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  • 95
    New England Biolabs micrococcal nuclease mnase digestion digestion
    AGO2 knock-down affects nucleosome occupancy at TSSs bound by SWI/SNF. ( a ) HeLaS3 cells were transfected with a control siRNA (siCTRL) or a pool of AGO2 siRNA (siAGO2). Down-regulation of AGO2 protein was verified by western blot. GAPDH was used as loading control. ( b ) Chromatin from siCTRL- or siAGO2-treated HeLaS3 cells was digested by <t>MNase</t> and recovered <t>DNA</t> fragments were sequenced. Nucleosome occupancy profile for siCTRL and siAGO2 cells was plotted for TSSs with at least 30 swiRNAs (siCTRL, black line; siAGO2, green line). The occupancy at the nucleosome +1 (arrow) is reduced in AGO2 knock-down cells. ( c ) Bars height represents percent reduction of nucleosome occupancy (siAGO2 versus siCTRL) at TSS ±150 nt overlapped by at least the indicated number of swiRNAs (green), IgG-IP ‘other sRNAs’ (black) and AGO1-associated ‘other sRNAs’ (purple). ** P value
    Micrococcal Nuclease Mnase Digestion Digestion, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore staphylococcus aureus mnase
    AGO2 knock-down affects nucleosome occupancy at TSSs bound by SWI/SNF. ( a ) HeLaS3 cells were transfected with a control siRNA (siCTRL) or a pool of AGO2 siRNA (siAGO2). Down-regulation of AGO2 protein was verified by western blot. GAPDH was used as loading control. ( b ) Chromatin from siCTRL- or siAGO2-treated HeLaS3 cells was digested by <t>MNase</t> and recovered <t>DNA</t> fragments were sequenced. Nucleosome occupancy profile for siCTRL and siAGO2 cells was plotted for TSSs with at least 30 swiRNAs (siCTRL, black line; siAGO2, green line). The occupancy at the nucleosome +1 (arrow) is reduced in AGO2 knock-down cells. ( c ) Bars height represents percent reduction of nucleosome occupancy (siAGO2 versus siCTRL) at TSS ±150 nt overlapped by at least the indicated number of swiRNAs (green), IgG-IP ‘other sRNAs’ (black) and AGO1-associated ‘other sRNAs’ (purple). ** P value
    Staphylococcus Aureus Mnase, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Millipore microccocal nuclease
    Structure and stability of H3.X- and H3.Y-containing nucleosomes. (A) In silico homology model of H3.X (purple, left) and H3.Y (light blue, right) protein structures in overlay with the crystal structure of H3.2 (dark blue). (B) Crystal structure of nucleosome with H3.2 exchanged by in silico homology models of H3.X (purple, left) and H3.Y (light blue, right), respectively. (C) IP of <t>mononucleosomes</t> generated from HeLa cells transfected with empty vector, HA-H3.1, -H3.X, and -H3.Y shows incorporation of novel H3 variants into nucleosomes. Bioanalyzer evaluation of purified DNA after IP of <t>MNase-treated</t> chromatin (unbound and bound material) shows digestion of chromatin to mononucleosomes and their successful precipitation (left; see also Fig. S2 A for DNA size and quality). Silver stain of 15% SDS-PAGE with α-HA IPs of mononucleosomes revealed successful binding of HA-tagged H3 variants (asterisks) and pull-down of core histones (top, right). Immunoblot of immunoprecipitates with α-HA (red) and α-H3 C-terminal (green) antibodies visualized by the Odyssey infrared imaging system (bottom, right). Notice that endogenous H3 is coimmunoprecipitated with all H3 variants analyzed. (D) FRAP experiments to evaluate nucleosomal stability of novel H3 variants using spinning disk confocal microscopy. HeLa Kyoto cells were transiently transfected with GFP, GFP-H3.1, -H3.3, -H3.X, and -H3.Y constructs. A small nuclear area was photobleached (box) and the recovery of the fluorescent signal was monitored over 1 min and up to 8 h (see Fig. S2, B–D, for long-term FRAP). Depicted is a short-term FRAP series (selected time points are shown) of GFP-tagged H3 variants compared with GFP alone. Bar, 5 µm. (E) Quantification of short-term FRAP experiment. Mean curves of 10–20 individual cells are shown. Standard deviations were very small (in the range of ± 0.02) and were omitted for clarity (for details see Fig. S2 D). All GFP-H3 variants show almost no recovery within the first 60 s after bleaching, which indicates that all expressed fusion protein was stably incorporated into nucleosomes. In contrast, GFP alone recovers to almost 100% within 5 s.
    Microccocal Nuclease, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 137 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher microccocal nuclease
    Digestion of different regions of the rDNA units with <t>MNase.</t> (A) Isolated 1- to 16-h embryonic nuclei were digested with MNase (0.5 U/μl) at <t>24°C</t> for 0, 1, or 4 min (lanes 2 to 4 of each panel). Purified (protein-free) genomic DNA was
    Microccocal Nuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Roche microccocal nuclease
    Bulk chromatin and centromeric chromatin were solubilized by <t>MNase</t> digestion of HeLa nuclei in 0.3 M NaCl. (A) Centromeric proteins CENP-A, -B, and -C were solubilized by MNase digestion. Isolated HeLa nuclei (2 × 10 8 ) were suspended with 1 ml of WB containing 0.3 M NaCl (sample a in lane 1 and sample c in lanes 4 to 6) or 0.6 M NaCl (sample b in lanes 2 and 3). Sample c was digested with 60 U of MNase per ml for 10 min at <t>37°C.</t> Soluble and insoluble materials from each sample were separated by centrifugation. ACA beads were added to the supernatant of sample c and incubated overnight at 4°C. Pellets were resuspended in 1 ml of SDS buffer by extensive sonication and 5 μl of each sample was separated by SDS-7.5% (for CENP-B and CENP-C) or 12.5% (for CENP-A) PAGE, and centromeric proteins were detected by immunostaining with ACA serum. Lane 1, supernatant fraction of a; lane 2, supernatant fraction of b; lane 3, pellet fraction of b; lane 4, supernatant fraction of c before addition of ACA beads; lane 5, supernatant fraction of c after addition of ACA beads; lane 6, pellet fraction of c. Lane M, marker centromeric proteins, CENP-A, CENP-B, and CENP-C. (B) Size distribution of DNA fragments from bulk chromatin after MNase digestion. HeLa nuclei were digested with MNase to various extents. The fragmented DNA in the soluble fractions was extracted with phenol and electrophoresed through 1% agarose gel. DNA was detected with ethidium bromide staining. Lane 1, 20 U/ml for 2 min (40 U/ml × min, sample 1); lane 2, 20 U/ml for 4 min (80 U/ml × min, sample 2); lane 3, 40 U/ml for 5 min (200 U/ml × min, sample 3); lane 4, 80 U/ml for 45 min (3,600 U/ml × min, sample 4). Positions of the DNA size markers are indicated at the left. (C) Detection of core histones and CENP-A in each fraction. Soluble (sup.) and insoluble (pellet) fractions were subjected to SDS-12.5% PAGE, and the separated core histones were stained with Coomassie brilliant blue (upper panel). The proteins were transferred to a membrane and immunolabeled with ACA serum (AK) (lower panel). Lane M in the lower panel is a recombinant CENP-A marker protein. Lanes 1 to 4 correspond to samples 1 to 4 of the soluble (sup.) fractions, and lanes 5 to 8 to samples 1 to 4 of the pellet fractions.
    Microccocal Nuclease, supplied by Roche, used in various techniques. Bioz Stars score: 79/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Worthington Biochemical microccocal nuclease mnase worthington
    RSC Maintains Open NFRs in Lowly Expressed Genes but Is Not Necessary for an Acute Transcriptional Response (A) Experiment outline (see Figure 2 A). (B) RNA fold change during Sth1 depletion and recovery. RNA level was normalized with K. lactis spike-in. Each row is a gene (5,529 genes), and each column is a sample. Heatmap is normalized to expression level prior to auxin addition (also mid-log). The levels of genes at this time are shown by the orange and purple columns. (C) NFR width per RNA level. NFR width per RNA percentile in each sample (Loess smoothed) (top). Percentage of NFRs that closed in the presence of auxin for 0.5 hr (orange line) and 2 hr (yellow line) out of the NFRs that were open in steady state, per RNA percentile at the same time point (bottom). (D) Stress experiment outline. Yeast cells were grown to mid-log in YPD. Auxin was added for 20 min, followed by salt addition (0.4 M KCl); samples were taken in time course and were subjected to <t>MNase-seq</t> and RNA sequencing (RNA-seq). Control samples without auxin or without KCL were performed. (E) Heatmap of RNA fold change in three treatments: auxin only, salt only, and both salt and auxin. RNA levels are normalized per library. 2,322 clustered genes that change in response to the treatments are shown as fold change with respect to the matching expression at T = 0. Time points are indicated in the experiment outline (A). (F) Metagene of subsets of stress-induced genes showing a typical response of chromatin structure to salt induction in time points in three treatments: auxin only, KCl only, and both KCl and auxin. Genes are positioned according to the nucleosome +1 center at T = 0. Black arrows mark location of changes. See also Figure S4 .
    Microccocal Nuclease Mnase Worthington, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    New England Biolabs microccocal nuclease
    Association of Vpr with chromatin correlates with the formation of nuclear foci. A ) HeLa cells were transfected with plasmids expressing HA-tagged Vpr (WT) or an empty plasmid used as negative control. Forty-eight hours after transfection, cells were harvested and lysed with 0.5% Triton X-100. The soluble fraction was used as input control (Soluble). Insoluble debris containing chromatin was treated with <t>microccocal</t> nuclease (+MNase) or with buffer alone (−MNase). The resulting solubilized fractions and input controls were resolved by SDS-PAGE and analyzed by western blot. Specific monoclonal antibodies were used to detect GAPDH (cytoplasmic marker) and HA-Vpr. Histone 3 (chromatin marker) and VPRBP were detected using rabbit polyclonal antibodies. * Denotes a non-specific band detected with the anti-HA antibody. B ) HeLa cells were transfected with plasmids expressing HA-tagged Vpr (WT), Vpr (Q65R), Vpr (R80A), and Vpr (1–78). Cell extracts were processed and analysed as in A). C ) HeLa cells were first transfected with scrambled siRNA or siRNA targeting VPRBP. Twenty-four hours after transfection, cells were transfected with a plasmid expressing HA-Vpr (WT) or an empty plasmid as negative control. Cell extracts were processed and analyzed as in A).
    Microccocal Nuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 89/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Roche microccocal nuclease buffer
    Association of Vpr with chromatin correlates with the formation of nuclear foci. A ) HeLa cells were transfected with plasmids expressing HA-tagged Vpr (WT) or an empty plasmid used as negative control. Forty-eight hours after transfection, cells were harvested and lysed with 0.5% Triton X-100. The soluble fraction was used as input control (Soluble). Insoluble debris containing chromatin was treated with <t>microccocal</t> nuclease (+MNase) or with buffer alone (−MNase). The resulting solubilized fractions and input controls were resolved by SDS-PAGE and analyzed by western blot. Specific monoclonal antibodies were used to detect GAPDH (cytoplasmic marker) and HA-Vpr. Histone 3 (chromatin marker) and VPRBP were detected using rabbit polyclonal antibodies. * Denotes a non-specific band detected with the anti-HA antibody. B ) HeLa cells were transfected with plasmids expressing HA-tagged Vpr (WT), Vpr (Q65R), Vpr (R80A), and Vpr (1–78). Cell extracts were processed and analysed as in A). C ) HeLa cells were first transfected with scrambled siRNA or siRNA targeting VPRBP. Twenty-four hours after transfection, cells were transfected with a plasmid expressing HA-Vpr (WT) or an empty plasmid as negative control. Cell extracts were processed and analyzed as in A).
    Microccocal Nuclease Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 82/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    TaKaRa mnase
    Frequencies of occurrence of DNA dinucleotide steps in the +1 <t>nucleosomes</t> of yeast and the sketch of <t>MNase-seq</t> 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.
    Mnase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Boehringer Mannheim mnase
    Frequencies of occurrence of DNA dinucleotide steps in the +1 <t>nucleosomes</t> of yeast and the sketch of <t>MNase-seq</t> 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.
    Mnase, supplied by Boehringer Mannheim, 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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    94
    Cell Signaling Technology Inc mnase
    Frequencies of occurrence of DNA dinucleotide steps in the +1 <t>nucleosomes</t> of yeast and the sketch of <t>MNase-seq</t> 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.
    Mnase, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore 25 u microccocal nuclease
    Frequencies of occurrence of DNA dinucleotide steps in the +1 <t>nucleosomes</t> of yeast and the sketch of <t>MNase-seq</t> 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.
    25 U Microccocal Nuclease, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    TaKaRa mnase i
    Frequencies of occurrence of DNA dinucleotide steps in the +1 <t>nucleosomes</t> of yeast and the sketch of <t>MNase-seq</t> 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.
    Mnase I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 80/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Thermo Fisher mnase enzyme
    Frequencies of occurrence of DNA dinucleotide steps in the +1 <t>nucleosomes</t> of yeast and the sketch of <t>MNase-seq</t> 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.
    Mnase Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 83/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Active Motif mnase cocktail
    Frequencies of occurrence of DNA dinucleotide steps in the +1 <t>nucleosomes</t> of yeast and the sketch of <t>MNase-seq</t> 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.
    Mnase Cocktail, supplied by Active Motif, used in various techniques. Bioz Stars score: 87/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mnase i
    Frequencies of occurrence of DNA dinucleotide steps in the +1 <t>nucleosomes</t> of yeast and the sketch of <t>MNase-seq</t> 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.
    Mnase I, supplied by Millipore, used in various techniques. Bioz Stars score: 83/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher usb mnase
    Frequencies of occurrence of DNA dinucleotide steps in the +1 <t>nucleosomes</t> of yeast and the sketch of <t>MNase-seq</t> 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.
    Usb Mnase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche mnase s7
    Frequencies of occurrence of DNA dinucleotide steps in the +1 <t>nucleosomes</t> of yeast and the sketch of <t>MNase-seq</t> 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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    Thermo Fisher mnase i
    Frequencies of occurrence of DNA dinucleotide steps in the +1 <t>nucleosomes</t> of yeast and the sketch of <t>MNase-seq</t> 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.
    Mnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare mnase
    Frequencies of occurrence of DNA dinucleotide steps in the +1 <t>nucleosomes</t> of yeast and the sketch of <t>MNase-seq</t> 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.
    Mnase, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mnase digestion buffer
    Frequencies of occurrence of DNA dinucleotide steps in the +1 <t>nucleosomes</t> of yeast and the sketch of <t>MNase-seq</t> 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.
    Mnase Digestion Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Diagenode mnase
    Frequencies of occurrence of DNA dinucleotide steps in the +1 <t>nucleosomes</t> of yeast and the sketch of <t>MNase-seq</t> 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.
    Mnase, supplied by Diagenode, used in various techniques. Bioz Stars score: 94/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Illumina Inc mnase
    Frequencies of occurrence of DNA dinucleotide steps in the +1 <t>nucleosomes</t> of yeast and the sketch of <t>MNase-seq</t> 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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    Boehringer Mannheim micrococcal nuclease mnase
    Frequencies of occurrence of DNA dinucleotide steps in the +1 <t>nucleosomes</t> of yeast and the sketch of <t>MNase-seq</t> 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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    Millipore micrococcal nuclease mnase
    shRNA-mediated inactivation of ATRX does not alter subtelomeric chromatin accessibility to <t>MNase.</t> (A) Chromatin isolated from 8-MG-BA glioma cells in which ATRX had been inactivated (shATRX) or not (shscrambled [shSCR]) was digested with MNase for the indicated times. (Left gel) Ethidium bromide (EtBr) staining of bulk chromatin. (Right gel) Southern blot with subtelomeric probe. (Far right) Quantification of the data. The signals obtained for mononucleosomes were normalized to the total signals measured for each time point (EtBr or Southern blot). (B) Chromatin samples from shATRX or shSCR 8-MG-BA cells were digested for 5 min with the indicated amounts of MNase (milliunits per microgram of DNA). (Far right) Quantification of the data.
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    Roche micrococcal nuclease mnase
    Nucleosome reassembly at the TSS is the initiating event in MLH1 resilencing. A , Regions assayed for nucleosome occupancy using <t>MNase-qPCR</t> (Regions I–IX) and NOMe-Seq (Regions N1 and N2). Shown beneath the gene schematic is NOMe-Seq data from untreated RKO cells at Region N1. Black arrows indicate the MLH1 and EPM2AIP1 TSS. Bottom panel represents GpC accessibility. Black circles = GpC dinucleotides methylated/accessible to the GpC methyltransferase M. Cvi PI. White circles = GpC dinucleotides not methylated/inaccessible to GpC methyltransferase. Pink shading indicates regions of inaccessibility of ≥150 bp. Asterix = region of M. Cvi PI accessibility. B , Relative nucleosome levels in untreated RKO cells at the indicated regions (black bars labeled Regions I–IX) as determined by MNase-qPCR. Drawn to scale with schematic shown in A. Error bars = SD. C and D , qPCR results showing changes in relative nucleosome levels at Regions III and VI following decitabine exposure. E and F , MLH1 gene expression (E) and promoter bisulfite pyrosequencing (F), reproduced from Figure 2A and D for ease of comparison with nucleosome levels. G–I , NOMe-Seq analysis of the MLH1 promoter at Region N2 in SW620 (F) and RKO (G,H) cells at the indicated treatment points. Black filled triangles = methylated CpG dinucleotides; white filled triangles = unmethylated CpG dinucleotides. See also Figure S2 .
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    ATUM mnase digested 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. <t>MNase</t> (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.
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    86
    Illumina Inc mnase digested chromatin
    Fun30 is required for normal CEN -flanking nucleosome positioning and/or CEN core particle structure. A) Genome browser trace of Fun30 ChIP enrichment and nucleosome dyad frequency centred on and surrounding yeast CEN1 . The upper trace shows Log 2 Fun30 ChIP-seq enrichment values binned at 10 bp intervals and smoothed with a 3 bin moving average. Wildtype (WT) and Δfun30 chromatin was digested with <t>MNase</t> and nuclease-protected DNA species sequenced using paired-end mode <t>Illumina</t> technology. Nucleosome sequencing data (nuc) traces were plotted as mirror images in the lower panel. The graph shows a map of the centre point positions of paired sequence reads with end-to-end distances of 150 bp+/−20% wild-type and Δfun30 mutant chromatin samples surrounding CEN1 . The frequency distributions, which effectively map chromatin particle dyads, were binned at 10 bp intervals, and smoothed by applying a 3 bin moving average. Peaks in the dyad distributions correspond to translationally-positioned nucleosomes in the original genome. The CEN core particle is also mapped using this method and can be visualised as a small peak centred on the CEN region marked with a grey box. Pink bars show the positions of ORFs (B–D) Genome browser plots of Fun30 ChIP-seq and nucleosome sequence distributions as described above for CEN10, 11 and 12 respectively. Fun30-dependent changes in the height of a nucleosome dyad or CEN core particle peak are marked with a red asterix. Fun30-dependent changes in the position of a CEN -flanking nucleosome dyad peak are marked with red arrows. Genome browser plots for all yeast CENs are shown in Figure S6 .
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    95
    New England Biolabs mnase buffer
    Fun30 is required for normal CEN -flanking nucleosome positioning and/or CEN core particle structure. A) Genome browser trace of Fun30 ChIP enrichment and nucleosome dyad frequency centred on and surrounding yeast CEN1 . The upper trace shows Log 2 Fun30 ChIP-seq enrichment values binned at 10 bp intervals and smoothed with a 3 bin moving average. Wildtype (WT) and Δfun30 chromatin was digested with <t>MNase</t> and nuclease-protected DNA species sequenced using paired-end mode <t>Illumina</t> technology. Nucleosome sequencing data (nuc) traces were plotted as mirror images in the lower panel. The graph shows a map of the centre point positions of paired sequence reads with end-to-end distances of 150 bp+/−20% wild-type and Δfun30 mutant chromatin samples surrounding CEN1 . The frequency distributions, which effectively map chromatin particle dyads, were binned at 10 bp intervals, and smoothed by applying a 3 bin moving average. Peaks in the dyad distributions correspond to translationally-positioned nucleosomes in the original genome. The CEN core particle is also mapped using this method and can be visualised as a small peak centred on the CEN region marked with a grey box. Pink bars show the positions of ORFs (B–D) Genome browser plots of Fun30 ChIP-seq and nucleosome sequence distributions as described above for CEN10, 11 and 12 respectively. Fun30-dependent changes in the height of a nucleosome dyad or CEN core particle peak are marked with a red asterix. Fun30-dependent changes in the position of a CEN -flanking nucleosome dyad peak are marked with red arrows. Genome browser plots for all yeast CENs are shown in Figure S6 .
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    New England Biolabs mnase i
    Fun30 is required for normal CEN -flanking nucleosome positioning and/or CEN core particle structure. A) Genome browser trace of Fun30 ChIP enrichment and nucleosome dyad frequency centred on and surrounding yeast CEN1 . The upper trace shows Log 2 Fun30 ChIP-seq enrichment values binned at 10 bp intervals and smoothed with a 3 bin moving average. Wildtype (WT) and Δfun30 chromatin was digested with <t>MNase</t> and nuclease-protected DNA species sequenced using paired-end mode <t>Illumina</t> technology. Nucleosome sequencing data (nuc) traces were plotted as mirror images in the lower panel. The graph shows a map of the centre point positions of paired sequence reads with end-to-end distances of 150 bp+/−20% wild-type and Δfun30 mutant chromatin samples surrounding CEN1 . The frequency distributions, which effectively map chromatin particle dyads, were binned at 10 bp intervals, and smoothed by applying a 3 bin moving average. Peaks in the dyad distributions correspond to translationally-positioned nucleosomes in the original genome. The CEN core particle is also mapped using this method and can be visualised as a small peak centred on the CEN region marked with a grey box. Pink bars show the positions of ORFs (B–D) Genome browser plots of Fun30 ChIP-seq and nucleosome sequence distributions as described above for CEN10, 11 and 12 respectively. Fun30-dependent changes in the height of a nucleosome dyad or CEN core particle peak are marked with a red asterix. Fun30-dependent changes in the position of a CEN -flanking nucleosome dyad peak are marked with red arrows. Genome browser plots for all yeast CENs are shown in Figure S6 .
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    Thermo Fisher micrococcal nuclease mnase
    VP2 binds to chromatin and interacts with MCM3 but this does not require dual phosphatase activity . ( A ) The soluble (S) and chromatin (C) fractions were prepared by using CSK buffer containing 0.5% Triton X-100 in CHO cells of transfected with VP2-GFP plasmid. The fractions were treated with 0 U (-) and 150 U (+) of <t>MNase</t> or the indicated concentrations of <t>NaCl.</t> Nuclear extracts (N) containing VP2-GFP were as positive control and all fractions were subjected to immunoblotting against with lamin B receptor, MCM3, and VP2 antibodies. ( B ) At 48 h post-transfection, the GFP (as Control) and Flag-VP2-GFP (as wild-type; WT) were found in the CHO cells. Next the lysates were immunoprecipitated using Flag M2 beads and immunoblotted against VP2 and MCM3 antibodies. ( C ) The WT and mutants (C95S, C97S, and C95S/C97S) of the Flag-VP2-GFP in CHO cells were also examined at 48 h post-transfection. The cell lysates were immunoprecipitated using Flag M2 beads and immunoblotted against with VP2 and MCM3 antibodies. The arrow head was indicated a none-specific band. The nuclear extracts containing VP2-GFP were designated as N
    Micrococcal Nuclease Mnase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mnase enzyme
    VP2 binds to chromatin and interacts with MCM3 but this does not require dual phosphatase activity . ( A ) The soluble (S) and chromatin (C) fractions were prepared by using CSK buffer containing 0.5% Triton X-100 in CHO cells of transfected with VP2-GFP plasmid. The fractions were treated with 0 U (-) and 150 U (+) of <t>MNase</t> or the indicated concentrations of <t>NaCl.</t> Nuclear extracts (N) containing VP2-GFP were as positive control and all fractions were subjected to immunoblotting against with lamin B receptor, MCM3, and VP2 antibodies. ( B ) At 48 h post-transfection, the GFP (as Control) and Flag-VP2-GFP (as wild-type; WT) were found in the CHO cells. Next the lysates were immunoprecipitated using Flag M2 beads and immunoblotted against VP2 and MCM3 antibodies. ( C ) The WT and mutants (C95S, C97S, and C95S/C97S) of the Flag-VP2-GFP in CHO cells were also examined at 48 h post-transfection. The cell lysates were immunoprecipitated using Flag M2 beads and immunoblotted against with VP2 and MCM3 antibodies. The arrow head was indicated a none-specific band. The nuclear extracts containing VP2-GFP were designated as N
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    Image Search Results


    AGO2 knock-down affects nucleosome occupancy at TSSs bound by SWI/SNF. ( a ) HeLaS3 cells were transfected with a control siRNA (siCTRL) or a pool of AGO2 siRNA (siAGO2). Down-regulation of AGO2 protein was verified by western blot. GAPDH was used as loading control. ( b ) Chromatin from siCTRL- or siAGO2-treated HeLaS3 cells was digested by MNase and recovered DNA fragments were sequenced. Nucleosome occupancy profile for siCTRL and siAGO2 cells was plotted for TSSs with at least 30 swiRNAs (siCTRL, black line; siAGO2, green line). The occupancy at the nucleosome +1 (arrow) is reduced in AGO2 knock-down cells. ( c ) Bars height represents percent reduction of nucleosome occupancy (siAGO2 versus siCTRL) at TSS ±150 nt overlapped by at least the indicated number of swiRNAs (green), IgG-IP ‘other sRNAs’ (black) and AGO1-associated ‘other sRNAs’ (purple). ** P value

    Journal: Nucleic Acids Research

    Article Title: ARGONAUTE2 cooperates with SWI/SNF complex to determine nucleosome occupancy at human Transcription Start Sites

    doi: 10.1093/nar/gku1387

    Figure Lengend Snippet: AGO2 knock-down affects nucleosome occupancy at TSSs bound by SWI/SNF. ( a ) HeLaS3 cells were transfected with a control siRNA (siCTRL) or a pool of AGO2 siRNA (siAGO2). Down-regulation of AGO2 protein was verified by western blot. GAPDH was used as loading control. ( b ) Chromatin from siCTRL- or siAGO2-treated HeLaS3 cells was digested by MNase and recovered DNA fragments were sequenced. Nucleosome occupancy profile for siCTRL and siAGO2 cells was plotted for TSSs with at least 30 swiRNAs (siCTRL, black line; siAGO2, green line). The occupancy at the nucleosome +1 (arrow) is reduced in AGO2 knock-down cells. ( c ) Bars height represents percent reduction of nucleosome occupancy (siAGO2 versus siCTRL) at TSS ±150 nt overlapped by at least the indicated number of swiRNAs (green), IgG-IP ‘other sRNAs’ (black) and AGO1-associated ‘other sRNAs’ (purple). ** P value

    Article Snippet: Isolation of nucleosomal DNA by micrococcal nuclease (MNase) digestion Digestion of chromatin from untreated, siCTRL- or siAGO2-treated HeLa S3 cells (2 × 106 ) was performed with 50 U of MNase (New England Biolabs) in 300 μl of permeabilization buffer (15 mM Tris–HCl pH 7.4, 300 mM sucrose, 60 mM KCl, 15 mM NaCl, 4 mM CaCl2 , 0.5 mM EGTA, 0.2% NP-40, 0.5 mM β-mercaptoethanol) for 20 min at 37°C.

    Techniques: Transfection, Western Blot

    Structure and stability of H3.X- and H3.Y-containing nucleosomes. (A) In silico homology model of H3.X (purple, left) and H3.Y (light blue, right) protein structures in overlay with the crystal structure of H3.2 (dark blue). (B) Crystal structure of nucleosome with H3.2 exchanged by in silico homology models of H3.X (purple, left) and H3.Y (light blue, right), respectively. (C) IP of mononucleosomes generated from HeLa cells transfected with empty vector, HA-H3.1, -H3.X, and -H3.Y shows incorporation of novel H3 variants into nucleosomes. Bioanalyzer evaluation of purified DNA after IP of MNase-treated chromatin (unbound and bound material) shows digestion of chromatin to mononucleosomes and their successful precipitation (left; see also Fig. S2 A for DNA size and quality). Silver stain of 15% SDS-PAGE with α-HA IPs of mononucleosomes revealed successful binding of HA-tagged H3 variants (asterisks) and pull-down of core histones (top, right). Immunoblot of immunoprecipitates with α-HA (red) and α-H3 C-terminal (green) antibodies visualized by the Odyssey infrared imaging system (bottom, right). Notice that endogenous H3 is coimmunoprecipitated with all H3 variants analyzed. (D) FRAP experiments to evaluate nucleosomal stability of novel H3 variants using spinning disk confocal microscopy. HeLa Kyoto cells were transiently transfected with GFP, GFP-H3.1, -H3.3, -H3.X, and -H3.Y constructs. A small nuclear area was photobleached (box) and the recovery of the fluorescent signal was monitored over 1 min and up to 8 h (see Fig. S2, B–D, for long-term FRAP). Depicted is a short-term FRAP series (selected time points are shown) of GFP-tagged H3 variants compared with GFP alone. Bar, 5 µm. (E) Quantification of short-term FRAP experiment. Mean curves of 10–20 individual cells are shown. Standard deviations were very small (in the range of ± 0.02) and were omitted for clarity (for details see Fig. S2 D). All GFP-H3 variants show almost no recovery within the first 60 s after bleaching, which indicates that all expressed fusion protein was stably incorporated into nucleosomes. In contrast, GFP alone recovers to almost 100% within 5 s.

    Journal: The Journal of Cell Biology

    Article Title: Identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y

    doi: 10.1083/jcb.201002043

    Figure Lengend Snippet: Structure and stability of H3.X- and H3.Y-containing nucleosomes. (A) In silico homology model of H3.X (purple, left) and H3.Y (light blue, right) protein structures in overlay with the crystal structure of H3.2 (dark blue). (B) Crystal structure of nucleosome with H3.2 exchanged by in silico homology models of H3.X (purple, left) and H3.Y (light blue, right), respectively. (C) IP of mononucleosomes generated from HeLa cells transfected with empty vector, HA-H3.1, -H3.X, and -H3.Y shows incorporation of novel H3 variants into nucleosomes. Bioanalyzer evaluation of purified DNA after IP of MNase-treated chromatin (unbound and bound material) shows digestion of chromatin to mononucleosomes and their successful precipitation (left; see also Fig. S2 A for DNA size and quality). Silver stain of 15% SDS-PAGE with α-HA IPs of mononucleosomes revealed successful binding of HA-tagged H3 variants (asterisks) and pull-down of core histones (top, right). Immunoblot of immunoprecipitates with α-HA (red) and α-H3 C-terminal (green) antibodies visualized by the Odyssey infrared imaging system (bottom, right). Notice that endogenous H3 is coimmunoprecipitated with all H3 variants analyzed. (D) FRAP experiments to evaluate nucleosomal stability of novel H3 variants using spinning disk confocal microscopy. HeLa Kyoto cells were transiently transfected with GFP, GFP-H3.1, -H3.3, -H3.X, and -H3.Y constructs. A small nuclear area was photobleached (box) and the recovery of the fluorescent signal was monitored over 1 min and up to 8 h (see Fig. S2, B–D, for long-term FRAP). Depicted is a short-term FRAP series (selected time points are shown) of GFP-tagged H3 variants compared with GFP alone. Bar, 5 µm. (E) Quantification of short-term FRAP experiment. Mean curves of 10–20 individual cells are shown. Standard deviations were very small (in the range of ± 0.02) and were omitted for clarity (for details see Fig. S2 D). All GFP-H3 variants show almost no recovery within the first 60 s after bleaching, which indicates that all expressed fusion protein was stably incorporated into nucleosomes. In contrast, GFP alone recovers to almost 100% within 5 s.

    Article Snippet: Mononucleosomes were generated by digestion of chromatin with 0.25 U MNase (Sigma-Aldrich) for 15 min in buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 , 0.34 M sucrose, 10% glycerol [vol/vol], 1 mM DTT, and protease inhibitor cocktail [Roche] plus 1 mM CaCl2 ) and stopped by the addition of EGTA (final concentration of 2 mM).

    Techniques: In Silico, Generated, Transfection, Plasmid Preparation, Purification, Silver Staining, SDS Page, Binding Assay, Imaging, Confocal Microscopy, Construct, Stable Transfection

    Digestion of different regions of the rDNA units with MNase. (A) Isolated 1- to 16-h embryonic nuclei were digested with MNase (0.5 U/μl) at 24°C for 0, 1, or 4 min (lanes 2 to 4 of each panel). Purified (protein-free) genomic DNA was

    Journal: Molecular and Cellular Biology

    Article Title: Chromatin Structure and Transcription of the R1- and R2-Inserted rRNA Genes of Drosophila melanogaster ▿

    doi: 10.1128/MCB.01409-06

    Figure Lengend Snippet: Digestion of different regions of the rDNA units with MNase. (A) Isolated 1- to 16-h embryonic nuclei were digested with MNase (0.5 U/μl) at 24°C for 0, 1, or 4 min (lanes 2 to 4 of each panel). Purified (protein-free) genomic DNA was

    Article Snippet: For micrococcal nuclease (MNase) digestion, the nucleus solutions were made 3 mM CaCl2 and 0.5 units/μl MNase (Fermentas) and incubated at 24°C for the specified time.

    Techniques: Isolation, Purification

    Bulk chromatin and centromeric chromatin were solubilized by MNase digestion of HeLa nuclei in 0.3 M NaCl. (A) Centromeric proteins CENP-A, -B, and -C were solubilized by MNase digestion. Isolated HeLa nuclei (2 × 10 8 ) were suspended with 1 ml of WB containing 0.3 M NaCl (sample a in lane 1 and sample c in lanes 4 to 6) or 0.6 M NaCl (sample b in lanes 2 and 3). Sample c was digested with 60 U of MNase per ml for 10 min at 37°C. Soluble and insoluble materials from each sample were separated by centrifugation. ACA beads were added to the supernatant of sample c and incubated overnight at 4°C. Pellets were resuspended in 1 ml of SDS buffer by extensive sonication and 5 μl of each sample was separated by SDS-7.5% (for CENP-B and CENP-C) or 12.5% (for CENP-A) PAGE, and centromeric proteins were detected by immunostaining with ACA serum. Lane 1, supernatant fraction of a; lane 2, supernatant fraction of b; lane 3, pellet fraction of b; lane 4, supernatant fraction of c before addition of ACA beads; lane 5, supernatant fraction of c after addition of ACA beads; lane 6, pellet fraction of c. Lane M, marker centromeric proteins, CENP-A, CENP-B, and CENP-C. (B) Size distribution of DNA fragments from bulk chromatin after MNase digestion. HeLa nuclei were digested with MNase to various extents. The fragmented DNA in the soluble fractions was extracted with phenol and electrophoresed through 1% agarose gel. DNA was detected with ethidium bromide staining. Lane 1, 20 U/ml for 2 min (40 U/ml × min, sample 1); lane 2, 20 U/ml for 4 min (80 U/ml × min, sample 2); lane 3, 40 U/ml for 5 min (200 U/ml × min, sample 3); lane 4, 80 U/ml for 45 min (3,600 U/ml × min, sample 4). Positions of the DNA size markers are indicated at the left. (C) Detection of core histones and CENP-A in each fraction. Soluble (sup.) and insoluble (pellet) fractions were subjected to SDS-12.5% PAGE, and the separated core histones were stained with Coomassie brilliant blue (upper panel). The proteins were transferred to a membrane and immunolabeled with ACA serum (AK) (lower panel). Lane M in the lower panel is a recombinant CENP-A marker protein. Lanes 1 to 4 correspond to samples 1 to 4 of the soluble (sup.) fractions, and lanes 5 to 8 to samples 1 to 4 of the pellet fractions.

    Journal: Molecular and Cellular Biology

    Article Title: CENP-A, -B, and -C Chromatin Complex That Contains the I-Type ?-Satellite Array Constitutes the Prekinetochore in HeLa Cells

    doi: 10.1128/MCB.22.7.2229-2241.2002

    Figure Lengend Snippet: Bulk chromatin and centromeric chromatin were solubilized by MNase digestion of HeLa nuclei in 0.3 M NaCl. (A) Centromeric proteins CENP-A, -B, and -C were solubilized by MNase digestion. Isolated HeLa nuclei (2 × 10 8 ) were suspended with 1 ml of WB containing 0.3 M NaCl (sample a in lane 1 and sample c in lanes 4 to 6) or 0.6 M NaCl (sample b in lanes 2 and 3). Sample c was digested with 60 U of MNase per ml for 10 min at 37°C. Soluble and insoluble materials from each sample were separated by centrifugation. ACA beads were added to the supernatant of sample c and incubated overnight at 4°C. Pellets were resuspended in 1 ml of SDS buffer by extensive sonication and 5 μl of each sample was separated by SDS-7.5% (for CENP-B and CENP-C) or 12.5% (for CENP-A) PAGE, and centromeric proteins were detected by immunostaining with ACA serum. Lane 1, supernatant fraction of a; lane 2, supernatant fraction of b; lane 3, pellet fraction of b; lane 4, supernatant fraction of c before addition of ACA beads; lane 5, supernatant fraction of c after addition of ACA beads; lane 6, pellet fraction of c. Lane M, marker centromeric proteins, CENP-A, CENP-B, and CENP-C. (B) Size distribution of DNA fragments from bulk chromatin after MNase digestion. HeLa nuclei were digested with MNase to various extents. The fragmented DNA in the soluble fractions was extracted with phenol and electrophoresed through 1% agarose gel. DNA was detected with ethidium bromide staining. Lane 1, 20 U/ml for 2 min (40 U/ml × min, sample 1); lane 2, 20 U/ml for 4 min (80 U/ml × min, sample 2); lane 3, 40 U/ml for 5 min (200 U/ml × min, sample 3); lane 4, 80 U/ml for 45 min (3,600 U/ml × min, sample 4). Positions of the DNA size markers are indicated at the left. (C) Detection of core histones and CENP-A in each fraction. Soluble (sup.) and insoluble (pellet) fractions were subjected to SDS-12.5% PAGE, and the separated core histones were stained with Coomassie brilliant blue (upper panel). The proteins were transferred to a membrane and immunolabeled with ACA serum (AK) (lower panel). Lane M in the lower panel is a recombinant CENP-A marker protein. Lanes 1 to 4 correspond to samples 1 to 4 of the soluble (sup.) fractions, and lanes 5 to 8 to samples 1 to 4 of the pellet fractions.

    Article Snippet: The nuclear suspension was digested with MNase (Roche Diagnostics) at 37°C after addition of CaCl2 to a final concentration of 2 mM.

    Techniques: Isolation, Western Blot, Centrifugation, Incubation, Sonication, Polyacrylamide Gel Electrophoresis, Immunostaining, Marker, Agarose Gel Electrophoresis, Staining, Immunolabeling, Recombinant

    RSC Maintains Open NFRs in Lowly Expressed Genes but Is Not Necessary for an Acute Transcriptional Response (A) Experiment outline (see Figure 2 A). (B) RNA fold change during Sth1 depletion and recovery. RNA level was normalized with K. lactis spike-in. Each row is a gene (5,529 genes), and each column is a sample. Heatmap is normalized to expression level prior to auxin addition (also mid-log). The levels of genes at this time are shown by the orange and purple columns. (C) NFR width per RNA level. NFR width per RNA percentile in each sample (Loess smoothed) (top). Percentage of NFRs that closed in the presence of auxin for 0.5 hr (orange line) and 2 hr (yellow line) out of the NFRs that were open in steady state, per RNA percentile at the same time point (bottom). (D) Stress experiment outline. Yeast cells were grown to mid-log in YPD. Auxin was added for 20 min, followed by salt addition (0.4 M KCl); samples were taken in time course and were subjected to MNase-seq and RNA sequencing (RNA-seq). Control samples without auxin or without KCL were performed. (E) Heatmap of RNA fold change in three treatments: auxin only, salt only, and both salt and auxin. RNA levels are normalized per library. 2,322 clustered genes that change in response to the treatments are shown as fold change with respect to the matching expression at T = 0. Time points are indicated in the experiment outline (A). (F) Metagene of subsets of stress-induced genes showing a typical response of chromatin structure to salt induction in time points in three treatments: auxin only, KCl only, and both KCl and auxin. Genes are positioned according to the nucleosome +1 center at T = 0. Black arrows mark location of changes. See also Figure S4 .

    Journal: Cell Reports

    Article Title: Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

    doi: 10.1016/j.celrep.2018.12.020

    Figure Lengend Snippet: RSC Maintains Open NFRs in Lowly Expressed Genes but Is Not Necessary for an Acute Transcriptional Response (A) Experiment outline (see Figure 2 A). (B) RNA fold change during Sth1 depletion and recovery. RNA level was normalized with K. lactis spike-in. Each row is a gene (5,529 genes), and each column is a sample. Heatmap is normalized to expression level prior to auxin addition (also mid-log). The levels of genes at this time are shown by the orange and purple columns. (C) NFR width per RNA level. NFR width per RNA percentile in each sample (Loess smoothed) (top). Percentage of NFRs that closed in the presence of auxin for 0.5 hr (orange line) and 2 hr (yellow line) out of the NFRs that were open in steady state, per RNA percentile at the same time point (bottom). (D) Stress experiment outline. Yeast cells were grown to mid-log in YPD. Auxin was added for 20 min, followed by salt addition (0.4 M KCl); samples were taken in time course and were subjected to MNase-seq and RNA sequencing (RNA-seq). Control samples without auxin or without KCL were performed. (E) Heatmap of RNA fold change in three treatments: auxin only, salt only, and both salt and auxin. RNA levels are normalized per library. 2,322 clustered genes that change in response to the treatments are shown as fold change with respect to the matching expression at T = 0. Time points are indicated in the experiment outline (A). (F) Metagene of subsets of stress-induced genes showing a typical response of chromatin structure to salt induction in time points in three treatments: auxin only, KCl only, and both KCl and auxin. Genes are positioned according to the nucleosome +1 center at T = 0. Black arrows mark location of changes. See also Figure S4 .

    Article Snippet: Spheroplasts were washed, resuspended in NP buffer and treated with MNase (Micrococcal nuclease, Worthington) to generate 80% mono-nucleosomes (1 unit for 2.5 OD initial culture, 37°C, 20 minutes).

    Techniques: Expressing, RNA Sequencing Assay

    Changes in the +1 Nucleosome Position Are Reflected in TSS Usage (A) Experimental outline (as in Figure 2 A). An example of the data representation showing RNA 5′ ends (black), MNase read centers (dark red), and coverage (light red) around the TSS. (B) Nucleosome positioning and 5′ RNA ends during Sth1 depletion in CDC8 and ATG27 promoters. Dashed lines represent peak centers before and 1 hr after auxin addition. (C) 5′ RNA level at each position over the genome before and after Sth1 depletion (normalized with K. lactis spike-in). (D) Median nucleosome positioning around mRNA 5′ ends before (top) and 1 hr after (bottom) auxin addition. mRNA 5′ positions are separated to groups according to their fold change following Sth1 depletion. (E) Change in expression (1 hr/0 hr) versus change in accessibility (1 hr/0 hr) for mRNA 5′ locations that are expressed ( Figure 5 C) and accessible ( Figure S5 B) before auxin addition. See also Figure S5 .

    Journal: Cell Reports

    Article Title: Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

    doi: 10.1016/j.celrep.2018.12.020

    Figure Lengend Snippet: Changes in the +1 Nucleosome Position Are Reflected in TSS Usage (A) Experimental outline (as in Figure 2 A). An example of the data representation showing RNA 5′ ends (black), MNase read centers (dark red), and coverage (light red) around the TSS. (B) Nucleosome positioning and 5′ RNA ends during Sth1 depletion in CDC8 and ATG27 promoters. Dashed lines represent peak centers before and 1 hr after auxin addition. (C) 5′ RNA level at each position over the genome before and after Sth1 depletion (normalized with K. lactis spike-in). (D) Median nucleosome positioning around mRNA 5′ ends before (top) and 1 hr after (bottom) auxin addition. mRNA 5′ positions are separated to groups according to their fold change following Sth1 depletion. (E) Change in expression (1 hr/0 hr) versus change in accessibility (1 hr/0 hr) for mRNA 5′ locations that are expressed ( Figure 5 C) and accessible ( Figure S5 B) before auxin addition. See also Figure S5 .

    Article Snippet: Spheroplasts were washed, resuspended in NP buffer and treated with MNase (Micrococcal nuclease, Worthington) to generate 80% mono-nucleosomes (1 unit for 2.5 OD initial culture, 37°C, 20 minutes).

    Techniques: Expressing

    Dynamics of Sth1 Depletion and Recovery Show Massive yet Reversible Disruptions in Chromatin Organization (A) Experimental outline. For depletion, auxin (IAA) was added to mid-log degron-Sth1 cells, and MNase-seq was performed at the indicated time points. For recovery, mid-log degron-Sth1 cells were incubated in the presence of auxin for 2 hr. Auxin was washed from the media, and MNase-seq was performed at the indicated time points. (B) Median MNase coverage positioned relative to the TSS (metagene) following Sth1 depletion (top) and recovery (bottom). (C) MNase read centers (lines, dark color) and coverage (shade, light color) following Sth1 depletion and recovery in the TAF6/NSA1 promoter area. Dashed lines represent the position of nucleosomes +1 and −1 center before depletion and after full recovery. (D) Distribution of NFR width (defined as the distance between the peak −/+1 nucleosomes) through sth1 depletion and recovery. (E) Average MNase coverage (metagene) before (red line) and 1 hr after (yellow line) Sth1 depletion in genes with a GRF-binding site (top) and without GRF binding but with a poly(A/T) tract (bottom). Genes were positioned relative to the GRF-binding site or poly(A/T) tract site. GRF-binding sites were obtained from Gutin et al. (2018) . (F) Distribution of NFR width throughout Sth1 depletion in the two groups as in (E). The distribution of all genes before auxin addition is shown in gray. (G) Comparison of NFR width before Sth1 depletion and after recovery. Each point is related to NFR of a gene. Genes with fuzzy +1 or −1 nucleosomes were excluded. See also Figure S2 .

    Journal: Cell Reports

    Article Title: Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

    doi: 10.1016/j.celrep.2018.12.020

    Figure Lengend Snippet: Dynamics of Sth1 Depletion and Recovery Show Massive yet Reversible Disruptions in Chromatin Organization (A) Experimental outline. For depletion, auxin (IAA) was added to mid-log degron-Sth1 cells, and MNase-seq was performed at the indicated time points. For recovery, mid-log degron-Sth1 cells were incubated in the presence of auxin for 2 hr. Auxin was washed from the media, and MNase-seq was performed at the indicated time points. (B) Median MNase coverage positioned relative to the TSS (metagene) following Sth1 depletion (top) and recovery (bottom). (C) MNase read centers (lines, dark color) and coverage (shade, light color) following Sth1 depletion and recovery in the TAF6/NSA1 promoter area. Dashed lines represent the position of nucleosomes +1 and −1 center before depletion and after full recovery. (D) Distribution of NFR width (defined as the distance between the peak −/+1 nucleosomes) through sth1 depletion and recovery. (E) Average MNase coverage (metagene) before (red line) and 1 hr after (yellow line) Sth1 depletion in genes with a GRF-binding site (top) and without GRF binding but with a poly(A/T) tract (bottom). Genes were positioned relative to the GRF-binding site or poly(A/T) tract site. GRF-binding sites were obtained from Gutin et al. (2018) . (F) Distribution of NFR width throughout Sth1 depletion in the two groups as in (E). The distribution of all genes before auxin addition is shown in gray. (G) Comparison of NFR width before Sth1 depletion and after recovery. Each point is related to NFR of a gene. Genes with fuzzy +1 or −1 nucleosomes were excluded. See also Figure S2 .

    Article Snippet: Spheroplasts were washed, resuspended in NP buffer and treated with MNase (Micrococcal nuclease, Worthington) to generate 80% mono-nucleosomes (1 unit for 2.5 OD initial culture, 37°C, 20 minutes).

    Techniques: Incubation, Binding Assay

    Sth1-Dependent NFR Clearing Is Replication Independent (A) Experimental outline in G1-arrested cells. For depletion, yeast cells were grown to mid-log in YPD and incubated with or without alpha factor for 2 hr. At the indicated time, cells were transferred to a new tube, and Sth1 depletion was induced by auxin addition. All samples were fixed at the same time. For recovery, yeast cells were grown to mid-log in YPD and incubated with alpha-factor and auxin for 2 hr. Cells were washed and resuspended with or without alpha factor. MNase-seq was performed at the indicated time points. (B) Distribution of NFR width in time course through Sth1 depletion (top) and recovery (bottom) in G1-arrested cells (right) and in unsynchronized cells (left). (C) Density scatter of the change in NFR width for all genes through Sth1 depletion (1 hr, top) and recovery (4 hr, bottom), in G1 arrested versus unsynchronized cells. See also Figure S3 .

    Journal: Cell Reports

    Article Title: Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

    doi: 10.1016/j.celrep.2018.12.020

    Figure Lengend Snippet: Sth1-Dependent NFR Clearing Is Replication Independent (A) Experimental outline in G1-arrested cells. For depletion, yeast cells were grown to mid-log in YPD and incubated with or without alpha factor for 2 hr. At the indicated time, cells were transferred to a new tube, and Sth1 depletion was induced by auxin addition. All samples were fixed at the same time. For recovery, yeast cells were grown to mid-log in YPD and incubated with alpha-factor and auxin for 2 hr. Cells were washed and resuspended with or without alpha factor. MNase-seq was performed at the indicated time points. (B) Distribution of NFR width in time course through Sth1 depletion (top) and recovery (bottom) in G1-arrested cells (right) and in unsynchronized cells (left). (C) Density scatter of the change in NFR width for all genes through Sth1 depletion (1 hr, top) and recovery (4 hr, bottom), in G1 arrested versus unsynchronized cells. See also Figure S3 .

    Article Snippet: Spheroplasts were washed, resuspended in NP buffer and treated with MNase (Micrococcal nuclease, Worthington) to generate 80% mono-nucleosomes (1 unit for 2.5 OD initial culture, 37°C, 20 minutes).

    Techniques: Incubation

    Induced Knockdown Screen of ATP-Dependent Chromatin Remodelers (A) An auxin-inducible degron (AID) system ( Morawska and Ulrich, 2013 ) yielding an auxin-inducible, rapid degradation of tagged chromatin remodelers. Plant hormone auxin (IAA) directly induces rapid degradation of the AID-tagged protein by mediating the interaction of a degron domain in the target protein with the substrate recognition domain of TIR1. (B) Experimental outline. AID-tagged chromatin remodeler strains were grown to mid-log in YPD. MNase-seq was performed to compare nucleosome positioning before and at two time points after auxin addition. (C) Average MNase coverage positioned relative to the transcription start site (TSS) (“metagene”) for each chromatin remodeler AID strain before and after auxin addition and in the relevant KO strains (if available) (top). Average of the change in MNase coverage before and after auxin addition (1 hr to 0 hr) positioned relative to the TSS for each chromatin remodeler AID strain (bottom). (D) Heatmaps representing the change in MNase coverage before and after auxin addition (1 hr to 0 hr) positioned relative to the TSS (in yellow) for each AID strain. Genes (rows) are sorted, in each strain, by the magnitude of changes in coverage following the depletion in the NFR area. See also Figure S1 .

    Journal: Cell Reports

    Article Title: Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

    doi: 10.1016/j.celrep.2018.12.020

    Figure Lengend Snippet: Induced Knockdown Screen of ATP-Dependent Chromatin Remodelers (A) An auxin-inducible degron (AID) system ( Morawska and Ulrich, 2013 ) yielding an auxin-inducible, rapid degradation of tagged chromatin remodelers. Plant hormone auxin (IAA) directly induces rapid degradation of the AID-tagged protein by mediating the interaction of a degron domain in the target protein with the substrate recognition domain of TIR1. (B) Experimental outline. AID-tagged chromatin remodeler strains were grown to mid-log in YPD. MNase-seq was performed to compare nucleosome positioning before and at two time points after auxin addition. (C) Average MNase coverage positioned relative to the transcription start site (TSS) (“metagene”) for each chromatin remodeler AID strain before and after auxin addition and in the relevant KO strains (if available) (top). Average of the change in MNase coverage before and after auxin addition (1 hr to 0 hr) positioned relative to the TSS for each chromatin remodeler AID strain (bottom). (D) Heatmaps representing the change in MNase coverage before and after auxin addition (1 hr to 0 hr) positioned relative to the TSS (in yellow) for each AID strain. Genes (rows) are sorted, in each strain, by the magnitude of changes in coverage following the depletion in the NFR area. See also Figure S1 .

    Article Snippet: Spheroplasts were washed, resuspended in NP buffer and treated with MNase (Micrococcal nuclease, Worthington) to generate 80% mono-nucleosomes (1 unit for 2.5 OD initial culture, 37°C, 20 minutes).

    Techniques:

    Association of Vpr with chromatin correlates with the formation of nuclear foci. A ) HeLa cells were transfected with plasmids expressing HA-tagged Vpr (WT) or an empty plasmid used as negative control. Forty-eight hours after transfection, cells were harvested and lysed with 0.5% Triton X-100. The soluble fraction was used as input control (Soluble). Insoluble debris containing chromatin was treated with microccocal nuclease (+MNase) or with buffer alone (−MNase). The resulting solubilized fractions and input controls were resolved by SDS-PAGE and analyzed by western blot. Specific monoclonal antibodies were used to detect GAPDH (cytoplasmic marker) and HA-Vpr. Histone 3 (chromatin marker) and VPRBP were detected using rabbit polyclonal antibodies. * Denotes a non-specific band detected with the anti-HA antibody. B ) HeLa cells were transfected with plasmids expressing HA-tagged Vpr (WT), Vpr (Q65R), Vpr (R80A), and Vpr (1–78). Cell extracts were processed and analysed as in A). C ) HeLa cells were first transfected with scrambled siRNA or siRNA targeting VPRBP. Twenty-four hours after transfection, cells were transfected with a plasmid expressing HA-Vpr (WT) or an empty plasmid as negative control. Cell extracts were processed and analyzed as in A).

    Journal: PLoS Pathogens

    Article Title: Formation of Mobile Chromatin-Associated Nuclear Foci Containing HIV-1 Vpr and VPRBP Is Critical for the Induction of G2 Cell Cycle Arrest

    doi: 10.1371/journal.ppat.1001080

    Figure Lengend Snippet: Association of Vpr with chromatin correlates with the formation of nuclear foci. A ) HeLa cells were transfected with plasmids expressing HA-tagged Vpr (WT) or an empty plasmid used as negative control. Forty-eight hours after transfection, cells were harvested and lysed with 0.5% Triton X-100. The soluble fraction was used as input control (Soluble). Insoluble debris containing chromatin was treated with microccocal nuclease (+MNase) or with buffer alone (−MNase). The resulting solubilized fractions and input controls were resolved by SDS-PAGE and analyzed by western blot. Specific monoclonal antibodies were used to detect GAPDH (cytoplasmic marker) and HA-Vpr. Histone 3 (chromatin marker) and VPRBP were detected using rabbit polyclonal antibodies. * Denotes a non-specific band detected with the anti-HA antibody. B ) HeLa cells were transfected with plasmids expressing HA-tagged Vpr (WT), Vpr (Q65R), Vpr (R80A), and Vpr (1–78). Cell extracts were processed and analysed as in A). C ) HeLa cells were first transfected with scrambled siRNA or siRNA targeting VPRBP. Twenty-four hours after transfection, cells were transfected with a plasmid expressing HA-Vpr (WT) or an empty plasmid as negative control. Cell extracts were processed and analyzed as in A).

    Article Snippet: Pellets were washed once with nuclease buffer (50 mM Tris pH 8.0, 5 mM CaCl2 , and 100 µg/ml BSA), split in two, and resuspended in nuclease buffer alone or nuclease buffer containing 200 U/ml microccocal nuclease (New England Biolabs, Ipswich, MA, USA).

    Techniques: Transfection, Expressing, Plasmid Preparation, Negative Control, SDS Page, Western Blot, Marker

    DNA nucleases induce NET digestion . (A) Migration profile of pure λDNA after digestion with 4 U/mL DNase, MNase, or Alu-I. (B) Alu-I, DNase, and MNase dose-effects on NET dsDNA obtained after A23187 stimulation of PMN. Incubation with the restriction enzymes lasted 20 min at 37°C. DNA migration took place in 0.8% agarose gel containing ethidium bromide.

    Journal: Frontiers in Immunology

    Article Title: An Improved Strategy to Recover Large Fragments of Functional Human Neutrophil Extracellular Traps

    doi: 10.3389/fimmu.2013.00166

    Figure Lengend Snippet: DNA nucleases induce NET digestion . (A) Migration profile of pure λDNA after digestion with 4 U/mL DNase, MNase, or Alu-I. (B) Alu-I, DNase, and MNase dose-effects on NET dsDNA obtained after A23187 stimulation of PMN. Incubation with the restriction enzymes lasted 20 min at 37°C. DNA migration took place in 0.8% agarose gel containing ethidium bromide.

    Article Snippet: NET digestion by nucleases In a first set of experiments, 4 μg of purified λDNA (Invitrogen) was treated with 4 U/mL DNase I (Sigma Aldrich), 4 U/mL MNase (New England BioLabs, France), or 4 U/mL Alu I (New England BioLabs) for 20 min at 37°C.

    Techniques: Migration, Incubation, Agarose Gel Electrophoresis

    Histone deacetylation contributes to stable gene silencing. ( A ) RT-PCR showed that 1 ng/ml TSA for 24 hr significantly relieved Ikaros-induced reduced repression of Igll and Myc primary transcripts. Mean ± SE, 3 independent biological replicates. ( B ) MNase PCR showed that 1 ng/ml TSA for 24 hr did not significantly affect protection of 80–120 bp amplicons (short, left) but significantly reduced protection of 130–140 bp amplicons (long, right) at the Igll1 promoter. Mean ± SE, 3 independent biological replicates. ( C ) ChIP-PCR to assess Ikaros-induced recruitment of histone H2B to the Igll1 promoter between control cells and cells treated with 1 ng/ml TSA for 24 hr. Enrichment was normalised to total H3. Mean ± SE, 3 independent biological replicates. TSA significantly blunted the Ikaros-induced increase the H2B/H3 ratio at the Igll1 promoter and TSS. ( D ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, cells were treated over night with TSA (1 ng/ml) and/or 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of TSA was statistically significant across replicates (p=9.54 × 10-18 GLM binomial logit). DOI: http://dx.doi.org/10.7554/eLife.22767.021 10.7554/eLife.22767.022 Numerical data used to generate Figure 6A,B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.022

    Journal: eLife

    Article Title: A high-resolution map of transcriptional repression

    doi: 10.7554/eLife.22767

    Figure Lengend Snippet: Histone deacetylation contributes to stable gene silencing. ( A ) RT-PCR showed that 1 ng/ml TSA for 24 hr significantly relieved Ikaros-induced reduced repression of Igll and Myc primary transcripts. Mean ± SE, 3 independent biological replicates. ( B ) MNase PCR showed that 1 ng/ml TSA for 24 hr did not significantly affect protection of 80–120 bp amplicons (short, left) but significantly reduced protection of 130–140 bp amplicons (long, right) at the Igll1 promoter. Mean ± SE, 3 independent biological replicates. ( C ) ChIP-PCR to assess Ikaros-induced recruitment of histone H2B to the Igll1 promoter between control cells and cells treated with 1 ng/ml TSA for 24 hr. Enrichment was normalised to total H3. Mean ± SE, 3 independent biological replicates. TSA significantly blunted the Ikaros-induced increase the H2B/H3 ratio at the Igll1 promoter and TSS. ( D ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, cells were treated over night with TSA (1 ng/ml) and/or 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of TSA was statistically significant across replicates (p=9.54 × 10-18 GLM binomial logit). DOI: http://dx.doi.org/10.7554/eLife.22767.021 10.7554/eLife.22767.022 Numerical data used to generate Figure 6A,B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.022

    Article Snippet: For MNase-seq, 50 ng DNA after MNase digestion (carrier ethanol treatment for 6 hr or 0.5 μM 4-OHT treatment for 6 hr) were used to prepare MNase-seq samples (Next Ultra, NEB) without size selection steps.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Fluorescence In Situ Hybridization

    Ikaros controls promoter accessibility through NuRD-associated chromatin remodeling. ( A ) Left: CHD4 expression in control and Chd4 shRNA cells by western blotting. Tubulin is a loading control. One of 5 independent biological replicates. Right: Experimental outline. ( B ) MNase-PCR at the Igll1 and Myc promoters in control (black) or Chd4 shRNA cells (red) at the indicated times after 4-OHT. Mean ± SE, 3 independent biological replicates. Chd4 shRNA significantly reduced the Ikaros-induced increase in nucleosome occupancy at 15, 30 and 120 min at the Igll1 promoter and at 30 and 120 min at the Myc promoter. ( C ) RNAP2 ChIP-PCR (top) and MNase-PCR (bottom) at the Igll1 and Myc promoters after 4-OHT in control (black) or Chd4 shRNA cells (red). Mean ± SE, 3 independent biological replicates. RNAP2 binding was significantly reduced in control cells but not in Chd4 shRNA-treated cells from 5 to 120 min after 4-OHT at the Igll1 and the Myc promoter. Primary transcripts were significantly reduced in control but not in Chd4 shRNA-treated cells at 15 and 30 min for Igll1 and at 30 and 120 min for Myc . ( D ) ChIP-PCR for CHD4 (black), MBD3 (grey) and BRG1 (orange) at the Igll1 promoters at the indicated times after 4-OHT. Mean ± SE, 5 independent biological replicates for CHD4 and BRG1, 3 independent biological replicates for MBD3. CHD4 and MBD3 binding at the Igll1 promoter were significantly increased from 5 to 60 min. BRG1 binding was significantly decreased from 30 to 120 min. DOI: http://dx.doi.org/10.7554/eLife.22767.015 10.7554/eLife.22767.016 Numerical data used to generate Figure 4B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.016

    Journal: eLife

    Article Title: A high-resolution map of transcriptional repression

    doi: 10.7554/eLife.22767

    Figure Lengend Snippet: Ikaros controls promoter accessibility through NuRD-associated chromatin remodeling. ( A ) Left: CHD4 expression in control and Chd4 shRNA cells by western blotting. Tubulin is a loading control. One of 5 independent biological replicates. Right: Experimental outline. ( B ) MNase-PCR at the Igll1 and Myc promoters in control (black) or Chd4 shRNA cells (red) at the indicated times after 4-OHT. Mean ± SE, 3 independent biological replicates. Chd4 shRNA significantly reduced the Ikaros-induced increase in nucleosome occupancy at 15, 30 and 120 min at the Igll1 promoter and at 30 and 120 min at the Myc promoter. ( C ) RNAP2 ChIP-PCR (top) and MNase-PCR (bottom) at the Igll1 and Myc promoters after 4-OHT in control (black) or Chd4 shRNA cells (red). Mean ± SE, 3 independent biological replicates. RNAP2 binding was significantly reduced in control cells but not in Chd4 shRNA-treated cells from 5 to 120 min after 4-OHT at the Igll1 and the Myc promoter. Primary transcripts were significantly reduced in control but not in Chd4 shRNA-treated cells at 15 and 30 min for Igll1 and at 30 and 120 min for Myc . ( D ) ChIP-PCR for CHD4 (black), MBD3 (grey) and BRG1 (orange) at the Igll1 promoters at the indicated times after 4-OHT. Mean ± SE, 5 independent biological replicates for CHD4 and BRG1, 3 independent biological replicates for MBD3. CHD4 and MBD3 binding at the Igll1 promoter were significantly increased from 5 to 60 min. BRG1 binding was significantly decreased from 30 to 120 min. DOI: http://dx.doi.org/10.7554/eLife.22767.015 10.7554/eLife.22767.016 Numerical data used to generate Figure 4B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.016

    Article Snippet: For MNase-seq, 50 ng DNA after MNase digestion (carrier ethanol treatment for 6 hr or 0.5 μM 4-OHT treatment for 6 hr) were used to prepare MNase-seq samples (Next Ultra, NEB) without size selection steps.

    Techniques: Expressing, shRNA, Western Blot, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Binding Assay

    Nuclear translocation of Ikaros by proteolytic cleavage of Ikaros fusion proteins. ( A ) Schematic representation of Ikaros translocation induced by the proteolytic cleavage of Ikaros-TEV-ERt2 by Tobacco Etch Virus (TEV) protease. Ikaros-TEV-ERt2 fusion proteins are retained in the cytompasm until cleavage. The separate N- and C-termini of TEV protease are fused to FKBP and FRB, respectively. TEV activity is restored by the addition of rapamycin (R) ( Wehr et al., 2006 ). Cleavage separates Ikaros from ERt2. The efficiency of inducible cleavage after rapamycin (2 hr, 25 nM) is monitored by western blotting using Ikaros antibodies (right). Actin was used as loading control. Representative of 3 independent biological replicates. ( B ) MNase digestion followed by PCR was used to determine changes in the accessibility of the Igll1 and Myc promoters after triptolide-induced removal of RNAP2. The experiment complements the data shown in Figure 3B and C , but Split-TEV was used instead of 4-OHT-induced nuclear translocation of Ikaros. Split-TEV was activated by the addition of rapamycin at 25 nM for 2 hr. Mean ± SE, 3 independent biological replicates. Ikaros induction and tripolide-mediated RNAP2 depletion may synergise in increasing the nucleosome occupancy of the Igll1 promoter. DOI: http://dx.doi.org/10.7554/eLife.22767.013 10.7554/eLife.22767.014 Numerical data used to generate Figure 3—figure supplement 1A,B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.014

    Journal: eLife

    Article Title: A high-resolution map of transcriptional repression

    doi: 10.7554/eLife.22767

    Figure Lengend Snippet: Nuclear translocation of Ikaros by proteolytic cleavage of Ikaros fusion proteins. ( A ) Schematic representation of Ikaros translocation induced by the proteolytic cleavage of Ikaros-TEV-ERt2 by Tobacco Etch Virus (TEV) protease. Ikaros-TEV-ERt2 fusion proteins are retained in the cytompasm until cleavage. The separate N- and C-termini of TEV protease are fused to FKBP and FRB, respectively. TEV activity is restored by the addition of rapamycin (R) ( Wehr et al., 2006 ). Cleavage separates Ikaros from ERt2. The efficiency of inducible cleavage after rapamycin (2 hr, 25 nM) is monitored by western blotting using Ikaros antibodies (right). Actin was used as loading control. Representative of 3 independent biological replicates. ( B ) MNase digestion followed by PCR was used to determine changes in the accessibility of the Igll1 and Myc promoters after triptolide-induced removal of RNAP2. The experiment complements the data shown in Figure 3B and C , but Split-TEV was used instead of 4-OHT-induced nuclear translocation of Ikaros. Split-TEV was activated by the addition of rapamycin at 25 nM for 2 hr. Mean ± SE, 3 independent biological replicates. Ikaros induction and tripolide-mediated RNAP2 depletion may synergise in increasing the nucleosome occupancy of the Igll1 promoter. DOI: http://dx.doi.org/10.7554/eLife.22767.013 10.7554/eLife.22767.014 Numerical data used to generate Figure 3—figure supplement 1A,B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.014

    Article Snippet: For MNase-seq, 50 ng DNA after MNase digestion (carrier ethanol treatment for 6 hr or 0.5 μM 4-OHT treatment for 6 hr) were used to prepare MNase-seq samples (Next Ultra, NEB) without size selection steps.

    Techniques: Translocation Assay, Activity Assay, Western Blot, Polymerase Chain Reaction

    Interdependence of silencing mechanisms leveraged by Ikaros. ( A ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, control or sh Chd4 cells were treated over night with 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of Chd4 knockdown was statistically significant across replicates (p=5.54×10-38 GLM binomial logit). ( B ) ChIP kinetics of Ikaros and EBF binding to the Igll1 promoter in control (left) and sh Chd4 cells (right). Increased binding of Ikaros to the Igll1 promoter was significant for both control and sh Chd4 cells, decreased binding of EBF1 was significant in control, but not in sh Chd4 cells. Mean ± SE, 3 independent biological replicates. Ikaros and EBF1 binding at 15, 30 and 120 min were significantly higher in sh Chd4 than control cells. ( C ) MNase-seq data from 3 independent biological replicates were integrated with Ikaros ChIP-seq data to show nucleosome occupancy at Ikaros binding peaks before and 6 hr after nuclear translocation of Ikaros. ( D ) Dynamics of Ikaros binding, RNAP2 eviction, loss of primary transcripts, nucleosome invasion, and histone deacetylation. DOI: http://dx.doi.org/10.7554/eLife.22767.023 10.7554/eLife.22767.024 Numerical data used to generate Figure 7A and B . DOI: http://dx.doi.org/10.7554/eLife.22767.024

    Journal: eLife

    Article Title: A high-resolution map of transcriptional repression

    doi: 10.7554/eLife.22767

    Figure Lengend Snippet: Interdependence of silencing mechanisms leveraged by Ikaros. ( A ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, control or sh Chd4 cells were treated over night with 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of Chd4 knockdown was statistically significant across replicates (p=5.54×10-38 GLM binomial logit). ( B ) ChIP kinetics of Ikaros and EBF binding to the Igll1 promoter in control (left) and sh Chd4 cells (right). Increased binding of Ikaros to the Igll1 promoter was significant for both control and sh Chd4 cells, decreased binding of EBF1 was significant in control, but not in sh Chd4 cells. Mean ± SE, 3 independent biological replicates. Ikaros and EBF1 binding at 15, 30 and 120 min were significantly higher in sh Chd4 than control cells. ( C ) MNase-seq data from 3 independent biological replicates were integrated with Ikaros ChIP-seq data to show nucleosome occupancy at Ikaros binding peaks before and 6 hr after nuclear translocation of Ikaros. ( D ) Dynamics of Ikaros binding, RNAP2 eviction, loss of primary transcripts, nucleosome invasion, and histone deacetylation. DOI: http://dx.doi.org/10.7554/eLife.22767.023 10.7554/eLife.22767.024 Numerical data used to generate Figure 7A and B . DOI: http://dx.doi.org/10.7554/eLife.22767.024

    Article Snippet: For MNase-seq, 50 ng DNA after MNase digestion (carrier ethanol treatment for 6 hr or 0.5 μM 4-OHT treatment for 6 hr) were used to prepare MNase-seq samples (Next Ultra, NEB) without size selection steps.

    Techniques: Fluorescence In Situ Hybridization, Chromatin Immunoprecipitation, Binding Assay, Translocation Assay

    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: The nucleosome mixture (94 nM), containing all types of nucleosomes, was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1, 3, 6 and 10 min, in 50 mM Tris–HCl (pH 8.0) buffer, containing 2.5 mM CaCl2 , 69 mM NaCl, 81 mM KCl and 1.9 mM dithiothreitol.

    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: The nucleosome mixture (94 nM), containing all types of nucleosomes, was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1, 3, 6 and 10 min, in 50 mM Tris–HCl (pH 8.0) buffer, containing 2.5 mM CaCl2 , 69 mM NaCl, 81 mM KCl and 1.9 mM dithiothreitol.

    Techniques: Incubation, Atomic Absorption Spectroscopy

    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: The nucleosome mixture (94 nM), containing all types of nucleosomes, was incubated with 5.5 units/ml of MNase (Takara) at 37°C for 1, 3, 6 and 10 min, in 50 mM Tris–HCl (pH 8.0) buffer, containing 2.5 mM CaCl2 , 69 mM NaCl, 81 mM KCl and 1.9 mM dithiothreitol.

    Techniques: Sequencing

    shRNA-mediated inactivation of ATRX does not alter subtelomeric chromatin accessibility to MNase. (A) Chromatin isolated from 8-MG-BA glioma cells in which ATRX had been inactivated (shATRX) or not (shscrambled [shSCR]) was digested with MNase for the indicated times. (Left gel) Ethidium bromide (EtBr) staining of bulk chromatin. (Right gel) Southern blot with subtelomeric probe. (Far right) Quantification of the data. The signals obtained for mononucleosomes were normalized to the total signals measured for each time point (EtBr or Southern blot). (B) Chromatin samples from shATRX or shSCR 8-MG-BA cells were digested for 5 min with the indicated amounts of MNase (milliunits per microgram of DNA). (Far right) Quantification of the data.

    Journal: Molecular and Cellular Biology

    Article Title: Genetic Inactivation of ATRX Leads to a Decrease in the Amount of Telomeric Cohesin and Level of Telomere Transcription in Human Glioma Cells

    doi: 10.1128/MCB.01317-14

    Figure Lengend Snippet: shRNA-mediated inactivation of ATRX does not alter subtelomeric chromatin accessibility to MNase. (A) Chromatin isolated from 8-MG-BA glioma cells in which ATRX had been inactivated (shATRX) or not (shscrambled [shSCR]) was digested with MNase for the indicated times. (Left gel) Ethidium bromide (EtBr) staining of bulk chromatin. (Right gel) Southern blot with subtelomeric probe. (Far right) Quantification of the data. The signals obtained for mononucleosomes were normalized to the total signals measured for each time point (EtBr or Southern blot). (B) Chromatin samples from shATRX or shSCR 8-MG-BA cells were digested for 5 min with the indicated amounts of MNase (milliunits per microgram of DNA). (Far right) Quantification of the data.

    Article Snippet: Nuclei isolated from 107 cells were digested with 8 mU of micrococcal nuclease (MNase) (Sigma)/μg DNA at 37°C for the indicated times, as described previously ( ).

    Techniques: shRNA, Isolation, Staining, Southern Blot

    Nucleosome reassembly at the TSS is the initiating event in MLH1 resilencing. A , Regions assayed for nucleosome occupancy using MNase-qPCR (Regions I–IX) and NOMe-Seq (Regions N1 and N2). Shown beneath the gene schematic is NOMe-Seq data from untreated RKO cells at Region N1. Black arrows indicate the MLH1 and EPM2AIP1 TSS. Bottom panel represents GpC accessibility. Black circles = GpC dinucleotides methylated/accessible to the GpC methyltransferase M. Cvi PI. White circles = GpC dinucleotides not methylated/inaccessible to GpC methyltransferase. Pink shading indicates regions of inaccessibility of ≥150 bp. Asterix = region of M. Cvi PI accessibility. B , Relative nucleosome levels in untreated RKO cells at the indicated regions (black bars labeled Regions I–IX) as determined by MNase-qPCR. Drawn to scale with schematic shown in A. Error bars = SD. C and D , qPCR results showing changes in relative nucleosome levels at Regions III and VI following decitabine exposure. E and F , MLH1 gene expression (E) and promoter bisulfite pyrosequencing (F), reproduced from Figure 2A and D for ease of comparison with nucleosome levels. G–I , NOMe-Seq analysis of the MLH1 promoter at Region N2 in SW620 (F) and RKO (G,H) cells at the indicated treatment points. Black filled triangles = methylated CpG dinucleotides; white filled triangles = unmethylated CpG dinucleotides. See also Figure S2 .

    Journal: PLoS Genetics

    Article Title: Reassembly of Nucleosomes at the MLH1 Promoter Initiates Resilencing Following Decitabine Exposure

    doi: 10.1371/journal.pgen.1003636

    Figure Lengend Snippet: Nucleosome reassembly at the TSS is the initiating event in MLH1 resilencing. A , Regions assayed for nucleosome occupancy using MNase-qPCR (Regions I–IX) and NOMe-Seq (Regions N1 and N2). Shown beneath the gene schematic is NOMe-Seq data from untreated RKO cells at Region N1. Black arrows indicate the MLH1 and EPM2AIP1 TSS. Bottom panel represents GpC accessibility. Black circles = GpC dinucleotides methylated/accessible to the GpC methyltransferase M. Cvi PI. White circles = GpC dinucleotides not methylated/inaccessible to GpC methyltransferase. Pink shading indicates regions of inaccessibility of ≥150 bp. Asterix = region of M. Cvi PI accessibility. B , Relative nucleosome levels in untreated RKO cells at the indicated regions (black bars labeled Regions I–IX) as determined by MNase-qPCR. Drawn to scale with schematic shown in A. Error bars = SD. C and D , qPCR results showing changes in relative nucleosome levels at Regions III and VI following decitabine exposure. E and F , MLH1 gene expression (E) and promoter bisulfite pyrosequencing (F), reproduced from Figure 2A and D for ease of comparison with nucleosome levels. G–I , NOMe-Seq analysis of the MLH1 promoter at Region N2 in SW620 (F) and RKO (G,H) cells at the indicated treatment points. Black filled triangles = methylated CpG dinucleotides; white filled triangles = unmethylated CpG dinucleotides. See also Figure S2 .

    Article Snippet: Isolation of mononucleosome DNA using micrococcal nuclease (MNase) and qPCR A total of 1×107 cells were lysed on ice in 50 mM Tris HCl pH 7.9, 100 mM KCl, 5 mM MgCl2 , 50% (v/v) glycerol, 1.5% (v/v) β-mercaptoethanol, 0.1% (w/v) Saponin and Complete Protease Inhibitor with EDTA (Roche) followed by equilibration in 50 mM Tris HCl pH 7.5, 0.32 mM sucrose, 4 mM MgCl2 , 1 mM CaCl2 , and EDTA-free Complete Protease Inhibitors (Roche).

    Techniques: Real-time Polymerase Chain Reaction, Gel Permeation Chromatography, Methylation, Labeling, Expressing

    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: Southern Detection: MNase digested DNA (20–30 μg) was run in 1.5% agarose at 80V for 6 hr and Southern blotted as previously described ( ).

    Techniques: End Labeling, Standard Deviation

    Fun30 is required for normal CEN -flanking nucleosome positioning and/or CEN core particle structure. A) Genome browser trace of Fun30 ChIP enrichment and nucleosome dyad frequency centred on and surrounding yeast CEN1 . The upper trace shows Log 2 Fun30 ChIP-seq enrichment values binned at 10 bp intervals and smoothed with a 3 bin moving average. Wildtype (WT) and Δfun30 chromatin was digested with MNase and nuclease-protected DNA species sequenced using paired-end mode Illumina technology. Nucleosome sequencing data (nuc) traces were plotted as mirror images in the lower panel. The graph shows a map of the centre point positions of paired sequence reads with end-to-end distances of 150 bp+/−20% wild-type and Δfun30 mutant chromatin samples surrounding CEN1 . The frequency distributions, which effectively map chromatin particle dyads, were binned at 10 bp intervals, and smoothed by applying a 3 bin moving average. Peaks in the dyad distributions correspond to translationally-positioned nucleosomes in the original genome. The CEN core particle is also mapped using this method and can be visualised as a small peak centred on the CEN region marked with a grey box. Pink bars show the positions of ORFs (B–D) Genome browser plots of Fun30 ChIP-seq and nucleosome sequence distributions as described above for CEN10, 11 and 12 respectively. Fun30-dependent changes in the height of a nucleosome dyad or CEN core particle peak are marked with a red asterix. Fun30-dependent changes in the position of a CEN -flanking nucleosome dyad peak are marked with red arrows. Genome browser plots for all yeast CENs are shown in Figure S6 .

    Journal: PLoS Genetics

    Article Title: SWI/SNF-Like Chromatin Remodeling Factor Fun30 Supports Point Centromere Function in S. cerevisiae

    doi: 10.1371/journal.pgen.1002974

    Figure Lengend Snippet: Fun30 is required for normal CEN -flanking nucleosome positioning and/or CEN core particle structure. A) Genome browser trace of Fun30 ChIP enrichment and nucleosome dyad frequency centred on and surrounding yeast CEN1 . The upper trace shows Log 2 Fun30 ChIP-seq enrichment values binned at 10 bp intervals and smoothed with a 3 bin moving average. Wildtype (WT) and Δfun30 chromatin was digested with MNase and nuclease-protected DNA species sequenced using paired-end mode Illumina technology. Nucleosome sequencing data (nuc) traces were plotted as mirror images in the lower panel. The graph shows a map of the centre point positions of paired sequence reads with end-to-end distances of 150 bp+/−20% wild-type and Δfun30 mutant chromatin samples surrounding CEN1 . The frequency distributions, which effectively map chromatin particle dyads, were binned at 10 bp intervals, and smoothed by applying a 3 bin moving average. Peaks in the dyad distributions correspond to translationally-positioned nucleosomes in the original genome. The CEN core particle is also mapped using this method and can be visualised as a small peak centred on the CEN region marked with a grey box. Pink bars show the positions of ORFs (B–D) Genome browser plots of Fun30 ChIP-seq and nucleosome sequence distributions as described above for CEN10, 11 and 12 respectively. Fun30-dependent changes in the height of a nucleosome dyad or CEN core particle peak are marked with a red asterix. Fun30-dependent changes in the position of a CEN -flanking nucleosome dyad peak are marked with red arrows. Genome browser plots for all yeast CENs are shown in Figure S6 .

    Article Snippet: MNase digested chromatin samples processed for paired-end mode Illumina DNA sequencing.

    Techniques: Chromatin Immunoprecipitation, Sequencing, Mutagenesis

    VP2 binds to chromatin and interacts with MCM3 but this does not require dual phosphatase activity . ( A ) The soluble (S) and chromatin (C) fractions were prepared by using CSK buffer containing 0.5% Triton X-100 in CHO cells of transfected with VP2-GFP plasmid. The fractions were treated with 0 U (-) and 150 U (+) of MNase or the indicated concentrations of NaCl. Nuclear extracts (N) containing VP2-GFP were as positive control and all fractions were subjected to immunoblotting against with lamin B receptor, MCM3, and VP2 antibodies. ( B ) At 48 h post-transfection, the GFP (as Control) and Flag-VP2-GFP (as wild-type; WT) were found in the CHO cells. Next the lysates were immunoprecipitated using Flag M2 beads and immunoblotted against VP2 and MCM3 antibodies. ( C ) The WT and mutants (C95S, C97S, and C95S/C97S) of the Flag-VP2-GFP in CHO cells were also examined at 48 h post-transfection. The cell lysates were immunoprecipitated using Flag M2 beads and immunoblotted against with VP2 and MCM3 antibodies. The arrow head was indicated a none-specific band. The nuclear extracts containing VP2-GFP were designated as N

    Journal: BMC Veterinary Research

    Article Title: Identification of the NLS and NES motifs of VP2 from chicken anemia virus and the interaction of VP2 with mini-chromosome maintenance protein 3

    doi: 10.1186/1746-6148-8-15

    Figure Lengend Snippet: VP2 binds to chromatin and interacts with MCM3 but this does not require dual phosphatase activity . ( A ) The soluble (S) and chromatin (C) fractions were prepared by using CSK buffer containing 0.5% Triton X-100 in CHO cells of transfected with VP2-GFP plasmid. The fractions were treated with 0 U (-) and 150 U (+) of MNase or the indicated concentrations of NaCl. Nuclear extracts (N) containing VP2-GFP were as positive control and all fractions were subjected to immunoblotting against with lamin B receptor, MCM3, and VP2 antibodies. ( B ) At 48 h post-transfection, the GFP (as Control) and Flag-VP2-GFP (as wild-type; WT) were found in the CHO cells. Next the lysates were immunoprecipitated using Flag M2 beads and immunoblotted against VP2 and MCM3 antibodies. ( C ) The WT and mutants (C95S, C97S, and C95S/C97S) of the Flag-VP2-GFP in CHO cells were also examined at 48 h post-transfection. The cell lysates were immunoprecipitated using Flag M2 beads and immunoblotted against with VP2 and MCM3 antibodies. The arrow head was indicated a none-specific band. The nuclear extracts containing VP2-GFP were designated as N

    Article Snippet: For micrococcal nuclease (MNase) (Fermentas, Canada) or NaCl treatment, the chromatin was resuspended in 100 μl of CSK buffer supplemented with 0.1 M, 0.3 M of NaCl or 0 U (-) and 150 U (+) MNase with 2 mM CaCl2 and incubated for 30 min at 37°C.

    Techniques: Activity Assay, Transfection, Plasmid Preparation, Positive Control, Immunoprecipitation