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  • 99
    Millipore mononucleosomal dna
    Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal <t>DNA</t> upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. <t>Mononucleosomal</t> DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.
    Mononucleosomal Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Illumina Inc mononucleosomal dna
    Position-specific weight matrix of nucleosomal signatures. Heat map representation of the position-specific weight matrix (PSWM) for the indicated four species. The x -axis indicates positions relative to the nucleosomal dyad; the y -axis indicates the log-odd score of the 16 dinucleotides along <t>mononucleosomal</t> <t>DNA</t> calculated as the ratio of their frequency at each position relative to their genomic frequency. Bars on the right represent a color scale associated with the log-odd score values.
    Mononucleosomal Dna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 315 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    EpiCypher hela mononucleosomes
    UHRF1 and UHRF2 diverge in their ability to ubiquitinate nucleosomal histones. ( A ) Chromatin association assays for FLAG-tagged UHRF2 (WT) or the indicated mutants from asynchronously growing <t>HeLa</t> cells. Mock treatment represents an empty vector control. ( B ) Comparative in vitro ubiquitination of H3 (1–32) K9me2 (left) and HeLa <t>mononucleosomes</t> (right) by UHRF1 and UHRF2. Moles of H3 are equivalent in these reactions.
    Hela Mononucleosomes, supplied by EpiCypher, used in various techniques. Bioz Stars score: 90/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    EpiCypher mononucleosomes
    Characterizing lysine prioritization of UHRF1 ubiquitylation on <t>mononucleosomes.</t> ( A ) Ubiquitylation of Hela mononucleosomes after 2 hr in the presence of HeDNA or UnDNA. ( B ) Ratio and normalized ratio (HeDNA/UnDNA) for H3 ubiquitylated peptides from propionylated samples. Samples were normalized to the mean ratio of free ubiquitin peptides. Quantification was performed with Skyline and values for each peptide charge state were summed. ( C ) Ratio and normalized ratio (HeDNA/UnDNA) for H3 ubiquitylated peptides from propionylated and unreacted samples. A ubiquitylated peptide from the PHD (marked with an asterisk) wa s enriched in the HeDNA sample. ( D ) Location of UHRF1 auto-ubiquitylation sites lining the TTD-PHD. TTD, pink; PHD, blue; modified lysines, red. The C-terminal atom on the H3 peptide (S10) is shown as a sphere. ( E ) Auto-ubiquitylation of UHRF1 in the absence of H3 peptides with either HeDNA or UnDNA when run under conditions to resolve higher molecular weight species. DOI: http://dx.doi.org/10.7554/eLife.17101.015
    Mononucleosomes, supplied by EpiCypher, used in various techniques. Bioz Stars score: 90/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    EpiCypher recombinant mononucleosomes
    BRPF1-KAT6 complexes catalyze H3K23 propionylation in vitro. ( A ) Molecular architecture of the tetrameric complexes. BRPF1 has two EPC (enhancer of polycomb)–like motifs: EPC-I is required for association with the MYST domain of KAT6A or KAT6B, whereas EPC-II is necessary and sufficient for interaction with ING5 (or the paralog ING4) and MEAF6. The BRPF-specific N-terminal (BN) domain also contributes to the association with the MYST domain. BRPF1 contains the PZP domain, bromodomain, and PWWP domain for chromatin association. Unlike its paralogs BRPF2 and BRPF3, BRPF1 has an Sfp1-like C2H2 zinc finger (SZ). NLS, nuclear localization signal; H1-like, histone H1–like domain; PZP, PHD–zinc knuckle–PHD; bromo, bromodomain; PWWP, Pro-Trp-Trp-Pro containing domain; SM, serine/methionine-rich ( 8 , 23 ). ( B ) BRPF1 promotes H3K23 propionylation. KAT6A was expressed in HEK293 cells as a FLAG-tagged fusion protein along with HA-tagged BRPF1, ING5, and MEAF6 as indicated. Affinity-purified proteins were used for acylation of HeLa oligonucleosomes in the presence of the respective acyl-CoA. Immunoblotting with antibodies recognizing histone H3 and its acylated forms was used to detect acylation states as indicated. See fig. S2A for immunoblotting analysis of the soluble extracts. Signals detected by the anti-H3K23cr and anti-H3K23bu antibodies need to be interpreted with caution due to cross-reactivity to H3K23ac and/or H3K23pr (see fig. S1B). ( C ) The H3K23 propionyltransferase activity is intrinsic to the MYST domain of KAT6A. Complex preparation and assays were performed as in (B) to compare KAT6A with its mutants. Recombinant <t>mononucleosomes</t> were used as substrate. See fig. S2B for immunoblotting analysis of the soluble extracts. Asterisks in (B) and (C) denote degraded products; the degradation varies from experiment to experiment. ( D ) Same as in (B) but ING4 and ING5 were compared. See fig. S2C for immunoblotting analysis of the soluble extracts. ( E ) Comparison of KAT6B fragments. Complex preparation and assays were performed as in (B) with the recombinant mononucleosome substrate, but KAT6B fragments were analyzed. Full-length KAT6B was difficult to express ( 21 ).
    Recombinant Mononucleosomes, supplied by EpiCypher, used in various techniques. Bioz Stars score: 92/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    ATUM mononucleosomal dna
    DMC treatment inhibits the interaction of proteins with the <t>DNA.</t> ( A ) Nuclei were isolated from MCF7 cells and treated with MNase to obtain mononucleosome fragmented DNA (150 bp). <t>Mononucleosomal</t> DNA was extracted and labeled with [ 32 P]. DNA-binding activity of nuclear proteins of untreated, 10 μM MC or DMC treated for 4 hours was analyzed by EMSA. ( B ) The cell cycle profile was checked by Fluorescence activated cell sorting (FACS). MCF7 cells left untreated, or treated with 10 μM MC or DMC for 4 hours and fixed in 30% ethanol and cellular DNA were stained with propidium iodide. ( C ) Chromatin immunoprecipitation (ChIP) was carried out in untreated, 10 μM MC or DMC treated for 4 hours and UV (50J/m 2 ) irradiated MCF7 cells. ChIP was performed in 400 μg of cross-linked and sonicated cell lysates and antibody against ATR. Immunoprecipitated DNA was amplified by real-time quantitative PCR with primers for p53 intron 7 gene. Non-specific IgG was used to subtract the background. Values were normalized to IgG and inputs, followed by normalization to the untreated samples.
    Mononucleosomal Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 90/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Illumina Inc mononucleosome library
    ∆ Lk gain produced by the <t>mononucleosome</t> library. a DNA topology of five individual minichromosomes of the nucleosomal library. In each case, the length (bp) of the inserted mononucleosomal DNA fragment, the nucleosome ID, genic positioning and stability are indicated. 2D gel-blots show a marker of Lk topoisomers (lane M), the minichromosome DNA extracted from fixed cells (lane 1) and after its relaxation with topo I (lane 2). 2D schemes show the relative position of Lk topoisomers visible in lanes 1 (orange dots) and 2 (green dots). The intensity plots of the above topoisomers showing the mean of the Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value were obtained as in Fig. 1e . ∆∆ LK is the ∆ Lk gain produced relative to YCp1.3 (∆ Lk = −5.81). b 1D and 2D gel-blots show the DNA of the pool of minichromosomes extracted from the entire library (lane 1) and the pooled DNA after its relaxation with topoisomerase I (lane 2). The 2D gel includes a marker of individual Lk topoisomers (lane M). Nicked (N) and linear (L) molecules are indicated. c Top, Lk distributions corresponding to the minichromosomes carrying the nucleosome library (orange) and their DNAs after relaxation with topo I (green). The mean of the pooled Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value of −7.07 ± 0.02 (mean ± s.d.) from four replicate experiments were determined as detailed in Supplementary Fig. 4 . The position of a hypothetical Lk distribution with ∆ Lk of − 6.81, which would have implied that the nucleosome library produced a ∆∆ Lk of −1.0, is illustrated (dashed gray). Bottom, Lk distributions and ∆ Lk of YCp1.3 are shown at the same scale and denote that ∆∆ Lk = −1.26
    Mononucleosome Library, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    EpiCypher recombinant human mononucleosomes
    ∆ Lk gain produced by the <t>mononucleosome</t> library. a DNA topology of five individual minichromosomes of the nucleosomal library. In each case, the length (bp) of the inserted mononucleosomal DNA fragment, the nucleosome ID, genic positioning and stability are indicated. 2D gel-blots show a marker of Lk topoisomers (lane M), the minichromosome DNA extracted from fixed cells (lane 1) and after its relaxation with topo I (lane 2). 2D schemes show the relative position of Lk topoisomers visible in lanes 1 (orange dots) and 2 (green dots). The intensity plots of the above topoisomers showing the mean of the Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value were obtained as in Fig. 1e . ∆∆ LK is the ∆ Lk gain produced relative to YCp1.3 (∆ Lk = −5.81). b 1D and 2D gel-blots show the DNA of the pool of minichromosomes extracted from the entire library (lane 1) and the pooled DNA after its relaxation with topoisomerase I (lane 2). The 2D gel includes a marker of individual Lk topoisomers (lane M). Nicked (N) and linear (L) molecules are indicated. c Top, Lk distributions corresponding to the minichromosomes carrying the nucleosome library (orange) and their DNAs after relaxation with topo I (green). The mean of the pooled Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value of −7.07 ± 0.02 (mean ± s.d.) from four replicate experiments were determined as detailed in Supplementary Fig. 4 . The position of a hypothetical Lk distribution with ∆ Lk of − 6.81, which would have implied that the nucleosome library produced a ∆∆ Lk of −1.0, is illustrated (dashed gray). Bottom, Lk distributions and ∆ Lk of YCp1.3 are shown at the same scale and denote that ∆∆ Lk = −1.26
    Recombinant Human Mononucleosomes, supplied by EpiCypher, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    EpiCypher recombinant xenopus mononucleosomes
    ∆ Lk gain produced by the <t>mononucleosome</t> library. a DNA topology of five individual minichromosomes of the nucleosomal library. In each case, the length (bp) of the inserted mononucleosomal DNA fragment, the nucleosome ID, genic positioning and stability are indicated. 2D gel-blots show a marker of Lk topoisomers (lane M), the minichromosome DNA extracted from fixed cells (lane 1) and after its relaxation with topo I (lane 2). 2D schemes show the relative position of Lk topoisomers visible in lanes 1 (orange dots) and 2 (green dots). The intensity plots of the above topoisomers showing the mean of the Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value were obtained as in Fig. 1e . ∆∆ LK is the ∆ Lk gain produced relative to YCp1.3 (∆ Lk = −5.81). b 1D and 2D gel-blots show the DNA of the pool of minichromosomes extracted from the entire library (lane 1) and the pooled DNA after its relaxation with topoisomerase I (lane 2). The 2D gel includes a marker of individual Lk topoisomers (lane M). Nicked (N) and linear (L) molecules are indicated. c Top, Lk distributions corresponding to the minichromosomes carrying the nucleosome library (orange) and their DNAs after relaxation with topo I (green). The mean of the pooled Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value of −7.07 ± 0.02 (mean ± s.d.) from four replicate experiments were determined as detailed in Supplementary Fig. 4 . The position of a hypothetical Lk distribution with ∆ Lk of − 6.81, which would have implied that the nucleosome library produced a ∆∆ Lk of −1.0, is illustrated (dashed gray). Bottom, Lk distributions and ∆ Lk of YCp1.3 are shown at the same scale and denote that ∆∆ Lk = −1.26
    Recombinant Xenopus Mononucleosomes, supplied by EpiCypher, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    EpiCypher biotinylated mononucleosomes
    ∆ Lk gain produced by the <t>mononucleosome</t> library. a DNA topology of five individual minichromosomes of the nucleosomal library. In each case, the length (bp) of the inserted mononucleosomal DNA fragment, the nucleosome ID, genic positioning and stability are indicated. 2D gel-blots show a marker of Lk topoisomers (lane M), the minichromosome DNA extracted from fixed cells (lane 1) and after its relaxation with topo I (lane 2). 2D schemes show the relative position of Lk topoisomers visible in lanes 1 (orange dots) and 2 (green dots). The intensity plots of the above topoisomers showing the mean of the Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value were obtained as in Fig. 1e . ∆∆ LK is the ∆ Lk gain produced relative to YCp1.3 (∆ Lk = −5.81). b 1D and 2D gel-blots show the DNA of the pool of minichromosomes extracted from the entire library (lane 1) and the pooled DNA after its relaxation with topoisomerase I (lane 2). The 2D gel includes a marker of individual Lk topoisomers (lane M). Nicked (N) and linear (L) molecules are indicated. c Top, Lk distributions corresponding to the minichromosomes carrying the nucleosome library (orange) and their DNAs after relaxation with topo I (green). The mean of the pooled Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value of −7.07 ± 0.02 (mean ± s.d.) from four replicate experiments were determined as detailed in Supplementary Fig. 4 . The position of a hypothetical Lk distribution with ∆ Lk of − 6.81, which would have implied that the nucleosome library produced a ∆∆ Lk of −1.0, is illustrated (dashed gray). Bottom, Lk distributions and ∆ Lk of YCp1.3 are shown at the same scale and denote that ∆∆ Lk = −1.26
    Biotinylated Mononucleosomes, supplied by EpiCypher, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    EpiCypher cenp a mononucleosome
    ∆ Lk gain produced by the <t>mononucleosome</t> library. a DNA topology of five individual minichromosomes of the nucleosomal library. In each case, the length (bp) of the inserted mononucleosomal DNA fragment, the nucleosome ID, genic positioning and stability are indicated. 2D gel-blots show a marker of Lk topoisomers (lane M), the minichromosome DNA extracted from fixed cells (lane 1) and after its relaxation with topo I (lane 2). 2D schemes show the relative position of Lk topoisomers visible in lanes 1 (orange dots) and 2 (green dots). The intensity plots of the above topoisomers showing the mean of the Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value were obtained as in Fig. 1e . ∆∆ LK is the ∆ Lk gain produced relative to YCp1.3 (∆ Lk = −5.81). b 1D and 2D gel-blots show the DNA of the pool of minichromosomes extracted from the entire library (lane 1) and the pooled DNA after its relaxation with topoisomerase I (lane 2). The 2D gel includes a marker of individual Lk topoisomers (lane M). Nicked (N) and linear (L) molecules are indicated. c Top, Lk distributions corresponding to the minichromosomes carrying the nucleosome library (orange) and their DNAs after relaxation with topo I (green). The mean of the pooled Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value of −7.07 ± 0.02 (mean ± s.d.) from four replicate experiments were determined as detailed in Supplementary Fig. 4 . The position of a hypothetical Lk distribution with ∆ Lk of − 6.81, which would have implied that the nucleosome library produced a ∆∆ Lk of −1.0, is illustrated (dashed gray). Bottom, Lk distributions and ∆ Lk of YCp1.3 are shown at the same scale and denote that ∆∆ Lk = −1.26
    Cenp A Mononucleosome, supplied by EpiCypher, 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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    91
    EpiCypher human biotinylated mononucleosomes
    ∆ Lk gain produced by the <t>mononucleosome</t> library. a DNA topology of five individual minichromosomes of the nucleosomal library. In each case, the length (bp) of the inserted mononucleosomal DNA fragment, the nucleosome ID, genic positioning and stability are indicated. 2D gel-blots show a marker of Lk topoisomers (lane M), the minichromosome DNA extracted from fixed cells (lane 1) and after its relaxation with topo I (lane 2). 2D schemes show the relative position of Lk topoisomers visible in lanes 1 (orange dots) and 2 (green dots). The intensity plots of the above topoisomers showing the mean of the Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value were obtained as in Fig. 1e . ∆∆ LK is the ∆ Lk gain produced relative to YCp1.3 (∆ Lk = −5.81). b 1D and 2D gel-blots show the DNA of the pool of minichromosomes extracted from the entire library (lane 1) and the pooled DNA after its relaxation with topoisomerase I (lane 2). The 2D gel includes a marker of individual Lk topoisomers (lane M). Nicked (N) and linear (L) molecules are indicated. c Top, Lk distributions corresponding to the minichromosomes carrying the nucleosome library (orange) and their DNAs after relaxation with topo I (green). The mean of the pooled Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value of −7.07 ± 0.02 (mean ± s.d.) from four replicate experiments were determined as detailed in Supplementary Fig. 4 . The position of a hypothetical Lk distribution with ∆ Lk of − 6.81, which would have implied that the nucleosome library produced a ∆∆ Lk of −1.0, is illustrated (dashed gray). Bottom, Lk distributions and ∆ Lk of YCp1.3 are shown at the same scale and denote that ∆∆ Lk = −1.26
    Human Biotinylated Mononucleosomes, supplied by EpiCypher, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher dsdna mononucleosome assembly oligonucleotides
    ∆ Lk gain produced by the <t>mononucleosome</t> library. a DNA topology of five individual minichromosomes of the nucleosomal library. In each case, the length (bp) of the inserted mononucleosomal DNA fragment, the nucleosome ID, genic positioning and stability are indicated. 2D gel-blots show a marker of Lk topoisomers (lane M), the minichromosome DNA extracted from fixed cells (lane 1) and after its relaxation with topo I (lane 2). 2D schemes show the relative position of Lk topoisomers visible in lanes 1 (orange dots) and 2 (green dots). The intensity plots of the above topoisomers showing the mean of the Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value were obtained as in Fig. 1e . ∆∆ LK is the ∆ Lk gain produced relative to YCp1.3 (∆ Lk = −5.81). b 1D and 2D gel-blots show the DNA of the pool of minichromosomes extracted from the entire library (lane 1) and the pooled DNA after its relaxation with topoisomerase I (lane 2). The 2D gel includes a marker of individual Lk topoisomers (lane M). Nicked (N) and linear (L) molecules are indicated. c Top, Lk distributions corresponding to the minichromosomes carrying the nucleosome library (orange) and their DNAs after relaxation with topo I (green). The mean of the pooled Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value of −7.07 ± 0.02 (mean ± s.d.) from four replicate experiments were determined as detailed in Supplementary Fig. 4 . The position of a hypothetical Lk distribution with ∆ Lk of − 6.81, which would have implied that the nucleosome library produced a ∆∆ Lk of −1.0, is illustrated (dashed gray). Bottom, Lk distributions and ∆ Lk of YCp1.3 are shown at the same scale and denote that ∆∆ Lk = −1.26
    Dsdna Mononucleosome Assembly Oligonucleotides, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Illumina Inc mononucleosomal dna sequencing technique
    ∆ Lk gain produced by the <t>mononucleosome</t> library. a DNA topology of five individual minichromosomes of the nucleosomal library. In each case, the length (bp) of the inserted mononucleosomal DNA fragment, the nucleosome ID, genic positioning and stability are indicated. 2D gel-blots show a marker of Lk topoisomers (lane M), the minichromosome DNA extracted from fixed cells (lane 1) and after its relaxation with topo I (lane 2). 2D schemes show the relative position of Lk topoisomers visible in lanes 1 (orange dots) and 2 (green dots). The intensity plots of the above topoisomers showing the mean of the Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value were obtained as in Fig. 1e . ∆∆ LK is the ∆ Lk gain produced relative to YCp1.3 (∆ Lk = −5.81). b 1D and 2D gel-blots show the DNA of the pool of minichromosomes extracted from the entire library (lane 1) and the pooled DNA after its relaxation with topoisomerase I (lane 2). The 2D gel includes a marker of individual Lk topoisomers (lane M). Nicked (N) and linear (L) molecules are indicated. c Top, Lk distributions corresponding to the minichromosomes carrying the nucleosome library (orange) and their DNAs after relaxation with topo I (green). The mean of the pooled Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value of −7.07 ± 0.02 (mean ± s.d.) from four replicate experiments were determined as detailed in Supplementary Fig. 4 . The position of a hypothetical Lk distribution with ∆ Lk of − 6.81, which would have implied that the nucleosome library produced a ∆∆ Lk of −1.0, is illustrated (dashed gray). Bottom, Lk distributions and ∆ Lk of YCp1.3 are shown at the same scale and denote that ∆∆ Lk = −1.26
    Mononucleosomal Dna Sequencing Technique, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Illumina Inc mononucleosomal fragments preparation
    ∆ Lk gain produced by the <t>mononucleosome</t> library. a DNA topology of five individual minichromosomes of the nucleosomal library. In each case, the length (bp) of the inserted mononucleosomal DNA fragment, the nucleosome ID, genic positioning and stability are indicated. 2D gel-blots show a marker of Lk topoisomers (lane M), the minichromosome DNA extracted from fixed cells (lane 1) and after its relaxation with topo I (lane 2). 2D schemes show the relative position of Lk topoisomers visible in lanes 1 (orange dots) and 2 (green dots). The intensity plots of the above topoisomers showing the mean of the Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value were obtained as in Fig. 1e . ∆∆ LK is the ∆ Lk gain produced relative to YCp1.3 (∆ Lk = −5.81). b 1D and 2D gel-blots show the DNA of the pool of minichromosomes extracted from the entire library (lane 1) and the pooled DNA after its relaxation with topoisomerase I (lane 2). The 2D gel includes a marker of individual Lk topoisomers (lane M). Nicked (N) and linear (L) molecules are indicated. c Top, Lk distributions corresponding to the minichromosomes carrying the nucleosome library (orange) and their DNAs after relaxation with topo I (green). The mean of the pooled Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value of −7.07 ± 0.02 (mean ± s.d.) from four replicate experiments were determined as detailed in Supplementary Fig. 4 . The position of a hypothetical Lk distribution with ∆ Lk of − 6.81, which would have implied that the nucleosome library produced a ∆∆ Lk of −1.0, is illustrated (dashed gray). Bottom, Lk distributions and ∆ Lk of YCp1.3 are shown at the same scale and denote that ∆∆ Lk = −1.26
    Mononucleosomal Fragments Preparation, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc mononucleosomal illumina sequencing coverage plots
    ∆ Lk gain produced by the <t>mononucleosome</t> library. a DNA topology of five individual minichromosomes of the nucleosomal library. In each case, the length (bp) of the inserted mononucleosomal DNA fragment, the nucleosome ID, genic positioning and stability are indicated. 2D gel-blots show a marker of Lk topoisomers (lane M), the minichromosome DNA extracted from fixed cells (lane 1) and after its relaxation with topo I (lane 2). 2D schemes show the relative position of Lk topoisomers visible in lanes 1 (orange dots) and 2 (green dots). The intensity plots of the above topoisomers showing the mean of the Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value were obtained as in Fig. 1e . ∆∆ LK is the ∆ Lk gain produced relative to YCp1.3 (∆ Lk = −5.81). b 1D and 2D gel-blots show the DNA of the pool of minichromosomes extracted from the entire library (lane 1) and the pooled DNA after its relaxation with topoisomerase I (lane 2). The 2D gel includes a marker of individual Lk topoisomers (lane M). Nicked (N) and linear (L) molecules are indicated. c Top, Lk distributions corresponding to the minichromosomes carrying the nucleosome library (orange) and their DNAs after relaxation with topo I (green). The mean of the pooled Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value of −7.07 ± 0.02 (mean ± s.d.) from four replicate experiments were determined as detailed in Supplementary Fig. 4 . The position of a hypothetical Lk distribution with ∆ Lk of − 6.81, which would have implied that the nucleosome library produced a ∆∆ Lk of −1.0, is illustrated (dashed gray). Bottom, Lk distributions and ∆ Lk of YCp1.3 are shown at the same scale and denote that ∆∆ Lk = −1.26
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    Solexa mononucleosome associated dna
    ∆ Lk gain produced by the <t>mononucleosome</t> library. a DNA topology of five individual minichromosomes of the nucleosomal library. In each case, the length (bp) of the inserted mononucleosomal DNA fragment, the nucleosome ID, genic positioning and stability are indicated. 2D gel-blots show a marker of Lk topoisomers (lane M), the minichromosome DNA extracted from fixed cells (lane 1) and after its relaxation with topo I (lane 2). 2D schemes show the relative position of Lk topoisomers visible in lanes 1 (orange dots) and 2 (green dots). The intensity plots of the above topoisomers showing the mean of the Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value were obtained as in Fig. 1e . ∆∆ LK is the ∆ Lk gain produced relative to YCp1.3 (∆ Lk = −5.81). b 1D and 2D gel-blots show the DNA of the pool of minichromosomes extracted from the entire library (lane 1) and the pooled DNA after its relaxation with topoisomerase I (lane 2). The 2D gel includes a marker of individual Lk topoisomers (lane M). Nicked (N) and linear (L) molecules are indicated. c Top, Lk distributions corresponding to the minichromosomes carrying the nucleosome library (orange) and their DNAs after relaxation with topo I (green). The mean of the pooled Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value of −7.07 ± 0.02 (mean ± s.d.) from four replicate experiments were determined as detailed in Supplementary Fig. 4 . The position of a hypothetical Lk distribution with ∆ Lk of − 6.81, which would have implied that the nucleosome library produced a ∆∆ Lk of −1.0, is illustrated (dashed gray). Bottom, Lk distributions and ∆ Lk of YCp1.3 are shown at the same scale and denote that ∆∆ Lk = −1.26
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    Illumina Inc mononucleosome dna isolation
    ∆ Lk gain produced by the <t>mononucleosome</t> library. a DNA topology of five individual minichromosomes of the nucleosomal library. In each case, the length (bp) of the inserted mononucleosomal DNA fragment, the nucleosome ID, genic positioning and stability are indicated. 2D gel-blots show a marker of Lk topoisomers (lane M), the minichromosome DNA extracted from fixed cells (lane 1) and after its relaxation with topo I (lane 2). 2D schemes show the relative position of Lk topoisomers visible in lanes 1 (orange dots) and 2 (green dots). The intensity plots of the above topoisomers showing the mean of the Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value were obtained as in Fig. 1e . ∆∆ LK is the ∆ Lk gain produced relative to YCp1.3 (∆ Lk = −5.81). b 1D and 2D gel-blots show the DNA of the pool of minichromosomes extracted from the entire library (lane 1) and the pooled DNA after its relaxation with topoisomerase I (lane 2). The 2D gel includes a marker of individual Lk topoisomers (lane M). Nicked (N) and linear (L) molecules are indicated. c Top, Lk distributions corresponding to the minichromosomes carrying the nucleosome library (orange) and their DNAs after relaxation with topo I (green). The mean of the pooled Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value of −7.07 ± 0.02 (mean ± s.d.) from four replicate experiments were determined as detailed in Supplementary Fig. 4 . The position of a hypothetical Lk distribution with ∆ Lk of − 6.81, which would have implied that the nucleosome library produced a ∆∆ Lk of −1.0, is illustrated (dashed gray). Bottom, Lk distributions and ∆ Lk of YCp1.3 are shown at the same scale and denote that ∆∆ Lk = −1.26
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    Thermo Fisher mononucleosome fraction
    ∆ Lk gain produced by the <t>mononucleosome</t> library. a DNA topology of five individual minichromosomes of the nucleosomal library. In each case, the length (bp) of the inserted mononucleosomal DNA fragment, the nucleosome ID, genic positioning and stability are indicated. 2D gel-blots show a marker of Lk topoisomers (lane M), the minichromosome DNA extracted from fixed cells (lane 1) and after its relaxation with topo I (lane 2). 2D schemes show the relative position of Lk topoisomers visible in lanes 1 (orange dots) and 2 (green dots). The intensity plots of the above topoisomers showing the mean of the Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value were obtained as in Fig. 1e . ∆∆ LK is the ∆ Lk gain produced relative to YCp1.3 (∆ Lk = −5.81). b 1D and 2D gel-blots show the DNA of the pool of minichromosomes extracted from the entire library (lane 1) and the pooled DNA after its relaxation with topoisomerase I (lane 2). The 2D gel includes a marker of individual Lk topoisomers (lane M). Nicked (N) and linear (L) molecules are indicated. c Top, Lk distributions corresponding to the minichromosomes carrying the nucleosome library (orange) and their DNAs after relaxation with topo I (green). The mean of the pooled Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value of −7.07 ± 0.02 (mean ± s.d.) from four replicate experiments were determined as detailed in Supplementary Fig. 4 . The position of a hypothetical Lk distribution with ∆ Lk of − 6.81, which would have implied that the nucleosome library produced a ∆∆ Lk of −1.0, is illustrated (dashed gray). Bottom, Lk distributions and ∆ Lk of YCp1.3 are shown at the same scale and denote that ∆∆ Lk = −1.26
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    Promega mononucleosome sized fragments
    ∆ Lk gain produced by the <t>mononucleosome</t> library. a DNA topology of five individual minichromosomes of the nucleosomal library. In each case, the length (bp) of the inserted mononucleosomal DNA fragment, the nucleosome ID, genic positioning and stability are indicated. 2D gel-blots show a marker of Lk topoisomers (lane M), the minichromosome DNA extracted from fixed cells (lane 1) and after its relaxation with topo I (lane 2). 2D schemes show the relative position of Lk topoisomers visible in lanes 1 (orange dots) and 2 (green dots). The intensity plots of the above topoisomers showing the mean of the Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value were obtained as in Fig. 1e . ∆∆ LK is the ∆ Lk gain produced relative to YCp1.3 (∆ Lk = −5.81). b 1D and 2D gel-blots show the DNA of the pool of minichromosomes extracted from the entire library (lane 1) and the pooled DNA after its relaxation with topoisomerase I (lane 2). The 2D gel includes a marker of individual Lk topoisomers (lane M). Nicked (N) and linear (L) molecules are indicated. c Top, Lk distributions corresponding to the minichromosomes carrying the nucleosome library (orange) and their DNAs after relaxation with topo I (green). The mean of the pooled Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value of −7.07 ± 0.02 (mean ± s.d.) from four replicate experiments were determined as detailed in Supplementary Fig. 4 . The position of a hypothetical Lk distribution with ∆ Lk of − 6.81, which would have implied that the nucleosome library produced a ∆∆ Lk of −1.0, is illustrated (dashed gray). Bottom, Lk distributions and ∆ Lk of YCp1.3 are shown at the same scale and denote that ∆∆ Lk = −1.26
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    Helicos mononucleosomes
    ∆ Lk gain produced by the <t>mononucleosome</t> library. a DNA topology of five individual minichromosomes of the nucleosomal library. In each case, the length (bp) of the inserted mononucleosomal DNA fragment, the nucleosome ID, genic positioning and stability are indicated. 2D gel-blots show a marker of Lk topoisomers (lane M), the minichromosome DNA extracted from fixed cells (lane 1) and after its relaxation with topo I (lane 2). 2D schemes show the relative position of Lk topoisomers visible in lanes 1 (orange dots) and 2 (green dots). The intensity plots of the above topoisomers showing the mean of the Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value were obtained as in Fig. 1e . ∆∆ LK is the ∆ Lk gain produced relative to YCp1.3 (∆ Lk = −5.81). b 1D and 2D gel-blots show the DNA of the pool of minichromosomes extracted from the entire library (lane 1) and the pooled DNA after its relaxation with topoisomerase I (lane 2). The 2D gel includes a marker of individual Lk topoisomers (lane M). Nicked (N) and linear (L) molecules are indicated. c Top, Lk distributions corresponding to the minichromosomes carrying the nucleosome library (orange) and their DNAs after relaxation with topo I (green). The mean of the pooled Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value of −7.07 ± 0.02 (mean ± s.d.) from four replicate experiments were determined as detailed in Supplementary Fig. 4 . The position of a hypothetical Lk distribution with ∆ Lk of − 6.81, which would have implied that the nucleosome library produced a ∆∆ Lk of −1.0, is illustrated (dashed gray). Bottom, Lk distributions and ∆ Lk of YCp1.3 are shown at the same scale and denote that ∆∆ Lk = −1.26
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    Image Search Results


    Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.

    Journal: eLife

    Article Title: Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126

    doi: 10.7554/eLife.53362

    Figure Lengend Snippet: Nucleosome positioning at a (CAG) 85 repeat is not altered in the absence of H2A.1 or H2A.2. A ) Indirect end-labeling of nucleosomal DNA upstream of the CAG repeat. MNase (0, 0.25, 2.5, and 7.5 units) digested DNA was run in 1.5% agarose with ethidium bromide (left) and Southern blotted (right) using a probe ~100 bp proximal to the CAG repeat (red line Figure 1—figure supplement 1A ). Ovals represent nucleosome positions. The experiment was repeated six times; a representative blot is shown. ( B ) Illumina array mapping of nucleosome protection at the CAG repeat. Mononucleosomal DNA from strains containing the (CAG) 85 repeats was hybridized to a custom array of 30-mer probes spanning 425 bp upstream of the repeat to 436 bp downstream of the repeat in YAC CF1. Probes 14–16 contain CAG repeats; probe 15 is composed purely of CAG repeats (probe sequences in Supplementary file 3 ). Error bars represent standard deviation of 2–3 independent experiments.

    Article Snippet: Mononucleosome positioning detection by Illumina bead array Chromatin was isolated and MNase digested as described above, except mononucleosomes were prepared by digesting the chromatin with 10 units of MNase for 15 min. Purified mononucleosomal DNA was amplified using the GenomePlex Whole Genome Amplification kit (Sigma).

    Techniques: End Labeling, Standard Deviation

    Position-specific weight matrix of nucleosomal signatures. Heat map representation of the position-specific weight matrix (PSWM) for the indicated four species. The x -axis indicates positions relative to the nucleosomal dyad; the y -axis indicates the log-odd score of the 16 dinucleotides along mononucleosomal DNA calculated as the ratio of their frequency at each position relative to their genomic frequency. Bars on the right represent a color scale associated with the log-odd score values.

    Journal: Genome Research

    Article Title: Nucleosomal signatures impose nucleosome positioning in coding and noncoding sequences in the genome

    doi: 10.1101/gr.207241.116

    Figure Lengend Snippet: Position-specific weight matrix of nucleosomal signatures. Heat map representation of the position-specific weight matrix (PSWM) for the indicated four species. The x -axis indicates positions relative to the nucleosomal dyad; the y -axis indicates the log-odd score of the 16 dinucleotides along mononucleosomal DNA calculated as the ratio of their frequency at each position relative to their genomic frequency. Bars on the right represent a color scale associated with the log-odd score values.

    Article Snippet: Libraries of mononucleosomal DNA were constructed following the Illumina protocol and were sequenced in an Illumina NextSeq500 platform using the paired-end protocol.

    Techniques:

    UHRF1 and UHRF2 diverge in their ability to ubiquitinate nucleosomal histones. ( A ) Chromatin association assays for FLAG-tagged UHRF2 (WT) or the indicated mutants from asynchronously growing HeLa cells. Mock treatment represents an empty vector control. ( B ) Comparative in vitro ubiquitination of H3 (1–32) K9me2 (left) and HeLa mononucleosomes (right) by UHRF1 and UHRF2. Moles of H3 are equivalent in these reactions.

    Journal: Nucleic Acids Research

    Article Title: Comparative biochemical analysis of UHRF proteins reveals molecular mechanisms that uncouple UHRF2 from DNA methylation maintenance

    doi: 10.1093/nar/gky151

    Figure Lengend Snippet: UHRF1 and UHRF2 diverge in their ability to ubiquitinate nucleosomal histones. ( A ) Chromatin association assays for FLAG-tagged UHRF2 (WT) or the indicated mutants from asynchronously growing HeLa cells. Mock treatment represents an empty vector control. ( B ) Comparative in vitro ubiquitination of H3 (1–32) K9me2 (left) and HeLa mononucleosomes (right) by UHRF1 and UHRF2. Moles of H3 are equivalent in these reactions.

    Article Snippet: For in vitro ubiquitination of HeLa mononucleosomes, reactions were run in ubiquitin assay buffer containing 1.5 μM E3, 50 nM E1 UBA1, 667 nM E2 UBCH5A, 5 μM TAMRA-ubiquitin, and 0.5 μM HeLa mononucleosomes (Epicypher).

    Techniques: Plasmid Preparation, In Vitro

    Characterizing lysine prioritization of UHRF1 ubiquitylation on mononucleosomes. ( A ) Ubiquitylation of Hela mononucleosomes after 2 hr in the presence of HeDNA or UnDNA. ( B ) Ratio and normalized ratio (HeDNA/UnDNA) for H3 ubiquitylated peptides from propionylated samples. Samples were normalized to the mean ratio of free ubiquitin peptides. Quantification was performed with Skyline and values for each peptide charge state were summed. ( C ) Ratio and normalized ratio (HeDNA/UnDNA) for H3 ubiquitylated peptides from propionylated and unreacted samples. A ubiquitylated peptide from the PHD (marked with an asterisk) wa s enriched in the HeDNA sample. ( D ) Location of UHRF1 auto-ubiquitylation sites lining the TTD-PHD. TTD, pink; PHD, blue; modified lysines, red. The C-terminal atom on the H3 peptide (S10) is shown as a sphere. ( E ) Auto-ubiquitylation of UHRF1 in the absence of H3 peptides with either HeDNA or UnDNA when run under conditions to resolve higher molecular weight species. DOI: http://dx.doi.org/10.7554/eLife.17101.015

    Journal: eLife

    Article Title: Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1

    doi: 10.7554/eLife.17101

    Figure Lengend Snippet: Characterizing lysine prioritization of UHRF1 ubiquitylation on mononucleosomes. ( A ) Ubiquitylation of Hela mononucleosomes after 2 hr in the presence of HeDNA or UnDNA. ( B ) Ratio and normalized ratio (HeDNA/UnDNA) for H3 ubiquitylated peptides from propionylated samples. Samples were normalized to the mean ratio of free ubiquitin peptides. Quantification was performed with Skyline and values for each peptide charge state were summed. ( C ) Ratio and normalized ratio (HeDNA/UnDNA) for H3 ubiquitylated peptides from propionylated and unreacted samples. A ubiquitylated peptide from the PHD (marked with an asterisk) wa s enriched in the HeDNA sample. ( D ) Location of UHRF1 auto-ubiquitylation sites lining the TTD-PHD. TTD, pink; PHD, blue; modified lysines, red. The C-terminal atom on the H3 peptide (S10) is shown as a sphere. ( E ) Auto-ubiquitylation of UHRF1 in the absence of H3 peptides with either HeDNA or UnDNA when run under conditions to resolve higher molecular weight species. DOI: http://dx.doi.org/10.7554/eLife.17101.015

    Article Snippet: Recombinant histone proteins and mononucleosomes were obtained commercially from Epicypher (H2A, #15–0301; H2B, 15–0302; and H3.1, #15–0303; mononucleosomes, #16–0002; Research Triangle Park, NC).

    Techniques: Modification, Molecular Weight

    UHRF1 ubiquitin ligase assays. ( A ) Time course assay of UHRF1 auto-ubiquitylation in the presence of UnDNA or HeDNA in the absence of a histone peptide substrate (left). Rates of UHRF1 auto-ubiquitylation (right) were quantified from blots using ImageQuant TL (GE Lifesciences). Quantified data was best described by a linear fit over the measured time scale. ( B ) UHRF1 ubiquitylation of HeLa mononucleosomes in the presence or absence of HeDNA reveals that mononucleosome ubiquitylation is significantly enhanced in the presence of HeDNA. ( C ) Ubiquitylation of H3 1-32 K9me2 by MBP-tagged UHRF1 and the indicated mutants. Mutant UHRF1 proteins display significant defects in HeDNA stimulated ubiquitylation activity with the exception of the D469G. ( D ) FP binding assays quantifying the interaction of the MBP-UHRF1 D469G with FAM-labeled HeDNA, SyDNA, or UnDNA. Error is represented as ± s.e.m. for two independent experiments. While the literature suggests a HeDNA binding defect for the D469G mutation, these assays reveal this mutant binds DNA similarly to wild-type (for comparison see Figure 1B ). DOI: http://dx.doi.org/10.7554/eLife.17101.010

    Journal: eLife

    Article Title: Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1

    doi: 10.7554/eLife.17101

    Figure Lengend Snippet: UHRF1 ubiquitin ligase assays. ( A ) Time course assay of UHRF1 auto-ubiquitylation in the presence of UnDNA or HeDNA in the absence of a histone peptide substrate (left). Rates of UHRF1 auto-ubiquitylation (right) were quantified from blots using ImageQuant TL (GE Lifesciences). Quantified data was best described by a linear fit over the measured time scale. ( B ) UHRF1 ubiquitylation of HeLa mononucleosomes in the presence or absence of HeDNA reveals that mononucleosome ubiquitylation is significantly enhanced in the presence of HeDNA. ( C ) Ubiquitylation of H3 1-32 K9me2 by MBP-tagged UHRF1 and the indicated mutants. Mutant UHRF1 proteins display significant defects in HeDNA stimulated ubiquitylation activity with the exception of the D469G. ( D ) FP binding assays quantifying the interaction of the MBP-UHRF1 D469G with FAM-labeled HeDNA, SyDNA, or UnDNA. Error is represented as ± s.e.m. for two independent experiments. While the literature suggests a HeDNA binding defect for the D469G mutation, these assays reveal this mutant binds DNA similarly to wild-type (for comparison see Figure 1B ). DOI: http://dx.doi.org/10.7554/eLife.17101.010

    Article Snippet: Recombinant histone proteins and mononucleosomes were obtained commercially from Epicypher (H2A, #15–0301; H2B, 15–0302; and H3.1, #15–0303; mononucleosomes, #16–0002; Research Triangle Park, NC).

    Techniques: Mutagenesis, Activity Assay, Binding Assay, Labeling

    BRPF1-KAT6 complexes catalyze H3K23 propionylation in vitro. ( A ) Molecular architecture of the tetrameric complexes. BRPF1 has two EPC (enhancer of polycomb)–like motifs: EPC-I is required for association with the MYST domain of KAT6A or KAT6B, whereas EPC-II is necessary and sufficient for interaction with ING5 (or the paralog ING4) and MEAF6. The BRPF-specific N-terminal (BN) domain also contributes to the association with the MYST domain. BRPF1 contains the PZP domain, bromodomain, and PWWP domain for chromatin association. Unlike its paralogs BRPF2 and BRPF3, BRPF1 has an Sfp1-like C2H2 zinc finger (SZ). NLS, nuclear localization signal; H1-like, histone H1–like domain; PZP, PHD–zinc knuckle–PHD; bromo, bromodomain; PWWP, Pro-Trp-Trp-Pro containing domain; SM, serine/methionine-rich ( 8 , 23 ). ( B ) BRPF1 promotes H3K23 propionylation. KAT6A was expressed in HEK293 cells as a FLAG-tagged fusion protein along with HA-tagged BRPF1, ING5, and MEAF6 as indicated. Affinity-purified proteins were used for acylation of HeLa oligonucleosomes in the presence of the respective acyl-CoA. Immunoblotting with antibodies recognizing histone H3 and its acylated forms was used to detect acylation states as indicated. See fig. S2A for immunoblotting analysis of the soluble extracts. Signals detected by the anti-H3K23cr and anti-H3K23bu antibodies need to be interpreted with caution due to cross-reactivity to H3K23ac and/or H3K23pr (see fig. S1B). ( C ) The H3K23 propionyltransferase activity is intrinsic to the MYST domain of KAT6A. Complex preparation and assays were performed as in (B) to compare KAT6A with its mutants. Recombinant mononucleosomes were used as substrate. See fig. S2B for immunoblotting analysis of the soluble extracts. Asterisks in (B) and (C) denote degraded products; the degradation varies from experiment to experiment. ( D ) Same as in (B) but ING4 and ING5 were compared. See fig. S2C for immunoblotting analysis of the soluble extracts. ( E ) Comparison of KAT6B fragments. Complex preparation and assays were performed as in (B) with the recombinant mononucleosome substrate, but KAT6B fragments were analyzed. Full-length KAT6B was difficult to express ( 21 ).

    Journal: Science Advances

    Article Title: Deficient histone H3 propionylation by BRPF1-KAT6 complexes in neurodevelopmental disorders and cancer

    doi: 10.1126/sciadv.aax0021

    Figure Lengend Snippet: BRPF1-KAT6 complexes catalyze H3K23 propionylation in vitro. ( A ) Molecular architecture of the tetrameric complexes. BRPF1 has two EPC (enhancer of polycomb)–like motifs: EPC-I is required for association with the MYST domain of KAT6A or KAT6B, whereas EPC-II is necessary and sufficient for interaction with ING5 (or the paralog ING4) and MEAF6. The BRPF-specific N-terminal (BN) domain also contributes to the association with the MYST domain. BRPF1 contains the PZP domain, bromodomain, and PWWP domain for chromatin association. Unlike its paralogs BRPF2 and BRPF3, BRPF1 has an Sfp1-like C2H2 zinc finger (SZ). NLS, nuclear localization signal; H1-like, histone H1–like domain; PZP, PHD–zinc knuckle–PHD; bromo, bromodomain; PWWP, Pro-Trp-Trp-Pro containing domain; SM, serine/methionine-rich ( 8 , 23 ). ( B ) BRPF1 promotes H3K23 propionylation. KAT6A was expressed in HEK293 cells as a FLAG-tagged fusion protein along with HA-tagged BRPF1, ING5, and MEAF6 as indicated. Affinity-purified proteins were used for acylation of HeLa oligonucleosomes in the presence of the respective acyl-CoA. Immunoblotting with antibodies recognizing histone H3 and its acylated forms was used to detect acylation states as indicated. See fig. S2A for immunoblotting analysis of the soluble extracts. Signals detected by the anti-H3K23cr and anti-H3K23bu antibodies need to be interpreted with caution due to cross-reactivity to H3K23ac and/or H3K23pr (see fig. S1B). ( C ) The H3K23 propionyltransferase activity is intrinsic to the MYST domain of KAT6A. Complex preparation and assays were performed as in (B) to compare KAT6A with its mutants. Recombinant mononucleosomes were used as substrate. See fig. S2B for immunoblotting analysis of the soluble extracts. Asterisks in (B) and (C) denote degraded products; the degradation varies from experiment to experiment. ( D ) Same as in (B) but ING4 and ING5 were compared. See fig. S2C for immunoblotting analysis of the soluble extracts. ( E ) Comparison of KAT6B fragments. Complex preparation and assays were performed as in (B) with the recombinant mononucleosome substrate, but KAT6B fragments were analyzed. Full-length KAT6B was difficult to express ( 21 ).

    Article Snippet: HeLa oligonucleosomes were purified as previously described , where recombinant mononucleosomes were purchased from EpiCypher (16-0009).

    Techniques: In Vitro, Affinity Purification, Activity Assay, Recombinant

    Functional impact of BRPF1 variants associated with neurodevelopmental disorders. ( A ) Cartoon representation of variants previously identified in 29 individuals with syndromic intellectual disability ( 12 , 33 – 36 ) or autism ( 43 ). See Fig. 1A for domain nomenclature. ( B ) Nucleosomal acylation assays. HeLa oligonucleosomes were used for acylation by the affinity-purified wild-type and mutant complexes. The complexes were prepared for assays as in Fig. 1B . See fig. S2D for immunoblotting analysis of the soluble extracts. ( C ) Nucleosomal histone acylation assays. Recombinant mononucleosomes were used for acylation by the affinity-purified wild-type and mutant complexes. ( D ) Assays were carried out similarly as in (C), and the MYST domain of KAT6A was used. See fig. S2E for immunoblotting analysis of the soluble extracts. ( E ) Immunoblotting to detect histone H3 acylation in extracts from control and the Pro370Ser LCLs. ( F ) Immunoblotting to detect histone H3 acylation in protein extracts from the control and Arg455* LCLs. ( G ) Immunoblotting to detect histone H3 acylation in extracts from the control and Arg455* fibroblasts. ( H ) Assays were carried out similarly as in (C), but the Tyr406His variant was analyzed. See fig. S2F for immunoblotting analysis of the soluble extracts.

    Journal: Science Advances

    Article Title: Deficient histone H3 propionylation by BRPF1-KAT6 complexes in neurodevelopmental disorders and cancer

    doi: 10.1126/sciadv.aax0021

    Figure Lengend Snippet: Functional impact of BRPF1 variants associated with neurodevelopmental disorders. ( A ) Cartoon representation of variants previously identified in 29 individuals with syndromic intellectual disability ( 12 , 33 – 36 ) or autism ( 43 ). See Fig. 1A for domain nomenclature. ( B ) Nucleosomal acylation assays. HeLa oligonucleosomes were used for acylation by the affinity-purified wild-type and mutant complexes. The complexes were prepared for assays as in Fig. 1B . See fig. S2D for immunoblotting analysis of the soluble extracts. ( C ) Nucleosomal histone acylation assays. Recombinant mononucleosomes were used for acylation by the affinity-purified wild-type and mutant complexes. ( D ) Assays were carried out similarly as in (C), and the MYST domain of KAT6A was used. See fig. S2E for immunoblotting analysis of the soluble extracts. ( E ) Immunoblotting to detect histone H3 acylation in extracts from control and the Pro370Ser LCLs. ( F ) Immunoblotting to detect histone H3 acylation in protein extracts from the control and Arg455* LCLs. ( G ) Immunoblotting to detect histone H3 acylation in extracts from the control and Arg455* fibroblasts. ( H ) Assays were carried out similarly as in (C), but the Tyr406His variant was analyzed. See fig. S2F for immunoblotting analysis of the soluble extracts.

    Article Snippet: HeLa oligonucleosomes were purified as previously described , where recombinant mononucleosomes were purchased from EpiCypher (16-0009).

    Techniques: Functional Assay, Affinity Purification, Mutagenesis, Recombinant, Variant Assay

    Functional impact of somatic BRPF1 mutations from cancer. ( A ) Cartoon representation of somatic mutants identified in cancer. While five mutations are missense and produce Pro20Leu, Glu253Gly, Leu298Pro, Trp348Arg, and Glu369Asp, the rest are nonsense or cause reading frameshift, resulting in C-terminal truncation. According to the published report on other truncation mutations of BRPF1 ( 12 ), these nonsense or frameshift mutations may not trigger nonsense-mediated mRNA decay. See Fig. 1A for domain nomenclature. ( B ) Sequence similarity among the C2H2 zinc fingers of BRPF1, the fly ortholog Br140, the yeast stress- and nutrient-sensing transcription factor Sfp1, the fly Sfp1-like protein CG12054, and the related human transcription factors JAZF1 and ZF609. The SZ of BRPF1 is similar to the first of two zinc fingers that Sfp1 has for DNA binding and homologous to the middle of three zinc fingers that JAZF1 uses to recognize DNA. The three key residues involved in known and potential DNA binding are highlighted in bold. ( C ) Nucleosomal acylation assays. Recombinant mononucleosomes were used for acylation by the affinity-purified wild-type and mutant complexes as in Fig. 1C . Acylation of histone H3 was detected with antibodies recognizing histone H3 and its acylated forms as indicated. See fig. S2G (lanes 1 to 3) for immunoblotting analysis of the soluble extracts. ( D ) Subcellular localization of wild-type BRPF1 and its N-terminal mutants 1-51 and 1-71. Mutant 1-51 contains the SZ, and mutant 1-71 has this finger along with adjacent NLS1. Expression plasmids for BRPF1 or its mutants were transiently expressed in HEK293 cells as green fluorescent protein (GFP) fusion proteins with or without coexpression of FLAG- or HA-tagged KAT6A, ING5, and MEAF6 as indicated. Live green fluorescence images were taken. Scale bar, 50 μm. ( E and F ) Same as (C) except different variants were compared to wild-type BRPF1. See fig. S2G (lane 4) for immunoblotting analysis of extracts from HEK293 cells expressing Glu253Gly.

    Journal: Science Advances

    Article Title: Deficient histone H3 propionylation by BRPF1-KAT6 complexes in neurodevelopmental disorders and cancer

    doi: 10.1126/sciadv.aax0021

    Figure Lengend Snippet: Functional impact of somatic BRPF1 mutations from cancer. ( A ) Cartoon representation of somatic mutants identified in cancer. While five mutations are missense and produce Pro20Leu, Glu253Gly, Leu298Pro, Trp348Arg, and Glu369Asp, the rest are nonsense or cause reading frameshift, resulting in C-terminal truncation. According to the published report on other truncation mutations of BRPF1 ( 12 ), these nonsense or frameshift mutations may not trigger nonsense-mediated mRNA decay. See Fig. 1A for domain nomenclature. ( B ) Sequence similarity among the C2H2 zinc fingers of BRPF1, the fly ortholog Br140, the yeast stress- and nutrient-sensing transcription factor Sfp1, the fly Sfp1-like protein CG12054, and the related human transcription factors JAZF1 and ZF609. The SZ of BRPF1 is similar to the first of two zinc fingers that Sfp1 has for DNA binding and homologous to the middle of three zinc fingers that JAZF1 uses to recognize DNA. The three key residues involved in known and potential DNA binding are highlighted in bold. ( C ) Nucleosomal acylation assays. Recombinant mononucleosomes were used for acylation by the affinity-purified wild-type and mutant complexes as in Fig. 1C . Acylation of histone H3 was detected with antibodies recognizing histone H3 and its acylated forms as indicated. See fig. S2G (lanes 1 to 3) for immunoblotting analysis of the soluble extracts. ( D ) Subcellular localization of wild-type BRPF1 and its N-terminal mutants 1-51 and 1-71. Mutant 1-51 contains the SZ, and mutant 1-71 has this finger along with adjacent NLS1. Expression plasmids for BRPF1 or its mutants were transiently expressed in HEK293 cells as green fluorescent protein (GFP) fusion proteins with or without coexpression of FLAG- or HA-tagged KAT6A, ING5, and MEAF6 as indicated. Live green fluorescence images were taken. Scale bar, 50 μm. ( E and F ) Same as (C) except different variants were compared to wild-type BRPF1. See fig. S2G (lane 4) for immunoblotting analysis of extracts from HEK293 cells expressing Glu253Gly.

    Article Snippet: HeLa oligonucleosomes were purified as previously described , where recombinant mononucleosomes were purchased from EpiCypher (16-0009).

    Techniques: Functional Assay, Sequencing, Zinc-Fingers, Binding Assay, Recombinant, Affinity Purification, Mutagenesis, Expressing, Fluorescence

    DMC treatment inhibits the interaction of proteins with the DNA. ( A ) Nuclei were isolated from MCF7 cells and treated with MNase to obtain mononucleosome fragmented DNA (150 bp). Mononucleosomal DNA was extracted and labeled with [ 32 P]. DNA-binding activity of nuclear proteins of untreated, 10 μM MC or DMC treated for 4 hours was analyzed by EMSA. ( B ) The cell cycle profile was checked by Fluorescence activated cell sorting (FACS). MCF7 cells left untreated, or treated with 10 μM MC or DMC for 4 hours and fixed in 30% ethanol and cellular DNA were stained with propidium iodide. ( C ) Chromatin immunoprecipitation (ChIP) was carried out in untreated, 10 μM MC or DMC treated for 4 hours and UV (50J/m 2 ) irradiated MCF7 cells. ChIP was performed in 400 μg of cross-linked and sonicated cell lysates and antibody against ATR. Immunoprecipitated DNA was amplified by real-time quantitative PCR with primers for p53 intron 7 gene. Non-specific IgG was used to subtract the background. Values were normalized to IgG and inputs, followed by normalization to the untreated samples.

    Journal: Cell Cycle

    Article Title: Decarbamoyl mitomycin C (DMC) activates p53-independent ataxia telangiectasia and rad3 related protein (ATR) chromatin eviction

    doi: 10.1080/15384101.2014.997517

    Figure Lengend Snippet: DMC treatment inhibits the interaction of proteins with the DNA. ( A ) Nuclei were isolated from MCF7 cells and treated with MNase to obtain mononucleosome fragmented DNA (150 bp). Mononucleosomal DNA was extracted and labeled with [ 32 P]. DNA-binding activity of nuclear proteins of untreated, 10 μM MC or DMC treated for 4 hours was analyzed by EMSA. ( B ) The cell cycle profile was checked by Fluorescence activated cell sorting (FACS). MCF7 cells left untreated, or treated with 10 μM MC or DMC for 4 hours and fixed in 30% ethanol and cellular DNA were stained with propidium iodide. ( C ) Chromatin immunoprecipitation (ChIP) was carried out in untreated, 10 μM MC or DMC treated for 4 hours and UV (50J/m 2 ) irradiated MCF7 cells. ChIP was performed in 400 μg of cross-linked and sonicated cell lysates and antibody against ATR. Immunoprecipitated DNA was amplified by real-time quantitative PCR with primers for p53 intron 7 gene. Non-specific IgG was used to subtract the background. Values were normalized to IgG and inputs, followed by normalization to the untreated samples.

    Article Snippet: Reaction mixtures for EMSA experiments (30 μl) were composed of 0.1 pmol of mononucleosomal DNA, 20 mM HEPES (pH 7.8), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 1 μg of sonicated salmon sperm DNA, and 10% glycerol.

    Techniques: Isolation, Labeling, Binding Assay, Activity Assay, Fluorescence, FACS, Staining, Chromatin Immunoprecipitation, Irradiation, Sonication, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

    ∆ Lk gain produced by the mononucleosome library. a DNA topology of five individual minichromosomes of the nucleosomal library. In each case, the length (bp) of the inserted mononucleosomal DNA fragment, the nucleosome ID, genic positioning and stability are indicated. 2D gel-blots show a marker of Lk topoisomers (lane M), the minichromosome DNA extracted from fixed cells (lane 1) and after its relaxation with topo I (lane 2). 2D schemes show the relative position of Lk topoisomers visible in lanes 1 (orange dots) and 2 (green dots). The intensity plots of the above topoisomers showing the mean of the Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value were obtained as in Fig. 1e . ∆∆ LK is the ∆ Lk gain produced relative to YCp1.3 (∆ Lk = −5.81). b 1D and 2D gel-blots show the DNA of the pool of minichromosomes extracted from the entire library (lane 1) and the pooled DNA after its relaxation with topoisomerase I (lane 2). The 2D gel includes a marker of individual Lk topoisomers (lane M). Nicked (N) and linear (L) molecules are indicated. c Top, Lk distributions corresponding to the minichromosomes carrying the nucleosome library (orange) and their DNAs after relaxation with topo I (green). The mean of the pooled Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value of −7.07 ± 0.02 (mean ± s.d.) from four replicate experiments were determined as detailed in Supplementary Fig. 4 . The position of a hypothetical Lk distribution with ∆ Lk of − 6.81, which would have implied that the nucleosome library produced a ∆∆ Lk of −1.0, is illustrated (dashed gray). Bottom, Lk distributions and ∆ Lk of YCp1.3 are shown at the same scale and denote that ∆∆ Lk = −1.26

    Journal: Nature Communications

    Article Title: Intracellular nucleosomes constrain a DNA linking number difference of −1.26 that reconciles the Lk paradox

    doi: 10.1038/s41467-018-06547-w

    Figure Lengend Snippet: ∆ Lk gain produced by the mononucleosome library. a DNA topology of five individual minichromosomes of the nucleosomal library. In each case, the length (bp) of the inserted mononucleosomal DNA fragment, the nucleosome ID, genic positioning and stability are indicated. 2D gel-blots show a marker of Lk topoisomers (lane M), the minichromosome DNA extracted from fixed cells (lane 1) and after its relaxation with topo I (lane 2). 2D schemes show the relative position of Lk topoisomers visible in lanes 1 (orange dots) and 2 (green dots). The intensity plots of the above topoisomers showing the mean of the Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value were obtained as in Fig. 1e . ∆∆ LK is the ∆ Lk gain produced relative to YCp1.3 (∆ Lk = −5.81). b 1D and 2D gel-blots show the DNA of the pool of minichromosomes extracted from the entire library (lane 1) and the pooled DNA after its relaxation with topoisomerase I (lane 2). The 2D gel includes a marker of individual Lk topoisomers (lane M). Nicked (N) and linear (L) molecules are indicated. c Top, Lk distributions corresponding to the minichromosomes carrying the nucleosome library (orange) and their DNAs after relaxation with topo I (green). The mean of the pooled Lk distributions ( Lk 0 and Lk CHR ) and the resulting ∆ Lk value of −7.07 ± 0.02 (mean ± s.d.) from four replicate experiments were determined as detailed in Supplementary Fig. 4 . The position of a hypothetical Lk distribution with ∆ Lk of − 6.81, which would have implied that the nucleosome library produced a ∆∆ Lk of −1.0, is illustrated (dashed gray). Bottom, Lk distributions and ∆ Lk of YCp1.3 are shown at the same scale and denote that ∆∆ Lk = −1.26

    Article Snippet: Electrophoresis of Lk topoisomers DNA from YCp1.3 (1341 bp) and from minichromosomes containing the mononucleosome library (about 1.57 kb) were loaded onto 1.4% (w/v) agarose gels.

    Techniques: Produced, Two-Dimensional Gel Electrophoresis, Marker

    Number of binding sites identified in treatment and control sample using different sets of parameters . (A - C) Box-plots show the ratios of predicted binding site counts in treatment and control achieved with different parameter choices. Lower values suggest higher specificity. (D) The number of predicted nucleosomes in the control sample is plotted versus the number of nucleosomes identified from the mononucleosome sample. Different colours correspond to different background rate cut-offs while different symbols indicate different cut-off levels for support region rates.

    Journal: BMC Bioinformatics

    Article Title: ChIPseqR: analysis of ChIP-seq experiments

    doi: 10.1186/1471-2105-12-39

    Figure Lengend Snippet: Number of binding sites identified in treatment and control sample using different sets of parameters . (A - C) Box-plots show the ratios of predicted binding site counts in treatment and control achieved with different parameter choices. Lower values suggest higher specificity. (D) The number of predicted nucleosomes in the control sample is plotted versus the number of nucleosomes identified from the mononucleosome sample. Different colours correspond to different background rate cut-offs while different symbols indicate different cut-off levels for support region rates.

    Article Snippet: The purified mononucleosome cores were then used to generate a library for Illumina GS sequencing (carried out by Geneworks, Adelaide, Australia) or used for qPCR quantification.

    Techniques: Binding Assay