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  • 99
    Millipore mnase
    rRNA genes are switched off in chromatin. ( A ) Reconstitution of mononucleosomes on a 330 bp (–175 to +155) rDNA fragment. The end-labelled DNA was reconstituted into mononucleosomes by salt dialysis reconstitution (lanes 1 and 2) and analysed by native PAGE. <t>Nucleosomal</t> DNA molecules harbouring a nucleosome on the transcription start site were selected by digestion with the restriction enzyme Rsa I (lane 3). The positions of the nucleosomal DNA, the undigested and digested DNA fragment are indicated. ( B ) Transcription assay with free and nucleosomal rDNA fragments. Increasing amounts of free DNA (lanes 1 and 2), a mixture of free DNA and nucleosomal DNA (lanes 3 and 4) and Rsa I-selected nucleosomal DNA (lanes 5–7) were incubated with the transcription extract. The radioactive labelled transcripts were analysed by native PAGE. The nucleosomal templates used for the transcription reactions are shown above the gel. The positions of the undigested or nucleosomal rDNA fragment, the digested free DNA and the 155-nt-long transcript are indicated on the right. ( C ) Analysis of nucleosome positions on the rDNA promoter. Mononucleosomal templates (–175 to +155) were digested with <t>MNase,</t> and the protected nucleosomal DNA was gel-purified, cloned and sequenced. The graph shows the positions of the nucleosomal dyad axis. The positions of the nucAct and nucRep nucleosomes observed in vivo are indicated with the 5′, 3′ and dyad axis positions relative to the rRNA gene transcription start site. E1 and E2 indicate the dyad axis positions of nucleosomes located at the end of the DNA fragment. ( D ) Prediction of nucleosome positioning by the probability of nucleosome occupancy and the probability of encountering a nucleosomal start site. rRNA sequences from position –5000 to +5000 relative to the transcription start site were used for computational analysis at http://genie.weizmann.ac.il/pubs/nucleosomes06/ ( 13 ). The graph displays a window of the calculated predictions, ranging from position –300 to +300 within the rDNA sequence. Peaks of high p (nucleosomal start) values, indicating a high probability for a nucleosomal start site, are indicated. The two nucleosomal positions identified on the rRNA gene in vivo are indicated [ nucAct –157 to –2 (green); the repressive nucleosome position –132 to +22 (red)].
    Mnase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 658 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Worthington Biochemical mnase
    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 .
    Mnase, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 96/100, based on 484 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher mnase
    <t>DKC1-associated</t> RNAs in recombinant DKC1 complexes are resistant to extensive <t>MNase</t> digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010
    Mnase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 269 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 nucleosomes 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 187 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mnase  (Roche)
    95
    Roche mnase
    Terminally unwrapped CENP-A <t>nucleosomes</t> and their conventional counterparts with wrapped termini are similarly phased at normal centromeres ( a,b ) The position of each individual CENP-A ( a ) or bulk nucleosome ( b ) along a dimerized α-satellite consensus sequence is indicated by a horizontal line. Each fragment is color-coded based on length, as indicated. ( c,d ) The midpoint positions of CENP-A ( c ) or bulk nucleosome ( d ) fragments along the dimer α-satellite consensus sequence. Solid vertical lines indicate the location of the 17 bp CENP-B box ( B ) in ( a–d ). ( e,f ) Models of the preferred positioning and <t>MNase</t> cleavage sites on CENP-A ( e ) and bulk ( f ) nucleosomes at normal centromeres.
    Mnase, supplied by Roche, used in various techniques. Bioz Stars score: 95/100, based on 222 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 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    New England Biolabs mnase seq samples
    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 ) <t>MNase</t> 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 <t>4-OHT.</t> 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
    Mnase Seq Samples, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 80/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Illumina Inc mnase digested chromatin
    CLOCK binds to <t>DNA</t> wrapped around nucleosomes. ( A ) CLOCK ChIP-seq signal on mononucleosome (i.e., mouse liver chromatin digested by <t>MNase)</t> at ZT22 (light blue; left ) and ZT06 (dark blue; right ) for the top 400 CLOCK:BMAL1 DNA-binding sites. The signal
    Mnase Digested Chromatin, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 79/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Partek sperm mnase tiling array data
    Aberrant chromatin composition in mouse models of altered PAR metabolism. Chromomycin A3 (CMA3) intercalation into the DNA indicates incomplete chromatin condensation in s perm from <t>Parg</t> (110) −/− (A) and PJ34-treated (C) males with histone retention. (B, D) Histogram of sperm CMA3-staining intensities reflects that severity of CM3A staining varied at the level of individual sperm and individual fathers (n > 200 nuclei/sample, 3 males/group). (E) Immunoblot analyses of sperm protein lysates showing increase in histone retention in PJ34 treated males. TUBA1A: alpha tubulin loading control. (F) Overlaps of genes identified as differentially histone associated in sperm from 3 individual Parg (110) −/− males (“PargA”, “PargB”, “PargC”, the fathers of the embryos analyzed below) by micrococcal nuclease digests (MND) compared to the wild-type controls. The “PargAll” data set contains all genes commonly identified as differentially <t>MNase-sensitive</t> across 10 Parg (110) −/− males compared with 9 wild-type control animals. The red circle indicates common genes that were differentially histone associated in all groups (1604+216 = 1820, red circle) compared with wild-type. (G) PJ34: differentially MNase-sensitive genes in three different males (like in E) and overlap with a surrogate dataset (“PJ34All”) consisting of data from all 4 PJ34-treated males compared with 9 wild-type control males. The overlap of 2,489 genes that were commonly differentially histone associated in sperm samples is indicated (blue circle). (H) Overlap of genes commonly affected by differential histone association between the Parg (110) −/− and the PJ34 models compared to wild-type controls (red and blue circles in F and G). A Pearson correlation examining significance of this overlap using a genetic background of 19,472 genes was calculated with a resulting P
    Sperm Mnase Tiling Array Data, supplied by Partek, used in various techniques. Bioz Stars score: 78/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    rRNA genes are switched off in chromatin. ( A ) Reconstitution of mononucleosomes on a 330 bp (–175 to +155) rDNA fragment. The end-labelled DNA was reconstituted into mononucleosomes by salt dialysis reconstitution (lanes 1 and 2) and analysed by native PAGE. Nucleosomal DNA molecules harbouring a nucleosome on the transcription start site were selected by digestion with the restriction enzyme Rsa I (lane 3). The positions of the nucleosomal DNA, the undigested and digested DNA fragment are indicated. ( B ) Transcription assay with free and nucleosomal rDNA fragments. Increasing amounts of free DNA (lanes 1 and 2), a mixture of free DNA and nucleosomal DNA (lanes 3 and 4) and Rsa I-selected nucleosomal DNA (lanes 5–7) were incubated with the transcription extract. The radioactive labelled transcripts were analysed by native PAGE. The nucleosomal templates used for the transcription reactions are shown above the gel. The positions of the undigested or nucleosomal rDNA fragment, the digested free DNA and the 155-nt-long transcript are indicated on the right. ( C ) Analysis of nucleosome positions on the rDNA promoter. Mononucleosomal templates (–175 to +155) were digested with MNase, and the protected nucleosomal DNA was gel-purified, cloned and sequenced. The graph shows the positions of the nucleosomal dyad axis. The positions of the nucAct and nucRep nucleosomes observed in vivo are indicated with the 5′, 3′ and dyad axis positions relative to the rRNA gene transcription start site. E1 and E2 indicate the dyad axis positions of nucleosomes located at the end of the DNA fragment. ( D ) Prediction of nucleosome positioning by the probability of nucleosome occupancy and the probability of encountering a nucleosomal start site. rRNA sequences from position –5000 to +5000 relative to the transcription start site were used for computational analysis at http://genie.weizmann.ac.il/pubs/nucleosomes06/ ( 13 ). The graph displays a window of the calculated predictions, ranging from position –300 to +300 within the rDNA sequence. Peaks of high p (nucleosomal start) values, indicating a high probability for a nucleosomal start site, are indicated. The two nucleosomal positions identified on the rRNA gene in vivo are indicated [ nucAct –157 to –2 (green); the repressive nucleosome position –132 to +22 (red)].

    Journal: Nucleic Acids Research

    Article Title: DNA sequence encoded repression of rRNA gene transcription in chromatin

    doi: 10.1093/nar/gkq263

    Figure Lengend Snippet: rRNA genes are switched off in chromatin. ( A ) Reconstitution of mononucleosomes on a 330 bp (–175 to +155) rDNA fragment. The end-labelled DNA was reconstituted into mononucleosomes by salt dialysis reconstitution (lanes 1 and 2) and analysed by native PAGE. Nucleosomal DNA molecules harbouring a nucleosome on the transcription start site were selected by digestion with the restriction enzyme Rsa I (lane 3). The positions of the nucleosomal DNA, the undigested and digested DNA fragment are indicated. ( B ) Transcription assay with free and nucleosomal rDNA fragments. Increasing amounts of free DNA (lanes 1 and 2), a mixture of free DNA and nucleosomal DNA (lanes 3 and 4) and Rsa I-selected nucleosomal DNA (lanes 5–7) were incubated with the transcription extract. The radioactive labelled transcripts were analysed by native PAGE. The nucleosomal templates used for the transcription reactions are shown above the gel. The positions of the undigested or nucleosomal rDNA fragment, the digested free DNA and the 155-nt-long transcript are indicated on the right. ( C ) Analysis of nucleosome positions on the rDNA promoter. Mononucleosomal templates (–175 to +155) were digested with MNase, and the protected nucleosomal DNA was gel-purified, cloned and sequenced. The graph shows the positions of the nucleosomal dyad axis. The positions of the nucAct and nucRep nucleosomes observed in vivo are indicated with the 5′, 3′ and dyad axis positions relative to the rRNA gene transcription start site. E1 and E2 indicate the dyad axis positions of nucleosomes located at the end of the DNA fragment. ( D ) Prediction of nucleosome positioning by the probability of nucleosome occupancy and the probability of encountering a nucleosomal start site. rRNA sequences from position –5000 to +5000 relative to the transcription start site were used for computational analysis at http://genie.weizmann.ac.il/pubs/nucleosomes06/ ( 13 ). The graph displays a window of the calculated predictions, ranging from position –300 to +300 within the rDNA sequence. Peaks of high p (nucleosomal start) values, indicating a high probability for a nucleosomal start site, are indicated. The two nucleosomal positions identified on the rRNA gene in vivo are indicated [ nucAct –157 to –2 (green); the repressive nucleosome position –132 to +22 (red)].

    Article Snippet: For high resolution mapping of nucleosome positions, purified nucleosomal arrays (300 ng) were digested with 1.5 U of MNase (Sigma) for 40 s. Reactions were conducted in 10 mM Tris–HCl (pH 7.6), 80 mM KCl, 1.5 mM MgCl2 , 10% glycerol, 0.5 mM ATP and 200 ng/μl of bovine serum albumin.

    Techniques: Clear Native PAGE, Incubation, Purification, Clone Assay, In Vivo, Sequencing

    PK inhibits nucleosome remodelling at the rDNA promoter. ( A ) MNase digestion of chromatin assembled on the rDNA minigene (pMrWT-T). Chromatin reconstituted with the Drosophila extract was digested with MNase for 0.5–3 min (lanes 1–3) or for 0.5–6 min (lanes 4–8) in the presence of 600 μM PK. The nucleosomal ladder (1n-5n) and the DNA marker (M; 1kb ladder) are indicated. ( B ) Chromatin assembled on pMrWT-T was incubated in the absence or presence of TTF-I and partially digested with MNase. Purified DNA was digested with EcoRI, separated on an agarose gel and transferred onto a nylon membrane. Chromatin configuration around the TTF-I-binding site (T 0 ) was visualized by indirect end-labelling (lanes 1 and 2). Chromatin remodelling was monitored in the presence of K (lanes 4 and 5; 300 and 600 μM) or PK (lanes 6 and 7, 300 and 600 μM). The position of the TTF-I-binding site is indicated by Sal I digestion of the template DNA (lane 3). Open circles mark non-positioned nucleosomes, whereas the gray circles indicate positioned nucleosomes. The position of the TTF-I-binding site (gray box), MNase-protected DNA regions (black triangles) and MNase-sensitive regions (white triangles) are indicated. The strong band in lane 6 (marked with an asterisk) arises due to the relatively lower MNase digestion of this sample.

    Journal: Nucleic Acids Research

    Article Title: DNA sequence encoded repression of rRNA gene transcription in chromatin

    doi: 10.1093/nar/gkq263

    Figure Lengend Snippet: PK inhibits nucleosome remodelling at the rDNA promoter. ( A ) MNase digestion of chromatin assembled on the rDNA minigene (pMrWT-T). Chromatin reconstituted with the Drosophila extract was digested with MNase for 0.5–3 min (lanes 1–3) or for 0.5–6 min (lanes 4–8) in the presence of 600 μM PK. The nucleosomal ladder (1n-5n) and the DNA marker (M; 1kb ladder) are indicated. ( B ) Chromatin assembled on pMrWT-T was incubated in the absence or presence of TTF-I and partially digested with MNase. Purified DNA was digested with EcoRI, separated on an agarose gel and transferred onto a nylon membrane. Chromatin configuration around the TTF-I-binding site (T 0 ) was visualized by indirect end-labelling (lanes 1 and 2). Chromatin remodelling was monitored in the presence of K (lanes 4 and 5; 300 and 600 μM) or PK (lanes 6 and 7, 300 and 600 μM). The position of the TTF-I-binding site is indicated by Sal I digestion of the template DNA (lane 3). Open circles mark non-positioned nucleosomes, whereas the gray circles indicate positioned nucleosomes. The position of the TTF-I-binding site (gray box), MNase-protected DNA regions (black triangles) and MNase-sensitive regions (white triangles) are indicated. The strong band in lane 6 (marked with an asterisk) arises due to the relatively lower MNase digestion of this sample.

    Article Snippet: For high resolution mapping of nucleosome positions, purified nucleosomal arrays (300 ng) were digested with 1.5 U of MNase (Sigma) for 40 s. Reactions were conducted in 10 mM Tris–HCl (pH 7.6), 80 mM KCl, 1.5 mM MgCl2 , 10% glycerol, 0.5 mM ATP and 200 ng/μl of bovine serum albumin.

    Techniques: Marker, Incubation, Purification, Agarose Gel Electrophoresis, Binding Assay

    TTF-I-dependent chromatin dynamics at the rRNA gene promoter. Reconstituted nucleosomal arrays were incubated for 90 min with the TxE, TTF-I, K (600 μM) or PK (600 μM) as indicated. Nucleosome positions at the rDNA promoter were mapped by partial MNase digestion and primer extension of the purified DNA. DNA fragments were resolved on 8% sequencing gels and quantified with a PhosphorImager. The graph shows the positions (relative to the transcription start site, +1; site is marked by boxes) and relative intensities of the MNase cleavage sites corresponding to the 3′ boundaries of positioned nucleosomes. Boxes highlight the MNase cleavage sites around the transcription start site, correlating with the nucleosome position nucAct . The position of the oligonucleotide used for primer extension and the major MNase-sensitive sites on the rDNA are indicated. The scan of the DNA marker (10-bp ladder) is shown below the graphs.

    Journal: Nucleic Acids Research

    Article Title: DNA sequence encoded repression of rRNA gene transcription in chromatin

    doi: 10.1093/nar/gkq263

    Figure Lengend Snippet: TTF-I-dependent chromatin dynamics at the rRNA gene promoter. Reconstituted nucleosomal arrays were incubated for 90 min with the TxE, TTF-I, K (600 μM) or PK (600 μM) as indicated. Nucleosome positions at the rDNA promoter were mapped by partial MNase digestion and primer extension of the purified DNA. DNA fragments were resolved on 8% sequencing gels and quantified with a PhosphorImager. The graph shows the positions (relative to the transcription start site, +1; site is marked by boxes) and relative intensities of the MNase cleavage sites corresponding to the 3′ boundaries of positioned nucleosomes. Boxes highlight the MNase cleavage sites around the transcription start site, correlating with the nucleosome position nucAct . The position of the oligonucleotide used for primer extension and the major MNase-sensitive sites on the rDNA are indicated. The scan of the DNA marker (10-bp ladder) is shown below the graphs.

    Article Snippet: For high resolution mapping of nucleosome positions, purified nucleosomal arrays (300 ng) were digested with 1.5 U of MNase (Sigma) for 40 s. Reactions were conducted in 10 mM Tris–HCl (pH 7.6), 80 mM KCl, 1.5 mM MgCl2 , 10% glycerol, 0.5 mM ATP and 200 ng/μl of bovine serum albumin.

    Techniques: Incubation, Purification, Sequencing, Marker

    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:

    Chromatin-remodeling (nucleosome insertion and rearrangement) on Nanog gene promoter. ( A ) ESCs were treated with RA for 3 days, then treated with 5, 10 and 30 U of MNase for 6 min at 37°C. Extracted chromatin DNA was separated on 1.5% agarose gels followed by Southern blot hybridization with 32 P-labeled 500-bp probes specific to three regions on the Nanog promoter. Positions of probes are depicted on the map. ( B ) Restriction enzyme accessibility of Nanog promoter region in ESCs. Asterisk marks the diagnostic band indicative of each sensitive site. The results are summarized and shown on the map above these blots. Restriction sites are labeled under the map. ( C ) N1, N2, N3 and N4 PCR fragments, amplified from mononucleosomal DNA using the primers a/b (N1), c/d (N2), e/f (N3) and g/h (N4). Primer sequences were listed in Supplementary Table S1 . Genomic DNAs isolated from 1 × 10 6 cells were used for N1 amplification as control (upper panel). ESCs were transfected with siRNA specific for Brm or siRNA control. Changes in mononucleosome formation on the Nanog gene promoter were monitored (bottom panel). ( D ) Upper: nucleosome occupancy on the Nanog promoter in ESCs with or without RA treatment. The gray-highlighted region (q8–12) represents the location of the N2 nucleosome formation. Lower: analysis of nucleosome occupancy at the q8–12 regions of Nanog in control and Brm-knockdown ES cell by the MNase resistance assay. Data points represent average qPCR signals from two independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Coordinated repressive chromatin-remodeling of Oct4 and Nanog genes in RA-induced differentiation of embryonic stem cells involves RIP140

    doi: 10.1093/nar/gku092

    Figure Lengend Snippet: Chromatin-remodeling (nucleosome insertion and rearrangement) on Nanog gene promoter. ( A ) ESCs were treated with RA for 3 days, then treated with 5, 10 and 30 U of MNase for 6 min at 37°C. Extracted chromatin DNA was separated on 1.5% agarose gels followed by Southern blot hybridization with 32 P-labeled 500-bp probes specific to three regions on the Nanog promoter. Positions of probes are depicted on the map. ( B ) Restriction enzyme accessibility of Nanog promoter region in ESCs. Asterisk marks the diagnostic band indicative of each sensitive site. The results are summarized and shown on the map above these blots. Restriction sites are labeled under the map. ( C ) N1, N2, N3 and N4 PCR fragments, amplified from mononucleosomal DNA using the primers a/b (N1), c/d (N2), e/f (N3) and g/h (N4). Primer sequences were listed in Supplementary Table S1 . Genomic DNAs isolated from 1 × 10 6 cells were used for N1 amplification as control (upper panel). ESCs were transfected with siRNA specific for Brm or siRNA control. Changes in mononucleosome formation on the Nanog gene promoter were monitored (bottom panel). ( D ) Upper: nucleosome occupancy on the Nanog promoter in ESCs with or without RA treatment. The gray-highlighted region (q8–12) represents the location of the N2 nucleosome formation. Lower: analysis of nucleosome occupancy at the q8–12 regions of Nanog in control and Brm-knockdown ES cell by the MNase resistance assay. Data points represent average qPCR signals from two independent experiments.

    Article Snippet: Nuclei isolated from ESCs were digested with 5 and 30 U of MNase (Worthington, Lakewood, NJ, USA, www.worthington-biochem.com ) at 37°C for 5 min, followed by proteinase K treatment at 37°C overnight.

    Techniques: Southern Blot, Hybridization, Labeling, Diagnostic Assay, Polymerase Chain Reaction, Amplification, Isolation, Transfection, Real-time Polymerase Chain Reaction

    DKC1-associated RNAs in recombinant DKC1 complexes are resistant to extensive MNase digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010

    Journal: eLife

    Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

    doi: 10.7554/eLife.03573

    Figure Lengend Snippet: DKC1-associated RNAs in recombinant DKC1 complexes are resistant to extensive MNase digestion. RNAs co-purified from mock and MNase-treated recombinant DKC1 complexes in Figure 4—figure supplement 1 are 5′ end radiolabeled and separated on a 6% urea-polyacrylamide gel as described in Figure 2—figure supplement 1 . Note that the prominently labeled 130–140 nucleotide (nt)-long RNA clusters are resistant to complete nuclease digestion. It appears that these RNAs are cut on average once by MNase to generate two new smaller clusters (80–90 nt and 30–55 nt) that remain stably associated with the DKC1 complexes. Increasing the amount of MNase and/or nuclease digestion time did not change the patterns or disrupt the integrity of the protein complexes (data not shown). DOI: http://dx.doi.org/10.7554/eLife.03573.010

    Article Snippet: Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

    Techniques: Recombinant, Purification, Labeling, Stable Transfection

    Micrococcal nuclease (MNase)-treated recombinant DKC1 complexes remain structurally intact. Recombinant wild-type (WT) and various mutant DKC1 complexes are mock treated (−) or digested extensively with MNase (+), and washed extensively to remove any dissociated RNAs prior to FLAG peptide elution. Eluted protein complexes are analyzed by Coomassie Blue staining. DOI: http://dx.doi.org/10.7554/eLife.03573.009

    Journal: eLife

    Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

    doi: 10.7554/eLife.03573

    Figure Lengend Snippet: Micrococcal nuclease (MNase)-treated recombinant DKC1 complexes remain structurally intact. Recombinant wild-type (WT) and various mutant DKC1 complexes are mock treated (−) or digested extensively with MNase (+), and washed extensively to remove any dissociated RNAs prior to FLAG peptide elution. Eluted protein complexes are analyzed by Coomassie Blue staining. DOI: http://dx.doi.org/10.7554/eLife.03573.009

    Article Snippet: Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

    Techniques: Recombinant, Mutagenesis, Staining

    MNase digestion moderately increases DKC1 coactivator activity. Mock (−) or MNase-treated (+) WT and Nop10 R34W DKC1 complexes are assayed in in vitro transcription reactions (over a fourfold concentration range) supplemented with OCT4, SOX2, recombinant XPC complex and SCC-B. DOI: http://dx.doi.org/10.7554/eLife.03573.011

    Journal: eLife

    Article Title: The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

    doi: 10.7554/eLife.03573

    Figure Lengend Snippet: MNase digestion moderately increases DKC1 coactivator activity. Mock (−) or MNase-treated (+) WT and Nop10 R34W DKC1 complexes are assayed in in vitro transcription reactions (over a fourfold concentration range) supplemented with OCT4, SOX2, recombinant XPC complex and SCC-B. DOI: http://dx.doi.org/10.7554/eLife.03573.011

    Article Snippet: Bound DKC1 complexes were treated with 300 U of MNase (Thermo Scientific, Waltham, MA) or buffer at room temperature and nutated for 1 hr.

    Techniques: Activity Assay, In Vitro, Concentration Assay, Recombinant

    Neutrophils release calprotectin by forming NETs. (A–F) Confocal images of human neutrophils without stimulation (A), after 0.5 h (B), 1 h (C), 2 h (D), 3 h (E) and 4 h (F) after activation. Samples were stained with antibodies specific for the calprotectin heteroduplex (red) and for MPO (green). DNA was stained with DRAQ5 (blue). Calprotectin localizes to the cytoplasm and partially to the nucleus (A, arrow). After stimulation for 0.5 h (B) the neutrophils flattened and formed numerous vacuoles. This reveals a granular staining for MPO and a more dispersed cytoplasmic staining for calprotectin. After stimulation for 1 h (C) the neutrophils round up slightly. The MPO and calprotectin stain partially overlap in the cytoplasm. After stimulation for 2 h (D), calprotectin, MPO and nuclear DNA start to colocalize in the decondensed nucleus (purple). After 3 h (E) and more so after 4 h (F) of stimulation, the cell membrane ruptures and calprotectin is released in NETs colocalizing with MPO and DNA. Scale bar = 10 µm; one experiment out of two is shown. (G–I) Subunits of calprotectin S100A8 and S100A9 are released after cell death during NET formation and not by degranulation. NET formation was induced with PMA and degranulation using formyl-met-leu-phe (f-MLP). (G) Neutrophil death was monitored by quantification of LDH activity in supernatants calculated as means±s.d. (n = 3). (H) Release of S100A8, S100A9, lactotransferrin (LTF) and myeloperoxidase (MPO) were analyzed by immunoblotting. one experiment out of two is shown. (I) Quantification of immunoblots using 2D densitometry analyzing S100A9 protein preparations from supernatants (lane 1), MNase-digested NETs (lane 2) and cell remnants indigestible for MNase (lane 3). Values were calculated as means±s.d. (n = 3) from one experiment out of two.

    Journal: PLoS Pathogens

    Article Title: Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans

    doi: 10.1371/journal.ppat.1000639

    Figure Lengend Snippet: Neutrophils release calprotectin by forming NETs. (A–F) Confocal images of human neutrophils without stimulation (A), after 0.5 h (B), 1 h (C), 2 h (D), 3 h (E) and 4 h (F) after activation. Samples were stained with antibodies specific for the calprotectin heteroduplex (red) and for MPO (green). DNA was stained with DRAQ5 (blue). Calprotectin localizes to the cytoplasm and partially to the nucleus (A, arrow). After stimulation for 0.5 h (B) the neutrophils flattened and formed numerous vacuoles. This reveals a granular staining for MPO and a more dispersed cytoplasmic staining for calprotectin. After stimulation for 1 h (C) the neutrophils round up slightly. The MPO and calprotectin stain partially overlap in the cytoplasm. After stimulation for 2 h (D), calprotectin, MPO and nuclear DNA start to colocalize in the decondensed nucleus (purple). After 3 h (E) and more so after 4 h (F) of stimulation, the cell membrane ruptures and calprotectin is released in NETs colocalizing with MPO and DNA. Scale bar = 10 µm; one experiment out of two is shown. (G–I) Subunits of calprotectin S100A8 and S100A9 are released after cell death during NET formation and not by degranulation. NET formation was induced with PMA and degranulation using formyl-met-leu-phe (f-MLP). (G) Neutrophil death was monitored by quantification of LDH activity in supernatants calculated as means±s.d. (n = 3). (H) Release of S100A8, S100A9, lactotransferrin (LTF) and myeloperoxidase (MPO) were analyzed by immunoblotting. one experiment out of two is shown. (I) Quantification of immunoblots using 2D densitometry analyzing S100A9 protein preparations from supernatants (lane 1), MNase-digested NETs (lane 2) and cell remnants indigestible for MNase (lane 3). Values were calculated as means±s.d. (n = 3) from one experiment out of two.

    Article Snippet: NETs from 10 wells were digested with 5 U/ml MNase (Fermentas), a non-processive nuclease that cuts DNA at linker sites.

    Techniques: Activation Assay, Staining, Activity Assay, Western Blot

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky502

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

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

    Techniques:

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky502

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

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

    Techniques: Incubation, Atomic Absorption Spectroscopy

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky502

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

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

    Techniques: Sequencing

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky502

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

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

    Techniques: Sequencing, Incubation

    Terminally unwrapped CENP-A nucleosomes and their conventional counterparts with wrapped termini are similarly phased at normal centromeres ( a,b ) The position of each individual CENP-A ( a ) or bulk nucleosome ( b ) along a dimerized α-satellite consensus sequence is indicated by a horizontal line. Each fragment is color-coded based on length, as indicated. ( c,d ) The midpoint positions of CENP-A ( c ) or bulk nucleosome ( d ) fragments along the dimer α-satellite consensus sequence. Solid vertical lines indicate the location of the 17 bp CENP-B box ( B ) in ( a–d ). ( e,f ) Models of the preferred positioning and MNase cleavage sites on CENP-A ( e ) and bulk ( f ) nucleosomes at normal centromeres.

    Journal: Nature structural & molecular biology

    Article Title: The octamer is the major form of CENP-A nucleosomes at human centromeres

    doi: 10.1038/nsmb.2562

    Figure Lengend Snippet: Terminally unwrapped CENP-A nucleosomes and their conventional counterparts with wrapped termini are similarly phased at normal centromeres ( a,b ) The position of each individual CENP-A ( a ) or bulk nucleosome ( b ) along a dimerized α-satellite consensus sequence is indicated by a horizontal line. Each fragment is color-coded based on length, as indicated. ( c,d ) The midpoint positions of CENP-A ( c ) or bulk nucleosome ( d ) fragments along the dimer α-satellite consensus sequence. Solid vertical lines indicate the location of the 17 bp CENP-B box ( B ) in ( a–d ). ( e,f ) Models of the preferred positioning and MNase cleavage sites on CENP-A ( e ) and bulk ( f ) nucleosomes at normal centromeres.

    Article Snippet: Tetrasomes, nucleosomes, or nucleosomal arrays were digested with 2 U/µg MNase (Roche) in the presence of 3 mM CaCl2 for 0.5 to 2 min. Each comparison shown between CENP-A-containing and H3-containing particles was performed in parallel under identical reaction conditions for the same length of time.

    Techniques: Sequencing

    CENP-A nucleosomes are less phased and gain symmetric MNase digestion on the Y chromosome centromere that lacks functional CENP-B boxes ( a–c ) Maps of chromosome X HOR-aligned CENP-A sequences. ( d–f ). Maps of chromosome Y HOR-aligned CENP-A sequences. Data and models are shown in the same manner as for the global analysis of CENP-A nucleosome-associated α-satellite sequences ( Fig. 5 ).

    Journal: Nature structural & molecular biology

    Article Title: The octamer is the major form of CENP-A nucleosomes at human centromeres

    doi: 10.1038/nsmb.2562

    Figure Lengend Snippet: CENP-A nucleosomes are less phased and gain symmetric MNase digestion on the Y chromosome centromere that lacks functional CENP-B boxes ( a–c ) Maps of chromosome X HOR-aligned CENP-A sequences. ( d–f ). Maps of chromosome Y HOR-aligned CENP-A sequences. Data and models are shown in the same manner as for the global analysis of CENP-A nucleosome-associated α-satellite sequences ( Fig. 5 ).

    Article Snippet: Tetrasomes, nucleosomes, or nucleosomal arrays were digested with 2 U/µg MNase (Roche) in the presence of 3 mM CaCl2 for 0.5 to 2 min. Each comparison shown between CENP-A-containing and H3-containing particles was performed in parallel under identical reaction conditions for the same length of time.

    Techniques: Functional Assay

    Nuclease digestion of native CENP-A-containing particles resembles that of octameric nucleosomes with loose termini ( a ) DNA length distributions of MNase-digested CENP-A native ChIP and bulk nucleosomes from the same preparation. ( b ) Fluorescence in situ hybridization using DNA from bulk nucleosomes or CENP-A native ChIP as probes. Bulk nucleosome DNA labels the entire chromosome whereas CENP-A probe labels solely centromeric regions, as expected. ( c ) Quantitative real-time PCR analysis comparing enrichment of CENP-A native ChIP DNA relative to bulk nucleosome DNA. CENP-A ChIP sequences are enriched for α-satellite regions (α-satellite1, α-satellite2), but not at pericentric or promoter (aldo) regions, as expected. Error bars represent s.e.m. from three independent replicates. ( d ) Standard digestion (red) or overdigestion (blue, threefold higher concentration of MNase used) of chromatin. ( e ) DNA length distributions of CENP-A native ChIP following standard digestion (red) or overdigestion (blue) of chromatin.

    Journal: Nature structural & molecular biology

    Article Title: The octamer is the major form of CENP-A nucleosomes at human centromeres

    doi: 10.1038/nsmb.2562

    Figure Lengend Snippet: Nuclease digestion of native CENP-A-containing particles resembles that of octameric nucleosomes with loose termini ( a ) DNA length distributions of MNase-digested CENP-A native ChIP and bulk nucleosomes from the same preparation. ( b ) Fluorescence in situ hybridization using DNA from bulk nucleosomes or CENP-A native ChIP as probes. Bulk nucleosome DNA labels the entire chromosome whereas CENP-A probe labels solely centromeric regions, as expected. ( c ) Quantitative real-time PCR analysis comparing enrichment of CENP-A native ChIP DNA relative to bulk nucleosome DNA. CENP-A ChIP sequences are enriched for α-satellite regions (α-satellite1, α-satellite2), but not at pericentric or promoter (aldo) regions, as expected. Error bars represent s.e.m. from three independent replicates. ( d ) Standard digestion (red) or overdigestion (blue, threefold higher concentration of MNase used) of chromatin. ( e ) DNA length distributions of CENP-A native ChIP following standard digestion (red) or overdigestion (blue) of chromatin.

    Article Snippet: Tetrasomes, nucleosomes, or nucleosomal arrays were digested with 2 U/µg MNase (Roche) in the presence of 3 mM CaCl2 for 0.5 to 2 min. Each comparison shown between CENP-A-containing and H3-containing particles was performed in parallel under identical reaction conditions for the same length of time.

    Techniques: Chromatin Immunoprecipitation, Fluorescence, In Situ, Hybridization, Real-time Polymerase Chain Reaction, Concentration Assay

    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, Hemagglutination Assay, Plasmid Preparation, Negative Control, SDS Page, Western Blot, Marker

    A possible model of low-level nucleoid organization. a A fragment of EM image of E. coli nucleoid (adapted from [ 21 ]). b Genomic DNA (gray) forms loops with A-tract clusters (cyan) located at apexes and MNase resistant fragments (black) occupying loop ‘stems’. c A blow-up of a DNA loop, containing several A-tracts (cyan) and two MNase resistant fragments (black)

    Journal: BMC Microbiology

    Article Title: Organization of DNA in a bacterial nucleoid

    doi: 10.1186/s12866-016-0637-3

    Figure Lengend Snippet: A possible model of low-level nucleoid organization. a A fragment of EM image of E. coli nucleoid (adapted from [ 21 ]). b Genomic DNA (gray) forms loops with A-tract clusters (cyan) located at apexes and MNase resistant fragments (black) occupying loop ‘stems’. c A blow-up of a DNA loop, containing several A-tracts (cyan) and two MNase resistant fragments (black)

    Article Snippet: The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C.

    Techniques: Electron Microscopy

    Analysis of the DNA fragments from in vivo MNase digestion of nucleoid in wild type E. coli ( a ) and in vitro MNase digestion of purified genomic DNA ( b ) in agarose gel. a Wild type cells with the empty vector (lanes 2, 3) and MNase-expressing vector (lanes 4-8) were supplemented with arabinose (lane 6), CaCl 2 (lane 5) or both (lanes 3, 7, 8). Digestion reactions were stopped 1 minute (lane 7) or 5 minutes (lanes 3, 5, 8) after CaCl 2 was added. Lanes 1 and 9 show DNA molecular weight marker. b Lane 1, purified wild type genomic DNA; lanes 2-6, a time course of in vitro MNase digestion of the wild type genomic DNA; lane 7, DNA molecular marker

    Journal: BMC Microbiology

    Article Title: Organization of DNA in a bacterial nucleoid

    doi: 10.1186/s12866-016-0637-3

    Figure Lengend Snippet: Analysis of the DNA fragments from in vivo MNase digestion of nucleoid in wild type E. coli ( a ) and in vitro MNase digestion of purified genomic DNA ( b ) in agarose gel. a Wild type cells with the empty vector (lanes 2, 3) and MNase-expressing vector (lanes 4-8) were supplemented with arabinose (lane 6), CaCl 2 (lane 5) or both (lanes 3, 7, 8). Digestion reactions were stopped 1 minute (lane 7) or 5 minutes (lanes 3, 5, 8) after CaCl 2 was added. Lanes 1 and 9 show DNA molecular weight marker. b Lane 1, purified wild type genomic DNA; lanes 2-6, a time course of in vitro MNase digestion of the wild type genomic DNA; lane 7, DNA molecular marker

    Article Snippet: The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C.

    Techniques: In Vivo, In Vitro, Purification, Agarose Gel Electrophoresis, Plasmid Preparation, Expressing, Molecular Weight, Marker

    Genome-wide distribution of sequenced tags. a Tag frequencies for the entire E. coli genome. Frequencies of tags mapped on the positive and negative strands are shown with red and blue bars respectively. a , c Schematic illustrations of tag cross-correlation ( b ) and auto-correlation analyses ( c ). MNase resistant fragments are shown with grey rectangles. Vertical red and blue arrows represent 5’-ends of the digestion fragments mapping to the DNA positive and negative strands respectively

    Journal: BMC Microbiology

    Article Title: Organization of DNA in a bacterial nucleoid

    doi: 10.1186/s12866-016-0637-3

    Figure Lengend Snippet: Genome-wide distribution of sequenced tags. a Tag frequencies for the entire E. coli genome. Frequencies of tags mapped on the positive and negative strands are shown with red and blue bars respectively. a , c Schematic illustrations of tag cross-correlation ( b ) and auto-correlation analyses ( c ). MNase resistant fragments are shown with grey rectangles. Vertical red and blue arrows represent 5’-ends of the digestion fragments mapping to the DNA positive and negative strands respectively

    Article Snippet: The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C.

    Techniques: Genome Wide

    Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using anti‐HA antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies. Source data are available online for this figure.

    Journal: EMBO Reports

    Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum

    doi: 10.15252/embr.201846331

    Figure Lengend Snippet: Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using anti‐HA antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies. Source data are available online for this figure.

    Article Snippet: Mononucleosomes were prepared from freshly isolated nuclei by digestion with 4 U MNase (NEB) for different amounts of time at 33°C; digestion was stopped by adding 10 mM EGTA pH 8.0.

    Techniques: Immunofluorescence, Staining, Isolation, Expressing, Purification, Agarose Gel Electrophoresis, Polyacrylamide Gel Electrophoresis, Immunoprecipitation, Transfection

    Isolation of cross-linked ORF57-RNA complexes for HITS-CLIP analysis. Top Nitrocellulose membrane from an ORF57 immunoprecipitation performed under HITS-CLIP conditions. Cross-linked RNA fragments were end-labeled with 32 P to be visualized by Phosphoimager. Cells were induced to undergo lytic reactivation, exposed to UV and/or treated with high or low concentrations of MNase as indicated. The samples lacking an ORF57 antibody (lane 1) were precipitated with pre-bleed antibodies from the same rabbit. Lanes 6 and 7 are a dark exposure of lanes 4 and 5. The dashed white box indicates the position of the ORF57 complex cut from the membrane for library preparation. Bottom Western blot of the same HITS-CLIP samples shown in the top panel. Affinity purified rabbit anti-ORF57 was used to detect ORF57. Positions of molecular weight markers are shown on the left. On the right, a single asterisk marks the position of a contaminating ~37 kDa protein; double and triple asterisks mark positions of putative ORF57 homodimers and homotrimers bound to the same RNA. The doublet is likely due to an ORF57 cleavage product [ 104 ].

    Journal: PLoS Pathogens

    Article Title: HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism

    doi: 10.1371/journal.ppat.1004652

    Figure Lengend Snippet: Isolation of cross-linked ORF57-RNA complexes for HITS-CLIP analysis. Top Nitrocellulose membrane from an ORF57 immunoprecipitation performed under HITS-CLIP conditions. Cross-linked RNA fragments were end-labeled with 32 P to be visualized by Phosphoimager. Cells were induced to undergo lytic reactivation, exposed to UV and/or treated with high or low concentrations of MNase as indicated. The samples lacking an ORF57 antibody (lane 1) were precipitated with pre-bleed antibodies from the same rabbit. Lanes 6 and 7 are a dark exposure of lanes 4 and 5. The dashed white box indicates the position of the ORF57 complex cut from the membrane for library preparation. Bottom Western blot of the same HITS-CLIP samples shown in the top panel. Affinity purified rabbit anti-ORF57 was used to detect ORF57. Positions of molecular weight markers are shown on the left. On the right, a single asterisk marks the position of a contaminating ~37 kDa protein; double and triple asterisks mark positions of putative ORF57 homodimers and homotrimers bound to the same RNA. The doublet is likely due to an ORF57 cleavage product [ 104 ].

    Article Snippet: For RNA digestion, MNase (New England Biolabs) was diluted to 10 gel units/μL in RIPA buffer and 5 μL of this freshly diluted 1:200 stock was added to the extract.

    Techniques: Isolation, Cross-linking Immunoprecipitation, Immunoprecipitation, Labeling, Western Blot, Affinity Purification, Molecular Weight

    CAL1 is a CENP-A–specific nucleosome assembly factor. (A) CENP-A 101–225 –H4 was assembled on circular relaxed plasmid (pCR2.1-4 [CEN3 + CEN6]) using increasing amounts of CAL1 1–96 , CAL1 1–132 , or CAL1 1–160 in the presence of topoisomerase I. The extracted DNA samples were analyzed on agarose gel and stained with SYBR gold. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lane 3: CENP-A 101–225 –H4, H2A–H2B but no CAL1; lanes 4–7: relaxed plasmid and CENP-A 101–225 –H4, H2A–H2B with CAL1 1–96 ; lanes 8–11: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–132 ; lanes 12–15: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–160 . (B) CENP-A–containing nucleosomes and histone H3 nucleosomes were assembled using histone proteins, CAL1 1–160 , or yeast Nap1 and pGEM3Z-601 plasmid. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lanes 3, 6, and 9: mock reaction without CAL1 1–160 or Nap1; lanes 4 and 5: H3–H4, H2A–H2B with CAL1 1–160 ; lanes 7 and 8: CENP-A 101–225 –H4, H2A-H2B with CAL1 1–160 ; lanes 10 and 11: H3–H4, H2A–H2B with Nap1; lanes 12 and 13: CENP-A 101–225 –H4, H2A–H2B with Nap1. (C) CAL1-assembled CENP-A nucleosomes are negatively supercoiled. CENP-A nucleosomes (lane CENP-A) were assembled by CAL1 1–160 in the presence of topoisomerase I and H2A–H2B dimers, whereas histone H3 nucleosomes (lane H3) were assembled by Nap1. Controls are: supercoiled plasmid (superc.), relaxed plasmid (relaxed), and relaxed plasmid without histones or Nap1, but treated and processed as in the H3 and CENP-A nucleosome assembly reactions (mock). The samples were analyzed on agarose gels with or without 1 µg/ml chloroquine. Boxes highlight the decrease in migration that occurs in the presence of chloroquine in both H3 and CENP-A nucleosomes. (A–C) S, supercoiled; R, relaxed plasmid. (D) Diagram depicting the expected migration patterns for negatively and positively supercoiled DNA separated by 2D gel electrophoresis ( Tachiwana et al., 2011 ). Left gel: H3 nucleosomes assembled with Nap1; right gel: CENP-A nucleosomes assembled with CAL1 1–160 . (E) Mononucleosomes were assembled on 147-bp Widom DNA with Nap1 or CAL1 1–160 and digested with MNase for 30 or 120 s. Native PAGE of samples shown. Lanes 1–3: DNA only; lanes 4 and 5: H3 nucleosomes assembled by Nap1; lanes 6 and 7: CENP-A nucleosomes assembled by Nap1; lanes 8 and 9: CENP-A nucleosomes assembled by CAL1 1–160 .

    Journal: The Journal of Cell Biology

    Article Title: CAL1 is the Drosophila CENP-A assembly factor

    doi: 10.1083/jcb.201305036

    Figure Lengend Snippet: CAL1 is a CENP-A–specific nucleosome assembly factor. (A) CENP-A 101–225 –H4 was assembled on circular relaxed plasmid (pCR2.1-4 [CEN3 + CEN6]) using increasing amounts of CAL1 1–96 , CAL1 1–132 , or CAL1 1–160 in the presence of topoisomerase I. The extracted DNA samples were analyzed on agarose gel and stained with SYBR gold. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lane 3: CENP-A 101–225 –H4, H2A–H2B but no CAL1; lanes 4–7: relaxed plasmid and CENP-A 101–225 –H4, H2A–H2B with CAL1 1–96 ; lanes 8–11: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–132 ; lanes 12–15: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–160 . (B) CENP-A–containing nucleosomes and histone H3 nucleosomes were assembled using histone proteins, CAL1 1–160 , or yeast Nap1 and pGEM3Z-601 plasmid. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lanes 3, 6, and 9: mock reaction without CAL1 1–160 or Nap1; lanes 4 and 5: H3–H4, H2A–H2B with CAL1 1–160 ; lanes 7 and 8: CENP-A 101–225 –H4, H2A-H2B with CAL1 1–160 ; lanes 10 and 11: H3–H4, H2A–H2B with Nap1; lanes 12 and 13: CENP-A 101–225 –H4, H2A–H2B with Nap1. (C) CAL1-assembled CENP-A nucleosomes are negatively supercoiled. CENP-A nucleosomes (lane CENP-A) were assembled by CAL1 1–160 in the presence of topoisomerase I and H2A–H2B dimers, whereas histone H3 nucleosomes (lane H3) were assembled by Nap1. Controls are: supercoiled plasmid (superc.), relaxed plasmid (relaxed), and relaxed plasmid without histones or Nap1, but treated and processed as in the H3 and CENP-A nucleosome assembly reactions (mock). The samples were analyzed on agarose gels with or without 1 µg/ml chloroquine. Boxes highlight the decrease in migration that occurs in the presence of chloroquine in both H3 and CENP-A nucleosomes. (A–C) S, supercoiled; R, relaxed plasmid. (D) Diagram depicting the expected migration patterns for negatively and positively supercoiled DNA separated by 2D gel electrophoresis ( Tachiwana et al., 2011 ). Left gel: H3 nucleosomes assembled with Nap1; right gel: CENP-A nucleosomes assembled with CAL1 1–160 . (E) Mononucleosomes were assembled on 147-bp Widom DNA with Nap1 or CAL1 1–160 and digested with MNase for 30 or 120 s. Native PAGE of samples shown. Lanes 1–3: DNA only; lanes 4 and 5: H3 nucleosomes assembled by Nap1; lanes 6 and 7: CENP-A nucleosomes assembled by Nap1; lanes 8 and 9: CENP-A nucleosomes assembled by CAL1 1–160 .

    Article Snippet: MNase digestion of nucleosomes was conducted by incubating nucleosomes (∼60 nM) with 2,000 U/ml MNase (New England Biolabs, Inc.) for 30–120 s. After the reactions were stopped by addition of 20 mM EDTA, the samples were treated with 2% SDS and 0.4 mg/ml proteinase K for 30 min at 55°C.

    Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Staining, Migration, Two-Dimensional Gel Electrophoresis, Electrophoresis, Clear Native PAGE

    Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.

    Journal: Nucleic Acids Research

    Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF

    doi: 10.1093/nar/gky562

    Figure Lengend Snippet: Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.

    Article Snippet: Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp.

    Techniques: Binding Assay, Genome Wide, Sequencing, Immunoprecipitation

    Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.

    Journal: Nucleic Acids Research

    Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF

    doi: 10.1093/nar/gky562

    Figure Lengend Snippet: Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.

    Article Snippet: Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp.

    Techniques: Binding Assay, Genome Wide, Sequencing, Immunoprecipitation

    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

    CLOCK binds to DNA wrapped around nucleosomes. ( A ) CLOCK ChIP-seq signal on mononucleosome (i.e., mouse liver chromatin digested by MNase) at ZT22 (light blue; left ) and ZT06 (dark blue; right ) for the top 400 CLOCK:BMAL1 DNA-binding sites. The signal

    Journal:

    Article Title: CLOCK:BMAL1 is a pioneer-like transcription factor

    doi: 10.1101/gad.228536.113

    Figure Lengend Snippet: CLOCK binds to DNA wrapped around nucleosomes. ( A ) CLOCK ChIP-seq signal on mononucleosome (i.e., mouse liver chromatin digested by MNase) at ZT22 (light blue; left ) and ZT06 (dark blue; right ) for the top 400 CLOCK:BMAL1 DNA-binding sites. The signal

    Article Snippet: Sequencing libraries were generated using 50 ng of DNA purified from the MNase-digested chromatin (Illumina TruSeq DNA sample prep kit) and size-selected to ensure an insert size of a mononucleosome.

    Techniques: Chromatin Immunoprecipitation, Binding Assay

    Rhythmic CLOCK binding on DNA is associated with rhythmic H2A.Z signal at CLOCK:BMAL1 DNA-binding sites. ( A ) H2A.Z ChIP-seq over input signal ratio on MNase-treated chromatin in wild-type mice during the light phase (green) and dark phase (orange/red)

    Journal:

    Article Title: CLOCK:BMAL1 is a pioneer-like transcription factor

    doi: 10.1101/gad.228536.113

    Figure Lengend Snippet: Rhythmic CLOCK binding on DNA is associated with rhythmic H2A.Z signal at CLOCK:BMAL1 DNA-binding sites. ( A ) H2A.Z ChIP-seq over input signal ratio on MNase-treated chromatin in wild-type mice during the light phase (green) and dark phase (orange/red)

    Article Snippet: Sequencing libraries were generated using 50 ng of DNA purified from the MNase-digested chromatin (Illumina TruSeq DNA sample prep kit) and size-selected to ensure an insert size of a mononucleosome.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Mouse Assay

    Aberrant chromatin composition in mouse models of altered PAR metabolism. Chromomycin A3 (CMA3) intercalation into the DNA indicates incomplete chromatin condensation in s perm from Parg (110) −/− (A) and PJ34-treated (C) males with histone retention. (B, D) Histogram of sperm CMA3-staining intensities reflects that severity of CM3A staining varied at the level of individual sperm and individual fathers (n > 200 nuclei/sample, 3 males/group). (E) Immunoblot analyses of sperm protein lysates showing increase in histone retention in PJ34 treated males. TUBA1A: alpha tubulin loading control. (F) Overlaps of genes identified as differentially histone associated in sperm from 3 individual Parg (110) −/− males (“PargA”, “PargB”, “PargC”, the fathers of the embryos analyzed below) by micrococcal nuclease digests (MND) compared to the wild-type controls. The “PargAll” data set contains all genes commonly identified as differentially MNase-sensitive across 10 Parg (110) −/− males compared with 9 wild-type control animals. The red circle indicates common genes that were differentially histone associated in all groups (1604+216 = 1820, red circle) compared with wild-type. (G) PJ34: differentially MNase-sensitive genes in three different males (like in E) and overlap with a surrogate dataset (“PJ34All”) consisting of data from all 4 PJ34-treated males compared with 9 wild-type control males. The overlap of 2,489 genes that were commonly differentially histone associated in sperm samples is indicated (blue circle). (H) Overlap of genes commonly affected by differential histone association between the Parg (110) −/− and the PJ34 models compared to wild-type controls (red and blue circles in F and G). A Pearson correlation examining significance of this overlap using a genetic background of 19,472 genes was calculated with a resulting P

    Journal: PLoS Genetics

    Article Title: Paternal Poly (ADP-ribose) Metabolism Modulates Retention of Inheritable Sperm Histones and Early Embryonic Gene Expression

    doi: 10.1371/journal.pgen.1004317

    Figure Lengend Snippet: Aberrant chromatin composition in mouse models of altered PAR metabolism. Chromomycin A3 (CMA3) intercalation into the DNA indicates incomplete chromatin condensation in s perm from Parg (110) −/− (A) and PJ34-treated (C) males with histone retention. (B, D) Histogram of sperm CMA3-staining intensities reflects that severity of CM3A staining varied at the level of individual sperm and individual fathers (n > 200 nuclei/sample, 3 males/group). (E) Immunoblot analyses of sperm protein lysates showing increase in histone retention in PJ34 treated males. TUBA1A: alpha tubulin loading control. (F) Overlaps of genes identified as differentially histone associated in sperm from 3 individual Parg (110) −/− males (“PargA”, “PargB”, “PargC”, the fathers of the embryos analyzed below) by micrococcal nuclease digests (MND) compared to the wild-type controls. The “PargAll” data set contains all genes commonly identified as differentially MNase-sensitive across 10 Parg (110) −/− males compared with 9 wild-type control animals. The red circle indicates common genes that were differentially histone associated in all groups (1604+216 = 1820, red circle) compared with wild-type. (G) PJ34: differentially MNase-sensitive genes in three different males (like in E) and overlap with a surrogate dataset (“PJ34All”) consisting of data from all 4 PJ34-treated males compared with 9 wild-type control males. The overlap of 2,489 genes that were commonly differentially histone associated in sperm samples is indicated (blue circle). (H) Overlap of genes commonly affected by differential histone association between the Parg (110) −/− and the PJ34 models compared to wild-type controls (red and blue circles in F and G). A Pearson correlation examining significance of this overlap using a genetic background of 19,472 genes was calculated with a resulting P

    Article Snippet: To visualize differences between wild-type, Parg (110)−/− and PJ34-treated males regarding their sperm MNase tiling array data sets, principal component analysis (PCA, PARTEK software package) was used as a simple eigenvector-based multivariate analyses routinely used to reveal the internal structure of the data that best explains the observed variance.

    Techniques: Staining