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  • 94
    Millipore h3k4me1
    Organization of histone modifications in nucleosomes present in 48-h wild-type minichromosomes by ChIP-Seq analysis. Minichromosomes obtained 48 h postinfection were subjected to chromatin immunoprecipitation using antibodies to RNAP II and the following histone modifications: HH3, HH4, <t>H3K4me1,</t> H3K4me2, H3K4me3, H3K9me1, H3K9me2, and H3K9me3. The DNA present in the ChIP samples was purified, and sequencing libraries were prepared using the NEB kit and protocol. The libraries were paired-end sequenced by NGS using an Illumina MiSeq. Following quality assurance and trimming, the reads obtained were mapped to the SV40 wild-type genome, which was linearized between nt 2666 and 2667 for display and comparison. The data from at least four biological replicates were merged, and heat maps were generated for each ChIP analysis. The actual numbers of biological replicates used were as follows: HH3, 4; HH4, 7; H3K4me1, 5; H3K4me2, 4; H3K4me3, 4; H3K9me1, 8; H3K9me2, 4; H3K9me3, 6; and RNAP II, 32. The nucleotide numbers are indicated for orientation with respect to the SV40 genome, along with the locations of potential regulatory regions.
    H3k4me1, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 363 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti h3k4me1
    The effect of LSD1 and HPV16 E7 gene modulation on epigenetic change on the Viemntin promoter A . Association of LSD1 with HPV16 E7 in C33A cells. Immunoprecipitation with an antibody against LSD1, followed by immunoblotting with an antibody against HPV16 E7, demonstrated that HPV16 E7 co-immunoprecipitated with LSD1. Association of Co-Rest with HPV16 E7 in C33A cells. Immunoprecipitation with an antibody against Co-REST, followed by immunoblotting with an antibody against HPV16 E7, demonstrated that HPV16 E7 co-immunoprecipitated with Co-REST. B . HPV16 E7 suppressed the recruitment of LSD1 to the Vimentin promoter. Occupancy of LSD1 at the Vimentin promoter was markedly lower in HPV16 E7 -overexpressing cells and siLSD1 C33A cells than in blank cells, as shown by ChIP analysis with an LSD1 antibody. C . The histone change caused by LSD1 and its relationship with HPV16 E7. In Western blotting, <t>H3K4me1</t> and H3K4me2 protein levels were reduced in LSD1 -overexpressing C33A cells, but not in HPV16 E7 -overexpressing cells. Knockdown of LSD1 induced the expression of H3K4me1 and H3K4me2. H3K9me2 protein levels remained the same in all groups. D . In ChIP assays, the levels of H3K4me1 and H3K4me2 at the Vimentin promoter were lower in LSD1 -overexpressing cells than in blank cells. The level of H3K4me1 at the Vimentin promoter was greater in HPV16 E7 -overexpressing cells than in blank cells and LSD1 -overexpressing cells. The level of H3K4me2 at the Vimentin promoter was greater in HPV16 E7 -overexpressing cells than in LSD1 -overexpressing cells. The level of H3K9me2 remained the same.
    Anti H3k4me1, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 974 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Active Motif anti h3k4me1
    Histone modifications at Cp and Qp. (A) Schematic of Cp and Qp locations on the EBV genome and positions of primer sets used for ChIP-qPCR. TR, terminal repeats. (B) ChIP-qPCR of histone H3, H3K9ac, H3K27ac or control IgG in MutuI cells (blue) or LCLs (red). (C) ChIP-qPCR of histones <t>H3K4me1,</t> H3K4me2, H3K4me3, H3K9me3, H3K27me3 in MutuI cells (blue) or LCLs (red). ChIP DNA is measured as percent input, and error bars represent standard deviations for three technical replicates.
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    Diagenode h3k4me1
    Gene expression and histone modifications in regulated broadly-expressed and stable tissue-specific genes at third instar larvae a, Diagrams of developmentally regulated genes broadly-expressed across multiple tissues at third instar-larvae L3 (left panel), and stable genes expressed in only one tissue at L3 (right panel). b, Gene expression levels at L3 measured by whole organism RNASeq (left panel). The number of genes in each category is given under the boxplots. The bottom and top of the boxes are the first and third quartiles, and the line within, the median. The whiskers denote the interval within 1.5 times the IQR from the median. Outliers are plotted as dots. Validation by qPCR of the expression at L3 of regulated broadly-expressed genes compared to a stable gene ( Bmcp ) and a silent gene ( CG5367 ) (right panel). Error bars represent the Standard Error of the Mean (SEM) from three independent replicates. c, Levels of H3K4me3, H3K9ac, <t>H3K4me1</t> and H3K27ac on whole L3 individuals. The seven regulated genes broadly-expressed at L3 are depicted as red dots within the boxplots. P-values were computed using the Wilcoxon test (two-sided). d, Validation by individual ChIPs and qPCR of H3K4me3 and H3K9ac in regulated genes broadly-expressed at L3. H3K4me3 and H3K9ac ChIPs are represented as enrichment of the marks over the silent gene ( CG5367 ). Error bars represent the SEM from three independent replicates.
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    Cell Signaling Technology Inc anti h3k4me1
    High concordance between mitotic and interphase histone modification patterns. ( A ) tracks showing the alignments for the indicated histone modification ChIP-seq results for interphase (blue) and mitosis (red) samples on an ∼315-kb region on Chromosome 12. The scale of each track was adjusted to the total number of reads using the Normalize Coverage Data option in IGV (see Methods). The ChromHMM annotation for each genomic region is shown below the plot using the same color code as in C . ( B ) Scatter plots showing the normalized read counts for all regions enriched in each modification (see Methods for more details) in mitosis versus interphase. It should be noted that ChIP-seq-based quantification may be less accurate than quantification based on mass spectrometry data due to inherent noise in the ChIP-seq method. Thus, in cases of a discrepancy, e.g., <t>H3K4me1,</t> we rely on the mass spectrometry results for quantification, and on the ChIP-seq results for localization. ( C ) Bar plots showing the percentage of reads in peak regions per ChromHMM category.
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    Merck KGaA h3k4me1
    High concordance between mitotic and interphase histone modification patterns. ( A ) tracks showing the alignments for the indicated histone modification ChIP-seq results for interphase (blue) and mitosis (red) samples on an ∼315-kb region on Chromosome 12. The scale of each track was adjusted to the total number of reads using the Normalize Coverage Data option in IGV (see Methods). The ChromHMM annotation for each genomic region is shown below the plot using the same color code as in C . ( B ) Scatter plots showing the normalized read counts for all regions enriched in each modification (see Methods for more details) in mitosis versus interphase. It should be noted that ChIP-seq-based quantification may be less accurate than quantification based on mass spectrometry data due to inherent noise in the ChIP-seq method. Thus, in cases of a discrepancy, e.g., <t>H3K4me1,</t> we rely on the mass spectrometry results for quantification, and on the ChIP-seq results for localization. ( C ) Bar plots showing the percentage of reads in peak regions per ChromHMM category.
    H3k4me1, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti h3k4me1
    <t>H3K4me1</t> profiles segregate discrete classes of transcription factor occupied loci. ( A ) Heatmaps of H3K4me1 read density in ±2-kb regions centered on FOXA2 (islets or liver), PDX1, and HNF4A peak maxima. Peak max locations are indicated by red
    Anti H3k4me1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Active Motif rabbit anti h3k4me1
    dBRWD3 is epistatic to trx in the ectopic expression of Ubx and Abd-B . (A and B) A ChIP-qPCR analysis of H3K27me3 levels at the enhancers, promoters, and transcription start sites of Antp (A) and Ubx (B) in Elav-GAL4 control, E(z) depleted, and E(z) , dBRWD3 doubly depleted brains. Black asterisks indicate control versus E(z) depletion. Blue asterisks indicate E(z) depletion versus E(z) , dBRWD3 double depletion. (C and D) A ChIP-qPCR analysis of H2AK118ub levels at the enhancers, promoters, and transcription start sites of Antp (C) and Ubx (D) in Elav-GAL4 control, Pc depleted, and Pc , dBRWD3 doubly depleted brains. Black asterisks indicate control versus Pc depletion. Blue asterisks indicates Pc depletion versus Pc , dBRWD3 double depletion. (E-G) trx was overexpressed under the control of ms-1096-GAL4 . The TRX-induced Abd-B expression (arrows) in wild-type (E), dBRWD3 depletion (F), and yem over-expression (G) backgrounds. Scale bars indicate 20μm. (H) A ChIP-qPCR analysis of <t>H3K4me1</t> levels at Ubx and Abd-B enhancers in the UAS-mCD8-GFP control, trx over-expression, and trx over-expression, dBRWD3 depleted wings as indicated. ns. indicates not significant. ChIP-qPCR Data are shown as means ± S.D from 4 technical replicates. *, **, *** indicate P
    Rabbit Anti H3k4me1, supplied by Active Motif, used in various techniques. Bioz Stars score: 90/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti h3k4me1
    Little evidence of Setd7 interacting with MyoD or acting as an <t>H3K4me1</t> histone methyltransferase in MuSCs (A) Immunostaining of Setd7 in plated MuSCs highlighting cytoplasmic localization. Scale bar = 50µm. (B) Western blot analysis of Setd7 in Cytoplasmic (cyto), nuclear (nuc) and chromatin (chrom) fractions from plated MuSCs and myotubes. (C) Western blot analysis of H3K4me1 in WT and Sets7KO plated MuSCs. (D) Western blot of H3K4me1, me2, me3 in plated MuSCs and myotubes treated with or without a Setd7 inhibitor (PFI-2). (E+F) H3K4me1 ChIPSeq scatter plot correlating mean peak density in plated MuSCs treated with or without PFI-2 across whole genome (E) or at TSS (+/− 5KB) sites (F). (G) Mean signal of normalized peak density surrounding TSSs for H3K4me1 ChIPSeq for control treated MuSCs (blue line) and PFI-2 treated MuSCs (red line) (H) Heatmap displaying single gene resolution of H3K4me1 ChIP-Seq TSS enrichment data in (G). (I) Comparisons in H3K4me1 enrichment (RPKM) at myoblast (MB) and myotube (MT) enhancer regions in MuSCs treated with or without PFI-2. (J) Setd7 and MyoD co-immunostaining in plated MuSCs highlighting lack of co-localization. Scale bar = 100µm. (K) Western blot analysis of Setd7 and MyoD in cytoplasmic (cyto) and nuclear (nuc) fractions of plated MuSCs. (L) Setd7 IP samples blotted and probed with an anti-MyoD antibody. (M) H3K4me1 genome browser tracks in proximal promoters and gene bodies of MyoD targets (MyoG, Myh1 and Myl1). Control MuSCs (blue line) and PFI-2 treated MuSCs (red line) R values calculated with Pearson’s correlation coefficient.
    Rabbit Anti H3k4me1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti h3k4me1 antibodies
    Little evidence of Setd7 interacting with MyoD or acting as an <t>H3K4me1</t> histone methyltransferase in MuSCs (A) Immunostaining of Setd7 in plated MuSCs highlighting cytoplasmic localization. Scale bar = 50µm. (B) Western blot analysis of Setd7 in Cytoplasmic (cyto), nuclear (nuc) and chromatin (chrom) fractions from plated MuSCs and myotubes. (C) Western blot analysis of H3K4me1 in WT and Sets7KO plated MuSCs. (D) Western blot of H3K4me1, me2, me3 in plated MuSCs and myotubes treated with or without a Setd7 inhibitor (PFI-2). (E+F) H3K4me1 ChIPSeq scatter plot correlating mean peak density in plated MuSCs treated with or without PFI-2 across whole genome (E) or at TSS (+/− 5KB) sites (F). (G) Mean signal of normalized peak density surrounding TSSs for H3K4me1 ChIPSeq for control treated MuSCs (blue line) and PFI-2 treated MuSCs (red line) (H) Heatmap displaying single gene resolution of H3K4me1 ChIP-Seq TSS enrichment data in (G). (I) Comparisons in H3K4me1 enrichment (RPKM) at myoblast (MB) and myotube (MT) enhancer regions in MuSCs treated with or without PFI-2. (J) Setd7 and MyoD co-immunostaining in plated MuSCs highlighting lack of co-localization. Scale bar = 100µm. (K) Western blot analysis of Setd7 and MyoD in cytoplasmic (cyto) and nuclear (nuc) fractions of plated MuSCs. (L) Setd7 IP samples blotted and probed with an anti-MyoD antibody. (M) H3K4me1 genome browser tracks in proximal promoters and gene bodies of MyoD targets (MyoG, Myh1 and Myl1). Control MuSCs (blue line) and PFI-2 treated MuSCs (red line) R values calculated with Pearson’s correlation coefficient.
    Anti H3k4me1 Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti h3k4me1
    Met-VEL clusters occur across metastatic cancers a . UCSC browser view of <t>H3K4me1</t> profiles in MG63.3 (metastatic) and MG63 (parental) cell lines illustrating an example of a gained (left) and lost (right) Met-VEL cluster. Met-VELs identified by black bars. 200kb Met-VEL clusters highlighted in gray. b . Genome-wide lost Met-VEL landscape for MG63.3 cell line. Rows represent scaled chromosomal coordinates. Peaks represent maximum gained Met-VEL counts in 200kb sliding windows. Predicted target genes for selected peaks are labeled. c . Gained and lost Met-VEL cluster counts in patient lung metastases/primary tumors and metastatic/parental cell line pairs. d . Percentage of total gained (top) and lost (bottom) Met-VELs within and outside of clusters in patient lung metastases/primary tumors and metastatic/parental cell line pairs.
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    Abcam h3k4me1 chip grade
    Identification of hypo-enhancers. a Heat map displaying H3K27Ac and DNA methylation at enhancers, called by being <t>H3K4me1-positive</t> and H3K4me3-negative at 4 hpf. The heat map was first sorted on DNA methylation, split ( yellow dashed line ), and independently sorted on H3K27ac. b Genome browser view of a hyper-enhancer close to the TSS of sox2 . ChIP-Seq data and DNA methylation data are displayed for embryos 4 hpf. Normalized ChIP-Seq enrichments and fractional methylation are indicated at the left side of the image. The blue box indicates the hyper-enhancer. The blue arrow indicates the orientation of the gene. Blue horizontal bars indicate HMRs. c Genome browser view of a hypo-enhancer close to the TSS of tlx3b . ChIP-Seq data and DNA methylation data are displayed for embryos 4 hpf. Normalized ChIP-Seq enrichments and fractional methylation are indicated at the left side of the image. The blue box indicates the hypo-enhancer. The blue arrow indicates the orientation of the gene. Blue horizontal bars indicate HMRs. d The overlap of the hypo- and hyper-enhancers with H3K27ac at the indicated developmental time points (* p
    H3k4me1 Chip Grade, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti h3k4me1
    Global E2A occupancy declines during β-selection. Genome-wide E2A occupancy and patterns of H3K4 mono-methylation were examined in thymocytes isolated from either untreated Rag2 –/– mice (DN3) or Rag2 –/– mice injected with anti-CD3ε antibody (DN4). ( a ) Venn diagram displaying the distribution of E2A binding sites in DN3 versus DN4. ( b ) Cis-regulatory sequences associated with E2A occupancy in DN3 and DN4, identified by comparing enriched peaks to randomly selected genomic DNA sequences. Letter size directly relates to nucleotide frequency. P values are shown and indicate enrichment for a given motif as compared to randomly selected regions. ( c ) E2A occupancy and patterns of <t>H3K4me1</t> across the Hes1, Ptcra, Notch1, Notch3, Rag2/1, Cd3e, Rorc and Gfi1 loci in the DN3 and DN4 compartments. E2A occupancy in DN3 is shown in gray versus black for the DN4 compartment. H3K4me1 is indicated in blue in DN3 versus green in DN4. ( d ) E2A occupancy and patterns of H3K4me1 across the Zap70 locus. ( e ) Expression of Zap70 in wild-type and Id3-deficient DN1-4 thymocyte compartments. ( f ) Expression of Zap70 and CD27 in day 16.5 dpc fetal wild-type and E2A-deficient DN3 (top) and DN4 as well as ISP (bottom) thymocytes.
    Anti H3k4me1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit polyclonal anti h3k4me1
    Global E2A occupancy declines during β-selection. Genome-wide E2A occupancy and patterns of H3K4 mono-methylation were examined in thymocytes isolated from either untreated Rag2 –/– mice (DN3) or Rag2 –/– mice injected with anti-CD3ε antibody (DN4). ( a ) Venn diagram displaying the distribution of E2A binding sites in DN3 versus DN4. ( b ) Cis-regulatory sequences associated with E2A occupancy in DN3 and DN4, identified by comparing enriched peaks to randomly selected genomic DNA sequences. Letter size directly relates to nucleotide frequency. P values are shown and indicate enrichment for a given motif as compared to randomly selected regions. ( c ) E2A occupancy and patterns of <t>H3K4me1</t> across the Hes1, Ptcra, Notch1, Notch3, Rag2/1, Cd3e, Rorc and Gfi1 loci in the DN3 and DN4 compartments. E2A occupancy in DN3 is shown in gray versus black for the DN4 compartment. H3K4me1 is indicated in blue in DN3 versus green in DN4. ( d ) E2A occupancy and patterns of H3K4me1 across the Zap70 locus. ( e ) Expression of Zap70 in wild-type and Id3-deficient DN1-4 thymocyte compartments. ( f ) Expression of Zap70 and CD27 in day 16.5 dpc fetal wild-type and E2A-deficient DN3 (top) and DN4 as well as ISP (bottom) thymocytes.
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    Abcam anti h3k4me1 polyclonal antibodies
    Set1/MLL complex components are largely responsible for H3K4me2/3 in embryos. Embryos were dissected from WT, mutant, or RNAi treated adult eri-1 ( mg366; enhanced RNAi) animals. RNAi mediated knockdown was used for ash-2, dpy-30, cfp-1 and wdr-82 . Embryos were probed with rabbit anti-H3K4me3 (Panels in A) or mouse anti-H3K4me2 monoclonal antibodies (Panels in B); DNA was counter-stained with DAPI. Exposure times were the same for each condition. Scale bars represent 10um. (A and B) H3K4me3 and H3K4me2 are present in chromatin of wild-type embryos, but both are dramatically decreased in chromatin of embryos from wdr-5.1 and rbbp-5 mutants, and embryos from ash2, dpy-30, or cfp-1 RNAi treated mothers. H3K4me3, but not H3K4me2, is reduced in set-2(tm1630) mutant embryos. RNAi mediated knockdown of wdr-82 did not affect either H3K4me3 or H3K4me2. (C) A wdr-5.1::GFP transgene rescues H3K4 methylation in the wdr-5.1(ok1417) mutant. wdr-5.1(ok1417);wdr-5.1::GFP transgenic embryos were probed with antibodies against GFP and H3K4me3/2 as indicated. H3K4me3/2 were restored in the chromatin of early embryos in which WDR-5.1:GFP expression was detected. (D) Western blot of lysates from mixed stage wdr-5.1(ok1417), wdr-5.2(ok1444), wdr-5.1;wdr-5.2 double mutant, and set-1(tm1630) mutant embryos. The blots were probed with anti-histone H3, <t>anti-H3K4me1,</t> mouse monoclonal anti-H3K4me2 or rabbit <t>polyclonal</t> anti-H3K4me3 antibodies, as indicated. H3K4me3/2 levels were dramatically decreased in strains with the wdr-5.1(ok1417) allele, whereas H3K4me3 levels were more strongly depleted than H3K4me2 in set-2(tm1630) . H3K4me1 levels were not noticeably affected in any mutant strain tested.
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    Image Search Results


    Organization of histone modifications in nucleosomes present in 48-h wild-type minichromosomes by ChIP-Seq analysis. Minichromosomes obtained 48 h postinfection were subjected to chromatin immunoprecipitation using antibodies to RNAP II and the following histone modifications: HH3, HH4, H3K4me1, H3K4me2, H3K4me3, H3K9me1, H3K9me2, and H3K9me3. The DNA present in the ChIP samples was purified, and sequencing libraries were prepared using the NEB kit and protocol. The libraries were paired-end sequenced by NGS using an Illumina MiSeq. Following quality assurance and trimming, the reads obtained were mapped to the SV40 wild-type genome, which was linearized between nt 2666 and 2667 for display and comparison. The data from at least four biological replicates were merged, and heat maps were generated for each ChIP analysis. The actual numbers of biological replicates used were as follows: HH3, 4; HH4, 7; H3K4me1, 5; H3K4me2, 4; H3K4me3, 4; H3K9me1, 8; H3K9me2, 4; H3K9me3, 6; and RNAP II, 32. The nucleotide numbers are indicated for orientation with respect to the SV40 genome, along with the locations of potential regulatory regions.

    Journal: Journal of Virology

    Article Title: Directed Nucleosome Sliding during the Formation of the Simian Virus 40 Particle Exposes DNA Sequences Required for Early Transcription

    doi: 10.1128/JVI.01678-18

    Figure Lengend Snippet: Organization of histone modifications in nucleosomes present in 48-h wild-type minichromosomes by ChIP-Seq analysis. Minichromosomes obtained 48 h postinfection were subjected to chromatin immunoprecipitation using antibodies to RNAP II and the following histone modifications: HH3, HH4, H3K4me1, H3K4me2, H3K4me3, H3K9me1, H3K9me2, and H3K9me3. The DNA present in the ChIP samples was purified, and sequencing libraries were prepared using the NEB kit and protocol. The libraries were paired-end sequenced by NGS using an Illumina MiSeq. Following quality assurance and trimming, the reads obtained were mapped to the SV40 wild-type genome, which was linearized between nt 2666 and 2667 for display and comparison. The data from at least four biological replicates were merged, and heat maps were generated for each ChIP analysis. The actual numbers of biological replicates used were as follows: HH3, 4; HH4, 7; H3K4me1, 5; H3K4me2, 4; H3K4me3, 4; H3K9me1, 8; H3K9me2, 4; H3K9me3, 6; and RNAP II, 32. The nucleotide numbers are indicated for orientation with respect to the SV40 genome, along with the locations of potential regulatory regions.

    Article Snippet: The antibodies included antibodies to RNAP II (05-623; Millipore), hyperacetylated H3 (06-599; Millipore), hyperacetylated H4 (06-866; Millipore), H3K4me1 (07-436; Millipore), H3K4me2 (39141; Active Motif), H3K4me3 (04-745; Millipore), H3K9me1 (ab9045; Abcam), H3K9me2 (ab1220; Abcam), H3K9me3 (ab8898; Abcam), and H4K20me1 (39175; Active Motif).

    Techniques: Chromatin Immunoprecipitation, Purification, Sequencing, Next-Generation Sequencing, Generated

    LSD1 restricts chromatin accessibility at naïve b cell enhancers in plasmablasts. (A) Overlap between DAR group comparisons 1A vs 6A and 2A vs 6A. obs/exp refers to the ratio of observed DAR overlap over expected overlap according to a permutation test. (B) ChIP-seq rpm enrichment of nB H3K4me1 (left) and H3K27ac (right) for DAR groups 1A, 2A, and 6A. (C) Log 2 odds ratios of DAR group enrichment with active enhancers and active promoters from six different cell types. (D) Boxplot of ChIP-seq enrichment of nB and PB H3K4me2 for nB enhancers mapping to 1A DAR and 6A DAR. (E) Top significantly enriched transcription factor motifs identified through HOMER de novo motif analysis for 1A nB enh and 6A nB enh. (F) Boxplot of ChIP-seq enrichment of nB and PB H3K4me2 for PU.1 binding sites, IRF4 binding sites, and Blimp-1 binding sites mapping to 6R DEG. (G) Boxplot of chromatin accessibility of the indicated sample groups at 6R PU.1, 6R IRF4, and 6R Blimp-1 regions. (H) Gene tracks of example transcription factor binding sites mapping to a 6R gene that exhibit significant increases in chromatin accessibility in PB CKO. Significance determined by Wilcoxon rank sum test (B), Fisher’s exact test (C), or Student’s two-tailed t-test (D,F,G). rppm, reads per peak per million; rpm, reads per million.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: The histone demethylase LSD1 regulates B cell proliferation and plasmablast differentiation

    doi: 10.4049/jimmunol.1800952

    Figure Lengend Snippet: LSD1 restricts chromatin accessibility at naïve b cell enhancers in plasmablasts. (A) Overlap between DAR group comparisons 1A vs 6A and 2A vs 6A. obs/exp refers to the ratio of observed DAR overlap over expected overlap according to a permutation test. (B) ChIP-seq rpm enrichment of nB H3K4me1 (left) and H3K27ac (right) for DAR groups 1A, 2A, and 6A. (C) Log 2 odds ratios of DAR group enrichment with active enhancers and active promoters from six different cell types. (D) Boxplot of ChIP-seq enrichment of nB and PB H3K4me2 for nB enhancers mapping to 1A DAR and 6A DAR. (E) Top significantly enriched transcription factor motifs identified through HOMER de novo motif analysis for 1A nB enh and 6A nB enh. (F) Boxplot of ChIP-seq enrichment of nB and PB H3K4me2 for PU.1 binding sites, IRF4 binding sites, and Blimp-1 binding sites mapping to 6R DEG. (G) Boxplot of chromatin accessibility of the indicated sample groups at 6R PU.1, 6R IRF4, and 6R Blimp-1 regions. (H) Gene tracks of example transcription factor binding sites mapping to a 6R gene that exhibit significant increases in chromatin accessibility in PB CKO. Significance determined by Wilcoxon rank sum test (B), Fisher’s exact test (C), or Student’s two-tailed t-test (D,F,G). rppm, reads per peak per million; rpm, reads per million.

    Article Snippet: For immunoprecipitations, anti-H3K4me1 (MilliporeSigma 07–436) and anti-IgG (MilliporeSigma 12–370) antibody was used.

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Two Tailed Test

    Aberrant accumulation of H3K4me1 at LSD1-regulated loci. (A, B) ChIP-qPCR for H3K4me1 enrichment displayed as % of input at the indicated genomic regions. Data are combined from two independent experiments using three mice per group. Error bars represent mean ± SD. Significance determined by Student’s two-tailed t-test. * P

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: The histone demethylase LSD1 regulates B cell proliferation and plasmablast differentiation

    doi: 10.4049/jimmunol.1800952

    Figure Lengend Snippet: Aberrant accumulation of H3K4me1 at LSD1-regulated loci. (A, B) ChIP-qPCR for H3K4me1 enrichment displayed as % of input at the indicated genomic regions. Data are combined from two independent experiments using three mice per group. Error bars represent mean ± SD. Significance determined by Student’s two-tailed t-test. * P

    Article Snippet: For immunoprecipitations, anti-H3K4me1 (MilliporeSigma 07–436) and anti-IgG (MilliporeSigma 12–370) antibody was used.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Mouse Assay, Two Tailed Test

    The effect of LSD1 and HPV16 E7 gene modulation on epigenetic change on the Viemntin promoter A . Association of LSD1 with HPV16 E7 in C33A cells. Immunoprecipitation with an antibody against LSD1, followed by immunoblotting with an antibody against HPV16 E7, demonstrated that HPV16 E7 co-immunoprecipitated with LSD1. Association of Co-Rest with HPV16 E7 in C33A cells. Immunoprecipitation with an antibody against Co-REST, followed by immunoblotting with an antibody against HPV16 E7, demonstrated that HPV16 E7 co-immunoprecipitated with Co-REST. B . HPV16 E7 suppressed the recruitment of LSD1 to the Vimentin promoter. Occupancy of LSD1 at the Vimentin promoter was markedly lower in HPV16 E7 -overexpressing cells and siLSD1 C33A cells than in blank cells, as shown by ChIP analysis with an LSD1 antibody. C . The histone change caused by LSD1 and its relationship with HPV16 E7. In Western blotting, H3K4me1 and H3K4me2 protein levels were reduced in LSD1 -overexpressing C33A cells, but not in HPV16 E7 -overexpressing cells. Knockdown of LSD1 induced the expression of H3K4me1 and H3K4me2. H3K9me2 protein levels remained the same in all groups. D . In ChIP assays, the levels of H3K4me1 and H3K4me2 at the Vimentin promoter were lower in LSD1 -overexpressing cells than in blank cells. The level of H3K4me1 at the Vimentin promoter was greater in HPV16 E7 -overexpressing cells than in blank cells and LSD1 -overexpressing cells. The level of H3K4me2 at the Vimentin promoter was greater in HPV16 E7 -overexpressing cells than in LSD1 -overexpressing cells. The level of H3K9me2 remained the same.

    Journal: Oncotarget

    Article Title: LSD1 binds to HPV16 E7 and promotes the epithelial-mesenchymal transition in cervical cancer by demethylating histones at the Vimentin promoter

    doi: 10.18632/oncotarget.13516

    Figure Lengend Snippet: The effect of LSD1 and HPV16 E7 gene modulation on epigenetic change on the Viemntin promoter A . Association of LSD1 with HPV16 E7 in C33A cells. Immunoprecipitation with an antibody against LSD1, followed by immunoblotting with an antibody against HPV16 E7, demonstrated that HPV16 E7 co-immunoprecipitated with LSD1. Association of Co-Rest with HPV16 E7 in C33A cells. Immunoprecipitation with an antibody against Co-REST, followed by immunoblotting with an antibody against HPV16 E7, demonstrated that HPV16 E7 co-immunoprecipitated with Co-REST. B . HPV16 E7 suppressed the recruitment of LSD1 to the Vimentin promoter. Occupancy of LSD1 at the Vimentin promoter was markedly lower in HPV16 E7 -overexpressing cells and siLSD1 C33A cells than in blank cells, as shown by ChIP analysis with an LSD1 antibody. C . The histone change caused by LSD1 and its relationship with HPV16 E7. In Western blotting, H3K4me1 and H3K4me2 protein levels were reduced in LSD1 -overexpressing C33A cells, but not in HPV16 E7 -overexpressing cells. Knockdown of LSD1 induced the expression of H3K4me1 and H3K4me2. H3K9me2 protein levels remained the same in all groups. D . In ChIP assays, the levels of H3K4me1 and H3K4me2 at the Vimentin promoter were lower in LSD1 -overexpressing cells than in blank cells. The level of H3K4me1 at the Vimentin promoter was greater in HPV16 E7 -overexpressing cells than in blank cells and LSD1 -overexpressing cells. The level of H3K4me2 at the Vimentin promoter was greater in HPV16 E7 -overexpressing cells than in LSD1 -overexpressing cells. The level of H3K9me2 remained the same.

    Article Snippet: Membranes were blocked with 5% skim milk for 2 hours and incubated for 15 hours with the following rabbit monoclonal primary antibodies: anti-LSD1 (diluted 1:500; Sigma, St. Louis, MO, USA), anti-HPV16 E7 (diluted 1:100; Bioss, Shanghai, China), anti-Vimentin (diluted 1:500; Cell Signaling Technology, Beverley, MA, USA), anti-E-cadherin (diluted 1:500; Cell Signaling Technology), anti-GAPDH (Epitomics), anti-H3K4me1 (Abcam, Cambridge, UK), anti-H3K4me2 (Abcam) and anti-H3K9me2 (Abcam), followed by 1 hour of incubation with the appropriate secondary antibody.

    Techniques: Immunoprecipitation, Chromatin Immunoprecipitation, Western Blot, Expressing

    A . After binding to the Vimentin promoter, LSD1 demethylated H3K4me1 and H3K4me2, activated the transcription of Vimentin, and induced the EMT. B . HPV16 E7 suppressed the enrichment of LSD1 on the Vimentin promoter and rescued the methylation of H3K4me1 and H3K4me2.

    Journal: Oncotarget

    Article Title: LSD1 binds to HPV16 E7 and promotes the epithelial-mesenchymal transition in cervical cancer by demethylating histones at the Vimentin promoter

    doi: 10.18632/oncotarget.13516

    Figure Lengend Snippet: A . After binding to the Vimentin promoter, LSD1 demethylated H3K4me1 and H3K4me2, activated the transcription of Vimentin, and induced the EMT. B . HPV16 E7 suppressed the enrichment of LSD1 on the Vimentin promoter and rescued the methylation of H3K4me1 and H3K4me2.

    Article Snippet: Membranes were blocked with 5% skim milk for 2 hours and incubated for 15 hours with the following rabbit monoclonal primary antibodies: anti-LSD1 (diluted 1:500; Sigma, St. Louis, MO, USA), anti-HPV16 E7 (diluted 1:100; Bioss, Shanghai, China), anti-Vimentin (diluted 1:500; Cell Signaling Technology, Beverley, MA, USA), anti-E-cadherin (diluted 1:500; Cell Signaling Technology), anti-GAPDH (Epitomics), anti-H3K4me1 (Abcam, Cambridge, UK), anti-H3K4me2 (Abcam) and anti-H3K9me2 (Abcam), followed by 1 hour of incubation with the appropriate secondary antibody.

    Techniques: Binding Assay, Methylation

    a Boxplot of PCNA staining levels in different groups of mice. b Scatter plot of lesion size vs. the PCNA staining levels in ectopic endometrium. The dashed line represents the regression line. Each alphabet represents one data point, where U, L, and H denote that the mouse was from group UT, L, and H, respectively. The numbers in the figures are p-values for statistical significance in testing the difference among different groups (Kruskal-Wallis test). c Boxplot of VEGF staining levels in different groups of mice. d Boxplot of the number of CD31-positive microvessels in different groups of mice. e Boxplot of E-cadherin staining levels in different groups of mice. f Boxplot of vimentin staining levels in different groups of mice. g Boxplot of H3K4me1 staining levels in different groups of mice. h Boxplot of H3K4me2 staining levels in different groups of mice. ‘*’, p

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: Tranylcypromine, a lysine-specific demethylase 1 (LSD1) inhibitor, suppresses lesion growth and improves generalized hyperalgesia in mouse with induced endometriosis

    doi: 10.1186/s12958-016-0154-0

    Figure Lengend Snippet: a Boxplot of PCNA staining levels in different groups of mice. b Scatter plot of lesion size vs. the PCNA staining levels in ectopic endometrium. The dashed line represents the regression line. Each alphabet represents one data point, where U, L, and H denote that the mouse was from group UT, L, and H, respectively. The numbers in the figures are p-values for statistical significance in testing the difference among different groups (Kruskal-Wallis test). c Boxplot of VEGF staining levels in different groups of mice. d Boxplot of the number of CD31-positive microvessels in different groups of mice. e Boxplot of E-cadherin staining levels in different groups of mice. f Boxplot of vimentin staining levels in different groups of mice. g Boxplot of H3K4me1 staining levels in different groups of mice. h Boxplot of H3K4me2 staining levels in different groups of mice. ‘*’, p

    Article Snippet: The rabbit polyclonal antibodies against PCNA (Thermo Littleton, CO, USA), H3K4me1 (Abcam, Cambridge, MA, USA), H3K4me2 (Abcam), VEGF (Santa Cruz, TX, USA), CD31 (Abcam), E-cadherin (CST, MA, USA), vimentin (Abcam) diluted to 1:100, 1:100, 1:100, 1:50, 1:50, 1:100 and 1:200, respectively, were used as primary antibodies.

    Techniques: Staining, Mouse Assay

    Representative micrographs showing immunoreactivity to PCNA, VEGF, CD31 (MVD), E-cadherin, vimentin, H3K4me1, and H3K4me2, in ectopic endometrium among different groups. The abbreviations used: Low-TC, tissue sample taken from a mouse treated with low-dose TC; High-TC, treated with high-dose TC; UT, untreated. Magnification in all figures: X400. The scale bar represents 125 μm

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: Tranylcypromine, a lysine-specific demethylase 1 (LSD1) inhibitor, suppresses lesion growth and improves generalized hyperalgesia in mouse with induced endometriosis

    doi: 10.1186/s12958-016-0154-0

    Figure Lengend Snippet: Representative micrographs showing immunoreactivity to PCNA, VEGF, CD31 (MVD), E-cadherin, vimentin, H3K4me1, and H3K4me2, in ectopic endometrium among different groups. The abbreviations used: Low-TC, tissue sample taken from a mouse treated with low-dose TC; High-TC, treated with high-dose TC; UT, untreated. Magnification in all figures: X400. The scale bar represents 125 μm

    Article Snippet: The rabbit polyclonal antibodies against PCNA (Thermo Littleton, CO, USA), H3K4me1 (Abcam, Cambridge, MA, USA), H3K4me2 (Abcam), VEGF (Santa Cruz, TX, USA), CD31 (Abcam), E-cadherin (CST, MA, USA), vimentin (Abcam) diluted to 1:100, 1:100, 1:100, 1:50, 1:50, 1:100 and 1:200, respectively, were used as primary antibodies.

    Techniques:

    Dermo1 as a mediator of Wnt signaling/β-catenin in dermal precursors. ( A ) (Left) A highly conserved region near 1.2 kb upstream of Dermo1 transcription start site contains a cluster of three consensus Tcf/Lef-binding sites. Two of the consensus Tcf/Lef-binding sites (magenta and blue) were conserved across mammalian species (adapted from UCSC Genome browser). (Right) The –2.2 kb site contains a putative Tcf/Lef-binding site (green) and is not conserved. ( B ) Tcf4 and H3K4me1 chromatin immunoprecipitation (ChIP) show enrichment at the –1.2 kb and –2.2 kb Tcf/Lef motifs compared with β-actin (16-fold enrichment in Tcf4 ChIP and 34-fold enrichment in H3K4me1 ChIP for –1.2 kb; 6 and 26-fold enrichment for –2.2 kb site, respectively) or nearby non-target sites (ninefold enrichment in Tcf4 ChIP and fourfold enrichment in H3K4me1 ChIP for –1.2 kb; four and threefold enrichment for –2.2 kb site, respectively). ( C ) Schematic of the luciferase reporter plasmids used in the luciferase assay. Dermo1 promoter element with –1.2 kb Tcf/Lef sites had substantial transactivation in the presence of wild-type β-catenin. The negative controls (empty pGL4.10 vector and the reverse construct Dermo1 Rev-Luc) showed comparable luciferase activity with and without β-catenin transactivation. ( D ) Proposed model for the role of Wnt signaling/β-catenin in the selection of cranial dermal cell fate. IP, immunoprecipitated; NT, non-target.

    Journal: Development (Cambridge, England)

    Article Title: Role of canonical Wnt signaling/?-catenin via Dermo1 in cranial dermal cell development

    doi: 10.1242/dev.056473

    Figure Lengend Snippet: Dermo1 as a mediator of Wnt signaling/β-catenin in dermal precursors. ( A ) (Left) A highly conserved region near 1.2 kb upstream of Dermo1 transcription start site contains a cluster of three consensus Tcf/Lef-binding sites. Two of the consensus Tcf/Lef-binding sites (magenta and blue) were conserved across mammalian species (adapted from UCSC Genome browser). (Right) The –2.2 kb site contains a putative Tcf/Lef-binding site (green) and is not conserved. ( B ) Tcf4 and H3K4me1 chromatin immunoprecipitation (ChIP) show enrichment at the –1.2 kb and –2.2 kb Tcf/Lef motifs compared with β-actin (16-fold enrichment in Tcf4 ChIP and 34-fold enrichment in H3K4me1 ChIP for –1.2 kb; 6 and 26-fold enrichment for –2.2 kb site, respectively) or nearby non-target sites (ninefold enrichment in Tcf4 ChIP and fourfold enrichment in H3K4me1 ChIP for –1.2 kb; four and threefold enrichment for –2.2 kb site, respectively). ( C ) Schematic of the luciferase reporter plasmids used in the luciferase assay. Dermo1 promoter element with –1.2 kb Tcf/Lef sites had substantial transactivation in the presence of wild-type β-catenin. The negative controls (empty pGL4.10 vector and the reverse construct Dermo1 Rev-Luc) showed comparable luciferase activity with and without β-catenin transactivation. ( D ) Proposed model for the role of Wnt signaling/β-catenin in the selection of cranial dermal cell fate. IP, immunoprecipitated; NT, non-target.

    Article Snippet: Either anti-Tcf4 antibody (Cell Signaling) or anti-H3K4me1 antibody (Abcam) was used.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Luciferase, Plasmid Preparation, Construct, Activity Assay, Selection, Immunoprecipitation

    Notch Induces Chromatin Opening and Promotes Assisted Loading (A–D″) Levels of H3K27ac—green in (A) and (B), white in (A″) and (B″)—and H3K4me1—green in (C) and (D), white in (C″) and (D″)—histone modifications at E(spl)-C (arrowheads) labeled by ParB::RFP—red in (A)–(D), white in (A′)–(D′)—in Control (A and C) and Notch-ON cells (B and D). Higher magnifications, insets in (A)–(D), show unchanged flanking loci (yellow asterisk) compared with increase at E(spl)-C locus (arrowheads). (E–J) Quantification of the indicated histone modifications across the E(spl)-C locus, both H3K27ac and H3K4me1 are increased in Notch-ON cells. n > 25; mean ± SEM. (I–J) Chromatin accessibility across E(spl)-C (I) measured by enrichment of fragments to transposon tagging with ATAC (J); positions of primers used in (J) are indicated in (I) relative to the gene models (dark blue) and Su(H)-binding profile in Kc cells (cyan). Rab11 intron and Eip78C EcR are predicted open chromatin control regions, while Negative1 and Mst87F are closed chromatin control regions, none of which are subject to Notch regulation. Shown is the fold enrichment compared with the Neg1 control region. n = 3; mean ± SEM. (K–L″) Increased Hairless-GFP recruitment—green in (K) and (L), white in (K″) and (L″)—at E(spl)-C —red in (K) and (L), white in (K′) and (L′)—in Notch-ON cells (L) (arrowhead) compared with Notch-OFF cells (K) (arrowhead). (M) Quantification of Hairless::GFP intensity across E(spl)-C . n > 25; mean ± SEM.

    Journal: Developmental Cell

    Article Title: Activation of the Notch Signaling Pathway In Vivo Elicits Changes in CSL Nuclear Dynamics

    doi: 10.1016/j.devcel.2018.01.020

    Figure Lengend Snippet: Notch Induces Chromatin Opening and Promotes Assisted Loading (A–D″) Levels of H3K27ac—green in (A) and (B), white in (A″) and (B″)—and H3K4me1—green in (C) and (D), white in (C″) and (D″)—histone modifications at E(spl)-C (arrowheads) labeled by ParB::RFP—red in (A)–(D), white in (A′)–(D′)—in Control (A and C) and Notch-ON cells (B and D). Higher magnifications, insets in (A)–(D), show unchanged flanking loci (yellow asterisk) compared with increase at E(spl)-C locus (arrowheads). (E–J) Quantification of the indicated histone modifications across the E(spl)-C locus, both H3K27ac and H3K4me1 are increased in Notch-ON cells. n > 25; mean ± SEM. (I–J) Chromatin accessibility across E(spl)-C (I) measured by enrichment of fragments to transposon tagging with ATAC (J); positions of primers used in (J) are indicated in (I) relative to the gene models (dark blue) and Su(H)-binding profile in Kc cells (cyan). Rab11 intron and Eip78C EcR are predicted open chromatin control regions, while Negative1 and Mst87F are closed chromatin control regions, none of which are subject to Notch regulation. Shown is the fold enrichment compared with the Neg1 control region. n = 3; mean ± SEM. (K–L″) Increased Hairless-GFP recruitment—green in (K) and (L), white in (K″) and (L″)—at E(spl)-C —red in (K) and (L), white in (K′) and (L′)—in Notch-ON cells (L) (arrowhead) compared with Notch-OFF cells (K) (arrowhead). (M) Quantification of Hairless::GFP intensity across E(spl)-C . n > 25; mean ± SEM.

    Article Snippet: These included Rb anti-PolII (1:500, Abcam 5095), Rb anti-H3K27ac (1:500, Abcam 4729), Rb anti-H3K4me1 (1:1000, Abcam 8895), Rb anti-Trr (1:100, ).

    Techniques: Labeling, Binding Assay

    Histone modifications at Cp and Qp. (A) Schematic of Cp and Qp locations on the EBV genome and positions of primer sets used for ChIP-qPCR. TR, terminal repeats. (B) ChIP-qPCR of histone H3, H3K9ac, H3K27ac or control IgG in MutuI cells (blue) or LCLs (red). (C) ChIP-qPCR of histones H3K4me1, H3K4me2, H3K4me3, H3K9me3, H3K27me3 in MutuI cells (blue) or LCLs (red). ChIP DNA is measured as percent input, and error bars represent standard deviations for three technical replicates.

    Journal: Journal of Virology

    Article Title: HCF1 and OCT2 Cooperate with EBNA1 To Enhance OriP-Dependent Transcription and Episome Maintenance of Latent Epstein-Barr Virus

    doi: 10.1128/JVI.00239-16

    Figure Lengend Snippet: Histone modifications at Cp and Qp. (A) Schematic of Cp and Qp locations on the EBV genome and positions of primer sets used for ChIP-qPCR. TR, terminal repeats. (B) ChIP-qPCR of histone H3, H3K9ac, H3K27ac or control IgG in MutuI cells (blue) or LCLs (red). (C) ChIP-qPCR of histones H3K4me1, H3K4me2, H3K4me3, H3K9me3, H3K27me3 in MutuI cells (blue) or LCLs (red). ChIP DNA is measured as percent input, and error bars represent standard deviations for three technical replicates.

    Article Snippet: Rabbit serum anti-H3K9me3 (catalog no. 39161), anti-H3K4me1 (catalog no. 61633), anti-H3K27me3 (catalog no. 39155)- and anti-H3K27acetyl (catalog no. 39685), and anti-H3K4me2 (catalog no. 39141) were from Active Motif.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Histone modifications at OriP. (A) Schematic of EBV OriP region and positions of primer sets used for ChIP-qPCR (primers A to K). (B) ChIP-qPCR of histone H3, H3K9ac, H3K27ac, or control IgG in MutuI cells (blue) or LCLs (red). (C) ChIP-qPCR of histones H3K4me1, H3K4me2, H3K4me3, H3K9me3, and H3K27me3 in MutuI cells (blue) or LCLs (red). ChIP DNA is measured as percent input, and error bars represent standard deviations for three technical replicates.

    Journal: Journal of Virology

    Article Title: HCF1 and OCT2 Cooperate with EBNA1 To Enhance OriP-Dependent Transcription and Episome Maintenance of Latent Epstein-Barr Virus

    doi: 10.1128/JVI.00239-16

    Figure Lengend Snippet: Histone modifications at OriP. (A) Schematic of EBV OriP region and positions of primer sets used for ChIP-qPCR (primers A to K). (B) ChIP-qPCR of histone H3, H3K9ac, H3K27ac, or control IgG in MutuI cells (blue) or LCLs (red). (C) ChIP-qPCR of histones H3K4me1, H3K4me2, H3K4me3, H3K9me3, and H3K27me3 in MutuI cells (blue) or LCLs (red). ChIP DNA is measured as percent input, and error bars represent standard deviations for three technical replicates.

    Article Snippet: Rabbit serum anti-H3K9me3 (catalog no. 39161), anti-H3K4me1 (catalog no. 61633), anti-H3K27me3 (catalog no. 39155)- and anti-H3K27acetyl (catalog no. 39685), and anti-H3K4me2 (catalog no. 39141) were from Active Motif.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Lack of Ccl1 results in decreased global H3K4me3. The Fusarium wild type strains ( F. fujikuroi – FfWT, F. graminearum – FgWT) and the respective CCL1 deletion mutants ( F. fujikuroi – Δ ffccl1 , F. graminearum – Δ fgccl1 ) were grown for 3 days on solid complete medium. Whole protein extracts were subsequently isolated from lyophilized mycelia and roughly 15 μg of proteins were used for SDS-Page and western blotting. (A) The following primary antibodies were used for detection: H3 C-Term, H3K4me1, H3K4me2 as well as three different H3K4me3. (B) Coomassie staining was performed as an additional loading control.

    Journal: Frontiers in Microbiology

    Article Title: Lack of the COMPASS Component Ccl1 Reduces H3K4 Trimethylation Levels and Affects Transcription of Secondary Metabolite Genes in Two Plant–Pathogenic Fusarium Species

    doi: 10.3389/fmicb.2016.02144

    Figure Lengend Snippet: Lack of Ccl1 results in decreased global H3K4me3. The Fusarium wild type strains ( F. fujikuroi – FfWT, F. graminearum – FgWT) and the respective CCL1 deletion mutants ( F. fujikuroi – Δ ffccl1 , F. graminearum – Δ fgccl1 ) were grown for 3 days on solid complete medium. Whole protein extracts were subsequently isolated from lyophilized mycelia and roughly 15 μg of proteins were used for SDS-Page and western blotting. (A) The following primary antibodies were used for detection: H3 C-Term, H3K4me1, H3K4me2 as well as three different H3K4me3. (B) Coomassie staining was performed as an additional loading control.

    Article Snippet: The membrane was probed with H3 C-Term (Active Motif AM39163), H3K4me1 (Active Motif AM39297), H3K4me2 (Active Motif AM39141) as well as three different H3K4me3 (Active Motif AM39159, abcam ab8580, Millipore MP#07-473) primary antibodies and anti-rabbit (Sigma A0545) HRP conjugated secondary antibody.

    Techniques: Isolation, SDS Page, Western Blot, Staining

    Gene expression and histone modifications in regulated broadly-expressed and stable tissue-specific genes at third instar larvae a, Diagrams of developmentally regulated genes broadly-expressed across multiple tissues at third instar-larvae L3 (left panel), and stable genes expressed in only one tissue at L3 (right panel). b, Gene expression levels at L3 measured by whole organism RNASeq (left panel). The number of genes in each category is given under the boxplots. The bottom and top of the boxes are the first and third quartiles, and the line within, the median. The whiskers denote the interval within 1.5 times the IQR from the median. Outliers are plotted as dots. Validation by qPCR of the expression at L3 of regulated broadly-expressed genes compared to a stable gene ( Bmcp ) and a silent gene ( CG5367 ) (right panel). Error bars represent the Standard Error of the Mean (SEM) from three independent replicates. c, Levels of H3K4me3, H3K9ac, H3K4me1 and H3K27ac on whole L3 individuals. The seven regulated genes broadly-expressed at L3 are depicted as red dots within the boxplots. P-values were computed using the Wilcoxon test (two-sided). d, Validation by individual ChIPs and qPCR of H3K4me3 and H3K9ac in regulated genes broadly-expressed at L3. H3K4me3 and H3K9ac ChIPs are represented as enrichment of the marks over the silent gene ( CG5367 ). Error bars represent the SEM from three independent replicates.

    Journal: Nature genetics

    Article Title: Absence of canonical active chromatin marks in developmentally regulated genes

    doi: 10.1038/ng.3381

    Figure Lengend Snippet: Gene expression and histone modifications in regulated broadly-expressed and stable tissue-specific genes at third instar larvae a, Diagrams of developmentally regulated genes broadly-expressed across multiple tissues at third instar-larvae L3 (left panel), and stable genes expressed in only one tissue at L3 (right panel). b, Gene expression levels at L3 measured by whole organism RNASeq (left panel). The number of genes in each category is given under the boxplots. The bottom and top of the boxes are the first and third quartiles, and the line within, the median. The whiskers denote the interval within 1.5 times the IQR from the median. Outliers are plotted as dots. Validation by qPCR of the expression at L3 of regulated broadly-expressed genes compared to a stable gene ( Bmcp ) and a silent gene ( CG5367 ) (right panel). Error bars represent the Standard Error of the Mean (SEM) from three independent replicates. c, Levels of H3K4me3, H3K9ac, H3K4me1 and H3K27ac on whole L3 individuals. The seven regulated genes broadly-expressed at L3 are depicted as red dots within the boxplots. P-values were computed using the Wilcoxon test (two-sided). d, Validation by individual ChIPs and qPCR of H3K4me3 and H3K9ac in regulated genes broadly-expressed at L3. H3K4me3 and H3K9ac ChIPs are represented as enrichment of the marks over the silent gene ( CG5367 ). Error bars represent the SEM from three independent replicates.

    Article Snippet: The antibodies used for ChIP were: H3 (Abcam/ab1791); H3K4me3 (Abcam/ab8580) (Millipore-Upstate/07-473), H3K9ac (Abcam/ab4441), H3K36me3 (Abcam/ab9050), H3K4me1 (Diagenode/CS-037-100), H3K27ac (Abcam/ab4729) and H3K27me3 (Upstate-Millipore/07-449).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Distribution of histone modification levels in stable, regulated and silent genes during fly development a , Expression of stable, regulated, and silent genes during fly development at the time point of maximum expression for each gene. Gene expression was computed as FPKMs by the modENCODE consortium. The bottom and top of the boxes are the first and third quartiles, and the line within, the median. The whiskers denote the interval within 1.5 times the Inter Quartile Range (IQR) from the median. Outliers are plotted as dots. b, Normalized levels of H3K4me3, H3K9ac, H3K4me1 and H3K27ac at the time point of maximum expression during D. melanogaster development. These values represent the maximum height of the ChIPSeq peak within the gene body. P-values were computed using the Wilcoxon text (two-sided). c , Profiles of H3K4me3 during the 12 fly developmental time points in CG8636 , a gene stably expressed during fly development, and CG16733 , a pupa-specific gene. The expression (measured as FPKMs) along these points for the two genes is given on the left. d , Levels of H3K27me3 and H3K9me3 at the time point of maximum expression, computed as the average height of the ChIPSeq signal within the gene body, in stable, regulated and silent genes.

    Journal: Nature genetics

    Article Title: Absence of canonical active chromatin marks in developmentally regulated genes

    doi: 10.1038/ng.3381

    Figure Lengend Snippet: Distribution of histone modification levels in stable, regulated and silent genes during fly development a , Expression of stable, regulated, and silent genes during fly development at the time point of maximum expression for each gene. Gene expression was computed as FPKMs by the modENCODE consortium. The bottom and top of the boxes are the first and third quartiles, and the line within, the median. The whiskers denote the interval within 1.5 times the Inter Quartile Range (IQR) from the median. Outliers are plotted as dots. b, Normalized levels of H3K4me3, H3K9ac, H3K4me1 and H3K27ac at the time point of maximum expression during D. melanogaster development. These values represent the maximum height of the ChIPSeq peak within the gene body. P-values were computed using the Wilcoxon text (two-sided). c , Profiles of H3K4me3 during the 12 fly developmental time points in CG8636 , a gene stably expressed during fly development, and CG16733 , a pupa-specific gene. The expression (measured as FPKMs) along these points for the two genes is given on the left. d , Levels of H3K27me3 and H3K9me3 at the time point of maximum expression, computed as the average height of the ChIPSeq signal within the gene body, in stable, regulated and silent genes.

    Article Snippet: The antibodies used for ChIP were: H3 (Abcam/ab1791); H3K4me3 (Abcam/ab8580) (Millipore-Upstate/07-473), H3K9ac (Abcam/ab4441), H3K36me3 (Abcam/ab9050), H3K4me1 (Diagenode/CS-037-100), H3K27ac (Abcam/ab4729) and H3K27me3 (Upstate-Millipore/07-449).

    Techniques: Modification, Expressing, Stable Transfection

    Chromatin modifications and accessibility within the Krox20 locus. (A) ChIP-seq was performed for the H3K4me1 mark on wild type E8.5 embryo heads (light blue) using biological duplicates. Only one set of data is shown. ATAC-seq was performed on dissected regions (r3, r5 and a more posterior region (“post”; see text) from wild type embryos at E8.5 (light blue) and E9.5 (dark blue) using biological duplicates. Only one set of data is shown. CTCF ChIP-seq data from E14.5 mouse brain (ENCODE) are indicated below (see Fig 3A ). Genes, cis -regulatory elements (orange) and a genomic scale are indicated at the top. (B) ATAC-seq was performed on dissected parts from wild type (light blue), and Krox20 ΔC/ΔC (green) and Krox20 ΔA/ΔA (red) E8.5 embryos using biological duplicates. Only one set of data is shown. Cis -regulatory elements (orange) and a genomic scale are indicated at the top. Arrowheads indicate the summits (defined by macs2 after peak calling, see Material and methods ) used for quantifications in (C). (C) Barplots showing signal intensity of ATAC-seq (normalized fragment counts) at the summit of each element (arrowheads in panel B) for wild type (WT, blue) and Krox20 ΔC/ΔC (ΔC, green) embryos at E8.5 for each dissected part. The statistical significance was calculated using a negative binomial Wald Test (R package DESeq2) on the 2 replicates, which are represented by dots. Star indicates p-value

    Journal: PLoS Genetics

    Article Title: Krox20 hindbrain regulation incorporates multiple modes of cooperation between cis-acting elements

    doi: 10.1371/journal.pgen.1006903

    Figure Lengend Snippet: Chromatin modifications and accessibility within the Krox20 locus. (A) ChIP-seq was performed for the H3K4me1 mark on wild type E8.5 embryo heads (light blue) using biological duplicates. Only one set of data is shown. ATAC-seq was performed on dissected regions (r3, r5 and a more posterior region (“post”; see text) from wild type embryos at E8.5 (light blue) and E9.5 (dark blue) using biological duplicates. Only one set of data is shown. CTCF ChIP-seq data from E14.5 mouse brain (ENCODE) are indicated below (see Fig 3A ). Genes, cis -regulatory elements (orange) and a genomic scale are indicated at the top. (B) ATAC-seq was performed on dissected parts from wild type (light blue), and Krox20 ΔC/ΔC (green) and Krox20 ΔA/ΔA (red) E8.5 embryos using biological duplicates. Only one set of data is shown. Cis -regulatory elements (orange) and a genomic scale are indicated at the top. Arrowheads indicate the summits (defined by macs2 after peak calling, see Material and methods ) used for quantifications in (C). (C) Barplots showing signal intensity of ATAC-seq (normalized fragment counts) at the summit of each element (arrowheads in panel B) for wild type (WT, blue) and Krox20 ΔC/ΔC (ΔC, green) embryos at E8.5 for each dissected part. The statistical significance was calculated using a negative binomial Wald Test (R package DESeq2) on the 2 replicates, which are represented by dots. Star indicates p-value

    Article Snippet: 5–10 μg of chromatin was used for each IP using 3 μg of the following antibodies: anti H3K4me1 (C15410037, Diagenode) and anti H3K27ac (ab4729, Abcam) in RIPA buffer.

    Techniques: Chromatin Immunoprecipitation

    Little evidence of Setd7 interacting with MyoD or acting as an H3K4me1 histone methyltransferase in MuSCs (A) Immunostaining of Setd7 in plated MuSCs highlighting cytoplasmic localization. Scale bar = 50µm. (B) Western blot analysis of Setd7 in Cytoplasmic (cyto), nuclear (nuc) and chromatin (chrom) fractions from plated MuSCs and myotubes. (C) Western blot analysis of H3K4me1 in WT and Sets7KO plated MuSCs. (D) Western blot of H3K4me1, me2, me3 in plated MuSCs and myotubes treated with or without a Setd7 inhibitor (PFI-2). (E+F) H3K4me1 ChIPSeq scatter plot correlating mean peak density in plated MuSCs treated with or without PFI-2 across whole genome (E) or at TSS (+/− 5KB) sites (F). (G) Mean signal of normalized peak density surrounding TSSs for H3K4me1 ChIPSeq for control treated MuSCs (blue line) and PFI-2 treated MuSCs (red line) (H) Heatmap displaying single gene resolution of H3K4me1 ChIP-Seq TSS enrichment data in (G). (I) Comparisons in H3K4me1 enrichment (RPKM) at myoblast (MB) and myotube (MT) enhancer regions in MuSCs treated with or without PFI-2. (J) Setd7 and MyoD co-immunostaining in plated MuSCs highlighting lack of co-localization. Scale bar = 100µm. (K) Western blot analysis of Setd7 and MyoD in cytoplasmic (cyto) and nuclear (nuc) fractions of plated MuSCs. (L) Setd7 IP samples blotted and probed with an anti-MyoD antibody. (M) H3K4me1 genome browser tracks in proximal promoters and gene bodies of MyoD targets (MyoG, Myh1 and Myl1). Control MuSCs (blue line) and PFI-2 treated MuSCs (red line) R values calculated with Pearson’s correlation coefficient.

    Journal: Cell stem cell

    Article Title: Inhibition of methyltransferase Setd7 allows the in vitro expansion of myogenic stem cells with improved therapeutic potential

    doi: 10.1016/j.stem.2017.12.010

    Figure Lengend Snippet: Little evidence of Setd7 interacting with MyoD or acting as an H3K4me1 histone methyltransferase in MuSCs (A) Immunostaining of Setd7 in plated MuSCs highlighting cytoplasmic localization. Scale bar = 50µm. (B) Western blot analysis of Setd7 in Cytoplasmic (cyto), nuclear (nuc) and chromatin (chrom) fractions from plated MuSCs and myotubes. (C) Western blot analysis of H3K4me1 in WT and Sets7KO plated MuSCs. (D) Western blot of H3K4me1, me2, me3 in plated MuSCs and myotubes treated with or without a Setd7 inhibitor (PFI-2). (E+F) H3K4me1 ChIPSeq scatter plot correlating mean peak density in plated MuSCs treated with or without PFI-2 across whole genome (E) or at TSS (+/− 5KB) sites (F). (G) Mean signal of normalized peak density surrounding TSSs for H3K4me1 ChIPSeq for control treated MuSCs (blue line) and PFI-2 treated MuSCs (red line) (H) Heatmap displaying single gene resolution of H3K4me1 ChIP-Seq TSS enrichment data in (G). (I) Comparisons in H3K4me1 enrichment (RPKM) at myoblast (MB) and myotube (MT) enhancer regions in MuSCs treated with or without PFI-2. (J) Setd7 and MyoD co-immunostaining in plated MuSCs highlighting lack of co-localization. Scale bar = 100µm. (K) Western blot analysis of Setd7 and MyoD in cytoplasmic (cyto) and nuclear (nuc) fractions of plated MuSCs. (L) Setd7 IP samples blotted and probed with an anti-MyoD antibody. (M) H3K4me1 genome browser tracks in proximal promoters and gene bodies of MyoD targets (MyoG, Myh1 and Myl1). Control MuSCs (blue line) and PFI-2 treated MuSCs (red line) R values calculated with Pearson’s correlation coefficient.

    Article Snippet: For ChIP, anti-H3K4me1 antibody (Diagenode, Cat# C15410037 ) was incubated with anti-IgA magnetic beads (Dynabeads, ThemoFisher) for 2 hours.

    Techniques: Immunostaining, Western Blot, Chromatin Immunoprecipitation

    High concordance between mitotic and interphase histone modification patterns. ( A ) tracks showing the alignments for the indicated histone modification ChIP-seq results for interphase (blue) and mitosis (red) samples on an ∼315-kb region on Chromosome 12. The scale of each track was adjusted to the total number of reads using the Normalize Coverage Data option in IGV (see Methods). The ChromHMM annotation for each genomic region is shown below the plot using the same color code as in C . ( B ) Scatter plots showing the normalized read counts for all regions enriched in each modification (see Methods for more details) in mitosis versus interphase. It should be noted that ChIP-seq-based quantification may be less accurate than quantification based on mass spectrometry data due to inherent noise in the ChIP-seq method. Thus, in cases of a discrepancy, e.g., H3K4me1, we rely on the mass spectrometry results for quantification, and on the ChIP-seq results for localization. ( C ) Bar plots showing the percentage of reads in peak regions per ChromHMM category.

    Journal: Genome Research

    Article Title: Study of mitotic chromatin supports a model of bookmarking by histone modifications and reveals nucleosome deposition patterns

    doi: 10.1101/gr.230300.117

    Figure Lengend Snippet: High concordance between mitotic and interphase histone modification patterns. ( A ) tracks showing the alignments for the indicated histone modification ChIP-seq results for interphase (blue) and mitosis (red) samples on an ∼315-kb region on Chromosome 12. The scale of each track was adjusted to the total number of reads using the Normalize Coverage Data option in IGV (see Methods). The ChromHMM annotation for each genomic region is shown below the plot using the same color code as in C . ( B ) Scatter plots showing the normalized read counts for all regions enriched in each modification (see Methods for more details) in mitosis versus interphase. It should be noted that ChIP-seq-based quantification may be less accurate than quantification based on mass spectrometry data due to inherent noise in the ChIP-seq method. Thus, in cases of a discrepancy, e.g., H3K4me1, we rely on the mass spectrometry results for quantification, and on the ChIP-seq results for localization. ( C ) Bar plots showing the percentage of reads in peak regions per ChromHMM category.

    Article Snippet: The following antibodies were used: anti-RNA polymerase II 8WG16 (ab817, Abcam), anti-H3K4me1 (D1A9, Cell Signaling Technology), anti-H3K4me3 (C42D8, Cell Signaling Technology), anti-H3K9ac (C5B11, Cell Signaling Technology), anti-H3K27ac (D5E4, Cell Signaling Technology), anti-H3K27me3 (C36B11, Cell Signaling Technology), anti-H3K36me3 (D5A7, Cell Signaling Technology), and anti-CTCF G.758.4 (MA5-11187, Invitrogen).

    Techniques: Modification, Chromatin Immunoprecipitation, Mass Spectrometry

    KDM3A regulates H3K27ac and TEAD1 binding on enhancers. ( A , B ) ChIP analysis shows TEAD1 enrichment in KDM3A WT and KO cells lines. KDM3A depletion attenuates TEAD1 recruitment to CTGF under all three conditions (A), while on CYR61 only during FBS treatment (B). ( C ) ChIP analysis shows H3K4me1 and H3K27ac enrichment on CYR61 enhancers in KDM3A WT and KO cells lines. The locations of two enhancers are shown at the bottom. ( D ) The average H3K9me2, H3K27ac and TEAD1 enrichment on all TEAD1 target sites. ( E ) Heat map shows the expression (FPKM) and clustering of TEAD1 target genes. ( F ) The average H3K9me2, H3K27ac and TEAD1 enrichment on enhancers of KDM3A-dependent genes. ( G ) ChIP assays to show p300 enrichment on CYR61 enhancer under starvation and FBS treatment. ( H ) Co-immunoprecipitation of p300 and KDM3A in HCT116 cells under starvation and FBS treatment. • labelled non-specific bands. * means P -value ≤ 0.05, ** for P -value ≤ 0.01. The results in all histograms represent the means (±SD) of at least three independent experiments. The sequencing data were obtained from two biological replicates.

    Journal: Nucleic Acids Research

    Article Title: Histone demethylase KDM3A is required for enhancer activation of hippo target genes in colorectal cancer

    doi: 10.1093/nar/gky1317

    Figure Lengend Snippet: KDM3A regulates H3K27ac and TEAD1 binding on enhancers. ( A , B ) ChIP analysis shows TEAD1 enrichment in KDM3A WT and KO cells lines. KDM3A depletion attenuates TEAD1 recruitment to CTGF under all three conditions (A), while on CYR61 only during FBS treatment (B). ( C ) ChIP analysis shows H3K4me1 and H3K27ac enrichment on CYR61 enhancers in KDM3A WT and KO cells lines. The locations of two enhancers are shown at the bottom. ( D ) The average H3K9me2, H3K27ac and TEAD1 enrichment on all TEAD1 target sites. ( E ) Heat map shows the expression (FPKM) and clustering of TEAD1 target genes. ( F ) The average H3K9me2, H3K27ac and TEAD1 enrichment on enhancers of KDM3A-dependent genes. ( G ) ChIP assays to show p300 enrichment on CYR61 enhancer under starvation and FBS treatment. ( H ) Co-immunoprecipitation of p300 and KDM3A in HCT116 cells under starvation and FBS treatment. • labelled non-specific bands. * means P -value ≤ 0.05, ** for P -value ≤ 0.01. The results in all histograms represent the means (±SD) of at least three independent experiments. The sequencing data were obtained from two biological replicates.

    Article Snippet: Reagents and cell lines Antibodies against KDM3A (Abcam ab91252), β-Actin (Abclonal AC006), hippo signaling antibody sampler kit (CST 8579, including LATS1, MST1, MST2, SAV1, pYAP S127, YAP, MOB1, pMOB1 Thr35), H3K9me2 (Abcam ab1220), TEAD1 (CST 12292), p300 (Santa Cruz sc-585x), H3K4me1 (CST 5326), H3K27ac (Abcam ab4729), H3K27me3 (Millipore 07-449), Pol II (Abcam ab817), H3K4me3(Millipore 04-745), were purchased from indicated commercial sources.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Expressing, Immunoprecipitation, Sequencing

    H3K4me1 profiles segregate discrete classes of transcription factor occupied loci. ( A ) Heatmaps of H3K4me1 read density in ±2-kb regions centered on FOXA2 (islets or liver), PDX1, and HNF4A peak maxima. Peak max locations are indicated by red

    Journal: Genome Research

    Article Title: Locus co-occupancy, nucleosome positioning, and H3K4me1 regulate the functionality of FOXA2-, HNF4A-, and PDX1-bound loci in islets and liver

    doi: 10.1101/gr.104356.109

    Figure Lengend Snippet: H3K4me1 profiles segregate discrete classes of transcription factor occupied loci. ( A ) Heatmaps of H3K4me1 read density in ±2-kb regions centered on FOXA2 (islets or liver), PDX1, and HNF4A peak maxima. Peak max locations are indicated by red

    Article Snippet: Perfused liver or purified E14.5 hepatocytes were obtained from C57BL6/J, and ChIP was performed as described ( ) using 3 μg of anti-FOXA2 (Santa Cruz), anti-PDX1 (Upstate), anti-HNF4A (Santa Cruz), anti-H3K4me1 (Santa Cruz), anti-H3K4me3 (Santa Cruz), or normal rabbit IgG (Santa Cruz).

    Techniques:

    H3K4me1-marked nucleosome occupancy regulates transcription factor bound cis -regulatory site function. Analysis of H3K4me1 transitions for transcription factor occupied loci during ( A–D ) an IFNG-stimulated signaling event, or ( E–H ) liver

    Journal: Genome Research

    Article Title: Locus co-occupancy, nucleosome positioning, and H3K4me1 regulate the functionality of FOXA2-, HNF4A-, and PDX1-bound loci in islets and liver

    doi: 10.1101/gr.104356.109

    Figure Lengend Snippet: H3K4me1-marked nucleosome occupancy regulates transcription factor bound cis -regulatory site function. Analysis of H3K4me1 transitions for transcription factor occupied loci during ( A–D ) an IFNG-stimulated signaling event, or ( E–H ) liver

    Article Snippet: Perfused liver or purified E14.5 hepatocytes were obtained from C57BL6/J, and ChIP was performed as described ( ) using 3 μg of anti-FOXA2 (Santa Cruz), anti-PDX1 (Upstate), anti-HNF4A (Santa Cruz), anti-H3K4me1 (Santa Cruz), anti-H3K4me3 (Santa Cruz), or normal rabbit IgG (Santa Cruz).

    Techniques:

    Bimodal sites regulate target gene expression. Box-whisker plots of islet or liver ( A ) Tag-seq counts or ( B ) specificity against 202 other mouse SAGE libraries, for expressed genes associated with a bimodal, monomodal, or low H3K4me1 site in the FOXA2

    Journal: Genome Research

    Article Title: Locus co-occupancy, nucleosome positioning, and H3K4me1 regulate the functionality of FOXA2-, HNF4A-, and PDX1-bound loci in islets and liver

    doi: 10.1101/gr.104356.109

    Figure Lengend Snippet: Bimodal sites regulate target gene expression. Box-whisker plots of islet or liver ( A ) Tag-seq counts or ( B ) specificity against 202 other mouse SAGE libraries, for expressed genes associated with a bimodal, monomodal, or low H3K4me1 site in the FOXA2

    Article Snippet: Perfused liver or purified E14.5 hepatocytes were obtained from C57BL6/J, and ChIP was performed as described ( ) using 3 μg of anti-FOXA2 (Santa Cruz), anti-PDX1 (Upstate), anti-HNF4A (Santa Cruz), anti-H3K4me1 (Santa Cruz), anti-H3K4me3 (Santa Cruz), or normal rabbit IgG (Santa Cruz).

    Techniques: Expressing, Whisker Assay

    Co-factors bind few of their available motifs at monomodal or low H3K4me1 sites. Venn diagrams showing the number of loci shared by ( A ) PDX1 and FOXA2 in islets or ( B ) FOXA2 and HNF4A in the liver. ( C ) Fraction of co-bound islet sites (FOXA2–PDX1)

    Journal: Genome Research

    Article Title: Locus co-occupancy, nucleosome positioning, and H3K4me1 regulate the functionality of FOXA2-, HNF4A-, and PDX1-bound loci in islets and liver

    doi: 10.1101/gr.104356.109

    Figure Lengend Snippet: Co-factors bind few of their available motifs at monomodal or low H3K4me1 sites. Venn diagrams showing the number of loci shared by ( A ) PDX1 and FOXA2 in islets or ( B ) FOXA2 and HNF4A in the liver. ( C ) Fraction of co-bound islet sites (FOXA2–PDX1)

    Article Snippet: Perfused liver or purified E14.5 hepatocytes were obtained from C57BL6/J, and ChIP was performed as described ( ) using 3 μg of anti-FOXA2 (Santa Cruz), anti-PDX1 (Upstate), anti-HNF4A (Santa Cruz), anti-H3K4me1 (Santa Cruz), anti-H3K4me3 (Santa Cruz), or normal rabbit IgG (Santa Cruz).

    Techniques:

    dBRWD3 is epistatic to trx in the ectopic expression of Ubx and Abd-B . (A and B) A ChIP-qPCR analysis of H3K27me3 levels at the enhancers, promoters, and transcription start sites of Antp (A) and Ubx (B) in Elav-GAL4 control, E(z) depleted, and E(z) , dBRWD3 doubly depleted brains. Black asterisks indicate control versus E(z) depletion. Blue asterisks indicate E(z) depletion versus E(z) , dBRWD3 double depletion. (C and D) A ChIP-qPCR analysis of H2AK118ub levels at the enhancers, promoters, and transcription start sites of Antp (C) and Ubx (D) in Elav-GAL4 control, Pc depleted, and Pc , dBRWD3 doubly depleted brains. Black asterisks indicate control versus Pc depletion. Blue asterisks indicates Pc depletion versus Pc , dBRWD3 double depletion. (E-G) trx was overexpressed under the control of ms-1096-GAL4 . The TRX-induced Abd-B expression (arrows) in wild-type (E), dBRWD3 depletion (F), and yem over-expression (G) backgrounds. Scale bars indicate 20μm. (H) A ChIP-qPCR analysis of H3K4me1 levels at Ubx and Abd-B enhancers in the UAS-mCD8-GFP control, trx over-expression, and trx over-expression, dBRWD3 depleted wings as indicated. ns. indicates not significant. ChIP-qPCR Data are shown as means ± S.D from 4 technical replicates. *, **, *** indicate P

    Journal: PLoS Genetics

    Article Title: dBRWD3 Regulates Tissue Overgrowth and Ectopic Gene Expression Caused by Polycomb Group Mutations

    doi: 10.1371/journal.pgen.1006262

    Figure Lengend Snippet: dBRWD3 is epistatic to trx in the ectopic expression of Ubx and Abd-B . (A and B) A ChIP-qPCR analysis of H3K27me3 levels at the enhancers, promoters, and transcription start sites of Antp (A) and Ubx (B) in Elav-GAL4 control, E(z) depleted, and E(z) , dBRWD3 doubly depleted brains. Black asterisks indicate control versus E(z) depletion. Blue asterisks indicate E(z) depletion versus E(z) , dBRWD3 double depletion. (C and D) A ChIP-qPCR analysis of H2AK118ub levels at the enhancers, promoters, and transcription start sites of Antp (C) and Ubx (D) in Elav-GAL4 control, Pc depleted, and Pc , dBRWD3 doubly depleted brains. Black asterisks indicate control versus Pc depletion. Blue asterisks indicates Pc depletion versus Pc , dBRWD3 double depletion. (E-G) trx was overexpressed under the control of ms-1096-GAL4 . The TRX-induced Abd-B expression (arrows) in wild-type (E), dBRWD3 depletion (F), and yem over-expression (G) backgrounds. Scale bars indicate 20μm. (H) A ChIP-qPCR analysis of H3K4me1 levels at Ubx and Abd-B enhancers in the UAS-mCD8-GFP control, trx over-expression, and trx over-expression, dBRWD3 depleted wings as indicated. ns. indicates not significant. ChIP-qPCR Data are shown as means ± S.D from 4 technical replicates. *, **, *** indicate P

    Article Snippet: Primary antibodies used in this study include mouse anti-H2AK118ub (1:100, Millipore, E6C5), rabbit anti-H3K27me3 (1:100, Millipore), rabbit anti-H3K4me1 (1:100, Active Motif), rabbit anti-H3S10ph (1:500, Millipore), mouse anti-Ubx (1:20, DSHB, Ubx), mouse anti-Antp (1:20, DSHB, 8C11), rabbit anti-upd3 (1:750), and mouse anti-β-Galactosidase (1:1000, Sigma, GAL-50).

    Techniques: Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Over Expression

    Little evidence of Setd7 interacting with MyoD or acting as an H3K4me1 histone methyltransferase in MuSCs (A) Immunostaining of Setd7 in plated MuSCs highlighting cytoplasmic localization. Scale bar = 50µm. (B) Western blot analysis of Setd7 in Cytoplasmic (cyto), nuclear (nuc) and chromatin (chrom) fractions from plated MuSCs and myotubes. (C) Western blot analysis of H3K4me1 in WT and Sets7KO plated MuSCs. (D) Western blot of H3K4me1, me2, me3 in plated MuSCs and myotubes treated with or without a Setd7 inhibitor (PFI-2). (E+F) H3K4me1 ChIPSeq scatter plot correlating mean peak density in plated MuSCs treated with or without PFI-2 across whole genome (E) or at TSS (+/− 5KB) sites (F). (G) Mean signal of normalized peak density surrounding TSSs for H3K4me1 ChIPSeq for control treated MuSCs (blue line) and PFI-2 treated MuSCs (red line) (H) Heatmap displaying single gene resolution of H3K4me1 ChIP-Seq TSS enrichment data in (G). (I) Comparisons in H3K4me1 enrichment (RPKM) at myoblast (MB) and myotube (MT) enhancer regions in MuSCs treated with or without PFI-2. (J) Setd7 and MyoD co-immunostaining in plated MuSCs highlighting lack of co-localization. Scale bar = 100µm. (K) Western blot analysis of Setd7 and MyoD in cytoplasmic (cyto) and nuclear (nuc) fractions of plated MuSCs. (L) Setd7 IP samples blotted and probed with an anti-MyoD antibody. (M) H3K4me1 genome browser tracks in proximal promoters and gene bodies of MyoD targets (MyoG, Myh1 and Myl1). Control MuSCs (blue line) and PFI-2 treated MuSCs (red line) R values calculated with Pearson’s correlation coefficient.

    Journal: Cell stem cell

    Article Title: Inhibition of methyltransferase Setd7 allows the in vitro expansion of myogenic stem cells with improved therapeutic potential

    doi: 10.1016/j.stem.2017.12.010

    Figure Lengend Snippet: Little evidence of Setd7 interacting with MyoD or acting as an H3K4me1 histone methyltransferase in MuSCs (A) Immunostaining of Setd7 in plated MuSCs highlighting cytoplasmic localization. Scale bar = 50µm. (B) Western blot analysis of Setd7 in Cytoplasmic (cyto), nuclear (nuc) and chromatin (chrom) fractions from plated MuSCs and myotubes. (C) Western blot analysis of H3K4me1 in WT and Sets7KO plated MuSCs. (D) Western blot of H3K4me1, me2, me3 in plated MuSCs and myotubes treated with or without a Setd7 inhibitor (PFI-2). (E+F) H3K4me1 ChIPSeq scatter plot correlating mean peak density in plated MuSCs treated with or without PFI-2 across whole genome (E) or at TSS (+/− 5KB) sites (F). (G) Mean signal of normalized peak density surrounding TSSs for H3K4me1 ChIPSeq for control treated MuSCs (blue line) and PFI-2 treated MuSCs (red line) (H) Heatmap displaying single gene resolution of H3K4me1 ChIP-Seq TSS enrichment data in (G). (I) Comparisons in H3K4me1 enrichment (RPKM) at myoblast (MB) and myotube (MT) enhancer regions in MuSCs treated with or without PFI-2. (J) Setd7 and MyoD co-immunostaining in plated MuSCs highlighting lack of co-localization. Scale bar = 100µm. (K) Western blot analysis of Setd7 and MyoD in cytoplasmic (cyto) and nuclear (nuc) fractions of plated MuSCs. (L) Setd7 IP samples blotted and probed with an anti-MyoD antibody. (M) H3K4me1 genome browser tracks in proximal promoters and gene bodies of MyoD targets (MyoG, Myh1 and Myl1). Control MuSCs (blue line) and PFI-2 treated MuSCs (red line) R values calculated with Pearson’s correlation coefficient.

    Article Snippet: Membranes were probed with the following primary antibodies overnight at 4°C: 1:500 mouse anti-Setd7 (Abcam, clone 2D10), 1:500 rabbit anti-Setd7 (Cell Signalling Technologies), 1:200 rabbit anti-MyoD (Santa-Cruz, clone C-20), 1:2000 mouse anti-MyHC (DSHB, clone MF20 concentrate), 1:1000 rabbit anti-alpha Tubulin (Abcam), 1:1000 rabbit anti-β-Actin (Abcam), rabbit anti-histone 3 (Cell Signalling Technologies), 1:2000 rabbit anti-H3K4me1 (Cell Signalling Technologies, Clone D1H2), 1:1000 rabbit anti-H3K4me2 (Cell Signalling Technologies, clone D1A9), 1:1000 rabbit anti-H3K4me3 (Cell Signalling Technologies), 1:200 mouse anti-β-catenin (Santa Cruz, clone E5).

    Techniques: Immunostaining, Western Blot, Chromatin Immunoprecipitation

    Met-VEL clusters occur across metastatic cancers a . UCSC browser view of H3K4me1 profiles in MG63.3 (metastatic) and MG63 (parental) cell lines illustrating an example of a gained (left) and lost (right) Met-VEL cluster. Met-VELs identified by black bars. 200kb Met-VEL clusters highlighted in gray. b . Genome-wide lost Met-VEL landscape for MG63.3 cell line. Rows represent scaled chromosomal coordinates. Peaks represent maximum gained Met-VEL counts in 200kb sliding windows. Predicted target genes for selected peaks are labeled. c . Gained and lost Met-VEL cluster counts in patient lung metastases/primary tumors and metastatic/parental cell line pairs. d . Percentage of total gained (top) and lost (bottom) Met-VELs within and outside of clusters in patient lung metastases/primary tumors and metastatic/parental cell line pairs.

    Journal: Nature medicine

    Article Title: Positively selected enhancer elements endow osteosarcoma cells with metastatic competence

    doi: 10.1038/nm.4475

    Figure Lengend Snippet: Met-VEL clusters occur across metastatic cancers a . UCSC browser view of H3K4me1 profiles in MG63.3 (metastatic) and MG63 (parental) cell lines illustrating an example of a gained (left) and lost (right) Met-VEL cluster. Met-VELs identified by black bars. 200kb Met-VEL clusters highlighted in gray. b . Genome-wide lost Met-VEL landscape for MG63.3 cell line. Rows represent scaled chromosomal coordinates. Peaks represent maximum gained Met-VEL counts in 200kb sliding windows. Predicted target genes for selected peaks are labeled. c . Gained and lost Met-VEL cluster counts in patient lung metastases/primary tumors and metastatic/parental cell line pairs. d . Percentage of total gained (top) and lost (bottom) Met-VELs within and outside of clusters in patient lung metastases/primary tumors and metastatic/parental cell line pairs.

    Article Snippet: The following antibodies were used for ChIP: rabbit anti-H3K4me1 (Abcam #8895), rabbit anti-H3K27ac (Abcam #4729), rabbit anti-c-Fos (Santa Cruz #sc-52), rabbit anti-FOSL1/Fra-1 (Santa Cruz #sc-605).

    Techniques: Genome Wide, Labeling

    Met-VEL profiles of osteosarcoma patient lung metastases and human osteosarcoma cell lines a , Aggregate plots showing H3K4me1 ChIP-seq and H3K27ac ChIP-seq signal +/− 3Kb from midpoints of gained and lost Met-VELs in paired patient lung metastases and primary tumors. b , Aggregate plots showing H3K4me1 ChIP-seq, H3K27ac ChIP-seq and DNase-seq signal +/− 3Kb from mid-points of gained and lost Met-VELs in metastatic/parental human osteosarcoma cell lines.

    Journal: Nature medicine

    Article Title: Positively selected enhancer elements endow osteosarcoma cells with metastatic competence

    doi: 10.1038/nm.4475

    Figure Lengend Snippet: Met-VEL profiles of osteosarcoma patient lung metastases and human osteosarcoma cell lines a , Aggregate plots showing H3K4me1 ChIP-seq and H3K27ac ChIP-seq signal +/− 3Kb from midpoints of gained and lost Met-VELs in paired patient lung metastases and primary tumors. b , Aggregate plots showing H3K4me1 ChIP-seq, H3K27ac ChIP-seq and DNase-seq signal +/− 3Kb from mid-points of gained and lost Met-VELs in metastatic/parental human osteosarcoma cell lines.

    Article Snippet: The following antibodies were used for ChIP: rabbit anti-H3K4me1 (Abcam #8895), rabbit anti-H3K27ac (Abcam #4729), rabbit anti-c-Fos (Santa Cruz #sc-52), rabbit anti-FOSL1/Fra-1 (Santa Cruz #sc-605).

    Techniques: Chromatin Immunoprecipitation

    Assessment of Tissue Factor ( F3 ) dysregulation in metastatic osteosarcoma a. IGV browser view of H3K27ac, H3K4me1, and DNase profiles at F3 gained Met-VEL cluster locus in MG63.3 (metastatic) and MG63 (parental) cell lines. Top of figure shows local contact profile analysis of F3 locus in MG63.3. In the top panel (main trend), the contact intensity (black line) is calculated by using a running mean analysis of normalized read counts with a 1kb sliding window. The 20th and 80th percentile are visualized as a gray trend graph. In the bottom panel, contact intensities are computed using linearly increasing sliding windows (scaled 2–50 kb) and are displayed as a color-coded heatmap of positive 4C signals (maximum of interaction set to 1). Local color changes are log-scaled to indicate changes of statistical enrichment of captured sequences, corresponding to the enhancer-promoter interaction. Areas of significant contact highlighted. b . Fold-change quantile normalized F3 FPKM values in 3 metastatic cell lines at 24hrs and 14 days of metastatic outgrowth in ex vivo lung culture relative to parental line at 24hrs. c . Tissue Factor ( F3 ) relative expression in human patient primary tumors and lung metastases normalized to expression in normal osteoblasts. d. IGV browser view of H3K27ac, H3K4me1, and DNase profiles at F3 gained Met-VEL cluster locus in MG63.3, MNNG, and 143B metastatic cell lines.

    Journal: Nature medicine

    Article Title: Positively selected enhancer elements endow osteosarcoma cells with metastatic competence

    doi: 10.1038/nm.4475

    Figure Lengend Snippet: Assessment of Tissue Factor ( F3 ) dysregulation in metastatic osteosarcoma a. IGV browser view of H3K27ac, H3K4me1, and DNase profiles at F3 gained Met-VEL cluster locus in MG63.3 (metastatic) and MG63 (parental) cell lines. Top of figure shows local contact profile analysis of F3 locus in MG63.3. In the top panel (main trend), the contact intensity (black line) is calculated by using a running mean analysis of normalized read counts with a 1kb sliding window. The 20th and 80th percentile are visualized as a gray trend graph. In the bottom panel, contact intensities are computed using linearly increasing sliding windows (scaled 2–50 kb) and are displayed as a color-coded heatmap of positive 4C signals (maximum of interaction set to 1). Local color changes are log-scaled to indicate changes of statistical enrichment of captured sequences, corresponding to the enhancer-promoter interaction. Areas of significant contact highlighted. b . Fold-change quantile normalized F3 FPKM values in 3 metastatic cell lines at 24hrs and 14 days of metastatic outgrowth in ex vivo lung culture relative to parental line at 24hrs. c . Tissue Factor ( F3 ) relative expression in human patient primary tumors and lung metastases normalized to expression in normal osteoblasts. d. IGV browser view of H3K27ac, H3K4me1, and DNase profiles at F3 gained Met-VEL cluster locus in MG63.3, MNNG, and 143B metastatic cell lines.

    Article Snippet: The following antibodies were used for ChIP: rabbit anti-H3K4me1 (Abcam #8895), rabbit anti-H3K27ac (Abcam #4729), rabbit anti-c-Fos (Santa Cruz #sc-52), rabbit anti-FOSL1/Fra-1 (Santa Cruz #sc-605).

    Techniques: Ex Vivo, Expressing

    H3K4me1 ChIP-seq identifies metastatic variant enhancer loci (Met-VELs) and Met-VEL clusters a , Schematic representation of human tumor and metastatic human osteosarcoma cell line cohort. b , UCSC browser views of H3K4me1 profiles from MG63.3 (metastatic) and MG63 (parental) cell lines illustrating an example of gained (top) and lost (bottom) Met-VEL(s). Met-VELs are boxed in red. c , Heatmap showing H3K4me1 ChIP-seq signal +/−5kb from H3K4me1 peak midpoints for all putative enhancers in MG63.3/MG63 pair sorted by differences in signal. Sub-panel shows heatmap for gained and lost Met-VELs alone. d , Aggregate plots showing H3K4me1 ChIP-seq and H3K27ac ChIP-seq signal +/− 3kb from mid-points of gained (left) and lost (right) Met-VELs for a representative matched primary/lung metastatic human tumor pair (top) and MG63.3/MG63 cell line pair (bottom). DNase-seq signal +/− 3kb from Met-VEL mid-points is also shown for the MG63.3 and MG63 cell lines. e. Percentage of enhancers gained and lost in metastatic samples relative to primary tumors or non-metastatic cell lines. f. Heatmap of fold change normalized RPKM in metastatic samples vs. primary tumor or non-metastatic cell lines for aggregated list of all gained and lost Met-VELs across all samples. H3K4me1 signal shown on the left, H3K27ac signal shown on the right. The samples from left to right are as follows M112, 143B, MNNG, MG63.3, LM7, Lung Met 1, Lung Met 2, Lung Met 3, Lung Met 4, Lung Met 5. Asterisks indicate 143B and MNNG samples. g. Genome-wide gained Met-VEL landscape for human osteosarcoma metastatic tumor (Lung Met 4) and MG63.3 cell line. Rows represent scaled chromosomal coordinates. Peaks represent maximum gained Met-VEL counts in 200kb sliding windows. Predicted target genes for selected peaks are labeled.

    Journal: Nature medicine

    Article Title: Positively selected enhancer elements endow osteosarcoma cells with metastatic competence

    doi: 10.1038/nm.4475

    Figure Lengend Snippet: H3K4me1 ChIP-seq identifies metastatic variant enhancer loci (Met-VELs) and Met-VEL clusters a , Schematic representation of human tumor and metastatic human osteosarcoma cell line cohort. b , UCSC browser views of H3K4me1 profiles from MG63.3 (metastatic) and MG63 (parental) cell lines illustrating an example of gained (top) and lost (bottom) Met-VEL(s). Met-VELs are boxed in red. c , Heatmap showing H3K4me1 ChIP-seq signal +/−5kb from H3K4me1 peak midpoints for all putative enhancers in MG63.3/MG63 pair sorted by differences in signal. Sub-panel shows heatmap for gained and lost Met-VELs alone. d , Aggregate plots showing H3K4me1 ChIP-seq and H3K27ac ChIP-seq signal +/− 3kb from mid-points of gained (left) and lost (right) Met-VELs for a representative matched primary/lung metastatic human tumor pair (top) and MG63.3/MG63 cell line pair (bottom). DNase-seq signal +/− 3kb from Met-VEL mid-points is also shown for the MG63.3 and MG63 cell lines. e. Percentage of enhancers gained and lost in metastatic samples relative to primary tumors or non-metastatic cell lines. f. Heatmap of fold change normalized RPKM in metastatic samples vs. primary tumor or non-metastatic cell lines for aggregated list of all gained and lost Met-VELs across all samples. H3K4me1 signal shown on the left, H3K27ac signal shown on the right. The samples from left to right are as follows M112, 143B, MNNG, MG63.3, LM7, Lung Met 1, Lung Met 2, Lung Met 3, Lung Met 4, Lung Met 5. Asterisks indicate 143B and MNNG samples. g. Genome-wide gained Met-VEL landscape for human osteosarcoma metastatic tumor (Lung Met 4) and MG63.3 cell line. Rows represent scaled chromosomal coordinates. Peaks represent maximum gained Met-VEL counts in 200kb sliding windows. Predicted target genes for selected peaks are labeled.

    Article Snippet: The following antibodies were used for ChIP: rabbit anti-H3K4me1 (Abcam #8895), rabbit anti-H3K27ac (Abcam #4729), rabbit anti-c-Fos (Santa Cruz #sc-52), rabbit anti-FOSL1/Fra-1 (Santa Cruz #sc-605).

    Techniques: Chromatin Immunoprecipitation, Variant Assay, Genome Wide, Labeling

    IRF8 and PU.1 binding and the H3K4me1 enhancer signature at the Klf4 gene locus. (A) UCSC genome browser image of tag density plots for IRF8, PU.1, and H3K4me1 ChIP-seq and input DNA at the Klf4 gene locus in empty MSCV-transduced Tot2 cells or IRF8-transduced

    Journal: Blood

    Article Title: Essential role of the IRF8-KLF4 transcription factor cascade in murine monocyte differentiation

    doi: 10.1182/blood-2012-06-437863

    Figure Lengend Snippet: IRF8 and PU.1 binding and the H3K4me1 enhancer signature at the Klf4 gene locus. (A) UCSC genome browser image of tag density plots for IRF8, PU.1, and H3K4me1 ChIP-seq and input DNA at the Klf4 gene locus in empty MSCV-transduced Tot2 cells or IRF8-transduced

    Article Snippet: Immunoprecipitation was then performed by using 10 µg of goat anti-IRF8 antibody (C-19; Santa Cruz Biotechnology, Santa Cruz, CA), 2 µg of rabbit anti-PU.1 antibody (T-21; Santa Cruz Biotechnology), or 2 µg of rabbit anti-H3K4me1 antibody (Ab8895, Abcam, Cambridge, UK).

    Techniques: Binding Assay, Chromatin Immunoprecipitation

    Identification of hypo-enhancers. a Heat map displaying H3K27Ac and DNA methylation at enhancers, called by being H3K4me1-positive and H3K4me3-negative at 4 hpf. The heat map was first sorted on DNA methylation, split ( yellow dashed line ), and independently sorted on H3K27ac. b Genome browser view of a hyper-enhancer close to the TSS of sox2 . ChIP-Seq data and DNA methylation data are displayed for embryos 4 hpf. Normalized ChIP-Seq enrichments and fractional methylation are indicated at the left side of the image. The blue box indicates the hyper-enhancer. The blue arrow indicates the orientation of the gene. Blue horizontal bars indicate HMRs. c Genome browser view of a hypo-enhancer close to the TSS of tlx3b . ChIP-Seq data and DNA methylation data are displayed for embryos 4 hpf. Normalized ChIP-Seq enrichments and fractional methylation are indicated at the left side of the image. The blue box indicates the hypo-enhancer. The blue arrow indicates the orientation of the gene. Blue horizontal bars indicate HMRs. d The overlap of the hypo- and hyper-enhancers with H3K27ac at the indicated developmental time points (* p

    Journal: Genome Biology

    Article Title: Enhancers reside in a unique epigenetic environment during early zebrafish development

    doi: 10.1186/s13059-016-1013-1

    Figure Lengend Snippet: Identification of hypo-enhancers. a Heat map displaying H3K27Ac and DNA methylation at enhancers, called by being H3K4me1-positive and H3K4me3-negative at 4 hpf. The heat map was first sorted on DNA methylation, split ( yellow dashed line ), and independently sorted on H3K27ac. b Genome browser view of a hyper-enhancer close to the TSS of sox2 . ChIP-Seq data and DNA methylation data are displayed for embryos 4 hpf. Normalized ChIP-Seq enrichments and fractional methylation are indicated at the left side of the image. The blue box indicates the hyper-enhancer. The blue arrow indicates the orientation of the gene. Blue horizontal bars indicate HMRs. c Genome browser view of a hypo-enhancer close to the TSS of tlx3b . ChIP-Seq data and DNA methylation data are displayed for embryos 4 hpf. Normalized ChIP-Seq enrichments and fractional methylation are indicated at the left side of the image. The blue box indicates the hypo-enhancer. The blue arrow indicates the orientation of the gene. Blue horizontal bars indicate HMRs. d The overlap of the hypo- and hyper-enhancers with H3K27ac at the indicated developmental time points (* p

    Article Snippet: The following antibodies were used in this study: H3K4me1 ChIP grade (abcam), H3K27ac ChIP grade (abcam), and H3K4me2 ChIP grade (abcam).

    Techniques: DNA Methylation Assay, Chromatin Immunoprecipitation, Methylation

    Hypo-enhancers are stable throughout zebrafish development. a Heat map displaying RPM-normalized H3K4me1 intensities of all hypo- and hyper-enhancers called throughout the first 24 h of development. The heat map was clustered using k-means clustering ( n = 6). Yellow dashed lines indicate cluster transitions. b , c Dynamics of H3K4me1-normalized read density of hypo- and hyper-enhancers at 4 and 8 hpf. Red circles indicate peaks where the normalized read density changes more than fourfold between the two time points. Black circles indicate peaks where the normalized read density changes less than fourfold between the two time points

    Journal: Genome Biology

    Article Title: Enhancers reside in a unique epigenetic environment during early zebrafish development

    doi: 10.1186/s13059-016-1013-1

    Figure Lengend Snippet: Hypo-enhancers are stable throughout zebrafish development. a Heat map displaying RPM-normalized H3K4me1 intensities of all hypo- and hyper-enhancers called throughout the first 24 h of development. The heat map was clustered using k-means clustering ( n = 6). Yellow dashed lines indicate cluster transitions. b , c Dynamics of H3K4me1-normalized read density of hypo- and hyper-enhancers at 4 and 8 hpf. Red circles indicate peaks where the normalized read density changes more than fourfold between the two time points. Black circles indicate peaks where the normalized read density changes less than fourfold between the two time points

    Article Snippet: The following antibodies were used in this study: H3K4me1 ChIP grade (abcam), H3K27ac ChIP grade (abcam), and H3K4me2 ChIP grade (abcam).

    Techniques:

    Global E2A occupancy declines during β-selection. Genome-wide E2A occupancy and patterns of H3K4 mono-methylation were examined in thymocytes isolated from either untreated Rag2 –/– mice (DN3) or Rag2 –/– mice injected with anti-CD3ε antibody (DN4). ( a ) Venn diagram displaying the distribution of E2A binding sites in DN3 versus DN4. ( b ) Cis-regulatory sequences associated with E2A occupancy in DN3 and DN4, identified by comparing enriched peaks to randomly selected genomic DNA sequences. Letter size directly relates to nucleotide frequency. P values are shown and indicate enrichment for a given motif as compared to randomly selected regions. ( c ) E2A occupancy and patterns of H3K4me1 across the Hes1, Ptcra, Notch1, Notch3, Rag2/1, Cd3e, Rorc and Gfi1 loci in the DN3 and DN4 compartments. E2A occupancy in DN3 is shown in gray versus black for the DN4 compartment. H3K4me1 is indicated in blue in DN3 versus green in DN4. ( d ) E2A occupancy and patterns of H3K4me1 across the Zap70 locus. ( e ) Expression of Zap70 in wild-type and Id3-deficient DN1-4 thymocyte compartments. ( f ) Expression of Zap70 and CD27 in day 16.5 dpc fetal wild-type and E2A-deficient DN3 (top) and DN4 as well as ISP (bottom) thymocytes.

    Journal: Nature immunology

    Article Title: The opposing roles of E2A and Id3 that orchestrate and enforce the na?ve T cell fate

    doi: 10.1038/ni.2086

    Figure Lengend Snippet: Global E2A occupancy declines during β-selection. Genome-wide E2A occupancy and patterns of H3K4 mono-methylation were examined in thymocytes isolated from either untreated Rag2 –/– mice (DN3) or Rag2 –/– mice injected with anti-CD3ε antibody (DN4). ( a ) Venn diagram displaying the distribution of E2A binding sites in DN3 versus DN4. ( b ) Cis-regulatory sequences associated with E2A occupancy in DN3 and DN4, identified by comparing enriched peaks to randomly selected genomic DNA sequences. Letter size directly relates to nucleotide frequency. P values are shown and indicate enrichment for a given motif as compared to randomly selected regions. ( c ) E2A occupancy and patterns of H3K4me1 across the Hes1, Ptcra, Notch1, Notch3, Rag2/1, Cd3e, Rorc and Gfi1 loci in the DN3 and DN4 compartments. E2A occupancy in DN3 is shown in gray versus black for the DN4 compartment. H3K4me1 is indicated in blue in DN3 versus green in DN4. ( d ) E2A occupancy and patterns of H3K4me1 across the Zap70 locus. ( e ) Expression of Zap70 in wild-type and Id3-deficient DN1-4 thymocyte compartments. ( f ) Expression of Zap70 and CD27 in day 16.5 dpc fetal wild-type and E2A-deficient DN3 (top) and DN4 as well as ISP (bottom) thymocytes.

    Article Snippet: Sonicated chromatin was immunprecipitated with 10 μg of anti-E2A (Santa Cruz Biotechnology) or 10 μg anti-H3K4me1 (ab8895) coupled to Dynal-beads.

    Techniques: Selection, Genome Wide, Methylation, Isolation, Mouse Assay, Injection, Binding Assay, Expressing

    Set1/MLL complex components are largely responsible for H3K4me2/3 in embryos. Embryos were dissected from WT, mutant, or RNAi treated adult eri-1 ( mg366; enhanced RNAi) animals. RNAi mediated knockdown was used for ash-2, dpy-30, cfp-1 and wdr-82 . Embryos were probed with rabbit anti-H3K4me3 (Panels in A) or mouse anti-H3K4me2 monoclonal antibodies (Panels in B); DNA was counter-stained with DAPI. Exposure times were the same for each condition. Scale bars represent 10um. (A and B) H3K4me3 and H3K4me2 are present in chromatin of wild-type embryos, but both are dramatically decreased in chromatin of embryos from wdr-5.1 and rbbp-5 mutants, and embryos from ash2, dpy-30, or cfp-1 RNAi treated mothers. H3K4me3, but not H3K4me2, is reduced in set-2(tm1630) mutant embryos. RNAi mediated knockdown of wdr-82 did not affect either H3K4me3 or H3K4me2. (C) A wdr-5.1::GFP transgene rescues H3K4 methylation in the wdr-5.1(ok1417) mutant. wdr-5.1(ok1417);wdr-5.1::GFP transgenic embryos were probed with antibodies against GFP and H3K4me3/2 as indicated. H3K4me3/2 were restored in the chromatin of early embryos in which WDR-5.1:GFP expression was detected. (D) Western blot of lysates from mixed stage wdr-5.1(ok1417), wdr-5.2(ok1444), wdr-5.1;wdr-5.2 double mutant, and set-1(tm1630) mutant embryos. The blots were probed with anti-histone H3, anti-H3K4me1, mouse monoclonal anti-H3K4me2 or rabbit polyclonal anti-H3K4me3 antibodies, as indicated. H3K4me3/2 levels were dramatically decreased in strains with the wdr-5.1(ok1417) allele, whereas H3K4me3 levels were more strongly depleted than H3K4me2 in set-2(tm1630) . H3K4me1 levels were not noticeably affected in any mutant strain tested.

    Journal: PLoS Genetics

    Article Title: A Role for Set1/MLL-Related Components in Epigenetic Regulation of the Caenorhabditis elegans Germ Line

    doi: 10.1371/journal.pgen.1001349

    Figure Lengend Snippet: Set1/MLL complex components are largely responsible for H3K4me2/3 in embryos. Embryos were dissected from WT, mutant, or RNAi treated adult eri-1 ( mg366; enhanced RNAi) animals. RNAi mediated knockdown was used for ash-2, dpy-30, cfp-1 and wdr-82 . Embryos were probed with rabbit anti-H3K4me3 (Panels in A) or mouse anti-H3K4me2 monoclonal antibodies (Panels in B); DNA was counter-stained with DAPI. Exposure times were the same for each condition. Scale bars represent 10um. (A and B) H3K4me3 and H3K4me2 are present in chromatin of wild-type embryos, but both are dramatically decreased in chromatin of embryos from wdr-5.1 and rbbp-5 mutants, and embryos from ash2, dpy-30, or cfp-1 RNAi treated mothers. H3K4me3, but not H3K4me2, is reduced in set-2(tm1630) mutant embryos. RNAi mediated knockdown of wdr-82 did not affect either H3K4me3 or H3K4me2. (C) A wdr-5.1::GFP transgene rescues H3K4 methylation in the wdr-5.1(ok1417) mutant. wdr-5.1(ok1417);wdr-5.1::GFP transgenic embryos were probed with antibodies against GFP and H3K4me3/2 as indicated. H3K4me3/2 were restored in the chromatin of early embryos in which WDR-5.1:GFP expression was detected. (D) Western blot of lysates from mixed stage wdr-5.1(ok1417), wdr-5.2(ok1444), wdr-5.1;wdr-5.2 double mutant, and set-1(tm1630) mutant embryos. The blots were probed with anti-histone H3, anti-H3K4me1, mouse monoclonal anti-H3K4me2 or rabbit polyclonal anti-H3K4me3 antibodies, as indicated. H3K4me3/2 levels were dramatically decreased in strains with the wdr-5.1(ok1417) allele, whereas H3K4me3 levels were more strongly depleted than H3K4me2 in set-2(tm1630) . H3K4me1 levels were not noticeably affected in any mutant strain tested.

    Article Snippet: Rabbit anti-H3K4me3 (1:1000) and anti-H3K4me1 polyclonal antibodies were purchased from Abcam (ab8580 and ab8895, respectively).

    Techniques: Mutagenesis, Staining, Methylation, Transgenic Assay, Expressing, Western Blot