

    Journal: bioRxiv

    Article Title: Nucleosome assembly and disassembly pathways

    doi: 10.1101/2020.04.01.020263

    Figure Lengend Snippet: The DNA/histone interface. In both A and B panels, the DNA/histone interface data were extracted and analysed from NCP simulations [ 33 ]. A: Schematic representation of DNA regions involved in the DNA/histone interface. The nucleotides interacting with the histone structured and unstructured domains are positioned along the 601 sequence, specifying the contacts with the different chains (A, B, C, etc…) of each histone type (H3 and H4 in red, H2A and H2B in blue). The lower banner summarises those regions contacted by H3-H4, H2A-H2B, both H3-H4 and H2A-H2B (black) or none of them (grey). The DNA sequence is labeled by SHL (Super Helical Location, defined in Material and Methods). The dotted lines correspond to intervals of 1 SHL. In the upper banner, the location of YY steps along the two strands of the 601 sequence is pictured by black bars. B: Quantification of the DNA/histone interactions. The average contact areas (CA av ) quantified the DNA/histone interactions at the base pair level of DNA regions, and uses the same color code as in panel A. The vertical error bars are standard deviations over the simulation trajectory. The data presented in panels A and B were extracted from the VLDM analysis of structures from a 1.2 μs simulation of a 601-nucleosome.

    Article Snippet: Nucleosomes were reconstituted with a salt dilution method according to manufacturer’s instructions (New England BioLabs) with a slight modification.

    Techniques: Sequencing, Labeling

    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

    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

    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

    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

    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 HS4 insulator is enriched with ubiquitinated histones. A) Sucrose gradient fractionation of native MNase-digested nucleosomes. Fractions containing di- and tri- nucleosomes (e.g., 5–7) are pooled for ChIP analysis. B) SDS-PAGE analysis of histone purity in di/tri-nucleosome preparations from 10 day chick embryo red cells (R) and whole brain (B). C) Western blot analysis of mono-ubiquitinated H2B present in immunoprecipitates from 6C2 cell di/tri-nucleosomes. D, E) Native ChIP of histone ubiquitination at sites across the chicken β-globin gene neighborhood in 10 day chick embryo red cells (D) and whole brain (E). The enrichment of each sequence is normalized to the background observed at the downstream inactive OR51M1 ( COR3′ ) gene. Significant ChIP enrichments are represented by asterisks (⋆ = p

    Journal: PLoS Genetics

    Article Title: Histone Crosstalk Directed by H2B Ubiquitination Is Required for Chromatin Boundary Integrity

    doi: 10.1371/journal.pgen.1002175

    Figure Lengend Snippet: The HS4 insulator is enriched with ubiquitinated histones. A) Sucrose gradient fractionation of native MNase-digested nucleosomes. Fractions containing di- and tri- nucleosomes (e.g., 5–7) are pooled for ChIP analysis. B) SDS-PAGE analysis of histone purity in di/tri-nucleosome preparations from 10 day chick embryo red cells (R) and whole brain (B). C) Western blot analysis of mono-ubiquitinated H2B present in immunoprecipitates from 6C2 cell di/tri-nucleosomes. D, E) Native ChIP of histone ubiquitination at sites across the chicken β-globin gene neighborhood in 10 day chick embryo red cells (D) and whole brain (E). The enrichment of each sequence is normalized to the background observed at the downstream inactive OR51M1 ( COR3′ ) gene. Significant ChIP enrichments are represented by asterisks (⋆ = p

    Article Snippet: Nucleosomes were exchanged into N-ChIP buffer (50 mM NaCl, 5 mM EDTA, 10 mM Tris pH 7.5) buffer using P-6DG Bio-Gel (BioRad).

    Techniques: Fractionation, Chromatin Immunoprecipitation, SDS Page, Western Blot, Sequencing

    Long term loss of H2B ubiquitination results in a breach of the HS4 chromatin boundary. Analyses of early erythroid 6C2 cells following forty days of doxycycline-induced knockdown of RNF20 expression. A) Western blotting of whole cell extracts with (+) and without (−) doxycycline-induced RNF20 knockdown. The expression level of RNF20 following knockdown (relative to the loading control TBP) is shown. B) Western blotting of histone modifications present on total nucleosomes with and without RNF20 knockdown. The levels of each modification after RNF20 knockdown (relative to unmodified H3) are shown. C–G) Native ChIP analyses following forty days of RNF20 knockdown. Enrichments of C) H2BK120ub1, D) H3K4me2, E) H3K4me3, F) H3K9me3 and G) H4K20me3 at sites across the chicken β-globin gene neighborhood in wild type (red bars) and RNF20 knockdown (blue bars) cells. The enrichment of each sequence is normalized to the background observed at the condensed region (15.850). Significant changes in ChIP enrichments following RNF20 depletion are represented by asterisks (⋆ = p

    Journal: PLoS Genetics

    Article Title: Histone Crosstalk Directed by H2B Ubiquitination Is Required for Chromatin Boundary Integrity

    doi: 10.1371/journal.pgen.1002175

    Figure Lengend Snippet: Long term loss of H2B ubiquitination results in a breach of the HS4 chromatin boundary. Analyses of early erythroid 6C2 cells following forty days of doxycycline-induced knockdown of RNF20 expression. A) Western blotting of whole cell extracts with (+) and without (−) doxycycline-induced RNF20 knockdown. The expression level of RNF20 following knockdown (relative to the loading control TBP) is shown. B) Western blotting of histone modifications present on total nucleosomes with and without RNF20 knockdown. The levels of each modification after RNF20 knockdown (relative to unmodified H3) are shown. C–G) Native ChIP analyses following forty days of RNF20 knockdown. Enrichments of C) H2BK120ub1, D) H3K4me2, E) H3K4me3, F) H3K9me3 and G) H4K20me3 at sites across the chicken β-globin gene neighborhood in wild type (red bars) and RNF20 knockdown (blue bars) cells. The enrichment of each sequence is normalized to the background observed at the condensed region (15.850). Significant changes in ChIP enrichments following RNF20 depletion are represented by asterisks (⋆ = p

    Article Snippet: Nucleosomes were exchanged into N-ChIP buffer (50 mM NaCl, 5 mM EDTA, 10 mM Tris pH 7.5) buffer using P-6DG Bio-Gel (BioRad).

    Techniques: Expressing, Western Blot, Modification, Chromatin Immunoprecipitation, Sequencing

    The ubiquitin ligase RNF20 mediates H2B ubiquitination at the HS4 insulator. Crosslinking ChIP analysis of (A) H2BK120ub1 and (B) RNF20 enrichment at the chicken β-globin locus in 6C2 cells. The enrichment of each sequence is normalized to the background observed at the condensed region (15.850). C–E) Analyses of early erythroid 6C2 cells following four days of doxycycline-induced knockdown of RNF20 expression. (C) Western blotting of whole cell extracts with (+) and without (−) doxycycline-induced RNF20 knockdown. The expression levels of each factor (relative to the loading control TBP) after RNF20 knockdown are shown. (D) Western blotting of histone modifications present on total nucleosomes with and without RNF20 knockdown. The levels of each modification after RNF20 knockdown (relative to unmodified H3 loading control) are shown. (E) Native ChIP of histone ubiquitination at sites across the chicken β-globin gene neighborhood in wild type (red bars) and RNF20 knockdown (blue bars) 6C2 cells. The enrichment of each sequence is normalized to the background observed downstream of the inactive ρ-globin gene. Significant changes in ChIP enrichments following RNF20 depletion are represented by asterisks (⋆ = p

    Journal: PLoS Genetics

    Article Title: Histone Crosstalk Directed by H2B Ubiquitination Is Required for Chromatin Boundary Integrity

    doi: 10.1371/journal.pgen.1002175

    Figure Lengend Snippet: The ubiquitin ligase RNF20 mediates H2B ubiquitination at the HS4 insulator. Crosslinking ChIP analysis of (A) H2BK120ub1 and (B) RNF20 enrichment at the chicken β-globin locus in 6C2 cells. The enrichment of each sequence is normalized to the background observed at the condensed region (15.850). C–E) Analyses of early erythroid 6C2 cells following four days of doxycycline-induced knockdown of RNF20 expression. (C) Western blotting of whole cell extracts with (+) and without (−) doxycycline-induced RNF20 knockdown. The expression levels of each factor (relative to the loading control TBP) after RNF20 knockdown are shown. (D) Western blotting of histone modifications present on total nucleosomes with and without RNF20 knockdown. The levels of each modification after RNF20 knockdown (relative to unmodified H3 loading control) are shown. (E) Native ChIP of histone ubiquitination at sites across the chicken β-globin gene neighborhood in wild type (red bars) and RNF20 knockdown (blue bars) 6C2 cells. The enrichment of each sequence is normalized to the background observed downstream of the inactive ρ-globin gene. Significant changes in ChIP enrichments following RNF20 depletion are represented by asterisks (⋆ = p

    Article Snippet: Nucleosomes were exchanged into N-ChIP buffer (50 mM NaCl, 5 mM EDTA, 10 mM Tris pH 7.5) buffer using P-6DG Bio-Gel (BioRad).

    Techniques: Chromatin Immunoprecipitation, Sequencing, Expressing, Western Blot, Modification

    H2B ubiquitination in the establishment of a chromatin boundary. A) HS4 lies at the boundary between the β-globin locus and an upstream region of condensed chromatin enriched in multiple repressive histone marks (blue). H2B ubiquitination at the HS4 insulator is mediated by the E3 ligase RNF20 and is dependent upon the USF binding site. H2Bub1 is required for H3K4me3 mediated by the SET1 complex and multiple histone acetylation at two or three nucleosomes around HS4 (green). B) Scale schematic diagram summarizing the extent of chromatin domains at the FOLR1 and β-globin loci mapped in wild type 6C2 cells (top panel) and cells following short-term (middle panel) and long-term (bottom panel) depletion of RNF20. Sequences enriched with H3K4me3, H4K20me3, H3K9me3 and H3K9me2 are represented by green, purple, dark blue and light blue rectangles, respectively. Faded green depicts the depletion of H3K4me3 at the boundary elements following RNF20 knockdown. Arrowheads depict the encroachment of heterochromatin marks.

    Journal: PLoS Genetics

    Article Title: Histone Crosstalk Directed by H2B Ubiquitination Is Required for Chromatin Boundary Integrity

    doi: 10.1371/journal.pgen.1002175

    Figure Lengend Snippet: H2B ubiquitination in the establishment of a chromatin boundary. A) HS4 lies at the boundary between the β-globin locus and an upstream region of condensed chromatin enriched in multiple repressive histone marks (blue). H2B ubiquitination at the HS4 insulator is mediated by the E3 ligase RNF20 and is dependent upon the USF binding site. H2Bub1 is required for H3K4me3 mediated by the SET1 complex and multiple histone acetylation at two or three nucleosomes around HS4 (green). B) Scale schematic diagram summarizing the extent of chromatin domains at the FOLR1 and β-globin loci mapped in wild type 6C2 cells (top panel) and cells following short-term (middle panel) and long-term (bottom panel) depletion of RNF20. Sequences enriched with H3K4me3, H4K20me3, H3K9me3 and H3K9me2 are represented by green, purple, dark blue and light blue rectangles, respectively. Faded green depicts the depletion of H3K4me3 at the boundary elements following RNF20 knockdown. Arrowheads depict the encroachment of heterochromatin marks.

    Article Snippet: Nucleosomes were exchanged into N-ChIP buffer (50 mM NaCl, 5 mM EDTA, 10 mM Tris pH 7.5) buffer using P-6DG Bio-Gel (BioRad).

    Techniques: Binding Assay

    Basic structure and folding of the nucleosomal filament. Panel I: ( A ) A nucleosomal filament with five nucleosomes. ( B ) Core particle. ( C ) Histone H1, ( D ) A nucleosomal repeat length (NRL) of 200 bp consisting of 146 bp of nucleosomal DNA (red) plus 54 bp of linker DNA (black). ( E ) The angle (α) between the entering and exiting linkers, the change in direction of the linkers (δ = 180°−α) and the dyad axis (a d ). ( F ) The rotational angle (β) between the flat faces of the nucleosomes. ( G ) The slope of the DNA (γ) and the angle (η 1 ) between the projection of the linkers into a plane through the axis of symmetry of the nucleosome (a s ) and the dyad axis, bisecting α. ( H ) The projection (α 0 ) of α in a chromatin fiber into a plane perpendicular to the fiber axis. Panel II: Unfolding the nucleosomal filament from α = 0° to α = 180°. Panel III: The effect of β on the direction and coiling of the filament. Rotation of the terminal nucleosomes of a trinucleosome of a polygon fiber ( A ) and a star fiber ( B ). The trinucleosomes are oriented with the dyad axis of the central nucleosome (yellow) in the viewing direction. Arrows indicate the direction of the filament from nucleosome No. 1→3. ( C ) Side-on view of a dinucleosomes showing the pitch ( p ) of the dinucleosomes, as defined by the height difference between the entry and exit sites of the terminal linkers in a plane perpendicular to the interconnecting linker. ( D ) Dinucleosomes oriented with the interconnecting linker in the viewing direction and nucleosome no. 1 in front. Clockwise and counter clockwise rotations are indicated by (+) and (−) and the pitch of the dinucleosomes is indicated by arrows. White and black asterisks indicate the entry and exit sites of the terminal linkers. Panel IV: Possible effects of binding of histone H1 on the geometry of the dinucleosome, as shown for NRL = 200 bp. ( A and B ) The entry and exit sites of the linkers on the nucleosomes approach each other, corresponding to two full coils of DNA around the core particles, while the size of α is decreased below 180° by bending of the linkers close to their entry/exit sites, causing the nucleosomes to rotate away from each other (arrows). ( C ) Rotation of the nucleosomes until α = 0°, forming a stem conformation, which occupies 25–30 bp of the linker. ( D – F) Bending of the linker may introduce a twist angle ( τ ) between the flat faces of the nucleosomes. 146 bp nucleosomal DNA (gray); 54 bp connecting linker DNA (blue). The angle (75°) between the terminals of the 146 bp nucleosomal DNA is shown by the white sectors on the core particles.

    Journal: Nucleic Acids Research

    Article Title: Choreography for nucleosomes: the conformational freedom of the nucleosomal filament and its limitations

    doi: 10.1093/nar/gkm560

    Figure Lengend Snippet: Basic structure and folding of the nucleosomal filament. Panel I: ( A ) A nucleosomal filament with five nucleosomes. ( B ) Core particle. ( C ) Histone H1, ( D ) A nucleosomal repeat length (NRL) of 200 bp consisting of 146 bp of nucleosomal DNA (red) plus 54 bp of linker DNA (black). ( E ) The angle (α) between the entering and exiting linkers, the change in direction of the linkers (δ = 180°−α) and the dyad axis (a d ). ( F ) The rotational angle (β) between the flat faces of the nucleosomes. ( G ) The slope of the DNA (γ) and the angle (η 1 ) between the projection of the linkers into a plane through the axis of symmetry of the nucleosome (a s ) and the dyad axis, bisecting α. ( H ) The projection (α 0 ) of α in a chromatin fiber into a plane perpendicular to the fiber axis. Panel II: Unfolding the nucleosomal filament from α = 0° to α = 180°. Panel III: The effect of β on the direction and coiling of the filament. Rotation of the terminal nucleosomes of a trinucleosome of a polygon fiber ( A ) and a star fiber ( B ). The trinucleosomes are oriented with the dyad axis of the central nucleosome (yellow) in the viewing direction. Arrows indicate the direction of the filament from nucleosome No. 1→3. ( C ) Side-on view of a dinucleosomes showing the pitch ( p ) of the dinucleosomes, as defined by the height difference between the entry and exit sites of the terminal linkers in a plane perpendicular to the interconnecting linker. ( D ) Dinucleosomes oriented with the interconnecting linker in the viewing direction and nucleosome no. 1 in front. Clockwise and counter clockwise rotations are indicated by (+) and (−) and the pitch of the dinucleosomes is indicated by arrows. White and black asterisks indicate the entry and exit sites of the terminal linkers. Panel IV: Possible effects of binding of histone H1 on the geometry of the dinucleosome, as shown for NRL = 200 bp. ( A and B ) The entry and exit sites of the linkers on the nucleosomes approach each other, corresponding to two full coils of DNA around the core particles, while the size of α is decreased below 180° by bending of the linkers close to their entry/exit sites, causing the nucleosomes to rotate away from each other (arrows). ( C ) Rotation of the nucleosomes until α = 0°, forming a stem conformation, which occupies 25–30 bp of the linker. ( D – F) Bending of the linker may introduce a twist angle ( τ ) between the flat faces of the nucleosomes. 146 bp nucleosomal DNA (gray); 54 bp connecting linker DNA (blue). The angle (75°) between the terminals of the 146 bp nucleosomal DNA is shown by the white sectors on the core particles.

    Article Snippet: In a fiber with β sequence (3033 )n , (3 ) swivel-linkers separate right-handed coils of four nucleosomes (•3•0•3•), providing sufficient space for the formation of a double helix in which swivel-linkers in one filament is alternately located on the inside and outside of swivel-linkers in the other filament ( B).

    Techniques: Binding Assay, Introduce

    Swivel-linkers and compaction of a polygon fiber (α = 108°). ( A ) A fiber with β sequence (0 33 033) n , in which β(0)-swivel-linkers (arrows) connect trinucleosomes, which alternate between left- (• 3 • 3 •) and right-handed (•3•3•) orientation. ( B–H ) Compaction of a fiber (B) with β sequence (33 3 ) n in which ( 3 )-swivel-linkers (arrows) are separating right-handed trinucleosomes (•3•3•), two of which are numbered 1–3 and 4–6. The fiber is compacted by decreasing α at the terminals of the swivel-linker ( C ) and in the trinucleosomes ( D ). (E ) Axial view of the compacted fiber showing the superposition of linkers of two consecutive trinucleosomes. ( F ) Compact fiber as viewed from the side. Nucleosomes with an orientation close to the fiber axis (blue arrows) are stacking edge-to-edge and nucleosomes perpendicular to the fiber axis (red arrows) have the potential to interact with nucleosomes in the same orientation in a neighboring fiber by interdigitation. ( G ) Interdigitation of two compact fibers as seen from the end and from the side. ( H ). Packing of three sheets (red, green, yellow), each consisting of three interdigitated fibers, as seen from the end. Rectangles delineate the cross-section area used for density calculation. Bar = 30 nm.

    Journal: Nucleic Acids Research

    Article Title: Choreography for nucleosomes: the conformational freedom of the nucleosomal filament and its limitations

    doi: 10.1093/nar/gkm560

    Figure Lengend Snippet: Swivel-linkers and compaction of a polygon fiber (α = 108°). ( A ) A fiber with β sequence (0 33 033) n , in which β(0)-swivel-linkers (arrows) connect trinucleosomes, which alternate between left- (• 3 • 3 •) and right-handed (•3•3•) orientation. ( B–H ) Compaction of a fiber (B) with β sequence (33 3 ) n in which ( 3 )-swivel-linkers (arrows) are separating right-handed trinucleosomes (•3•3•), two of which are numbered 1–3 and 4–6. The fiber is compacted by decreasing α at the terminals of the swivel-linker ( C ) and in the trinucleosomes ( D ). (E ) Axial view of the compacted fiber showing the superposition of linkers of two consecutive trinucleosomes. ( F ) Compact fiber as viewed from the side. Nucleosomes with an orientation close to the fiber axis (blue arrows) are stacking edge-to-edge and nucleosomes perpendicular to the fiber axis (red arrows) have the potential to interact with nucleosomes in the same orientation in a neighboring fiber by interdigitation. ( G ) Interdigitation of two compact fibers as seen from the end and from the side. ( H ). Packing of three sheets (red, green, yellow), each consisting of three interdigitated fibers, as seen from the end. Rectangles delineate the cross-section area used for density calculation. Bar = 30 nm.

    Article Snippet: In a fiber with β sequence (3033 )n , (3 ) swivel-linkers separate right-handed coils of four nucleosomes (•3•0•3•), providing sufficient space for the formation of a double helix in which swivel-linkers in one filament is alternately located on the inside and outside of swivel-linkers in the other filament ( B).

    Techniques: Sequencing

    Four types of chromatin double fibers formed by polygon fibers (red and white). ( A ) Two filaments with β sequence (33 3 ) n and α = 108° (as shown in Figure 4 B) align and interlock like a zip, in which nucleosomes in one fiber fit spatially into the space provided by a swivel-linker between two trinucleosomes in the other fiber (framed). ( B ) Two fibers with closed, negative linker configuration, β sequence (303 3 ) n , α = 108°, coil right-handed around each other forming a double helix. The swivel-linkers are parallel to the axis of the fiber, with alternating positions of the red and the white filament on the outside of the fiber. ( C ) Two fibers coil left-handed around each other by intercalation of every second nucleosome, α varying between 85 and 120° and β between 72 and 160°. ( D ) The positions of the intercalated core particles in the fiber in ( C ). ( E – G ) Two nucleosomal filaments with β(5) (180°) placed one on top of the other. ( E ) Nucleosomes stacking face-to-face with every second linker (red and green) touching each other at two crossover points (arrows), which are symmetrically positioned relative to the midpoint of the linker. (F and G) Parallel staggering of the ribbons along the fiber axis causes one crossover to be displaced toward the middle of the linker and the other toward the nucleosomal DNA of every second nucleosome, which is located at one edge of the ribbon (arrows). ( H and I ) A left-handed double helix formed by two filaments with alternating negative and positive linker configuration folded from a filament with open linker configuration; ∼90°

    Journal: Nucleic Acids Research

    Article Title: Choreography for nucleosomes: the conformational freedom of the nucleosomal filament and its limitations

    doi: 10.1093/nar/gkm560

    Figure Lengend Snippet: Four types of chromatin double fibers formed by polygon fibers (red and white). ( A ) Two filaments with β sequence (33 3 ) n and α = 108° (as shown in Figure 4 B) align and interlock like a zip, in which nucleosomes in one fiber fit spatially into the space provided by a swivel-linker between two trinucleosomes in the other fiber (framed). ( B ) Two fibers with closed, negative linker configuration, β sequence (303 3 ) n , α = 108°, coil right-handed around each other forming a double helix. The swivel-linkers are parallel to the axis of the fiber, with alternating positions of the red and the white filament on the outside of the fiber. ( C ) Two fibers coil left-handed around each other by intercalation of every second nucleosome, α varying between 85 and 120° and β between 72 and 160°. ( D ) The positions of the intercalated core particles in the fiber in ( C ). ( E – G ) Two nucleosomal filaments with β(5) (180°) placed one on top of the other. ( E ) Nucleosomes stacking face-to-face with every second linker (red and green) touching each other at two crossover points (arrows), which are symmetrically positioned relative to the midpoint of the linker. (F and G) Parallel staggering of the ribbons along the fiber axis causes one crossover to be displaced toward the middle of the linker and the other toward the nucleosomal DNA of every second nucleosome, which is located at one edge of the ribbon (arrows). ( H and I ) A left-handed double helix formed by two filaments with alternating negative and positive linker configuration folded from a filament with open linker configuration; ∼90°

    Article Snippet: In a fiber with β sequence (3033 )n , (3 ) swivel-linkers separate right-handed coils of four nucleosomes (•3•0•3•), providing sufficient space for the formation of a double helix in which swivel-linkers in one filament is alternately located on the inside and outside of swivel-linkers in the other filament ( B).

    Techniques: Sequencing

    Selected conformations of star and polygon fibers with repeated β sequences. ( A ) A star fiber with β sequence ( 1 1) n showing collisions between every fifth nucleosome. (B – I ) Polygon fibers. ( B and C ) (2 2 ) n , (C) in a compact form as seen from the side and from the end. ( D ) A coil formed by (4 4 ) n . ( E and F ) A polygon fiber with the approximate β sequence ( 22 2) n , as shown in side-on view from two angles differing by 90° (E), and in a more compact form (F). ( G – I ) The fiber (11 11 ) n as seen in side view from two angles and from the end (G). One and the same nucleosome is marked by an asterisk. (H) Model of the fiber in (G, F) with partially overlapping hexanucleosomal loops. The first four nucleosomes (•1•1• 1 • 1 ) in each repeat are painted in same color in the sequence red, green, blue, yellow and red (top to bottom) and numbered 1–4. (I) A loop formed by β sequence (33 33 ) n . α = 36 and 108° in the star- and polygon fibers, respectively, except in (E, F) where α = 90°. Bar = 30 nm.

    Journal: Nucleic Acids Research

    Article Title: Choreography for nucleosomes: the conformational freedom of the nucleosomal filament and its limitations

    doi: 10.1093/nar/gkm560

    Figure Lengend Snippet: Selected conformations of star and polygon fibers with repeated β sequences. ( A ) A star fiber with β sequence ( 1 1) n showing collisions between every fifth nucleosome. (B – I ) Polygon fibers. ( B and C ) (2 2 ) n , (C) in a compact form as seen from the side and from the end. ( D ) A coil formed by (4 4 ) n . ( E and F ) A polygon fiber with the approximate β sequence ( 22 2) n , as shown in side-on view from two angles differing by 90° (E), and in a more compact form (F). ( G – I ) The fiber (11 11 ) n as seen in side view from two angles and from the end (G). One and the same nucleosome is marked by an asterisk. (H) Model of the fiber in (G, F) with partially overlapping hexanucleosomal loops. The first four nucleosomes (•1•1• 1 • 1 ) in each repeat are painted in same color in the sequence red, green, blue, yellow and red (top to bottom) and numbered 1–4. (I) A loop formed by β sequence (33 33 ) n . α = 36 and 108° in the star- and polygon fibers, respectively, except in (E, F) where α = 90°. Bar = 30 nm.

    Article Snippet: In a fiber with β sequence (3033 )n , (3 ) swivel-linkers separate right-handed coils of four nucleosomes (•3•0•3•), providing sufficient space for the formation of a double helix in which swivel-linkers in one filament is alternately located on the inside and outside of swivel-linkers in the other filament ( B).

    Techniques: Sequencing

    The influence of β on the conformation of a star fiber ( A ) and a polygon fiber ( B ) with pentagonal axial symmetry (α = 36 and 108°, respectively). Each fiber contains 10 nucleosomes. The first nucleosome in each fiber is indicated by an asterisk. The nucleosomal repeat length [205 bp (A) and 196 bp (B)] is kept independent of β, which is varied in steps of 36°, corresponding to ∼10 bp/turn of the DNA, and numbered according to the number of increments of 36°. Large, black arrowheads point towards values of β that are not allowed for sterical reasons. Circular, white arrows indicate the coiling direction. The fibers in A and B are oriented with the connecting linker of the first dinucleosome in the viewing direction ( y -axis). As the shifting directions of the fibers do not follow the plane of the paper, as indicated by the thick fibers in gray scale, except for β = 0°, their true length cannot be inferred from the figure. ( C ) Polygon fiber with NRL 210 bp at β(−1). ( D ) Stacking of nucleosomes at β(5) into two columns with alternating positive and negative linker orientations by reducing the size of α. The fiber in ( D ) has NRL = 230 bp and α = ∼36°. ( E and F ) Compressed polygon fibers formed by reducing the size of α. (E) β(+4) is shown from the end (top) and from the side as seen from two different angles. (F) β(−4) seen from the end (top) and from the side. ( G ) Axial symmetries of the polygon fiber as a function of β at η 2 = 10 and 0°. Bar = 30 nm.

    Journal: Nucleic Acids Research

    Article Title: Choreography for nucleosomes: the conformational freedom of the nucleosomal filament and its limitations

    doi: 10.1093/nar/gkm560

    Figure Lengend Snippet: The influence of β on the conformation of a star fiber ( A ) and a polygon fiber ( B ) with pentagonal axial symmetry (α = 36 and 108°, respectively). Each fiber contains 10 nucleosomes. The first nucleosome in each fiber is indicated by an asterisk. The nucleosomal repeat length [205 bp (A) and 196 bp (B)] is kept independent of β, which is varied in steps of 36°, corresponding to ∼10 bp/turn of the DNA, and numbered according to the number of increments of 36°. Large, black arrowheads point towards values of β that are not allowed for sterical reasons. Circular, white arrows indicate the coiling direction. The fibers in A and B are oriented with the connecting linker of the first dinucleosome in the viewing direction ( y -axis). As the shifting directions of the fibers do not follow the plane of the paper, as indicated by the thick fibers in gray scale, except for β = 0°, their true length cannot be inferred from the figure. ( C ) Polygon fiber with NRL 210 bp at β(−1). ( D ) Stacking of nucleosomes at β(5) into two columns with alternating positive and negative linker orientations by reducing the size of α. The fiber in ( D ) has NRL = 230 bp and α = ∼36°. ( E and F ) Compressed polygon fibers formed by reducing the size of α. (E) β(+4) is shown from the end (top) and from the side as seen from two different angles. (F) β(−4) seen from the end (top) and from the side. ( G ) Axial symmetries of the polygon fiber as a function of β at η 2 = 10 and 0°. Bar = 30 nm.

    Article Snippet: In a fiber with β sequence (3033 )n , (3 ) swivel-linkers separate right-handed coils of four nucleosomes (•3•0•3•), providing sufficient space for the formation of a double helix in which swivel-linkers in one filament is alternately located on the inside and outside of swivel-linkers in the other filament ( B).

    Techniques:

    Polygon fiber of 65 nucleosomes (α = 90°) with a random β sequence (shown in Figure 8 ). ( A ) Relaxed fiber. ( B ) Stretched fiber. ( C ) Loop formation by interaction of nucleosome No. 1–5 with nucleosome No. 65. Stable loop clustering is indicated by brackets. ( D – F ) Nucleosome interactions in (A, B, C). Clusters of stable and flexible loops are shown in red and blue regions, respectively, and nucleosomes with relative stable and variable mutual positions are black and gray, respectively. ( G – I ) Compaction of nucleosome No. 1–20 (G), 1–40 (H) and 1–65 (I) after chromatin remodeling and twisting of the linkers to form β sequence (33 3 ) n , as shown in Figure 8 . Bar = 30 nm.

    Journal: Nucleic Acids Research

    Article Title: Choreography for nucleosomes: the conformational freedom of the nucleosomal filament and its limitations

    doi: 10.1093/nar/gkm560

    Figure Lengend Snippet: Polygon fiber of 65 nucleosomes (α = 90°) with a random β sequence (shown in Figure 8 ). ( A ) Relaxed fiber. ( B ) Stretched fiber. ( C ) Loop formation by interaction of nucleosome No. 1–5 with nucleosome No. 65. Stable loop clustering is indicated by brackets. ( D – F ) Nucleosome interactions in (A, B, C). Clusters of stable and flexible loops are shown in red and blue regions, respectively, and nucleosomes with relative stable and variable mutual positions are black and gray, respectively. ( G – I ) Compaction of nucleosome No. 1–20 (G), 1–40 (H) and 1–65 (I) after chromatin remodeling and twisting of the linkers to form β sequence (33 3 ) n , as shown in Figure 8 . Bar = 30 nm.

    Article Snippet: In a fiber with β sequence (3033 )n , (3 ) swivel-linkers separate right-handed coils of four nucleosomes (•3•0•3•), providing sufficient space for the formation of a double helix in which swivel-linkers in one filament is alternately located on the inside and outside of swivel-linkers in the other filament ( B).

    Techniques: Sequencing

    β sequences of the polygon fiber shown in Figure 7 . The original sequence was remodeled by moving nucleosomes the number of base pairs indicated in the directions shown by the arrows. The resulting β sequence is shown in the second row, the changed β values being indicated by a gray background. The third row shows the β sequence (33 3 ) n after twisting of the linkers within the twist constraints.

    Journal: Nucleic Acids Research

    Article Title: Choreography for nucleosomes: the conformational freedom of the nucleosomal filament and its limitations

    doi: 10.1093/nar/gkm560

    Figure Lengend Snippet: β sequences of the polygon fiber shown in Figure 7 . The original sequence was remodeled by moving nucleosomes the number of base pairs indicated in the directions shown by the arrows. The resulting β sequence is shown in the second row, the changed β values being indicated by a gray background. The third row shows the β sequence (33 3 ) n after twisting of the linkers within the twist constraints.

    Article Snippet: In a fiber with β sequence (3033 )n , (3 ) swivel-linkers separate right-handed coils of four nucleosomes (•3•0•3•), providing sufficient space for the formation of a double helix in which swivel-linkers in one filament is alternately located on the inside and outside of swivel-linkers in the other filament ( B).

    Techniques: Sequencing

    Loops of hexa- and octanucleosomes with different β combinations. ( A and B ) Hexanucleosomal loops formed by β = 0°, ±90° and 180° (0, +, −, 5). Arrows indicate the direction of the filament from nucleosome No. 1→6. The flat faces of the core particles nearest to the entering linkers are dark while the faces nearest to the exiting linkers are light. The line diagrams show the orientation of the five linkers in each loop. ( A1 – 3 ) Closed loops; ( B1 – 4 ) locked loops. ( C and D ) Hexanucleosomes; (2 3 1 3 1) (C); ( 4 1 4 1 4 ) (D). ( E ) A nucleosome is added to each terminal linker of the loop in (D) (asterisks). ( F and G ) Loops of six plus two nucleosomes as shown singly and in series of three. ( F ) A trimer, 1 (1 3 1 3 2)2, connected by β(2); ( G ) A trimer, 3( 33 1 23 )5, connected by β(5). Values of β are approximate. α = 90°. Bar = 30 nm (C–G).

    Journal: Nucleic Acids Research

    Article Title: Choreography for nucleosomes: the conformational freedom of the nucleosomal filament and its limitations

    doi: 10.1093/nar/gkm560

    Figure Lengend Snippet: Loops of hexa- and octanucleosomes with different β combinations. ( A and B ) Hexanucleosomal loops formed by β = 0°, ±90° and 180° (0, +, −, 5). Arrows indicate the direction of the filament from nucleosome No. 1→6. The flat faces of the core particles nearest to the entering linkers are dark while the faces nearest to the exiting linkers are light. The line diagrams show the orientation of the five linkers in each loop. ( A1 – 3 ) Closed loops; ( B1 – 4 ) locked loops. ( C and D ) Hexanucleosomes; (2 3 1 3 1) (C); ( 4 1 4 1 4 ) (D). ( E ) A nucleosome is added to each terminal linker of the loop in (D) (asterisks). ( F and G ) Loops of six plus two nucleosomes as shown singly and in series of three. ( F ) A trimer, 1 (1 3 1 3 2)2, connected by β(2); ( G ) A trimer, 3( 33 1 23 )5, connected by β(5). Values of β are approximate. α = 90°. Bar = 30 nm (C–G).

    Article Snippet: In a fiber with β sequence (3033 )n , (3 ) swivel-linkers separate right-handed coils of four nucleosomes (•3•0•3•), providing sufficient space for the formation of a double helix in which swivel-linkers in one filament is alternately located on the inside and outside of swivel-linkers in the other filament ( B).

    Techniques:

    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