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  • 99
    Millipore histone h4
    P49/STRAP reduced <t>histone</t> H4 acetylation and repressed the PGC-1α gene in C2C12 cells. 2A. p49/STRAP reduced the acetylation of histone H4 protein at lysine 16 (H4K16). P49/STRAP siRNA increased the H4K16 protein acetylation. Total histone H4 protein was used as a loading control. 2B. Quantitation of H4K16 protein relative to total histone H4 protein. Note that * refers P
    Histone H4, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 795 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam histone h4
    MP binding to NETs. NETs were generated from PMA-stimulated bone marrow neutrophils co-incubated with ( A ) vehicle ( B ) mutated annexin V (An Vm), ( C ) wild-type annexin V (An V) or ( D ) polysialic acid (PSA). Scanning electron microscopy showing MPs attached to neutrophil-derived NETs. Scale bar = 2 μm. ( E ) Aggregate data on MP binding to NETs. ( F ) Surface plasmon resonance with <t>histone</t> H4 immobilized to a CM5 sensorchip. Indicated concentrations of MPs were applied in a flow over the sensorchip surface as described in Methods. Data are presented as mean ± SEM and n = 5. * P
    Histone H4, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 560 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc histone h3
    TSA or MCP30 increases the decreased acetylation of Topo IIα promoter in benzene poisoning mice. After all mice were killed, bone marrow mononuclear cells were separated and histone acetylation of Topo IIα promoter was assessed with chromatin immunoprecipitation (ChIP) assay using anti-acetylated <t>histone</t> H3 and anti-acetylated histone H4. ( A ) Representatives were shown for acetylation levels of histone H3 and histone H4 in the Topo IIα promoter. ( B ) Statistical data showed the acetylation levels of histone H3 in the Topo IIα promoter were shown. ( C ) Statistical data showed the acetylation levels of histone H4 in the Topo IIα promoter. ** P
    Histone H3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 5201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti histone h3
    PPAD stabilizes gingipains and modulates the levels of phagocytosis-related proteins. (a) Relative (Rel.) levels of gingipains (RgpA/RgpB) in infected neutrophils. (b) Time course of <t>histone</t> H3 degradation by P. gingivalis proteases in the presence or absence of PPAD, as determined by Western blotting (SN, culture supernatant). (c and d) Quantification of significant changes in the amounts of phagocytosis-related proteins (c) and the antimicrobial histone H2B (d) in infected neutrophils, as approximated by mass spectrometry. (a, c, and d) Data are means of three replicates of one neutrophil donor. * * , P
    Anti Histone H3, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 4255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology histone h3
    Inactivation of the LOX core promoter in NNK treated cells. ChIP and PCR assays were performed to elucidate the active status of the LOX core promoter in treated cells by assessing acetylated <t>histone</t> H3 binding to the core promoter region. DNAs were isolated from control and NNK treated cells each with 2 × 10 6 , sonicated and immunoprecipitated with an antibody against acetylated histone H3 or RNA-PolyII. Using immunoprecipitated DNA as a template, the PCR with primer pairs as shown under Methods amplified the acetylated histone H3-bound LOX core promoter region with 136 bp, and the RNA-Poly II bound fragment of the GAPDH promoter (an internal control) with 90 bp, respectively. PCR products were analyzed on 2.2% agarose gels and densities of DNA bands measured by the 1D Scan software.
    Histone H3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1550 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Upstate Biotechnology Inc histone h4
    Recovery of histone acetylation by overexpression of wt p300 HAT. HS27/vIRF cells were transfected with wt p300 (lane 3) or p300 ΔHAT mutant (lane 4). HS27/cDNA3 (lane 1) and HS27/vIRF (lane 2) were included as controls. Identical amounts of proteins from cell lysates were used for immunoblotting analysis with an antibody specific for the acetylated histone H4. The bottom panel shows the amount of cellular <t>histone</t> H4 protein in each lane, detected by an anti-H4 antibody. Arrows indicate the acetylated form of histone H4 (Ac-H4) or total histone H4 (H4). Numbers at left are molecular masses in kilodaltons.
    Histone H4, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 621 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti histone h3
    PU.1 positively regulates DR5 expression and sensitizes AML cells to apoptosis. ( a–c ) Flow cytometry analysis of DR5 surface expression, DR5 and PU.1 western blot and quantitative RT-PCR (qPCR) analysis in NB4, HL60 as well as MOLM-13 control and PU.1-knockdown cells. Total protein was used as a loading control for western blot data. qPCR values were normalized to the housekeeping gene  HMBS . Results are given as  n -fold changes compared with control cells. ( d ) DR5 qPCR, DR5, cleaved PARP as well as PU.1 western blot and DR5 FACS analysis of NB4 control and PU.1-knockdown cells upon TRAIL treatment. Analysis as in ( a ). One out of three independent experiments is shown. ( e ) PU.1 and p65 ChIP analysis of three putative, neighboring PU.1/p65-binding sites upstream of the transcriptional start site of the  DR5  gene.  In vivo  binding of PU.1 and p65 to the indicated sites was shown by ChIP in NB4 cells using antibodies against PU.1 and p65. Antibodies against acetyl-histone H3 and IgG were used as a positive and negative control, respectively. GAPDH amplification was shown as a negative control for the different pulldowns. ( f ) DR5 expression analysis of NB4 cells expressing an inducible PU.1-estrogen receptor (PU.1-ER) fusion protein. DR5 qPCR, western blot and FACS analysis of NB4 control and PU.1-knockdown cells 48 h after 4-hydroxy-tamoxifen (4-OH-T) stimulation from three independent experiments. ( g ) Viability of NB4 control and PU.1-ER-expressing cells upon PU.1 activation for 48 h with 4-OH-T stimulation. Cells were incubated with rhTRAIL (500 ng/ml) 24 h after 4-OH-T addition. Percent of Annexin V +  cells from three independent experiments are shown (upper panel) and immunoblotting for pro- and cleaved caspase-8, cleaved caspase-3 and cleaved PARP are shown (lower panel). Total protein was used as a loading control. ( h ) Viability of NB4 control and PU.1-ER-expressing cells treated as in ( f ). Percent of Annexin V +  cells from three independent experiments are shown. Immunoblotting for cleaved PARP, FLIP L  and FLIP S , Bcl-2 and Mcl-1 is shown. Analysis as in ( g ). ** P
    Anti Histone H3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1887 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti histone h3 antibody nuclear loading control and chip grade
    At after infection, HSV-1 DNA was protected from serial MCN digestion to sizes of mono to poly-nucleosomes that fractionated in complexes with the hydrodynamic ratios of mono to poly-nucleosomes and contain histone H3. Nuclei of infected cells were subjected to serial MCN digestion and then cross-linked. (A) Cross-linked soluble chromatin separated by hydrodynamic ratios in a 0–10% sucrose gradient, and resolved in 2% agarose gel electrophoresis, stained with EtBr. The left pointing arrowheads at the right indicate the migration of mono- to octa-nucleosome-sized DNA. (B) Chromatin immunoprecipitation of HSV-1 DNA with histone H3. EtBr stained agarose gels of saturating PCR-amplified cellular (GAPDH) or HSV-1 (UL25, UL46, ICP4, or gE) DNA co-immunoprecipitated with histone H3. (C) Cartoon presenting the positions of the PCR amplicons in the HSV-1 genome. In, input (2%); H3, anti-histone H3 antibody; IgG, isotype antibody control (labeled only in the No Dig sample for simplicity); No Dig, undigested unfractionated chromatin; Ins, insoluble chromatin.
    Anti Histone H3 Antibody Nuclear Loading Control And Chip Grade, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 621 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam h3k4me3
    Subgenome-specific recruitment of p300 is associated with TEs. Subgenome-specific p300 peaks are enriched for TEs carrying transcription factor (TF) motifs active in early development. a Differential regulation of the slc2a2 homeologs at stage 10.5. Shown are the genomic profiles of <t>H3K4me3</t> ( green ), RNA Polymerase II (RNAPII; purple ), H3K36me3 ( blue ), and p300 ( yellow ) ChIP-seq tracks, as well as DNA methylation levels determined by WGBS ( gray ). The top panel shows slc2a2.L, which is highly expressed, as evidenced by RNAPII and H3K36me3, and has a number of active enhancers ( a – g ), while slc2a2.S, shown in the bottom panel , is expressed at a lower rate. The conservation between the L and S genomic sequence is shown in gray between the panels. Differential enhancers between L and S are highlighted in yellow , which illustrates lost enhancer function ( a , b ), conserved enhancer function ( c – e ), and deleted enhancers ( f , g ). b Subgenome-specific p300 peaks are associated with DNA transposon repeats (threshold p ≤ 10e-4, twofold enrichment compared to all X. laevis peaks and present at least in 15% of the peaks). The barplots show the frequency of occurrence of each of the three repeat types per megabase in the three (sub)genomes. Over the bars is represented the percentage of subgenome-specific peaks overlapping with the corresponding repeat. c TF found to be enriched in the subgenome-specific p300 peaks (threshold p ≤ 10e-4, threefold enrichment compared to all X. laevis peaks and present at least in 20% of the peaks)
    H3k4me3, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 3837 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho histone h3
    Effects of UTP and BzATP on the stimulation of the mitosis marker, <t>phospho-histone</t> 3, by FGF2. Quiescent cultures of rat cortical astrocytes were treated with FGF2 (25 ng/ml) alone or in combination with UTP (100 uM) or BzATP (100 uM) for 22 hr. Western
    Phospho Histone H3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam h3k27ac
    Polymer Simulations Predict Chromosome Interactions in Different Cell Lines (A) Simulated Capture-C profiles (colored lines) are shown for three cell lines, for three different viewpoints (at the URR, DRR, and Pax6 , indicated by arrows). The corresponding experimental data are shown as gray bars. Above each set of plots, a line of points show how beads were colored as non-binding (gray) or binding (red) for TFs. In simulations, Pax6 interacts strongly with a broad acetylated region downstream of the gene (red stars); this is not observed in the experimental data. (B) Plot showing the level of interaction between a viewpoint and a specified 10-kbp region. The arrow indicates the viewpoint (arrow base), and the interacting region (arrowhead). The height of the bar shows the number of interactions with the 10-kbp region as a percentage of interactions with the locus as a whole (chromosome 2 [chr2]: 105,200,000–105,800,00). (C) Similar plots were obtained from experimental Capture-C data. D) were used to infer TF-binding sites instead of <t>H3K27ac.</t> The erroneous interactions marked in (A) are now absent (red stars). (E) Similar plots to those in (B) but from the simulations using ATAC-seq data. .
    H3k27ac, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 3193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti acetyl histone h3
    SCFAs reduce the autoantibody response and autoimmunity in lupus-prone mice. Female MRL/ Fas lpr/lpr mice fed fiber diet were given SCFAs water (SCFAs) or plain water (Nil) starting at the age of 5 weeks and killed at 17 weeks of age. a Titers of circulating anti-dsDNA IgM, and anti-dsDNA, anti-RNP/Sm, <t>anti-histone,</t> and anti-RNA IgG1 and IgG2a (relative units, RU), as analyzed by specific ELISAs. Each symbol represents an individual mouse ( n = 4 per group, pooled from two experiments). The bars represent the mean ± SD. b ANA visualized by indirect immunofluorescence on HEp-2 cells that were incubated with sera (1:400 dilution) from the MRL/ Fas lp/lpr mice using FITC-labeled rat mAbs to mouse IgG1 and IgG2a. Data are from one of three independent experiments yielding comparable results. c , d AID and Blimp1 expression as analyzed by intracellular staining followed by fluorescence microscopy ( c ) and flow cytometry ( d ). e Spleen surface CD138 + plasmablasts/plasma cells, and intracellular CD138 + IgG1 + and CD138 + IgG2a + plasmablasts/plasma cells as analyzed by flow cytometry. Data are representative of three independent experiments yielding comparable results. f Dorsal skin lesions (left panels), kidney sections stained with H E (middle panels), and kidney sections stained with FITC-labeled rat mAbs to mouse IgG1 and IgG2a (right panels). Data are representative of three independent experiments. * p
    Anti Acetyl Histone H3, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1216 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    h3  (Abcam)
    99
    Abcam h3
    SCFAs reduce the autoantibody response and autoimmunity in lupus-prone mice. Female MRL/ Fas lpr/lpr mice fed fiber diet were given SCFAs water (SCFAs) or plain water (Nil) starting at the age of 5 weeks and killed at 17 weeks of age. a Titers of circulating anti-dsDNA IgM, and anti-dsDNA, anti-RNP/Sm, <t>anti-histone,</t> and anti-RNA IgG1 and IgG2a (relative units, RU), as analyzed by specific ELISAs. Each symbol represents an individual mouse ( n = 4 per group, pooled from two experiments). The bars represent the mean ± SD. b ANA visualized by indirect immunofluorescence on HEp-2 cells that were incubated with sera (1:400 dilution) from the MRL/ Fas lp/lpr mice using FITC-labeled rat mAbs to mouse IgG1 and IgG2a. Data are from one of three independent experiments yielding comparable results. c , d AID and Blimp1 expression as analyzed by intracellular staining followed by fluorescence microscopy ( c ) and flow cytometry ( d ). e Spleen surface CD138 + plasmablasts/plasma cells, and intracellular CD138 + IgG1 + and CD138 + IgG2a + plasmablasts/plasma cells as analyzed by flow cytometry. Data are representative of three independent experiments yielding comparable results. f Dorsal skin lesions (left panels), kidney sections stained with H E (middle panels), and kidney sections stained with FITC-labeled rat mAbs to mouse IgG1 and IgG2a (right panels). Data are representative of three independent experiments. * p
    H3, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2301 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam anti histone h3 antibody
    KLF6 transcriptionally activates autophagy-related genes in a p53-dependent manner. Transfection with the KLF6 -expression vector pcIneo-KLF6 induced KLF6 mRNA ( A ) and protein ( B ) expression. KLF6-over-expression induces autophagy in HepG2 cells as assessed by LC3 Western blotting ( C ) and quantification of LC3-II-bands normalized to loading control GAPDH ( D ; fold change versus control shown as mean ± SEM of n = 3 independent cell culture experiments; full length Western blot images are given in Supplementary information.). Autophagic flux was assessed by LC3-II and p62 Western blotting in HepG2 and pcIneo-KLF6 transfected cells in absence (−) or presence ( + ) of 100 µM chloroquine for 24 h (( C ) shows representative Western blot images, ( D) shows fold change versus control, shown as mean ± SEM of n = 3 independent experiments). Activation of the ATG7 and BECLIN1 promoter was quantified by luciferase activity in a co-transfection experiment of pcIneo-KLF6 with specific promoter reporter luciferase plasmids in HepG2 ( E ) and in p53-deficient HepG2-303 cells ( F ). The interaction of KLF6 with the ATG7 and BECLIN1 promoter containing putative KLF6-binding sites was confirmed by chromosomal immunoprecipitation (ChIP) in HepG2 cells ( G ) using two different KLF6 antibodies, IgG as negative controls, <t>Histone-H3</t> antibody was used as a positive control for ChIP; full length images of agarose gels are given in Supplementary information.
    Anti Histone H3 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 655 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam h3k9me3
    Histone acetylation and methylation in HDAC1 wt and HDAC1 dn mESCs induced into cardiomyocytes and treated with HDACi. The level of H3K9ac, <t>H3K9me3,</t> H4ac, H4K20ac, pan-acetylated lysines (K-ac), and α-actinin in ( A ) HDAC1 wt mESCs and ( B ) HDAC1 dn mESCs. In three biological replicates, Western blots were performed on one gel. For the data presented in panel A or B, the gel was separated by Photoshop to show samples that were compared in one relevant subset. Data on histone levels were normalized to the level of histone H3 and non-histone proteins were normalized and quantified to the level of GAPDH ( C ). In wt and HDAC1 dn non-treated cells and in TSA-, SAHA-, or VPA-treated mESCs, panel ( Ca ) shows the levels of H4ac, ( Cb ) shows H4K20ac, and ( Cc ) shows the levels of α-actinin. The total protein levels were measured using a µQuant spectrophotometer for each sample, and an identical protein amount was loaded on the gels. In panel ( A , B ), the levels of histone markers are also shown for embryonic hearts (e15). Quantification of the protein levels in panel ( C ) was performed using ImageJ software (NIH, freeware). Statistical analyses were performed using Student’s t -test; asterisks (*) in panel ( Ca – c ) show statistically significant differences at p ≤ 0.05. Note that the y -axis-scale in panel ( Ca ) is different (red frames) for the wt and HDAC1 dn cells for technical purposes. In panel ( Ca ), the level of H4ac is significantly less in the wt mESCs when compared with the HDAC1 dn cells ( Cb ).
    H3k9me3, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 3047 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore phospho histone h3
    Directional migration of stratified epithelium is not a result of localized cell proliferation. (A-H) Cell proliferation as detected by <t>phospho-histone</t> 3 (pH3) immunofluorescence (green) during the timecourse of mammary epithelial migration toward beads soaked in BSA (A-D) or FGF10 (E-H). Cell proliferation was quantified in one of the three evenly divided regions of an organoid – the front (f), middle (m) or rear (r) – depending on the distance between the region and the bead (asterisk). Scale bars: 100 µm. (I,J) Quantification of cell proliferation in different regions of mammary organoids during epithelial migration. Only signals that overlap with nuclei were counted as proliferating cells, and background noise was discounted. No statistically significant differences were apparent among the different regions at the times indicated (unpaired two-tailed Student's t -test). Values shown are the mean±s.d. from at least three independent experiments.
    Phospho Histone H3, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abcam rabbit anti histone h3
    Model of How Cellular Heterogeneities at the 4-Cell Stage Regulate Cell Fate (A–C) In 4-cell embryos, CARM1, which methylates <t>histone</t> H3R26, is differentially expressed ( Torres-Padilla et al., 2007 ). We hypothesize that higher levels of histone H3R26me facilitate the binding to DNA of pluripotency regulators such as Oct4 and Sox2, resulting in increased transcription of pluripotency-related target genes, such as Sox21 , Nanog , and Esrrb , biasing these cells to contribute to the pluripotent lineage. Conversely, in cells with lower levels of histone H3R26me, pluripotency regulators are only able to bind to DNA for shorter periods of time, and therefore their target genes are not as highly expressed. These cells have lower levels of pluripotency and are thus more likely to initiate expression of differentiation genes, such as Cdx2 , and initiate development into the extra-embryonic TE.
    Rabbit Anti Histone H3, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 895 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology anti histone h1
    Hyperactivation of NF-κB in Shn-2 −/− CD4 T cells. (A and B) Splenic CD4 T cells were incubated with medium alone overnight and then stimulated with immobilized anti–TCR-αβ mAb in the presence or absence of agonistic anti-CD28 antibody for 3 h. Nuclear extracts of the cultured cells were prepared and subjected to EMSAs with NF-κB probes. The supershift assays were performed with antibodies specific for NF-κB p50 and p65 subunit detection. Three independent experiments were performed with similar results. (C) Splenic CD4 T cells were incubated with medium alone overnight and then stimulated with immobilized anti–TCR-β mAb for 3 h. Subsequently, both cytosol and nuclear extracts were prepared and these were subjected to immunoblotting using anti-p50, anti-p65, anti–tubulin-α, and <t>anti–histone</t> H1 antibodies. Arbitrary densitometric units are shown under each band. (D) RNAs were prepared from fresh splenic CD4 T cells, resting CD4 T cells that were incubated with medium alone overnight, and anti-TCR–stimulated CD4 T cells, and then quantitative PCR assay was performed. The expression levels of p50 and p65 were normalized with 18S expression. Two independent experiments were performed with similar results.
    Anti Histone H1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 314 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam h3k4me1
    Epigenome annotation of variants from genome sequencing identifies a shared variant in a putative enhancer element Variant identified by whole genome sequencing, plus an additional five variants in patients with pancreatic agenesis map to a 25 Kb region downstream of PTF1A , which contains a single candidate pancreatic progenitor-specific enhancer within a highly conserved 400bp element. The top panel depicts ChIP-seq density plots for the enhancer mark <t>H3K4me1,</t> the second and third show occupancy for FOXA2 and PDX1. A broad panel of embryonic and adult human tissues do not show active chromatin marks in this region ( Supplementary Figure 5 ). Vertebrate Conservation and mammalian conservation tracks (as measured by the GERP Score) tracks illustrate the high conservation of this element. The red line depicts the approximate location of a 7.6 kb deletion in this region, and red arrows indicate point mutations, using the final 3 digits from the hg19 coordinates as labels (referring to positions 23508305A > G, 23508363A > G, 23508365A > G, 23508437A > G and 23508446A > C on chromosome 10 respectively).
    H3k4me1, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 1918 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    P49/STRAP reduced histone H4 acetylation and repressed the PGC-1α gene in C2C12 cells. 2A. p49/STRAP reduced the acetylation of histone H4 protein at lysine 16 (H4K16). P49/STRAP siRNA increased the H4K16 protein acetylation. Total histone H4 protein was used as a loading control. 2B. Quantitation of H4K16 protein relative to total histone H4 protein. Note that * refers P

    Journal: Experimental gerontology

    Article Title: Does p49/STRAP, a SRF-binding protein (SRFBP1), Modulate Cardiac Mitochondrial Function in Aging?

    doi: 10.1016/j.exger.2016.06.008

    Figure Lengend Snippet: P49/STRAP reduced histone H4 acetylation and repressed the PGC-1α gene in C2C12 cells. 2A. p49/STRAP reduced the acetylation of histone H4 protein at lysine 16 (H4K16). P49/STRAP siRNA increased the H4K16 protein acetylation. Total histone H4 protein was used as a loading control. 2B. Quantitation of H4K16 protein relative to total histone H4 protein. Note that * refers P

    Article Snippet: Acetylation of histone H4 on lysine 16 (H4K16) is a prevalent and reversible epigenetic, posttranslational chromatin modification in eukaryotes, which affects various gene expression levels ( ).

    Techniques: Pyrolysis Gas Chromatography, Quantitation Assay

    The KDM2A PHD domain does not contribute significantly to nucleosome binding. (A) KDM2A proteins encoding a wild-type (WT) or mutant PHD domain (C645A) were expressed and purified. (B) Both the WT and C645A KDM2A proteins bind to naked 216-bp nucleosome positioning DNA with the same level of efficiency, as determined by EMSA (left-hand lanes). Mutating the PHD domain did not inhibit binding to the 216-bp mononucleosome, whereas mutation of the ZF-CXXC DNA biding domain (K601A) completely abrogated binding. (C) Histone H3K4me3 was installed specifically into histone H3 and the incorporation verified by Western blotting of the recombinant histone with antibodies against H3K4me3 or histone H3. (D) Mononucleosomes (216 bp) were reconstituted with histones containing H3K4me3 as indicated by the green dots on the nucleosome cartoon above the EMSA panels. The addition of H3K4me3 to the mononucleosome did not increase binding of KDM2A, nor did mutation of the PHD domain inhibit binding to the nucleosome, indicating that H3K4me3 does not contribute significantly to nucleosome recognition by KDM2A. (E) Multiple sequence alignment of PHD domains that bind either to H3K4me3 (BPTF, ING2, and PHF8) or H3K4me0 (BHC80 and DNMT3L) and KDM2A. Gray boxes indicate conserved zinc-coordinating cysteines/histidines, and orange boxes indicate methyl-lysine- or lysine-interacting residues. Although KDM2A has conserved zinc-coordinating residues, lysine interaction residues are absent.

    Journal: Molecular and Cellular Biology

    Article Title: Recognition of CpG Island Chromatin by KDM2A Requires Direct and Specific Interaction with Linker DNA

    doi: 10.1128/MCB.06332-11

    Figure Lengend Snippet: The KDM2A PHD domain does not contribute significantly to nucleosome binding. (A) KDM2A proteins encoding a wild-type (WT) or mutant PHD domain (C645A) were expressed and purified. (B) Both the WT and C645A KDM2A proteins bind to naked 216-bp nucleosome positioning DNA with the same level of efficiency, as determined by EMSA (left-hand lanes). Mutating the PHD domain did not inhibit binding to the 216-bp mononucleosome, whereas mutation of the ZF-CXXC DNA biding domain (K601A) completely abrogated binding. (C) Histone H3K4me3 was installed specifically into histone H3 and the incorporation verified by Western blotting of the recombinant histone with antibodies against H3K4me3 or histone H3. (D) Mononucleosomes (216 bp) were reconstituted with histones containing H3K4me3 as indicated by the green dots on the nucleosome cartoon above the EMSA panels. The addition of H3K4me3 to the mononucleosome did not increase binding of KDM2A, nor did mutation of the PHD domain inhibit binding to the nucleosome, indicating that H3K4me3 does not contribute significantly to nucleosome recognition by KDM2A. (E) Multiple sequence alignment of PHD domains that bind either to H3K4me3 (BPTF, ING2, and PHF8) or H3K4me0 (BHC80 and DNMT3L) and KDM2A. Gray boxes indicate conserved zinc-coordinating cysteines/histidines, and orange boxes indicate methyl-lysine- or lysine-interacting residues. Although KDM2A has conserved zinc-coordinating residues, lysine interaction residues are absent.

    Article Snippet: Antibodies used for Western blotting were anti-KDM2A , anti-histone H3 (Ab1791), anti-histone H1 (Millipore AE-4), and anti-DNMT3B (Enzo Life Sciences 52A1018).

    Techniques: Binding Assay, Mutagenesis, Purification, Western Blot, Recombinant, Sequencing

    Pharmacological inhibition of HDAC1/HDAC2. ( A ) DUX4 and DUX4 target gene expression as determined by RT-qPCR in MB200 FSHD2 myoblasts treated with 2.5 µM MS-275 for the indicated times. Statistical significance was calculated by comparing the mRNA level at each time point to that at 0 hr using a two-tailed, two-sample Mann-Whitney U test. ( B ) ChIP-qPCR enrichment of histone H4 acetylation (H4Ac) along the D4Z4 repeat in MB200 FSHD2 myoblasts treated with 2.5 µM MS-275 for 24 hr. Statistical significance was calculated by comparing the H4Ac signal in untreated versus MS-275-treated cells at each site using a one-tailed, one-sample Wilcoxon signed-rank test. *, p≤0.05; ns, not significant, p > 0.05. Error bars denote the standard deviation from the mean of three (or six, for the 0 hr and 12 hr time points in ( A )) biological replicates. See also Figure 2—source data 1 .

    Journal: eLife

    Article Title: NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated by DUX4-induced MBD3L proteins

    doi: 10.7554/eLife.31023

    Figure Lengend Snippet: Pharmacological inhibition of HDAC1/HDAC2. ( A ) DUX4 and DUX4 target gene expression as determined by RT-qPCR in MB200 FSHD2 myoblasts treated with 2.5 µM MS-275 for the indicated times. Statistical significance was calculated by comparing the mRNA level at each time point to that at 0 hr using a two-tailed, two-sample Mann-Whitney U test. ( B ) ChIP-qPCR enrichment of histone H4 acetylation (H4Ac) along the D4Z4 repeat in MB200 FSHD2 myoblasts treated with 2.5 µM MS-275 for 24 hr. Statistical significance was calculated by comparing the H4Ac signal in untreated versus MS-275-treated cells at each site using a one-tailed, one-sample Wilcoxon signed-rank test. *, p≤0.05; ns, not significant, p > 0.05. Error bars denote the standard deviation from the mean of three (or six, for the 0 hr and 12 hr time points in ( A )) biological replicates. See also Figure 2—source data 1 .

    Article Snippet: Antibodies The following antibodies were used: α-Tubulin (T9026); Acetyl-Histone H4 (06–866 lot#2554112, EMD Millipore (Billerica, MA)); CHD4 (A301-081A, Bethyl Laboratories (Montgomery, TX)); FITC anti-mouse (715-095-150 lot#115855, Jackson ImmunoResearch (West Grove, PA)); FLAG M2 (F1804 lot#SLBG5673V and lot#124K6106); FLAG M2 (F3165 lot#SLBL1237V); HDAC2 (ab7029, lot#GR88809-7, Abcam (Cambridge, UK)); HRP anti-mouse (115-035-146, Jackson ImmunoResearch); MBD2 (A301-632A, Bethyl); mouse IgG (315-005-003 lot#120058, Jackson ImmunoResearch); MTA2 (ab8106 lot#GR185489-3, Abcam); TRITC anti-rabbit (711-025-152 lot#114768, Jackson ImmunoResearch); rabbit monoclonal antibodies against DUX4 (E5-5 and E14-3) were produced in collaboration with Epitomics and are described elsewhere ( ).

    Techniques: Inhibition, Expressing, Quantitative RT-PCR, Mass Spectrometry, Two Tailed Test, MANN-WHITNEY, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, One-tailed Test, Standard Deviation

    MP binding to NETs. NETs were generated from PMA-stimulated bone marrow neutrophils co-incubated with ( A ) vehicle ( B ) mutated annexin V (An Vm), ( C ) wild-type annexin V (An V) or ( D ) polysialic acid (PSA). Scanning electron microscopy showing MPs attached to neutrophil-derived NETs. Scale bar = 2 μm. ( E ) Aggregate data on MP binding to NETs. ( F ) Surface plasmon resonance with histone H4 immobilized to a CM5 sensorchip. Indicated concentrations of MPs were applied in a flow over the sensorchip surface as described in Methods. Data are presented as mean ± SEM and n = 5. * P

    Journal: Scientific Reports

    Article Title: Neutrophil extracellular trap-microparticle complexes enhance thrombin generation via the intrinsic pathway of coagulation in mice

    doi: 10.1038/s41598-018-22156-5

    Figure Lengend Snippet: MP binding to NETs. NETs were generated from PMA-stimulated bone marrow neutrophils co-incubated with ( A ) vehicle ( B ) mutated annexin V (An Vm), ( C ) wild-type annexin V (An V) or ( D ) polysialic acid (PSA). Scanning electron microscopy showing MPs attached to neutrophil-derived NETs. Scale bar = 2 μm. ( E ) Aggregate data on MP binding to NETs. ( F ) Surface plasmon resonance with histone H4 immobilized to a CM5 sensorchip. Indicated concentrations of MPs were applied in a flow over the sensorchip surface as described in Methods. Data are presented as mean ± SEM and n = 5. * P

    Article Snippet: Histone H4 (Abcam, Cambridge, UK) was diluted in sodium acetate (10 mM, pH 5.5) and immobilized via amine coupling to flow cell 2.

    Techniques: Binding Assay, Generated, Incubation, Electron Microscopy, Derivative Assay, SPR Assay, Flow Cytometry

    NET formation in abdominal sepsis. ( A ) Representative western blot and aggregate data on citrullinated histone 3 in neutrophils isolated from the blood from sham and CLP animals. Plasma levels of ( B ) cf-DNA and ( C ) DNA-histone complexes were determined 24 h after CLP. ( D ) TG over time, ( E ) peak and ( F ) total levels of TG were determined as described in Material and Methods. Mice underwent CLP or the identical laparotomy and resuscitation procedures, but the cecum was neither ligated nor punctured (Sham). Animals received intraperitoneal injections of DNAse (5 mg/kg), an anti-Ly-6G antibody (Anti-Ly-6G ab, 20 mg/kg) or vehicle. Data are presented as mean ± SEM and n = 5. * P

    Journal: Scientific Reports

    Article Title: Neutrophil extracellular trap-microparticle complexes enhance thrombin generation via the intrinsic pathway of coagulation in mice

    doi: 10.1038/s41598-018-22156-5

    Figure Lengend Snippet: NET formation in abdominal sepsis. ( A ) Representative western blot and aggregate data on citrullinated histone 3 in neutrophils isolated from the blood from sham and CLP animals. Plasma levels of ( B ) cf-DNA and ( C ) DNA-histone complexes were determined 24 h after CLP. ( D ) TG over time, ( E ) peak and ( F ) total levels of TG were determined as described in Material and Methods. Mice underwent CLP or the identical laparotomy and resuscitation procedures, but the cecum was neither ligated nor punctured (Sham). Animals received intraperitoneal injections of DNAse (5 mg/kg), an anti-Ly-6G antibody (Anti-Ly-6G ab, 20 mg/kg) or vehicle. Data are presented as mean ± SEM and n = 5. * P

    Article Snippet: Histone H4 (Abcam, Cambridge, UK) was diluted in sodium acetate (10 mM, pH 5.5) and immobilized via amine coupling to flow cell 2.

    Techniques: Western Blot, Isolation, Mouse Assay

    NETs regulate TG via the intrinsic pathway of coagulation. NETs generated from PMA-stimulated bone marrow neutrophils were incubated in wild-type, factor VII- (FVII −  plasma) and factor XII-deficient (FXII −  plasma) PPP. ( A ) TG over time, ( B ) peak and ( C ) total levels of TG were determined as described in Material and Methods. Transmission electron microscopy showing NETs and MPs incubated with a gold-labeled anti-histone H4 antibody (small gold particles, black arrowhead) and ( D ) gold-labeled annexin V (large gold particles, black arrow) or ( E ) a gold-labeled anti-FXII antibody (large gold particles, black arrow). Scale bar = 0.25 µm. Data are presented as mean ± SEM and  n  = 5. * P

    Journal: Scientific Reports

    Article Title: Neutrophil extracellular trap-microparticle complexes enhance thrombin generation via the intrinsic pathway of coagulation in mice

    doi: 10.1038/s41598-018-22156-5

    Figure Lengend Snippet: NETs regulate TG via the intrinsic pathway of coagulation. NETs generated from PMA-stimulated bone marrow neutrophils were incubated in wild-type, factor VII- (FVII − plasma) and factor XII-deficient (FXII − plasma) PPP. ( A ) TG over time, ( B ) peak and ( C ) total levels of TG were determined as described in Material and Methods. Transmission electron microscopy showing NETs and MPs incubated with a gold-labeled anti-histone H4 antibody (small gold particles, black arrowhead) and ( D ) gold-labeled annexin V (large gold particles, black arrow) or ( E ) a gold-labeled anti-FXII antibody (large gold particles, black arrow). Scale bar = 0.25 µm. Data are presented as mean ± SEM and n  = 5. * P

    Article Snippet: Histone H4 (Abcam, Cambridge, UK) was diluted in sodium acetate (10 mM, pH 5.5) and immobilized via amine coupling to flow cell 2.

    Techniques: Coagulation, Generated, Incubation, Transmission Assay, Electron Microscopy, Labeling

    TSA or MCP30 increases the decreased acetylation of Topo IIα promoter in benzene poisoning mice. After all mice were killed, bone marrow mononuclear cells were separated and histone acetylation of Topo IIα promoter was assessed with chromatin immunoprecipitation (ChIP) assay using anti-acetylated histone H3 and anti-acetylated histone H4. ( A ) Representatives were shown for acetylation levels of histone H3 and histone H4 in the Topo IIα promoter. ( B ) Statistical data showed the acetylation levels of histone H3 in the Topo IIα promoter were shown. ( C ) Statistical data showed the acetylation levels of histone H4 in the Topo IIα promoter. ** P

    Journal: PLoS ONE

    Article Title: Histone Deacetylase Inhibitors Trichostatin A and MCP30 Relieve Benzene-Induced Hematotoxicity via Restoring Topoisomerase IIα

    doi: 10.1371/journal.pone.0153330

    Figure Lengend Snippet: TSA or MCP30 increases the decreased acetylation of Topo IIα promoter in benzene poisoning mice. After all mice were killed, bone marrow mononuclear cells were separated and histone acetylation of Topo IIα promoter was assessed with chromatin immunoprecipitation (ChIP) assay using anti-acetylated histone H3 and anti-acetylated histone H4. ( A ) Representatives were shown for acetylation levels of histone H3 and histone H4 in the Topo IIα promoter. ( B ) Statistical data showed the acetylation levels of histone H3 in the Topo IIα promoter were shown. ( C ) Statistical data showed the acetylation levels of histone H4 in the Topo IIα promoter. ** P

    Article Snippet: In vivo treatment with TSA or MCP30 also significantly increased the reduced expression and activity of Topo IIα in bone marrow mononuclear cells from benzene-treated mice, but still less than those of the control mice, confirming that HDAC inhibitors can restore the expression and activity of Topo IIα by increasing the acetylation levels of histone H3 and histone H4 in its promoter.

    Techniques: Mouse Assay, Chromatin Immunoprecipitation

    TSA or MCP30 restores the HQ-induced increased HDAC activity, decreased Topo IIα expression, and the resulting apoptosis in human bone marrow mononuclear cells. ( A—C ) Mononuclear cells were isolated from bone marrow aspirates from four healthy donors and subsequently treated with or without 100 μM HQ, in the presence or absence of TSA (0.5 μM) or MCP30 (1 μg/ml). After 24 h, the activity of HDAC ( A ), the expression of Topo IIα ( B ), and apoptosis ( C ) were determined using HDAC activity assay kit, western blot, and Annexin V/PI double staining, respectively. NS stands for normal saline. Histone H3 was used as a loading control for nuclear protein in western blot analysis. (D) Human bone marrow mononuclear cells were treated with or without a Topo IIα inhibitor daunorubicin (200 nM) for 48 h and apoptosis was measured using Annexin V staining. Statistical data and representatives of four independent experiments from different healthy donors were shown. * P

    Journal: PLoS ONE

    Article Title: Histone Deacetylase Inhibitors Trichostatin A and MCP30 Relieve Benzene-Induced Hematotoxicity via Restoring Topoisomerase IIα

    doi: 10.1371/journal.pone.0153330

    Figure Lengend Snippet: TSA or MCP30 restores the HQ-induced increased HDAC activity, decreased Topo IIα expression, and the resulting apoptosis in human bone marrow mononuclear cells. ( A—C ) Mononuclear cells were isolated from bone marrow aspirates from four healthy donors and subsequently treated with or without 100 μM HQ, in the presence or absence of TSA (0.5 μM) or MCP30 (1 μg/ml). After 24 h, the activity of HDAC ( A ), the expression of Topo IIα ( B ), and apoptosis ( C ) were determined using HDAC activity assay kit, western blot, and Annexin V/PI double staining, respectively. NS stands for normal saline. Histone H3 was used as a loading control for nuclear protein in western blot analysis. (D) Human bone marrow mononuclear cells were treated with or without a Topo IIα inhibitor daunorubicin (200 nM) for 48 h and apoptosis was measured using Annexin V staining. Statistical data and representatives of four independent experiments from different healthy donors were shown. * P

    Article Snippet: In vivo treatment with TSA or MCP30 also significantly increased the reduced expression and activity of Topo IIα in bone marrow mononuclear cells from benzene-treated mice, but still less than those of the control mice, confirming that HDAC inhibitors can restore the expression and activity of Topo IIα by increasing the acetylation levels of histone H3 and histone H4 in its promoter.

    Techniques: Activity Assay, Expressing, Isolation, HDAC Activity Assay, Western Blot, Double Staining, Staining

    PPAD stabilizes gingipains and modulates the levels of phagocytosis-related proteins. (a) Relative (Rel.) levels of gingipains (RgpA/RgpB) in infected neutrophils. (b) Time course of histone H3 degradation by P. gingivalis proteases in the presence or absence of PPAD, as determined by Western blotting (SN, culture supernatant). (c and d) Quantification of significant changes in the amounts of phagocytosis-related proteins (c) and the antimicrobial histone H2B (d) in infected neutrophils, as approximated by mass spectrometry. (a, c, and d) Data are means of three replicates of one neutrophil donor. * * , P

    Journal: mBio

    Article Title: A Secreted Bacterial Peptidylarginine Deiminase Can Neutralize Human Innate Immune Defenses

    doi: 10.1128/mBio.01704-18

    Figure Lengend Snippet: PPAD stabilizes gingipains and modulates the levels of phagocytosis-related proteins. (a) Relative (Rel.) levels of gingipains (RgpA/RgpB) in infected neutrophils. (b) Time course of histone H3 degradation by P. gingivalis proteases in the presence or absence of PPAD, as determined by Western blotting (SN, culture supernatant). (c and d) Quantification of significant changes in the amounts of phagocytosis-related proteins (c) and the antimicrobial histone H2B (d) in infected neutrophils, as approximated by mass spectrometry. (a, c, and d) Data are means of three replicates of one neutrophil donor. * * , P

    Article Snippet: Primary rabbit anti-RgpA/B, rabbit anti-PPAD antibodies ( , ) or anti-histone H3 (ab18521; Abcam) in PBS-T (1:2,000) were added, and the blot was incubated for 1 h at RT.

    Techniques: Infection, Western Blot, Mass Spectrometry

    PPAD citrullinates histone H3. In vitro citrullination of histone H3. Citrullination by human PAD2 was used as a positive control. (a) Schematic representation of citrullinated arginine residues in histone H3 upon incubation with PPAD or PAD2, as determined by mass spectrometry. (b) Western blot analysis of citrullinated histone H3. Quantification of band intensity in three independent experiments is shown. * , P

    Journal: mBio

    Article Title: A Secreted Bacterial Peptidylarginine Deiminase Can Neutralize Human Innate Immune Defenses

    doi: 10.1128/mBio.01704-18

    Figure Lengend Snippet: PPAD citrullinates histone H3. In vitro citrullination of histone H3. Citrullination by human PAD2 was used as a positive control. (a) Schematic representation of citrullinated arginine residues in histone H3 upon incubation with PPAD or PAD2, as determined by mass spectrometry. (b) Western blot analysis of citrullinated histone H3. Quantification of band intensity in three independent experiments is shown. * , P

    Article Snippet: Primary rabbit anti-RgpA/B, rabbit anti-PPAD antibodies ( , ) or anti-histone H3 (ab18521; Abcam) in PBS-T (1:2,000) were added, and the blot was incubated for 1 h at RT.

    Techniques: In Vitro, Positive Control, Incubation, Mass Spectrometry, Western Blot

    PPAD impacts on histone H3 citrullination and allows P. gingivalis to evade and survive capture in neutrophil extracellular traps (NETs). (a to d) Representative fluorescence microscopy images of NETosis and citrullinated histone H3 levels in the presence of P. gingivalis . PMA was applied at a concentration of 20 nM to induce NETosis. DNA was stained with DAPI (blue), P. gingivalis was labeled with FITC (green), and citrullinated histone H3 (citH3; red) was visualized with a specific antibody (scale bars, 200 µm in regular images and 50 µm in enlarged images). (e) Quantification of live bacteria present in isolated NETs.

    Journal: mBio

    Article Title: A Secreted Bacterial Peptidylarginine Deiminase Can Neutralize Human Innate Immune Defenses

    doi: 10.1128/mBio.01704-18

    Figure Lengend Snippet: PPAD impacts on histone H3 citrullination and allows P. gingivalis to evade and survive capture in neutrophil extracellular traps (NETs). (a to d) Representative fluorescence microscopy images of NETosis and citrullinated histone H3 levels in the presence of P. gingivalis . PMA was applied at a concentration of 20 nM to induce NETosis. DNA was stained with DAPI (blue), P. gingivalis was labeled with FITC (green), and citrullinated histone H3 (citH3; red) was visualized with a specific antibody (scale bars, 200 µm in regular images and 50 µm in enlarged images). (e) Quantification of live bacteria present in isolated NETs.

    Article Snippet: Primary rabbit anti-RgpA/B, rabbit anti-PPAD antibodies ( , ) or anti-histone H3 (ab18521; Abcam) in PBS-T (1:2,000) were added, and the blot was incubated for 1 h at RT.

    Techniques: Fluorescence, Microscopy, Concentration Assay, Staining, Labeling, Isolation

    Overexpression and purification of the COMPASS complex from budding yeast S . cerevisiae . (A) COMPASS overexpression scheme. Full length SET1 and the Cps subunits of COMPASS ( CPS60 , CPS50 , CPS40 , CPS35 , CPS30 , and CPS25 ) together with GAL4 and GAL1-10 were cloned into pRS30-series vectors. A TAP tag was fused to the N-terminus of Set1. (B) SDS-PAGE analysis of the purified COMPASS complex. Purified Set1 consistently runs on SDS/PAGE as a doublet, the reasons behind this property of Set1 or its biological significance remain unclear 7 . (C) In vitro H3K4 mono-, di-, and tri-methylation activities of COMPASS complex, examined by western blotting using H3K4me1, me2, and me3 antibody and anti-H3 antibody.

    Journal: Scientific Reports

    Article Title: Architecture and subunit arrangement of the complete Saccharomyces cerevisiae COMPASS complex

    doi: 10.1038/s41598-018-35609-8

    Figure Lengend Snippet: Overexpression and purification of the COMPASS complex from budding yeast S . cerevisiae . (A) COMPASS overexpression scheme. Full length SET1 and the Cps subunits of COMPASS ( CPS60 , CPS50 , CPS40 , CPS35 , CPS30 , and CPS25 ) together with GAL4 and GAL1-10 were cloned into pRS30-series vectors. A TAP tag was fused to the N-terminus of Set1. (B) SDS-PAGE analysis of the purified COMPASS complex. Purified Set1 consistently runs on SDS/PAGE as a doublet, the reasons behind this property of Set1 or its biological significance remain unclear 7 . (C) In vitro H3K4 mono-, di-, and tri-methylation activities of COMPASS complex, examined by western blotting using H3K4me1, me2, and me3 antibody and anti-H3 antibody.

    Article Snippet: The extend of methylation of histone H3 was examined using Western analysis with anti-H3K4me1 (Abcam, ab8895), anti-H3K4me2 (Abcam, ab7766), anti-H3K4me3 (Abcam, ab8580), and anti-H3 (Abcam, ab18521) antibodies.

    Techniques: Over Expression, Purification, Clone Assay, SDS Page, In Vitro, Methylation, Western Blot

    Inactivation of the LOX core promoter in NNK treated cells. ChIP and PCR assays were performed to elucidate the active status of the LOX core promoter in treated cells by assessing acetylated histone H3 binding to the core promoter region. DNAs were isolated from control and NNK treated cells each with 2 × 10 6 , sonicated and immunoprecipitated with an antibody against acetylated histone H3 or RNA-PolyII. Using immunoprecipitated DNA as a template, the PCR with primer pairs as shown under Methods amplified the acetylated histone H3-bound LOX core promoter region with 136 bp, and the RNA-Poly II bound fragment of the GAPDH promoter (an internal control) with 90 bp, respectively. PCR products were analyzed on 2.2% agarose gels and densities of DNA bands measured by the 1D Scan software.

    Journal: International Journal of Environmental Research and Public Health

    Article Title: NNK, a Tobacco-Specific Carcinogen, Inhibits the Expression of Lysyl Oxidase, a Tumor Suppressor

    doi: 10.3390/ijerph120100064

    Figure Lengend Snippet: Inactivation of the LOX core promoter in NNK treated cells. ChIP and PCR assays were performed to elucidate the active status of the LOX core promoter in treated cells by assessing acetylated histone H3 binding to the core promoter region. DNAs were isolated from control and NNK treated cells each with 2 × 10 6 , sonicated and immunoprecipitated with an antibody against acetylated histone H3 or RNA-PolyII. Using immunoprecipitated DNA as a template, the PCR with primer pairs as shown under Methods amplified the acetylated histone H3-bound LOX core promoter region with 136 bp, and the RNA-Poly II bound fragment of the GAPDH promoter (an internal control) with 90 bp, respectively. PCR products were analyzed on 2.2% agarose gels and densities of DNA bands measured by the 1D Scan software.

    Article Snippet: As shown in , in comparison to the internal control, the GAPDH fragment bound with the RNA-PolyII, the histone H3 acetylated at the tested region of the LOX promoter in NNK-treated cells was reduced to 74, 41, 11 and 5% of the control for cells treated with 10, 30, 100 and 300 µM NNK, respectively.

    Techniques: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Isolation, Sonication, Immunoprecipitation, Amplification, Software

    Recovery of histone acetylation by overexpression of wt p300 HAT. HS27/vIRF cells were transfected with wt p300 (lane 3) or p300 ΔHAT mutant (lane 4). HS27/cDNA3 (lane 1) and HS27/vIRF (lane 2) were included as controls. Identical amounts of proteins from cell lysates were used for immunoblotting analysis with an antibody specific for the acetylated histone H4. The bottom panel shows the amount of cellular histone H4 protein in each lane, detected by an anti-H4 antibody. Arrows indicate the acetylated form of histone H4 (Ac-H4) or total histone H4 (H4). Numbers at left are molecular masses in kilodaltons.

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

    doi:

    Figure Lengend Snippet: Recovery of histone acetylation by overexpression of wt p300 HAT. HS27/vIRF cells were transfected with wt p300 (lane 3) or p300 ΔHAT mutant (lane 4). HS27/cDNA3 (lane 1) and HS27/vIRF (lane 2) were included as controls. Identical amounts of proteins from cell lysates were used for immunoblotting analysis with an antibody specific for the acetylated histone H4. The bottom panel shows the amount of cellular histone H4 protein in each lane, detected by an anti-H4 antibody. Arrows indicate the acetylated form of histone H4 (Ac-H4) or total histone H4 (H4). Numbers at left are molecular masses in kilodaltons.

    Article Snippet: Purified full-length p300 was mixed with histone H4 and 3 H-labeled acetyl-CoA in the presence or absence of purified vIRF protein.

    Techniques: Over Expression, HAT Assay, Transfection, Mutagenesis

    Immunofluorescence test of in vivo histone H3 and H4 acetylation. HS27/cDNA3 and HS27/vIRF cells were stained with antibodies which specifically reacted with the acetylated forms of histones H3 and H4. Cells were visualized with Nomarski optics. Immunofluorescence testing was performed with a Leica confocal immunofluorescence microscope.

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

    doi:

    Figure Lengend Snippet: Immunofluorescence test of in vivo histone H3 and H4 acetylation. HS27/cDNA3 and HS27/vIRF cells were stained with antibodies which specifically reacted with the acetylated forms of histones H3 and H4. Cells were visualized with Nomarski optics. Immunofluorescence testing was performed with a Leica confocal immunofluorescence microscope.

    Article Snippet: Purified full-length p300 was mixed with histone H4 and 3 H-labeled acetyl-CoA in the presence or absence of purified vIRF protein.

    Techniques: Immunofluorescence, In Vivo, Staining, Microscopy

    Inhibition of p300 HAT activity by vIRF. Recombinant baculovirus containing the flag-tagged p300, vIRF, or v-cyclin was used to purify each protein from insect cells. Purified p300 protein (30 nM) was mixed with [ 3 H]acetyl-CoA and histone H4 serving as substrates in the presence of increasing nanomolar amounts of vIRF or v-cyclin as indicated at the bottom of panel A. After 5 min, p300 HAT activity was measured by immunoblotting with an antibody specific for acetylated histone H4 (A) and quantitating radioactivity of 3 H-labeled histone H4 (B). In lane 7, p300 protein was first mixed with the substrates for 5 min, followed by incubation with vIRF protein (150 nM) for an additional 25 min. The bottom panel of panel A shows the amount of histone H4 protein used in each reaction, detected by an anti-H4 antibody. The values in panel B represent the averages of three independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

    doi:

    Figure Lengend Snippet: Inhibition of p300 HAT activity by vIRF. Recombinant baculovirus containing the flag-tagged p300, vIRF, or v-cyclin was used to purify each protein from insect cells. Purified p300 protein (30 nM) was mixed with [ 3 H]acetyl-CoA and histone H4 serving as substrates in the presence of increasing nanomolar amounts of vIRF or v-cyclin as indicated at the bottom of panel A. After 5 min, p300 HAT activity was measured by immunoblotting with an antibody specific for acetylated histone H4 (A) and quantitating radioactivity of 3 H-labeled histone H4 (B). In lane 7, p300 protein was first mixed with the substrates for 5 min, followed by incubation with vIRF protein (150 nM) for an additional 25 min. The bottom panel of panel A shows the amount of histone H4 protein used in each reaction, detected by an anti-H4 antibody. The values in panel B represent the averages of three independent experiments.

    Article Snippet: Purified full-length p300 was mixed with histone H4 and 3 H-labeled acetyl-CoA in the presence or absence of purified vIRF protein.

    Techniques: Inhibition, HAT Assay, Activity Assay, Recombinant, Purification, Radioactivity, Labeling, Incubation

    Alteration of in vivo histone H3 and H4 acetylation by vIRF expression or butyric acid treatment. Identical amounts of proteins from HS27/cDNA3 cells (lanes 1 and 3) and HS27/vIRF cells (lanes 2 and 4) treated with butyric acid overnight (lanes 3 and 4) or mock treated (lanes 1 and 2) were used for immunoblotting analysis with antibodies specific for the acetylated histone H3 (A) or H4 (B). Arrows indicate acetylated histones H3 (Ac-H3) and H4 (Ac-H4). Numbers at left of each panel show sizes in kilodaltons.

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

    doi:

    Figure Lengend Snippet: Alteration of in vivo histone H3 and H4 acetylation by vIRF expression or butyric acid treatment. Identical amounts of proteins from HS27/cDNA3 cells (lanes 1 and 3) and HS27/vIRF cells (lanes 2 and 4) treated with butyric acid overnight (lanes 3 and 4) or mock treated (lanes 1 and 2) were used for immunoblotting analysis with antibodies specific for the acetylated histone H3 (A) or H4 (B). Arrows indicate acetylated histones H3 (Ac-H3) and H4 (Ac-H4). Numbers at left of each panel show sizes in kilodaltons.

    Article Snippet: Purified full-length p300 was mixed with histone H4 and 3 H-labeled acetyl-CoA in the presence or absence of purified vIRF protein.

    Techniques: In Vivo, Expressing

    PU.1 positively regulates DR5 expression and sensitizes AML cells to apoptosis. ( a–c ) Flow cytometry analysis of DR5 surface expression, DR5 and PU.1 western blot and quantitative RT-PCR (qPCR) analysis in NB4, HL60 as well as MOLM-13 control and PU.1-knockdown cells. Total protein was used as a loading control for western blot data. qPCR values were normalized to the housekeeping gene  HMBS . Results are given as  n -fold changes compared with control cells. ( d ) DR5 qPCR, DR5, cleaved PARP as well as PU.1 western blot and DR5 FACS analysis of NB4 control and PU.1-knockdown cells upon TRAIL treatment. Analysis as in ( a ). One out of three independent experiments is shown. ( e ) PU.1 and p65 ChIP analysis of three putative, neighboring PU.1/p65-binding sites upstream of the transcriptional start site of the  DR5  gene.  In vivo  binding of PU.1 and p65 to the indicated sites was shown by ChIP in NB4 cells using antibodies against PU.1 and p65. Antibodies against acetyl-histone H3 and IgG were used as a positive and negative control, respectively. GAPDH amplification was shown as a negative control for the different pulldowns. ( f ) DR5 expression analysis of NB4 cells expressing an inducible PU.1-estrogen receptor (PU.1-ER) fusion protein. DR5 qPCR, western blot and FACS analysis of NB4 control and PU.1-knockdown cells 48 h after 4-hydroxy-tamoxifen (4-OH-T) stimulation from three independent experiments. ( g ) Viability of NB4 control and PU.1-ER-expressing cells upon PU.1 activation for 48 h with 4-OH-T stimulation. Cells were incubated with rhTRAIL (500 ng/ml) 24 h after 4-OH-T addition. Percent of Annexin V +  cells from three independent experiments are shown (upper panel) and immunoblotting for pro- and cleaved caspase-8, cleaved caspase-3 and cleaved PARP are shown (lower panel). Total protein was used as a loading control. ( h ) Viability of NB4 control and PU.1-ER-expressing cells treated as in ( f ). Percent of Annexin V +  cells from three independent experiments are shown. Immunoblotting for cleaved PARP, FLIP L  and FLIP S , Bcl-2 and Mcl-1 is shown. Analysis as in ( g ). ** P

    Journal: Cell Death and Differentiation

    Article Title: PU.1 supports TRAIL-induced cell death by inhibiting NF-κB-mediated cell survival and inducing DR5 expression

    doi: 10.1038/cdd.2017.40

    Figure Lengend Snippet: PU.1 positively regulates DR5 expression and sensitizes AML cells to apoptosis. ( a–c ) Flow cytometry analysis of DR5 surface expression, DR5 and PU.1 western blot and quantitative RT-PCR (qPCR) analysis in NB4, HL60 as well as MOLM-13 control and PU.1-knockdown cells. Total protein was used as a loading control for western blot data. qPCR values were normalized to the housekeeping gene HMBS . Results are given as n -fold changes compared with control cells. ( d ) DR5 qPCR, DR5, cleaved PARP as well as PU.1 western blot and DR5 FACS analysis of NB4 control and PU.1-knockdown cells upon TRAIL treatment. Analysis as in ( a ). One out of three independent experiments is shown. ( e ) PU.1 and p65 ChIP analysis of three putative, neighboring PU.1/p65-binding sites upstream of the transcriptional start site of the DR5 gene. In vivo binding of PU.1 and p65 to the indicated sites was shown by ChIP in NB4 cells using antibodies against PU.1 and p65. Antibodies against acetyl-histone H3 and IgG were used as a positive and negative control, respectively. GAPDH amplification was shown as a negative control for the different pulldowns. ( f ) DR5 expression analysis of NB4 cells expressing an inducible PU.1-estrogen receptor (PU.1-ER) fusion protein. DR5 qPCR, western blot and FACS analysis of NB4 control and PU.1-knockdown cells 48 h after 4-hydroxy-tamoxifen (4-OH-T) stimulation from three independent experiments. ( g ) Viability of NB4 control and PU.1-ER-expressing cells upon PU.1 activation for 48 h with 4-OH-T stimulation. Cells were incubated with rhTRAIL (500 ng/ml) 24 h after 4-OH-T addition. Percent of Annexin V + cells from three independent experiments are shown (upper panel) and immunoblotting for pro- and cleaved caspase-8, cleaved caspase-3 and cleaved PARP are shown (lower panel). Total protein was used as a loading control. ( h ) Viability of NB4 control and PU.1-ER-expressing cells treated as in ( f ). Percent of Annexin V + cells from three independent experiments are shown. Immunoblotting for cleaved PARP, FLIP L and FLIP S , Bcl-2 and Mcl-1 is shown. Analysis as in ( g ). ** P

    Article Snippet: The primary antibodies used were anti-PU.1 (no. 2258; Cell Signaling, Switzerland or sc-365208; Santa Cruz, Switzerland), anti-cFLIP (F9800; Sigma) and (AG-20B-0056; Adipogen AG, Switzerland), anti-XIAP (M044-3; MBL, Switzerland), anti-caspase-3 (235412; Calbiochem, Switzerland) and (no. 9661; Cell Signaling), anti-PARP (nos 9542, 9541; Cell Signaling) anti-cleaved caspase-8 (no. 9496; Cell Signaling), anti-caspase-8 (ALX-804-242; Enzo Life Sciences (ELS) AG, Switzerland), anti-Bcl-2 (SAB4500003; Sigma), anti-Mcl-1 (559027; BD Biosciences, Switzerland), anti-DR5 (no. 8074; Cell Signaling), anti-DcR2 (no. 8049; Cell Signaling), anti-p65 (no. 8242; Cell Signaling), anti-phospho-p65 (no. 3033; Cell Signaling), anti-I κ B α (no. 4814; Cell Signaling) anti-phospho-I κ B α (no. 2859; Cell Signaling), anti-RIPK1 (no. 3493; Cell Signaling) anti- α -tubulin (no. 3873; Cell Signaling, anti-Myc tag (ab13836; Abcam), anti-FLAG tag (no. 2368; Cell Signaling)) and anti-histone H3 (no. 9715; Cell Signaling).

    Techniques: Expressing, Flow Cytometry, Cytometry, Western Blot, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, FACS, Chromatin Immunoprecipitation, Binding Assay, In Vivo, Negative Control, Amplification, Activation Assay, Incubation

    At after infection, HSV-1 DNA was protected from serial MCN digestion to sizes of mono to poly-nucleosomes that fractionated in complexes with the hydrodynamic ratios of mono to poly-nucleosomes and contain histone H3. Nuclei of infected cells were subjected to serial MCN digestion and then cross-linked. (A) Cross-linked soluble chromatin separated by hydrodynamic ratios in a 0–10% sucrose gradient, and resolved in 2% agarose gel electrophoresis, stained with EtBr. The left pointing arrowheads at the right indicate the migration of mono- to octa-nucleosome-sized DNA. (B) Chromatin immunoprecipitation of HSV-1 DNA with histone H3. EtBr stained agarose gels of saturating PCR-amplified cellular (GAPDH) or HSV-1 (UL25, UL46, ICP4, or gE) DNA co-immunoprecipitated with histone H3. (C) Cartoon presenting the positions of the PCR amplicons in the HSV-1 genome. In, input (2%); H3, anti-histone H3 antibody; IgG, isotype antibody control (labeled only in the No Dig sample for simplicity); No Dig, undigested unfractionated chromatin; Ins, insoluble chromatin.

    Journal: PLoS Pathogens

    Article Title: Chromatin dynamics and the transcriptional competence of HSV-1 genomes during lytic infections

    doi: 10.1371/journal.ppat.1008076

    Figure Lengend Snippet: At after infection, HSV-1 DNA was protected from serial MCN digestion to sizes of mono to poly-nucleosomes that fractionated in complexes with the hydrodynamic ratios of mono to poly-nucleosomes and contain histone H3. Nuclei of infected cells were subjected to serial MCN digestion and then cross-linked. (A) Cross-linked soluble chromatin separated by hydrodynamic ratios in a 0–10% sucrose gradient, and resolved in 2% agarose gel electrophoresis, stained with EtBr. The left pointing arrowheads at the right indicate the migration of mono- to octa-nucleosome-sized DNA. (B) Chromatin immunoprecipitation of HSV-1 DNA with histone H3. EtBr stained agarose gels of saturating PCR-amplified cellular (GAPDH) or HSV-1 (UL25, UL46, ICP4, or gE) DNA co-immunoprecipitated with histone H3. (C) Cartoon presenting the positions of the PCR amplicons in the HSV-1 genome. In, input (2%); H3, anti-histone H3 antibody; IgG, isotype antibody control (labeled only in the No Dig sample for simplicity); No Dig, undigested unfractionated chromatin; Ins, insoluble chromatin.

    Article Snippet: Antibodies against histone H2A (Abcam, rabbit, Cat. ab88770), H2B (Abcam, mouse, Cat. ab52484), H3 (Abcam, rabbit, Cat. ab1791), H4 (Abcam, mouse, Cat. ab31830) were diluted 1:1000 in 10 ml PBS with 0.05% tween-20.

    Techniques: Infection, Agarose Gel Electrophoresis, Staining, Migration, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Immunoprecipitation, Labeling

    Subgenome-specific recruitment of p300 is associated with TEs. Subgenome-specific p300 peaks are enriched for TEs carrying transcription factor (TF) motifs active in early development. a Differential regulation of the slc2a2 homeologs at stage 10.5. Shown are the genomic profiles of H3K4me3 ( green ), RNA Polymerase II (RNAPII; purple ), H3K36me3 ( blue ), and p300 ( yellow ) ChIP-seq tracks, as well as DNA methylation levels determined by WGBS ( gray ). The top panel shows slc2a2.L, which is highly expressed, as evidenced by RNAPII and H3K36me3, and has a number of active enhancers ( a – g ), while slc2a2.S, shown in the bottom panel , is expressed at a lower rate. The conservation between the L and S genomic sequence is shown in gray between the panels. Differential enhancers between L and S are highlighted in yellow , which illustrates lost enhancer function ( a , b ), conserved enhancer function ( c – e ), and deleted enhancers ( f , g ). b Subgenome-specific p300 peaks are associated with DNA transposon repeats (threshold p ≤ 10e-4, twofold enrichment compared to all X. laevis peaks and present at least in 15% of the peaks). The barplots show the frequency of occurrence of each of the three repeat types per megabase in the three (sub)genomes. Over the bars is represented the percentage of subgenome-specific peaks overlapping with the corresponding repeat. c TF found to be enriched in the subgenome-specific p300 peaks (threshold p ≤ 10e-4, threefold enrichment compared to all X. laevis peaks and present at least in 20% of the peaks)

    Journal: Genome Biology

    Article Title: Regulatory remodeling in the allo-tetraploid frog Xenopus laevis

    doi: 10.1186/s13059-017-1335-7

    Figure Lengend Snippet: Subgenome-specific recruitment of p300 is associated with TEs. Subgenome-specific p300 peaks are enriched for TEs carrying transcription factor (TF) motifs active in early development. a Differential regulation of the slc2a2 homeologs at stage 10.5. Shown are the genomic profiles of H3K4me3 ( green ), RNA Polymerase II (RNAPII; purple ), H3K36me3 ( blue ), and p300 ( yellow ) ChIP-seq tracks, as well as DNA methylation levels determined by WGBS ( gray ). The top panel shows slc2a2.L, which is highly expressed, as evidenced by RNAPII and H3K36me3, and has a number of active enhancers ( a – g ), while slc2a2.S, shown in the bottom panel , is expressed at a lower rate. The conservation between the L and S genomic sequence is shown in gray between the panels. Differential enhancers between L and S are highlighted in yellow , which illustrates lost enhancer function ( a , b ), conserved enhancer function ( c – e ), and deleted enhancers ( f , g ). b Subgenome-specific p300 peaks are associated with DNA transposon repeats (threshold p ≤ 10e-4, twofold enrichment compared to all X. laevis peaks and present at least in 15% of the peaks). The barplots show the frequency of occurrence of each of the three repeat types per megabase in the three (sub)genomes. Over the bars is represented the percentage of subgenome-specific peaks overlapping with the corresponding repeat. c TF found to be enriched in the subgenome-specific p300 peaks (threshold p ≤ 10e-4, threefold enrichment compared to all X. laevis peaks and present at least in 20% of the peaks)

    Article Snippet: The following antibodies were used: H3K4me3 (Abcam ab8580), H3K4me1 (Abcam ab8895), p300 (C-20, Santa Cruz sc-585), H3K36me3 (Abcam ab9050), and RNA polymerase II (Diagenode C15200004).

    Techniques: Chromatin Immunoprecipitation, DNA Methylation Assay, Sequencing

    Alignment of a region on chromosome 8 in X. tropicalis and the X. laevis L and S subgenomes annotated with experimental ChIP-seq data (gastrula-stage embryos; NF stage 10.5). Shown are the gene annotation ( black ), repeats ( gray ), ChIP-seq profiles for H3K4me3 ( green ), p300 ( yellow ), RNA Polymerase II (RNAPII; brown ), and H3K36me3 ( dark green ). The sequence conservation is indicated by gray lines . Conserved H3K4me3 and p300 peaks are denoted by green and yellow lines , respectively. The anp32e gene is expressed in X. tropicalis and both the L and S subgenome of X. laevis . The plekho1 gene, on the other hand, has lost promoter and enhancer activity on the X. laevis S locus and shows no experimental evidence of being expressed

    Journal: Genome Biology

    Article Title: Regulatory remodeling in the allo-tetraploid frog Xenopus laevis

    doi: 10.1186/s13059-017-1335-7

    Figure Lengend Snippet: Alignment of a region on chromosome 8 in X. tropicalis and the X. laevis L and S subgenomes annotated with experimental ChIP-seq data (gastrula-stage embryos; NF stage 10.5). Shown are the gene annotation ( black ), repeats ( gray ), ChIP-seq profiles for H3K4me3 ( green ), p300 ( yellow ), RNA Polymerase II (RNAPII; brown ), and H3K36me3 ( dark green ). The sequence conservation is indicated by gray lines . Conserved H3K4me3 and p300 peaks are denoted by green and yellow lines , respectively. The anp32e gene is expressed in X. tropicalis and both the L and S subgenome of X. laevis . The plekho1 gene, on the other hand, has lost promoter and enhancer activity on the X. laevis S locus and shows no experimental evidence of being expressed

    Article Snippet: The following antibodies were used: H3K4me3 (Abcam ab8580), H3K4me1 (Abcam ab8895), p300 (C-20, Santa Cruz sc-585), H3K36me3 (Abcam ab9050), and RNA polymerase II (Diagenode C15200004).

    Techniques: Chromatin Immunoprecipitation, Sequencing, Activity Assay

    The S subgenome has more and larger deletions than L. a Size frequency distribution of deletions ( top ) and size ratio of LΔS deletions relative to SΔL deletions as a function of deletion size ( bottom ). b An example of a gene ( grlx2 ) that has lost the promoter on the S genome due to a deletion. Shown are the gene annotation ( black ), ChIP-seq profiles for H3K4me3 ( green ), RNAPII ( brown ), and H3K36me3 ( dark green ). The sequence conservation is indicated by gray lines . c The log2 fold difference between the observed number of deleted basepairs and the expected number (mean of 1000 randomizations). The fold difference is calculated per chromosome and summarized in a boxplot. Intergenic 1 kb distance from a gene, Intronic introns, Exonic UTRs + CDS, IntronicTx introns from genes actively transcribed, ExonicTx exons from genes actively transcribed, p300 genomic fragments having a p300 peak, H3K4me3 genomic fragments having a H3K4me3 peak. The asterisks mark significant differences between the L and S chromosomes ( p

    Journal: Genome Biology

    Article Title: Regulatory remodeling in the allo-tetraploid frog Xenopus laevis

    doi: 10.1186/s13059-017-1335-7

    Figure Lengend Snippet: The S subgenome has more and larger deletions than L. a Size frequency distribution of deletions ( top ) and size ratio of LΔS deletions relative to SΔL deletions as a function of deletion size ( bottom ). b An example of a gene ( grlx2 ) that has lost the promoter on the S genome due to a deletion. Shown are the gene annotation ( black ), ChIP-seq profiles for H3K4me3 ( green ), RNAPII ( brown ), and H3K36me3 ( dark green ). The sequence conservation is indicated by gray lines . c The log2 fold difference between the observed number of deleted basepairs and the expected number (mean of 1000 randomizations). The fold difference is calculated per chromosome and summarized in a boxplot. Intergenic 1 kb distance from a gene, Intronic introns, Exonic UTRs + CDS, IntronicTx introns from genes actively transcribed, ExonicTx exons from genes actively transcribed, p300 genomic fragments having a p300 peak, H3K4me3 genomic fragments having a H3K4me3 peak. The asterisks mark significant differences between the L and S chromosomes ( p

    Article Snippet: The following antibodies were used: H3K4me3 (Abcam ab8580), H3K4me1 (Abcam ab8895), p300 (C-20, Santa Cruz sc-585), H3K36me3 (Abcam ab9050), and RNA polymerase II (Diagenode C15200004).

    Techniques: Chromatin Immunoprecipitation, Sequencing

    TRIM28 bound to the Il17-Il17f gene locus in Th17 cells. a−d Naive CD4 + T cells were cultured under Th17 condition (TGF-β plus IL-6) for 1 day and then prepared for ChIP-seq or ChIP-qPCR assay using anti-TRIM28 antibody. a Distribution of TRIM28-binding peaks in Th17 cells. b Alignment of TRIM28 binding peaks with histone modification and DNA methylation/demethylation markers in Th17 cells. c IGV browser view of TRIM28 binding peaks with H3K4me3, H3K27me3, 5hmc and 5mc markers at the Il17-Il17f gene locus. d ChIP-qPCR analysis of TRIM28 binding at representative gene loci; the data shown are a representative result for more than three independent experiments. e WT or Trim28 −/− naive CD4 + T cells were cultured at Th17 condition (TGF-β plus IL-6) for 3 days, and then prepared for ChIP-qPCR assay performed by anti-H3K4Me3 antibody or MeDIP assay performed by 5hmc antibody. All error bars represent SDs. The experiments were repeated twice with consistent results

    Journal: Nature Communications

    Article Title: Epigenetic activation during T helper 17 cell differentiation is mediated by Tripartite motif containing 28

    doi: 10.1038/s41467-018-03852-2

    Figure Lengend Snippet: TRIM28 bound to the Il17-Il17f gene locus in Th17 cells. a−d Naive CD4 + T cells were cultured under Th17 condition (TGF-β plus IL-6) for 1 day and then prepared for ChIP-seq or ChIP-qPCR assay using anti-TRIM28 antibody. a Distribution of TRIM28-binding peaks in Th17 cells. b Alignment of TRIM28 binding peaks with histone modification and DNA methylation/demethylation markers in Th17 cells. c IGV browser view of TRIM28 binding peaks with H3K4me3, H3K27me3, 5hmc and 5mc markers at the Il17-Il17f gene locus. d ChIP-qPCR analysis of TRIM28 binding at representative gene loci; the data shown are a representative result for more than three independent experiments. e WT or Trim28 −/− naive CD4 + T cells were cultured at Th17 condition (TGF-β plus IL-6) for 3 days, and then prepared for ChIP-qPCR assay performed by anti-H3K4Me3 antibody or MeDIP assay performed by 5hmc antibody. All error bars represent SDs. The experiments were repeated twice with consistent results

    Article Snippet: The antibodies used for ChIP including: anti-TRIM28 (Active Motif, 61173), rabbit IgG (Abcam, ab37415), anti-pSTAT3 (CST, 9134), anti-RORγt (Abcam, ab78007), anti-p300 (Santa Cruz, sc-585), anti-phospho-PolII(Ser5)(CST, 13523), anti-H3K4me3 (Abcam, ab8580), anti-H3K27me3 (Millipore, 07-499), anti-H3K4me1 (homemade by Dr Wei Xie’s lab at Tsinghua University) and anti-H3K27ac (Active Motif, 39133).

    Techniques: Cell Culture, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Modification, DNA Methylation Assay, Methylated DNA Immunoprecipitation

    Effects of UTP and BzATP on the stimulation of the mitosis marker, phospho-histone 3, by FGF2. Quiescent cultures of rat cortical astrocytes were treated with FGF2 (25 ng/ml) alone or in combination with UTP (100 uM) or BzATP (100 uM) for 22 hr. Western

    Journal: Journal of neuroscience research

    Article Title: OPPOSING EFFECTS OF P2X7 AND P2Y PURINE/PYRIMIDINE-PREFERRING RECEPTORS ON PROLIFERATION OF ASTROCYTES INDUCED BY FIBROBLAST GROWTH FACTOR-2: IMPLICATIONS FOR CNS DEVELOPMENT, INJURY AND REPAIR

    doi: 10.1002/jnr.21765

    Figure Lengend Snippet: Effects of UTP and BzATP on the stimulation of the mitosis marker, phospho-histone 3, by FGF2. Quiescent cultures of rat cortical astrocytes were treated with FGF2 (25 ng/ml) alone or in combination with UTP (100 uM) or BzATP (100 uM) for 22 hr. Western

    Article Snippet: Immunoblotting was performed using antibodies specific for phospho-histone H3 (Upstate Cell Signaling Solutions, Lake Placid, NY), at a 1/500 dilution.

    Techniques: Marker, Western Blot

    Polymer Simulations Predict Chromosome Interactions in Different Cell Lines (A) Simulated Capture-C profiles (colored lines) are shown for three cell lines, for three different viewpoints (at the URR, DRR, and Pax6 , indicated by arrows). The corresponding experimental data are shown as gray bars. Above each set of plots, a line of points show how beads were colored as non-binding (gray) or binding (red) for TFs. In simulations, Pax6 interacts strongly with a broad acetylated region downstream of the gene (red stars); this is not observed in the experimental data. (B) Plot showing the level of interaction between a viewpoint and a specified 10-kbp region. The arrow indicates the viewpoint (arrow base), and the interacting region (arrowhead). The height of the bar shows the number of interactions with the 10-kbp region as a percentage of interactions with the locus as a whole (chromosome 2 [chr2]: 105,200,000–105,800,00). (C) Similar plots were obtained from experimental Capture-C data. D) were used to infer TF-binding sites instead of H3K27ac. The erroneous interactions marked in (A) are now absent (red stars). (E) Similar plots to those in (B) but from the simulations using ATAC-seq data. .

    Journal: Molecular Cell

    Article Title: Polymer Simulations of Heteromorphic Chromatin Predict the 3D Folding of Complex Genomic Loci

    doi: 10.1016/j.molcel.2018.09.016

    Figure Lengend Snippet: Polymer Simulations Predict Chromosome Interactions in Different Cell Lines (A) Simulated Capture-C profiles (colored lines) are shown for three cell lines, for three different viewpoints (at the URR, DRR, and Pax6 , indicated by arrows). The corresponding experimental data are shown as gray bars. Above each set of plots, a line of points show how beads were colored as non-binding (gray) or binding (red) for TFs. In simulations, Pax6 interacts strongly with a broad acetylated region downstream of the gene (red stars); this is not observed in the experimental data. (B) Plot showing the level of interaction between a viewpoint and a specified 10-kbp region. The arrow indicates the viewpoint (arrow base), and the interacting region (arrowhead). The height of the bar shows the number of interactions with the 10-kbp region as a percentage of interactions with the locus as a whole (chromosome 2 [chr2]: 105,200,000–105,800,00). (C) Similar plots were obtained from experimental Capture-C data. D) were used to infer TF-binding sites instead of H3K27ac. The erroneous interactions marked in (A) are now absent (red stars). (E) Similar plots to those in (B) but from the simulations using ATAC-seq data. .

    Article Snippet: Each IP was performed with 25 μl of Protein G Dyna Beads (ThermoFisher), pre-washed with 2x 1 mL PBS/BSA, and incubated in 200 μl PBS/BSA with 10 μl of capture antibody (CTCF antibody (D31H2 XP Rabbit mAb #3418), Rad21 antibody (Abcam Ab992), H3K27ac (Abcam, ab4729) or control Rabbit IgG (I5006 Sigma)) for 3 hr, rotating at 4°C.

    Techniques: Capture-C, Binding Assay

    SCFAs reduce the autoantibody response and autoimmunity in lupus-prone mice. Female MRL/ Fas lpr/lpr mice fed fiber diet were given SCFAs water (SCFAs) or plain water (Nil) starting at the age of 5 weeks and killed at 17 weeks of age. a Titers of circulating anti-dsDNA IgM, and anti-dsDNA, anti-RNP/Sm, anti-histone, and anti-RNA IgG1 and IgG2a (relative units, RU), as analyzed by specific ELISAs. Each symbol represents an individual mouse ( n = 4 per group, pooled from two experiments). The bars represent the mean ± SD. b ANA visualized by indirect immunofluorescence on HEp-2 cells that were incubated with sera (1:400 dilution) from the MRL/ Fas lp/lpr mice using FITC-labeled rat mAbs to mouse IgG1 and IgG2a. Data are from one of three independent experiments yielding comparable results. c , d AID and Blimp1 expression as analyzed by intracellular staining followed by fluorescence microscopy ( c ) and flow cytometry ( d ). e Spleen surface CD138 + plasmablasts/plasma cells, and intracellular CD138 + IgG1 + and CD138 + IgG2a + plasmablasts/plasma cells as analyzed by flow cytometry. Data are representative of three independent experiments yielding comparable results. f Dorsal skin lesions (left panels), kidney sections stained with H E (middle panels), and kidney sections stained with FITC-labeled rat mAbs to mouse IgG1 and IgG2a (right panels). Data are representative of three independent experiments. * p

    Journal: Nature Communications

    Article Title: B cell-intrinsic epigenetic modulation of antibody responses by dietary fiber-derived short-chain fatty acids

    doi: 10.1038/s41467-019-13603-6

    Figure Lengend Snippet: SCFAs reduce the autoantibody response and autoimmunity in lupus-prone mice. Female MRL/ Fas lpr/lpr mice fed fiber diet were given SCFAs water (SCFAs) or plain water (Nil) starting at the age of 5 weeks and killed at 17 weeks of age. a Titers of circulating anti-dsDNA IgM, and anti-dsDNA, anti-RNP/Sm, anti-histone, and anti-RNA IgG1 and IgG2a (relative units, RU), as analyzed by specific ELISAs. Each symbol represents an individual mouse ( n = 4 per group, pooled from two experiments). The bars represent the mean ± SD. b ANA visualized by indirect immunofluorescence on HEp-2 cells that were incubated with sera (1:400 dilution) from the MRL/ Fas lp/lpr mice using FITC-labeled rat mAbs to mouse IgG1 and IgG2a. Data are from one of three independent experiments yielding comparable results. c , d AID and Blimp1 expression as analyzed by intracellular staining followed by fluorescence microscopy ( c ) and flow cytometry ( d ). e Spleen surface CD138 + plasmablasts/plasma cells, and intracellular CD138 + IgG1 + and CD138 + IgG2a + plasmablasts/plasma cells as analyzed by flow cytometry. Data are representative of three independent experiments yielding comparable results. f Dorsal skin lesions (left panels), kidney sections stained with H E (middle panels), and kidney sections stained with FITC-labeled rat mAbs to mouse IgG1 and IgG2a (right panels). Data are representative of three independent experiments. * p

    Article Snippet: After blocking and overnight incubation at 4 °C with anti-AID (ZA001, Invitrogen), anti-Blimp1 (6D3, eBioscience), anti-acetyl-histone H3 (H3K9ac/K14ac, 17–615, Millipore), anti-histone H3 (601901, BioLegend), or anti-β-Actin mAb (AC-15, Sigma-Aldrich), the membranes were incubated with HRP-conjugated secondary Abs.

    Techniques: Mouse Assay, Immunofluorescence, Incubation, Labeling, Expressing, Staining, Fluorescence, Microscopy, Flow Cytometry, Cytometry

    SCFAs increase expression of Aicda- and Prdm1- targeting miRNAs by enhancing histone acetylation of related miRNA host genes. a Primary mouse B cells, CH12F3 B cells, and human B cells were stimulated with indicated stimuli for 60 h in the presence of nil, butyrate (500 μM), or propionate (2000 μM). Ex vivo B cells were isolated from mice injected with NP-LPS and on plain water or SCFA water for 21 days. Acetylated-histone H3 (H3K9ac), histone H3, and β-actin proteins as detected by immunoblotting. Data are one representative of three independent experiments. b Relative densities of the Ac-histone H3 bands normalized to histone H3 and β - actin levels. c , d B cells stimulated with LPS plus IL-4 in the presence of nil or butyrate for 60 h. Expression of the Aicda- and Prdm1- targeting miRNAs, and irrelevant miRNAs as analyzed by qRT-PCR ( c ). Relative abundance of H3K9ac/K14ac in miRNA host genes (HGs) as analyzed by ChIP-qPCR ( d ). e Schematic diagram of the luciferase reporter constructs-containing 3′UTRs of Aicda and Prdm1 mRNAs and their mutant (mut) counterparts. f Surface CD19 and IgA on CH12F3 B cells stimulated for 96 h in the presence of nil, butyrate (500 μM), propionate (2000 μM), or VPA (500 μM) as analyzed by flow cytometry. Data are one representative of 3 independent experiments yielding comparable results. g , miRNA expression in CH12F3 cells stimulated for 24 h in the presence of nil or butyrate, as analyzed by qRT-PCR. Data in c , d , and g are ratios to stimulated B cells cultured with nil (set as 1; means ± SEM of three independent experiments). h Luciferase activity in CH12F3 cells transfected with luciferase reporter vectors-containing wild-type or mutated Aicda or Prdm1 3′UTRs after 24 h treatment with nil or butyrate. Luciferase activity was measured 6 h after transfection, and normalized relative to luciferase activity in B cells cultured with nil. Data in h are ratios to transfected B cells cultured with nil (set as 100%; means ± SEM of three independent experiments). * p

    Journal: Nature Communications

    Article Title: B cell-intrinsic epigenetic modulation of antibody responses by dietary fiber-derived short-chain fatty acids

    doi: 10.1038/s41467-019-13603-6

    Figure Lengend Snippet: SCFAs increase expression of Aicda- and Prdm1- targeting miRNAs by enhancing histone acetylation of related miRNA host genes. a Primary mouse B cells, CH12F3 B cells, and human B cells were stimulated with indicated stimuli for 60 h in the presence of nil, butyrate (500 μM), or propionate (2000 μM). Ex vivo B cells were isolated from mice injected with NP-LPS and on plain water or SCFA water for 21 days. Acetylated-histone H3 (H3K9ac), histone H3, and β-actin proteins as detected by immunoblotting. Data are one representative of three independent experiments. b Relative densities of the Ac-histone H3 bands normalized to histone H3 and β - actin levels. c , d B cells stimulated with LPS plus IL-4 in the presence of nil or butyrate for 60 h. Expression of the Aicda- and Prdm1- targeting miRNAs, and irrelevant miRNAs as analyzed by qRT-PCR ( c ). Relative abundance of H3K9ac/K14ac in miRNA host genes (HGs) as analyzed by ChIP-qPCR ( d ). e Schematic diagram of the luciferase reporter constructs-containing 3′UTRs of Aicda and Prdm1 mRNAs and their mutant (mut) counterparts. f Surface CD19 and IgA on CH12F3 B cells stimulated for 96 h in the presence of nil, butyrate (500 μM), propionate (2000 μM), or VPA (500 μM) as analyzed by flow cytometry. Data are one representative of 3 independent experiments yielding comparable results. g , miRNA expression in CH12F3 cells stimulated for 24 h in the presence of nil or butyrate, as analyzed by qRT-PCR. Data in c , d , and g are ratios to stimulated B cells cultured with nil (set as 1; means ± SEM of three independent experiments). h Luciferase activity in CH12F3 cells transfected with luciferase reporter vectors-containing wild-type or mutated Aicda or Prdm1 3′UTRs after 24 h treatment with nil or butyrate. Luciferase activity was measured 6 h after transfection, and normalized relative to luciferase activity in B cells cultured with nil. Data in h are ratios to transfected B cells cultured with nil (set as 100%; means ± SEM of three independent experiments). * p

    Article Snippet: After blocking and overnight incubation at 4 °C with anti-AID (ZA001, Invitrogen), anti-Blimp1 (6D3, eBioscience), anti-acetyl-histone H3 (H3K9ac/K14ac, 17–615, Millipore), anti-histone H3 (601901, BioLegend), or anti-β-Actin mAb (AC-15, Sigma-Aldrich), the membranes were incubated with HRP-conjugated secondary Abs.

    Techniques: Expressing, Ex Vivo, Isolation, Mouse Assay, Injection, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Luciferase, Construct, Mutagenesis, Flow Cytometry, Cytometry, Cell Culture, Activity Assay, Transfection

    KLF6 transcriptionally activates autophagy-related genes in a p53-dependent manner. Transfection with the KLF6 -expression vector pcIneo-KLF6 induced KLF6 mRNA ( A ) and protein ( B ) expression. KLF6-over-expression induces autophagy in HepG2 cells as assessed by LC3 Western blotting ( C ) and quantification of LC3-II-bands normalized to loading control GAPDH ( D ; fold change versus control shown as mean ± SEM of n = 3 independent cell culture experiments; full length Western blot images are given in Supplementary information.). Autophagic flux was assessed by LC3-II and p62 Western blotting in HepG2 and pcIneo-KLF6 transfected cells in absence (−) or presence ( + ) of 100 µM chloroquine for 24 h (( C ) shows representative Western blot images, ( D) shows fold change versus control, shown as mean ± SEM of n = 3 independent experiments). Activation of the ATG7 and BECLIN1 promoter was quantified by luciferase activity in a co-transfection experiment of pcIneo-KLF6 with specific promoter reporter luciferase plasmids in HepG2 ( E ) and in p53-deficient HepG2-303 cells ( F ). The interaction of KLF6 with the ATG7 and BECLIN1 promoter containing putative KLF6-binding sites was confirmed by chromosomal immunoprecipitation (ChIP) in HepG2 cells ( G ) using two different KLF6 antibodies, IgG as negative controls, Histone-H3 antibody was used as a positive control for ChIP; full length images of agarose gels are given in Supplementary information.

    Journal: Scientific Reports

    Article Title: Krüppel-like factor 6 is a transcriptional activator of autophagy in acute liver injury

    doi: 10.1038/s41598-017-08680-w

    Figure Lengend Snippet: KLF6 transcriptionally activates autophagy-related genes in a p53-dependent manner. Transfection with the KLF6 -expression vector pcIneo-KLF6 induced KLF6 mRNA ( A ) and protein ( B ) expression. KLF6-over-expression induces autophagy in HepG2 cells as assessed by LC3 Western blotting ( C ) and quantification of LC3-II-bands normalized to loading control GAPDH ( D ; fold change versus control shown as mean ± SEM of n = 3 independent cell culture experiments; full length Western blot images are given in Supplementary information.). Autophagic flux was assessed by LC3-II and p62 Western blotting in HepG2 and pcIneo-KLF6 transfected cells in absence (−) or presence ( + ) of 100 µM chloroquine for 24 h (( C ) shows representative Western blot images, ( D) shows fold change versus control, shown as mean ± SEM of n = 3 independent experiments). Activation of the ATG7 and BECLIN1 promoter was quantified by luciferase activity in a co-transfection experiment of pcIneo-KLF6 with specific promoter reporter luciferase plasmids in HepG2 ( E ) and in p53-deficient HepG2-303 cells ( F ). The interaction of KLF6 with the ATG7 and BECLIN1 promoter containing putative KLF6-binding sites was confirmed by chromosomal immunoprecipitation (ChIP) in HepG2 cells ( G ) using two different KLF6 antibodies, IgG as negative controls, Histone-H3 antibody was used as a positive control for ChIP; full length images of agarose gels are given in Supplementary information.

    Article Snippet: Samples were immunoprecipitated with 10 µg of anti-KLF6 antibody (polyclonal antibody KLF6 (R-173) or monoclonal antibody KLF6 (E-10) (Santa Cruz Biotechnologies, Dallas, TX, USA), anti-histone H3 antibody (Abcam, Cambridge, UK) or control IgG (Abcam) and protein-A/G agarose beads (Santa Cruz Biotechnologies).

    Techniques: Transfection, Expressing, Plasmid Preparation, Over Expression, Western Blot, Cell Culture, Activation Assay, Luciferase, Activity Assay, Cotransfection, Binding Assay, Immunoprecipitation, Chromatin Immunoprecipitation, Positive Control

    Histone acetylation and methylation in HDAC1 wt and HDAC1 dn mESCs induced into cardiomyocytes and treated with HDACi. The level of H3K9ac, H3K9me3, H4ac, H4K20ac, pan-acetylated lysines (K-ac), and α-actinin in ( A ) HDAC1 wt mESCs and ( B ) HDAC1 dn mESCs. In three biological replicates, Western blots were performed on one gel. For the data presented in panel A or B, the gel was separated by Photoshop to show samples that were compared in one relevant subset. Data on histone levels were normalized to the level of histone H3 and non-histone proteins were normalized and quantified to the level of GAPDH ( C ). In wt and HDAC1 dn non-treated cells and in TSA-, SAHA-, or VPA-treated mESCs, panel ( Ca ) shows the levels of H4ac, ( Cb ) shows H4K20ac, and ( Cc ) shows the levels of α-actinin. The total protein levels were measured using a µQuant spectrophotometer for each sample, and an identical protein amount was loaded on the gels. In panel ( A , B ), the levels of histone markers are also shown for embryonic hearts (e15). Quantification of the protein levels in panel ( C ) was performed using ImageJ software (NIH, freeware). Statistical analyses were performed using Student’s t -test; asterisks (*) in panel ( Ca – c ) show statistically significant differences at p ≤ 0.05. Note that the y -axis-scale in panel ( Ca ) is different (red frames) for the wt and HDAC1 dn cells for technical purposes. In panel ( Ca ), the level of H4ac is significantly less in the wt mESCs when compared with the HDAC1 dn cells ( Cb ).

    Journal: International Journal of Molecular Sciences

    Article Title: Deacetylation of Histone H4 Accompanying Cardiomyogenesis is Weakened in HDAC1-Depleted ES Cells

    doi: 10.3390/ijms19082425

    Figure Lengend Snippet: Histone acetylation and methylation in HDAC1 wt and HDAC1 dn mESCs induced into cardiomyocytes and treated with HDACi. The level of H3K9ac, H3K9me3, H4ac, H4K20ac, pan-acetylated lysines (K-ac), and α-actinin in ( A ) HDAC1 wt mESCs and ( B ) HDAC1 dn mESCs. In three biological replicates, Western blots were performed on one gel. For the data presented in panel A or B, the gel was separated by Photoshop to show samples that were compared in one relevant subset. Data on histone levels were normalized to the level of histone H3 and non-histone proteins were normalized and quantified to the level of GAPDH ( C ). In wt and HDAC1 dn non-treated cells and in TSA-, SAHA-, or VPA-treated mESCs, panel ( Ca ) shows the levels of H4ac, ( Cb ) shows H4K20ac, and ( Cc ) shows the levels of α-actinin. The total protein levels were measured using a µQuant spectrophotometer for each sample, and an identical protein amount was loaded on the gels. In panel ( A , B ), the levels of histone markers are also shown for embryonic hearts (e15). Quantification of the protein levels in panel ( C ) was performed using ImageJ software (NIH, freeware). Statistical analyses were performed using Student’s t -test; asterisks (*) in panel ( Ca – c ) show statistically significant differences at p ≤ 0.05. Note that the y -axis-scale in panel ( Ca ) is different (red frames) for the wt and HDAC1 dn cells for technical purposes. In panel ( Ca ), the level of H4ac is significantly less in the wt mESCs when compared with the HDAC1 dn cells ( Cb ).

    Article Snippet: For this research, we used antibodies against the following proteins: α-actinin (#A-7811, Sigma-Aldrich), H3K9ac (#06-942, Merc Millipore), H3K9me3 (#ab8898, Abcam), H4ac (#382160, Merc Millipore), H4K20ac (#720087, Thermo Fisher Scientific), acetylated Lysin (K) (#ab21623, Abcam), histone H3 (#ab1791, Abcam), and GAPDH (#sc-365062, Santa Cruz Biotechnology, Dallas, TX, USA).

    Techniques: Methylation, Western Blot, Spectrophotometry, Software

    Histone post-translational modifications studied in mouse embryonic hearts (e15) treated with HDACi. ( A ) Western blots showed changes in H3K9ac, H3K9me3, H4ac, H4K20ac, pan-acetylated lysines (K-ac), and α-actinin in e15 embryonic hearts treated with HDACi (TSA, SAHA, and VPA). Data on histone levels were normalized to the level of histone H3 and non-histone proteins were normalized to the level of GAPDH. An identical protein amount for each experimental event was loaded on the gel. ( B ) Data from panel ( A ) were normalized to the relevant reference protein GAPDH, and the density of Western blot fragments was statistically analyzed using Student’s t -test; asterisks show statistically significant differences at p ≤ 0.05. GAPDH was used for data normalization, and α-actinin was used as a marker of cardiomyocytes. ( C ) The distribution pattern of H3K9ac (red) in the e15 mouse embryonic hearts is shown. DAPI (blue) was used as a counterstain of the cell nuclei. Arrows show the accumulation of H3K9ac in ventricular portions.

    Journal: International Journal of Molecular Sciences

    Article Title: Deacetylation of Histone H4 Accompanying Cardiomyogenesis is Weakened in HDAC1-Depleted ES Cells

    doi: 10.3390/ijms19082425

    Figure Lengend Snippet: Histone post-translational modifications studied in mouse embryonic hearts (e15) treated with HDACi. ( A ) Western blots showed changes in H3K9ac, H3K9me3, H4ac, H4K20ac, pan-acetylated lysines (K-ac), and α-actinin in e15 embryonic hearts treated with HDACi (TSA, SAHA, and VPA). Data on histone levels were normalized to the level of histone H3 and non-histone proteins were normalized to the level of GAPDH. An identical protein amount for each experimental event was loaded on the gel. ( B ) Data from panel ( A ) were normalized to the relevant reference protein GAPDH, and the density of Western blot fragments was statistically analyzed using Student’s t -test; asterisks show statistically significant differences at p ≤ 0.05. GAPDH was used for data normalization, and α-actinin was used as a marker of cardiomyocytes. ( C ) The distribution pattern of H3K9ac (red) in the e15 mouse embryonic hearts is shown. DAPI (blue) was used as a counterstain of the cell nuclei. Arrows show the accumulation of H3K9ac in ventricular portions.

    Article Snippet: For this research, we used antibodies against the following proteins: α-actinin (#A-7811, Sigma-Aldrich), H3K9ac (#06-942, Merc Millipore), H3K9me3 (#ab8898, Abcam), H4ac (#382160, Merc Millipore), H4K20ac (#720087, Thermo Fisher Scientific), acetylated Lysin (K) (#ab21623, Abcam), histone H3 (#ab1791, Abcam), and GAPDH (#sc-365062, Santa Cruz Biotechnology, Dallas, TX, USA).

    Techniques: Western Blot, Marker

    Directional migration of stratified epithelium is not a result of localized cell proliferation. (A-H) Cell proliferation as detected by phospho-histone 3 (pH3) immunofluorescence (green) during the timecourse of mammary epithelial migration toward beads soaked in BSA (A-D) or FGF10 (E-H). Cell proliferation was quantified in one of the three evenly divided regions of an organoid – the front (f), middle (m) or rear (r) – depending on the distance between the region and the bead (asterisk). Scale bars: 100 µm. (I,J) Quantification of cell proliferation in different regions of mammary organoids during epithelial migration. Only signals that overlap with nuclei were counted as proliferating cells, and background noise was discounted. No statistically significant differences were apparent among the different regions at the times indicated (unpaired two-tailed Student's t -test). Values shown are the mean±s.d. from at least three independent experiments.

    Journal: Development (Cambridge, England)

    Article Title: FGF ligands of the postnatal mammary stroma regulate distinct aspects of epithelial morphogenesis

    doi: 10.1242/dev.106732

    Figure Lengend Snippet: Directional migration of stratified epithelium is not a result of localized cell proliferation. (A-H) Cell proliferation as detected by phospho-histone 3 (pH3) immunofluorescence (green) during the timecourse of mammary epithelial migration toward beads soaked in BSA (A-D) or FGF10 (E-H). Cell proliferation was quantified in one of the three evenly divided regions of an organoid – the front (f), middle (m) or rear (r) – depending on the distance between the region and the bead (asterisk). Scale bars: 100 µm. (I,J) Quantification of cell proliferation in different regions of mammary organoids during epithelial migration. Only signals that overlap with nuclei were counted as proliferating cells, and background noise was discounted. No statistically significant differences were apparent among the different regions at the times indicated (unpaired two-tailed Student's t -test). Values shown are the mean±s.d. from at least three independent experiments.

    Article Snippet: Primary antibodies were against smooth muscle actin (Sigma, 1A4, C6198), K8 (DSHB, Troma-1), K14 (Covance, PRB-155P), phospho-histone H3 (Upstate, 06-570), β1-integrin (Millipore, MAB1997), E-cadherin (BD Biosciences, 610181) and ZO-1 (Millipore, MABT11).

    Techniques: Migration, Immunofluorescence, Two Tailed Test

    Model of How Cellular Heterogeneities at the 4-Cell Stage Regulate Cell Fate (A–C) In 4-cell embryos, CARM1, which methylates histone H3R26, is differentially expressed ( Torres-Padilla et al., 2007 ). We hypothesize that higher levels of histone H3R26me facilitate the binding to DNA of pluripotency regulators such as Oct4 and Sox2, resulting in increased transcription of pluripotency-related target genes, such as Sox21 , Nanog , and Esrrb , biasing these cells to contribute to the pluripotent lineage. Conversely, in cells with lower levels of histone H3R26me, pluripotency regulators are only able to bind to DNA for shorter periods of time, and therefore their target genes are not as highly expressed. These cells have lower levels of pluripotency and are thus more likely to initiate expression of differentiation genes, such as Cdx2 , and initiate development into the extra-embryonic TE.

    Journal: Cell

    Article Title: Heterogeneity in Oct4 and Sox2 Targets Biases Cell Fate in 4-Cell Mouse Embryos

    doi: 10.1016/j.cell.2016.01.047

    Figure Lengend Snippet: Model of How Cellular Heterogeneities at the 4-Cell Stage Regulate Cell Fate (A–C) In 4-cell embryos, CARM1, which methylates histone H3R26, is differentially expressed ( Torres-Padilla et al., 2007 ). We hypothesize that higher levels of histone H3R26me facilitate the binding to DNA of pluripotency regulators such as Oct4 and Sox2, resulting in increased transcription of pluripotency-related target genes, such as Sox21 , Nanog , and Esrrb , biasing these cells to contribute to the pluripotent lineage. Conversely, in cells with lower levels of histone H3R26me, pluripotency regulators are only able to bind to DNA for shorter periods of time, and therefore their target genes are not as highly expressed. These cells have lower levels of pluripotency and are thus more likely to initiate expression of differentiation genes, such as Cdx2 , and initiate development into the extra-embryonic TE.

    Article Snippet: Immunofluorescence Primary antibodies used were goat anti-Sox17 (R & D Systems), rabbit anti-Nanog (Abcam), mouse anti-Cdx2 (Biogenex), rabbit anti-Histone H3 (Abcam), rabbit anti-Sox2 (Cell Signaling Technology), rabbit anti-Sox21 (R & D Systems), and rabbit anti-Histone H3 (symmetric di methyl R26; Abcam).

    Techniques: Binding Assay, Expressing

    Hyperactivation of NF-κB in Shn-2 −/− CD4 T cells. (A and B) Splenic CD4 T cells were incubated with medium alone overnight and then stimulated with immobilized anti–TCR-αβ mAb in the presence or absence of agonistic anti-CD28 antibody for 3 h. Nuclear extracts of the cultured cells were prepared and subjected to EMSAs with NF-κB probes. The supershift assays were performed with antibodies specific for NF-κB p50 and p65 subunit detection. Three independent experiments were performed with similar results. (C) Splenic CD4 T cells were incubated with medium alone overnight and then stimulated with immobilized anti–TCR-β mAb for 3 h. Subsequently, both cytosol and nuclear extracts were prepared and these were subjected to immunoblotting using anti-p50, anti-p65, anti–tubulin-α, and anti–histone H1 antibodies. Arbitrary densitometric units are shown under each band. (D) RNAs were prepared from fresh splenic CD4 T cells, resting CD4 T cells that were incubated with medium alone overnight, and anti-TCR–stimulated CD4 T cells, and then quantitative PCR assay was performed. The expression levels of p50 and p65 were normalized with 18S expression. Two independent experiments were performed with similar results.

    Journal: The Journal of Experimental Medicine

    Article Title: Regulation of T helper type 2 cell differentiation by murine Schnurri-2

    doi: 10.1084/jem.20040733

    Figure Lengend Snippet: Hyperactivation of NF-κB in Shn-2 −/− CD4 T cells. (A and B) Splenic CD4 T cells were incubated with medium alone overnight and then stimulated with immobilized anti–TCR-αβ mAb in the presence or absence of agonistic anti-CD28 antibody for 3 h. Nuclear extracts of the cultured cells were prepared and subjected to EMSAs with NF-κB probes. The supershift assays were performed with antibodies specific for NF-κB p50 and p65 subunit detection. Three independent experiments were performed with similar results. (C) Splenic CD4 T cells were incubated with medium alone overnight and then stimulated with immobilized anti–TCR-β mAb for 3 h. Subsequently, both cytosol and nuclear extracts were prepared and these were subjected to immunoblotting using anti-p50, anti-p65, anti–tubulin-α, and anti–histone H1 antibodies. Arbitrary densitometric units are shown under each band. (D) RNAs were prepared from fresh splenic CD4 T cells, resting CD4 T cells that were incubated with medium alone overnight, and anti-TCR–stimulated CD4 T cells, and then quantitative PCR assay was performed. The expression levels of p50 and p65 were normalized with 18S expression. Two independent experiments were performed with similar results.

    Article Snippet: For NF-κB detection, anti-p65 (F-6), anti-p50 (E-10), and anti–histone H1 (AE-4, all from Santa Cruz Biotechnology, Inc.) antibodies were used.

    Techniques: Incubation, Cell Culture, Real-time Polymerase Chain Reaction, Expressing

    Epigenome annotation of variants from genome sequencing identifies a shared variant in a putative enhancer element Variant identified by whole genome sequencing, plus an additional five variants in patients with pancreatic agenesis map to a 25 Kb region downstream of PTF1A , which contains a single candidate pancreatic progenitor-specific enhancer within a highly conserved 400bp element. The top panel depicts ChIP-seq density plots for the enhancer mark H3K4me1, the second and third show occupancy for FOXA2 and PDX1. A broad panel of embryonic and adult human tissues do not show active chromatin marks in this region ( Supplementary Figure 5 ). Vertebrate Conservation and mammalian conservation tracks (as measured by the GERP Score) tracks illustrate the high conservation of this element. The red line depicts the approximate location of a 7.6 kb deletion in this region, and red arrows indicate point mutations, using the final 3 digits from the hg19 coordinates as labels (referring to positions 23508305A > G, 23508363A > G, 23508365A > G, 23508437A > G and 23508446A > C on chromosome 10 respectively).

    Journal: Nature genetics

    Article Title: Recessive mutations in a distal PTF1A enhancer cause isolated pancreatic agenesis

    doi: 10.1038/ng.2826

    Figure Lengend Snippet: Epigenome annotation of variants from genome sequencing identifies a shared variant in a putative enhancer element Variant identified by whole genome sequencing, plus an additional five variants in patients with pancreatic agenesis map to a 25 Kb region downstream of PTF1A , which contains a single candidate pancreatic progenitor-specific enhancer within a highly conserved 400bp element. The top panel depicts ChIP-seq density plots for the enhancer mark H3K4me1, the second and third show occupancy for FOXA2 and PDX1. A broad panel of embryonic and adult human tissues do not show active chromatin marks in this region ( Supplementary Figure 5 ). Vertebrate Conservation and mammalian conservation tracks (as measured by the GERP Score) tracks illustrate the high conservation of this element. The red line depicts the approximate location of a 7.6 kb deletion in this region, and red arrows indicate point mutations, using the final 3 digits from the hg19 coordinates as labels (referring to positions 23508305A > G, 23508363A > G, 23508365A > G, 23508437A > G and 23508446A > C on chromosome 10 respectively).

    Article Snippet: ChIP-Seq maps of pancreatic progenitor regulatory elements Chromatin immunoprecipitations (ChIPs) for H3K4me1 (Abcam ab-8895; n=2), FOXA2 (Santa Cruz Biotechnology sc-6554, n=2), GATA6 (Santa Cruz Biotechnology sc-9055X; n=1), HNF1β (Santa Cruz Biotechnology sc-22840-X, n=1), ONECUT1 (Santa Cruz Biotechnology sc-13050, n=1) and PDX1 (BCBC AB2027; n=1) were performed essentially as described , using ~10 million artificially derived pancreatic progenitors for each experiment.

    Techniques: Sequencing, Variant Assay, Chromatin Immunoprecipitation