histone h3  (Active Motif)

 
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  • 99
    Name:
    Histone H3K4me3 antibody pAb
    Description:

    Catalog Number:
    39159
    Price:
    455
    Host:
    Rabbit
    Category:
    Primary Antibodies
    Antibody Type:
    Polyclonal
    Isotype:
    Serum
    Reactivity:
    Budding Yeast Human Mouse Wide Range Predicted
    Format:
    100 µl
    Buy from Supplier


    Structured Review

    Active Motif histone h3
    The Paf1 complex and methylation of H3 K4 positively regulate ECM3 expression. (A) Representative northern blot analysis of ECM3 transcript levels in a wild-type strain (FY4) or strains lacking one of the five subunits of the Paf1 complex, either paf1 ∆ (YJ807), ctr9 ∆ (KY2170), rtf1 ∆ (YJ788), cdc73 ∆ (KY2171), or leo1 ∆ (KY1805). (B) Representative northern blot analysis of ECM3 transcript levels in a wild-type strain (FY4) and strains where the genes encoding <t>histone</t> modifiers that work in concert with the Paf1 complex have been deleted ( rad6 ∆, KY1712; bre1 ∆, KY1713; set1 ∆, KY2720; set2 ∆, KY2723; and dot1 ∆, KY2725). (C) Representative northern blot analysis of ECM3 transcript levels in a wild-type control strain, lacking one copy of the genes for H3 and H4 (JDY86), and derivatives of JDY86 in which the only copy of the H3-H4 genes encodes the indicated amino acid substitution in H3. (D) Representative northern blot analysis of ECM3 transcript levels in strains lacking a subunit of the Set3 HDAC complex ( set3 ∆, KY2782), the SAGA HAT complex ( gcn5 ∆, KY1743), or the NuA3 HAT complex ( sas3 ∆, ECY394) compared to wild-type levels (YJ1125). Quantitation below shows the average ECM3 mRNA levels relative to WT (set to 1) from at least three biological replicates. Error bars represent the SEM. SCR1 serves as a loading control. mRNA, messenger RNA; WT, wild-type.

    https://www.bioz.com/result/histone h3/product/Active Motif
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    histone h3 - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Evidence for Regulation of ECM3 Expression by Methylation of Histone H3 Lysine 4 and Intergenic Transcription in Saccharomyces cerevisiae"

    Article Title: Evidence for Regulation of ECM3 Expression by Methylation of Histone H3 Lysine 4 and Intergenic Transcription in Saccharomyces cerevisiae

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.116.033118

    The Paf1 complex and methylation of H3 K4 positively regulate ECM3 expression. (A) Representative northern blot analysis of ECM3 transcript levels in a wild-type strain (FY4) or strains lacking one of the five subunits of the Paf1 complex, either paf1 ∆ (YJ807), ctr9 ∆ (KY2170), rtf1 ∆ (YJ788), cdc73 ∆ (KY2171), or leo1 ∆ (KY1805). (B) Representative northern blot analysis of ECM3 transcript levels in a wild-type strain (FY4) and strains where the genes encoding histone modifiers that work in concert with the Paf1 complex have been deleted ( rad6 ∆, KY1712; bre1 ∆, KY1713; set1 ∆, KY2720; set2 ∆, KY2723; and dot1 ∆, KY2725). (C) Representative northern blot analysis of ECM3 transcript levels in a wild-type control strain, lacking one copy of the genes for H3 and H4 (JDY86), and derivatives of JDY86 in which the only copy of the H3-H4 genes encodes the indicated amino acid substitution in H3. (D) Representative northern blot analysis of ECM3 transcript levels in strains lacking a subunit of the Set3 HDAC complex ( set3 ∆, KY2782), the SAGA HAT complex ( gcn5 ∆, KY1743), or the NuA3 HAT complex ( sas3 ∆, ECY394) compared to wild-type levels (YJ1125). Quantitation below shows the average ECM3 mRNA levels relative to WT (set to 1) from at least three biological replicates. Error bars represent the SEM. SCR1 serves as a loading control. mRNA, messenger RNA; WT, wild-type.
    Figure Legend Snippet: The Paf1 complex and methylation of H3 K4 positively regulate ECM3 expression. (A) Representative northern blot analysis of ECM3 transcript levels in a wild-type strain (FY4) or strains lacking one of the five subunits of the Paf1 complex, either paf1 ∆ (YJ807), ctr9 ∆ (KY2170), rtf1 ∆ (YJ788), cdc73 ∆ (KY2171), or leo1 ∆ (KY1805). (B) Representative northern blot analysis of ECM3 transcript levels in a wild-type strain (FY4) and strains where the genes encoding histone modifiers that work in concert with the Paf1 complex have been deleted ( rad6 ∆, KY1712; bre1 ∆, KY1713; set1 ∆, KY2720; set2 ∆, KY2723; and dot1 ∆, KY2725). (C) Representative northern blot analysis of ECM3 transcript levels in a wild-type control strain, lacking one copy of the genes for H3 and H4 (JDY86), and derivatives of JDY86 in which the only copy of the H3-H4 genes encodes the indicated amino acid substitution in H3. (D) Representative northern blot analysis of ECM3 transcript levels in strains lacking a subunit of the Set3 HDAC complex ( set3 ∆, KY2782), the SAGA HAT complex ( gcn5 ∆, KY1743), or the NuA3 HAT complex ( sas3 ∆, ECY394) compared to wild-type levels (YJ1125). Quantitation below shows the average ECM3 mRNA levels relative to WT (set to 1) from at least three biological replicates. Error bars represent the SEM. SCR1 serves as a loading control. mRNA, messenger RNA; WT, wild-type.

    Techniques Used: Methylation, Expressing, Northern Blot, HAT Assay, Quantitation Assay

    2) Product Images from "Sequence-Specific Promoter Elements Regulate Temporal-Specific Changes in Chromatin Required for Testis-Specific Activation of the Pgk2 Gene"

    Article Title: Sequence-Specific Promoter Elements Regulate Temporal-Specific Changes in Chromatin Required for Testis-Specific Activation of the Pgk2 Gene

    Journal: Reproduction (Cambridge, England)

    doi: 10.1530/REP-13-0311

    Chromatin immunoprecipitation (ChIP) analysis of histone acetylation in Pgk2 transgenes
    Figure Legend Snippet: Chromatin immunoprecipitation (ChIP) analysis of histone acetylation in Pgk2 transgenes

    Techniques Used: Chromatin Immunoprecipitation

    3) Product Images from "Sequence-Specific Promoter Elements Regulate Temporal-Specific Changes in Chromatin Required for Testis-Specific Activation of the Pgk2 Gene"

    Article Title: Sequence-Specific Promoter Elements Regulate Temporal-Specific Changes in Chromatin Required for Testis-Specific Activation of the Pgk2 Gene

    Journal: Reproduction (Cambridge, England)

    doi: 10.1530/REP-13-0311

    Chromatin immunoprecipitation (ChIP) analysis of histone acetylation in Pgk2 transgenes
    Figure Legend Snippet: Chromatin immunoprecipitation (ChIP) analysis of histone acetylation in Pgk2 transgenes

    Techniques Used: Chromatin Immunoprecipitation

    4) Product Images from "Microbiota derived short chain fatty acids promote histone crotonylation in the colon through histone deacetylases"

    Article Title: Microbiota derived short chain fatty acids promote histone crotonylation in the colon through histone deacetylases

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02651-5

    Histone crotonylation is cell cycle regulated by class I HDACs. Cell cycle block and release experiment on HCT116 cells using CDK4/6 inhibitor abemaciclib with and without MS275. Lanes 1 and 9: asynchronous cells, lane 2: G1 arrested cells, lanes 3–8: increase in histone crotonylation (Kcr), H3K18cr and H3K18ac upon release into S phase. Lanes 10–16: histone crotonylation, H3K18cr and H3K18ac are upregulated during a G1 arrest and S phase when class I HDACs are inhibited with MS275. For the experiments in lanes 9–16, cells were blocked in G1 using 15 nM abemaciclib in the presence of 5 μM MS275 and released into S phase in the presence of 1 μM MS275. Cell cycle profiles are shown at the top, western blots in the middle, and quantifications of those, as calculated relative to H3 and normalized to the DMSO (vehicle) sample, at the bottom. Representative of two experiments is shown
    Figure Legend Snippet: Histone crotonylation is cell cycle regulated by class I HDACs. Cell cycle block and release experiment on HCT116 cells using CDK4/6 inhibitor abemaciclib with and without MS275. Lanes 1 and 9: asynchronous cells, lane 2: G1 arrested cells, lanes 3–8: increase in histone crotonylation (Kcr), H3K18cr and H3K18ac upon release into S phase. Lanes 10–16: histone crotonylation, H3K18cr and H3K18ac are upregulated during a G1 arrest and S phase when class I HDACs are inhibited with MS275. For the experiments in lanes 9–16, cells were blocked in G1 using 15 nM abemaciclib in the presence of 5 μM MS275 and released into S phase in the presence of 1 μM MS275. Cell cycle profiles are shown at the top, western blots in the middle, and quantifications of those, as calculated relative to H3 and normalized to the DMSO (vehicle) sample, at the bottom. Representative of two experiments is shown

    Techniques Used: Blocking Assay, Western Blot

    Class I HDACs are histone decrotonylases. a Histone H3 decrotonylation and deacetylation in vitro by HDAC1, HDAC2, or HDAC3/Ncor1 complex; 5.65 μM histones were crotonylated or acetylated in vitro and then subjected to removal of the modification by the indicated HDACs. HDAC1 was 0.25, 0.12, 0.06, and 0.03 μM. HDAC2 was 0.18, 0.09, 0.05, and 0.02 μM. HDAC3/Ncor1 complex was 0.45, 0.23, 0.11, and 0.06 μM. b Comparative kinetics of HDAC1 decrotonylation and deacetylation; 5.65 µM histones were crotonylated or acetylated and then subjected to removal of the modification by 0.03 µM HDAC1 for different lengths of time. Samples were analyzed by dot blotting and initial rates of reaction were determined by plotting substrate removal over time. Kinetic parameters V max , K m , and K cat , error bars are SEM, n = 3. c Effect of HDAC inhibitors TSA, crotonate, and butyrate on deacetylation and decrotonylation by HDAC1 in vitro. Representative blots of two repeat experiments are shown. d Histone crotonylation by HDAC1 using crotonate in vitro. Incubation of crotonate, acetate, or butyrate with or without HDAC1 followed by western blotting analysis with anti-H3K18ac/bt/cr. Western blot of HDAC1 and crotonate assay is representative of two western blots
    Figure Legend Snippet: Class I HDACs are histone decrotonylases. a Histone H3 decrotonylation and deacetylation in vitro by HDAC1, HDAC2, or HDAC3/Ncor1 complex; 5.65 μM histones were crotonylated or acetylated in vitro and then subjected to removal of the modification by the indicated HDACs. HDAC1 was 0.25, 0.12, 0.06, and 0.03 μM. HDAC2 was 0.18, 0.09, 0.05, and 0.02 μM. HDAC3/Ncor1 complex was 0.45, 0.23, 0.11, and 0.06 μM. b Comparative kinetics of HDAC1 decrotonylation and deacetylation; 5.65 µM histones were crotonylated or acetylated and then subjected to removal of the modification by 0.03 µM HDAC1 for different lengths of time. Samples were analyzed by dot blotting and initial rates of reaction were determined by plotting substrate removal over time. Kinetic parameters V max , K m , and K cat , error bars are SEM, n = 3. c Effect of HDAC inhibitors TSA, crotonate, and butyrate on deacetylation and decrotonylation by HDAC1 in vitro. Representative blots of two repeat experiments are shown. d Histone crotonylation by HDAC1 using crotonate in vitro. Incubation of crotonate, acetate, or butyrate with or without HDAC1 followed by western blotting analysis with anti-H3K18ac/bt/cr. Western blot of HDAC1 and crotonate assay is representative of two western blots

    Techniques Used: In Vitro, Modification, Incubation, Western Blot

    Histone crotonylation is found in the intestine. a Western blot analysis of whole cell extracts from several mouse tissues using indicated antibodies shows that histone crotonylation is particularly abundant in the brain and colon; the analysis of tissues from two mice is shown. b Relative abundance of H3K18cr in the intestinal epithelium cell fractions, n = 3, error bars are standard deviation. c , d Immunofluorescence microscopy with anti-pan crotonyl antibody (green, left panels) and DAPI counterstaining (cyan, right panels) of a mouse colon ( c ) and small intestinal ( d ) tissue sections, scale bars 40 μm
    Figure Legend Snippet: Histone crotonylation is found in the intestine. a Western blot analysis of whole cell extracts from several mouse tissues using indicated antibodies shows that histone crotonylation is particularly abundant in the brain and colon; the analysis of tissues from two mice is shown. b Relative abundance of H3K18cr in the intestinal epithelium cell fractions, n = 3, error bars are standard deviation. c , d Immunofluorescence microscopy with anti-pan crotonyl antibody (green, left panels) and DAPI counterstaining (cyan, right panels) of a mouse colon ( c ) and small intestinal ( d ) tissue sections, scale bars 40 μm

    Techniques Used: Western Blot, Mouse Assay, Standard Deviation, Immunofluorescence, Microscopy

    H3K18cr ChIP-seq from colon epithelium analysis. ChIP-sequencing on isolated colon epithelial cells from two mice. a Browser view of a segment from chromosome 1 showing a representative profile of the distribution of H3K18cr peaks with relationship to genes. Relative enrichment of the combined replicate sets of ChIP and input in linear scale are shown, probes are 500 bp, 250 bp overlap. b Average distribution of ChIP-seq normalized read counts with relation to genes shows that histone H3K18cr is highly enriched over transcription start sites (TSS) in colon epithelial cells. c Link between H3K4me3 and H3K18cr, using MACS peak quantification and an aligned probe plot. Probes were ranked according to H3K4me3 signal strength and span 5 kbp around MACS peaks. d Average distribution of reads in linear scale with relation to genes' TSS, showing enrichment over these sites. e Relationship between H3K18cr enrichment over TSS and mRNA levels of the corresponding genes from cells isolated from the mouse colon epithelium were quantified using mRNA-seq (three biological replicates) and the normalized read counts over genes were divided into percentile bins as indicated, from lowly expressed genes (0–25 percentile) to very highly expressed genes (99–100 percentile). H3K18cr over TSS ±0.5 kbp of genes belonging to the expression bins was quantified and is shown in box-whisker plots. f KEGG pathway terms and their adjusted p -values of significance of genes with the highest 10 percentile H3K18cr associated (MACS) peaks. Only results with –log 10 ( p ) > 6 are shown, see Supplementary Fig. 6 for all results. Cancer pathways are highlighted (red terms)
    Figure Legend Snippet: H3K18cr ChIP-seq from colon epithelium analysis. ChIP-sequencing on isolated colon epithelial cells from two mice. a Browser view of a segment from chromosome 1 showing a representative profile of the distribution of H3K18cr peaks with relationship to genes. Relative enrichment of the combined replicate sets of ChIP and input in linear scale are shown, probes are 500 bp, 250 bp overlap. b Average distribution of ChIP-seq normalized read counts with relation to genes shows that histone H3K18cr is highly enriched over transcription start sites (TSS) in colon epithelial cells. c Link between H3K4me3 and H3K18cr, using MACS peak quantification and an aligned probe plot. Probes were ranked according to H3K4me3 signal strength and span 5 kbp around MACS peaks. d Average distribution of reads in linear scale with relation to genes' TSS, showing enrichment over these sites. e Relationship between H3K18cr enrichment over TSS and mRNA levels of the corresponding genes from cells isolated from the mouse colon epithelium were quantified using mRNA-seq (three biological replicates) and the normalized read counts over genes were divided into percentile bins as indicated, from lowly expressed genes (0–25 percentile) to very highly expressed genes (99–100 percentile). H3K18cr over TSS ±0.5 kbp of genes belonging to the expression bins was quantified and is shown in box-whisker plots. f KEGG pathway terms and their adjusted p -values of significance of genes with the highest 10 percentile H3K18cr associated (MACS) peaks. Only results with –log 10 ( p ) > 6 are shown, see Supplementary Fig. 6 for all results. Cancer pathways are highlighted (red terms)

    Techniques Used: Chromatin Immunoprecipitation, Sequencing, Isolation, Mouse Assay, Magnetic Cell Separation, Expressing, Whisker Assay

    Butyrate and class I HDAC inhibition promote histone crotonylation. a Western blot analysis with indicated antibodies of whole cell extracts of small intestinal organoids treated for 48 h with indicated amounts of SCFAs. Representative western blot of two repeat experiments. b HCT116 cells were treated with MS275 or DMSO (vehicle) for 18 h, whole cell extracts collected, and analyzed by western blot using indicated antibodies; anti-Kcr: anti-crotonyl-lysine antibody, NT: not treated. c Increase in histone H3K18cr over promoters of indicated genes and repetitive, heterochromatic sites (alpha-satellite sequences, NBL2) upon MS275 treatment of HCT116 cells for 18 h. Summary of ChIP-qPCR data of three repeat experiments, error bars are SEM
    Figure Legend Snippet: Butyrate and class I HDAC inhibition promote histone crotonylation. a Western blot analysis with indicated antibodies of whole cell extracts of small intestinal organoids treated for 48 h with indicated amounts of SCFAs. Representative western blot of two repeat experiments. b HCT116 cells were treated with MS275 or DMSO (vehicle) for 18 h, whole cell extracts collected, and analyzed by western blot using indicated antibodies; anti-Kcr: anti-crotonyl-lysine antibody, NT: not treated. c Increase in histone H3K18cr over promoters of indicated genes and repetitive, heterochromatic sites (alpha-satellite sequences, NBL2) upon MS275 treatment of HCT116 cells for 18 h. Summary of ChIP-qPCR data of three repeat experiments, error bars are SEM

    Techniques Used: Inhibition, Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Microbiota depletion affects colonic histone crotonylation and HDAC2. Antibiotic treatment led to a decrease in luminal and serum SCFA levels in mice ( n ≥ 3, from experiment 2). a Acetate, propionate, and butyrate concentrations were measured in the colon lumen and serum by gas chromatography. Unpaired t -tests were conducted, * p- value
    Figure Legend Snippet: Microbiota depletion affects colonic histone crotonylation and HDAC2. Antibiotic treatment led to a decrease in luminal and serum SCFA levels in mice ( n ≥ 3, from experiment 2). a Acetate, propionate, and butyrate concentrations were measured in the colon lumen and serum by gas chromatography. Unpaired t -tests were conducted, * p- value

    Techniques Used: Mouse Assay, Gas Chromatography

    5) Product Images from "The JmjN Domain of Jhd2 Is Important for Its Protein Stability, and the Plant Homeodomain (PHD) Finger Mediates Its Chromatin Association Independent of H3K4 Methylation *"

    Article Title: The JmjN Domain of Jhd2 Is Important for Its Protein Stability, and the Plant Homeodomain (PHD) Finger Mediates Its Chromatin Association Independent of H3K4 Methylation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.117333

    Jhd2, an H3K4-specific demethylase, functions during both active transcription and repression. A , shown are the results from Western blot analysis of H3K4 methylation levels in crude nuclear extracts obtained from yeast strains with overexpressed JmjC domain-containing proteins. B , changes in H3K4me3 levels at the PMA1 locus were analyzed by ChIP assay. The graphs depict the data obtained from the WT control and a strain lacking Jhd2 ( jhd2 Δ) ( upper panel ) and from yeast strains overexpressing JHD2 ( JHD2 OE ) from a high copy plasmid or containing the plasmid vector alone ( lower panel ). The H3K4me3 ChIP signals were first normalized to total histone H3 ChIP signals (H3K4me3/H3), and then the H3K4me3/H3 values at the promoter ( P ), 5′-ORF, or 3′-ORF regions in the controls and test samples were normalized to the H3K4me3/H3 value obtained for the promoter region in the WT ( upper panel ) or vector control ( lower panel ), which was set as 1. Error bars denote S.E. from two independent experiments. C , the levels of Jhd2-LexA at PMA1 were analyzed by ChIP using anti-LexA antibody. LexA immunoprecipitation/input values obtained from the no-tag control (background) were subtracted from those obtained from yeast cells with overexpressed JHD2 -LexA, and the resulting differences were defined as Jhd2-LexA occupancy. The Jhd2-LexA occupancies at the 5′- and 3′-ORF regions were normalized to the promoter region, which was arbitrarily set as 1. Error bars denote S.E. from two independent experiments. D , temporal changes in H3K4me3 following the re-repression of INO1 were measured by ChIP assay. WT and jhd2 Δ strains were grown in inositol/choline medium for 4 h to repress INO1 expression ( R ) and then grown in inositol-free medium for 2 h to induce INO1 expression ( In ), and INO1 was repressed again by growing cells in inositol/choline medium for the indicated time periods ( Re-repression ). Cells at different time points were subjected to ChIP analysis using anti-H3K4me3 and anti-H3 antibodies. The levels of H3K4me3/H3 at the 5′-ORF of INO1 in the WT or jhd2 Δ mutant at different time points are shown as -fold changes relative to the H3K4me3/H3 value obtained at the repressed state for the WT, which was set as 1. Error bars denote S.E. from two independent experiments. E , temporal changes in Jhd2–9Myc occupancy following the re-repression of INO1 were measured by ChIP assay performed essentially as described for D . Chromatin-bound Jhd2–9Myc was immunoprecipitated using anti-Myc antibody. Myc immunoprecipitation/input values obtained from the no-tag control (background) were subtracted from those obtained for yeast expressing JHD2 -9Myc, and the resulting difference was defined as Jhd2–9Myc occupancy. The Jhd2–9Myc occupancies at the 5′-ORF of INO1 at different time points are shown as fold changes relative to the Jhd2–9Myc occupancy obtained at the initial repressed state ( R ), which was set as 1. Error bars denote S.E. obtained from two independent experiments.
    Figure Legend Snippet: Jhd2, an H3K4-specific demethylase, functions during both active transcription and repression. A , shown are the results from Western blot analysis of H3K4 methylation levels in crude nuclear extracts obtained from yeast strains with overexpressed JmjC domain-containing proteins. B , changes in H3K4me3 levels at the PMA1 locus were analyzed by ChIP assay. The graphs depict the data obtained from the WT control and a strain lacking Jhd2 ( jhd2 Δ) ( upper panel ) and from yeast strains overexpressing JHD2 ( JHD2 OE ) from a high copy plasmid or containing the plasmid vector alone ( lower panel ). The H3K4me3 ChIP signals were first normalized to total histone H3 ChIP signals (H3K4me3/H3), and then the H3K4me3/H3 values at the promoter ( P ), 5′-ORF, or 3′-ORF regions in the controls and test samples were normalized to the H3K4me3/H3 value obtained for the promoter region in the WT ( upper panel ) or vector control ( lower panel ), which was set as 1. Error bars denote S.E. from two independent experiments. C , the levels of Jhd2-LexA at PMA1 were analyzed by ChIP using anti-LexA antibody. LexA immunoprecipitation/input values obtained from the no-tag control (background) were subtracted from those obtained from yeast cells with overexpressed JHD2 -LexA, and the resulting differences were defined as Jhd2-LexA occupancy. The Jhd2-LexA occupancies at the 5′- and 3′-ORF regions were normalized to the promoter region, which was arbitrarily set as 1. Error bars denote S.E. from two independent experiments. D , temporal changes in H3K4me3 following the re-repression of INO1 were measured by ChIP assay. WT and jhd2 Δ strains were grown in inositol/choline medium for 4 h to repress INO1 expression ( R ) and then grown in inositol-free medium for 2 h to induce INO1 expression ( In ), and INO1 was repressed again by growing cells in inositol/choline medium for the indicated time periods ( Re-repression ). Cells at different time points were subjected to ChIP analysis using anti-H3K4me3 and anti-H3 antibodies. The levels of H3K4me3/H3 at the 5′-ORF of INO1 in the WT or jhd2 Δ mutant at different time points are shown as -fold changes relative to the H3K4me3/H3 value obtained at the repressed state for the WT, which was set as 1. Error bars denote S.E. from two independent experiments. E , temporal changes in Jhd2–9Myc occupancy following the re-repression of INO1 were measured by ChIP assay performed essentially as described for D . Chromatin-bound Jhd2–9Myc was immunoprecipitated using anti-Myc antibody. Myc immunoprecipitation/input values obtained from the no-tag control (background) were subtracted from those obtained for yeast expressing JHD2 -9Myc, and the resulting difference was defined as Jhd2–9Myc occupancy. The Jhd2–9Myc occupancies at the 5′-ORF of INO1 at different time points are shown as fold changes relative to the Jhd2–9Myc occupancy obtained at the initial repressed state ( R ), which was set as 1. Error bars denote S.E. obtained from two independent experiments.

    Techniques Used: Western Blot, Methylation, Chromatin Immunoprecipitation, Plasmid Preparation, Immunoprecipitation, Expressing, Mutagenesis

    Related Articles

    Magnetic Beads:

    Article Title: Fosb Induction in Nucleus Accumbens by Cocaine Is Regulated by E2F3a
    Article Snippet: .. Sheared chromatin was incubated overnight in a 0.1% SDS solution with the following antibodies previously bound to magnetic beads following the Dynabead protocol (Dynabeads M-280, Life Technologies): antibody to E2F3 [Santa Cruz Biotechnology, E2F-3 (C-18) × (sc-878X)], antibody to Rabbit-IgG [Santa Cruz Biotechnology, normal rabbit IgG × (sc-2027X)], antibody to H3K4me3 [ActiveMotif, Histone H3K4me3 antibody (pAb); 39915]. .. 1 µg of DNA was used for E2F3 and IgG IPs, 250 ng for H3K4me3.

    Isolation:

    Article Title: Paradoxical Changes Underscore Epigenetic Reprogramming During Adult Zebrafish Extraocular Muscle Regeneration
    Article Snippet: .. Other aliquots with 2 to 4 μg chromatin were precleared with protein A agarose beads (Invitrogen, Carlsbad, CA, USA), and regions of interest were isolated using 4 μg antibody against H3K27me3 (07-449; Millipore, Burlington, MA, USA), H3K27Ac (39133; Active Motif), or 3 μg antibody against H3K4me3 (39159; Active Motif). .. Similar to the input samples, the resulting complexes were washed, eluted with SDS buffer, and treated with RNase and proteinase K. They were incubated overnight at 65°C to reverse crosslinks, and the ChIP DNA products were purified with phenol-chloroform extraction and ethanol precipitation.

    Immunoprecipitation:

    Article Title: IL-17+ CD8+ T cell suppression by dimethyl fumarate associates with clinical response in multiple sclerosis
    Article Snippet: .. For immunoprecipitation, 2.5–4 µg of the following Abs were used: anti-H4ac (Millipore, 06–866), anti-H3K4me3 (Active Motif, 39159), anti-H3K27me3 (Active Motif, 39155), anti-H3K27ac (Abcam, ab4729) or control IgG (Cell Signaling, 2729). .. For immunoprecipitation, 2.5–4 µg of the following Abs were used: anti-H4ac (Millipore, 06–866), anti-H3K4me3 (Active Motif, 39159), anti-H3K27me3 (Active Motif, 39155), anti-H3K27ac (Abcam, ab4729) or control IgG (Cell Signaling, 2729).

    Incubation:

    Article Title: Fosb Induction in Nucleus Accumbens by Cocaine Is Regulated by E2F3a
    Article Snippet: .. Sheared chromatin was incubated overnight in a 0.1% SDS solution with the following antibodies previously bound to magnetic beads following the Dynabead protocol (Dynabeads M-280, Life Technologies): antibody to E2F3 [Santa Cruz Biotechnology, E2F-3 (C-18) × (sc-878X)], antibody to Rabbit-IgG [Santa Cruz Biotechnology, normal rabbit IgG × (sc-2027X)], antibody to H3K4me3 [ActiveMotif, Histone H3K4me3 antibody (pAb); 39915]. .. 1 µg of DNA was used for E2F3 and IgG IPs, 250 ng for H3K4me3.

    other:

    Article Title: Automated in situ profiling of chromatin modifications resolves cell types and gene regulatory programs
    Article Snippet: AntibodiesWe used Rabbit anti-CTCF (1:100, Millipore Cat#07-729), Rabbit anti-NPAT (1:100, Termo Fisher Cat#PA5-66839), Rabbit anti-H3K4me1 (1:100, Abcam Cat#ab8895), Rabbit anti-H3K4me2 (1:100, Millipore Cat#07-030), Rabbit anti-H3K4me3 (1:100, Active Motif Cat#39159), Rabbit anti-H3K27me3 (1:100, Cell Signaling Tech Cat#9733S).

    Modification:

    Article Title: Misregulation of the IgH Locus in Thymocytes
    Article Snippet: .. Modified histone antibodies were purchased from Active Motif: anti-H3K4me3 (Cat # 39519), anti-H3K9ac (Cat # 39137), anti-H3K27me3 (Cat # 39155). .. Antibodies for transcription factors were as follows: anti-E2A (Cat # Sc-349), anti-YY1 (Cat # Sc-1703), anti-Ets-1 (Cat # Sc-350), Anti-HEB (Cat # Sc-357) were from Santa Cruz Biotechnology and anti-Runx1 (Cat # ab23980), anti-Rad21 (Cat # ab992) were from Abcam; anti-CTCF (Cat # 07-729) was purchased from Millipore.

    Chromatin Immunoprecipitation:

    Article Title: Set1/COMPASS repels heterochromatin invasion at euchromatic sites by disrupting Suv39/Clr4 activity and nucleosome stability
    Article Snippet: .. One microliter of the following antibodies was added per ChIP sample: H3K9me2 (Abcam, ab1220), H3K4me3 (Active Motif 39159), H3K4me2 (Active Motif, 39141), H3K9ac (Active Motif, 39137), H3(pan)ac (Active Motif, 39064), H4(pan)ac (Active Motif, 39140), HA (Abcam, ab9110); 1.5 µL of anti-Flag M2 antibody (Sigma) was added per ChIP sample, and 1.4 μg of H3 antibody (Active Motif, 39064) was added per ChIP sample. .. Immune complexes were collected with Protein A Dynabeads (Thermo Fisher) for all ChIP samples except the anti-Flag ChIP samples, which were collected with Protein G Dynabeads (Thermo Fisher).

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  • 88
    Active Motif histone h3k27me3
    Knockout of EZH2 gene by CRISPR/Cas9 and stable infection of EBV. (A) Loss of EZH2 expression in the EZH2-KO Akata(−) cells. Two independent clones were used (named cl1 and cl2) for both WT and KO. Levels of EZH2, <t>H3K27me3,</t> histone H3, and β-actin in the samples from WT and EZH2-KO cells were examined by Western blotting. (B) Growth kinetics of EZH2-KO Akata(−) cells. A quantity of 1.0 × 10 5 cells/ml was seeded and cultured. Cell numbers were counted after the indicated number of days. (C) Reduced growth speed in the EZH2-KO cells. The WT and EZH2-KO Akata cells were infected with EBV and cultured in the presence of G418. At 0, 4, 8, and 12 weeks postinfection, cells were seeded at 1.0 × 10 5 cells/ml, and cell numbers were counted after 3 days. The average and SD from 2 independent replicates are shown as a ratio relative to the value of the starting point (0 week). Student’s t test was performed, and asterisks indicate statistical significance (*, P
    Histone H3k27me3, supplied by Active Motif, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Active Motif scwat
    Complementary mechanisms for thermogenic gene induction in acute and chronic cold stress via overlapping, but distinct mechanisms of JMJD1A. Brown fat cells mediate acute and robust thermogenic activation of Ucp1 , while <t>scWAT-derived</t> beige fat cells contribute to an adaptive response against chronic cold exposure (top). The acute response in <t>BAT</t> requires a BAR-dependent phosphorylation of JMJD1A that facilitates long-range enhancer-promoter interactions and stimulate thermogenic gene expressions, but this does not require the intrinsic H3K9me2 demethylation activity of JMJD1A (left bottom). The chronic adaptation in beigeing requires both phosphorylation-dependent chromatin recruitment and H3K9me2 demethylation activity of JMJD1A (right bottom). These histone demethylation-independent acute Ucp1 induction in BAT and demethylation-dependent chronic Ucp1 expression in beige scWAT ensure an ordered transition between acute and chronic adaptation to cold stress. TXN transcription
    Scwat, supplied by Active Motif, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Active Motif hdac phosphorylation
    Histone deacetylase <t>(HDAC)</t> phosphorylation and myocyte enhancer factor 2 (MEF2) activities in hearts of <t>E16.5</t> Ryr2 +/+ and Ryr2 ADA/ADA mice. A : [ 32 P] incorporation ( left , top) and Coomassie brilliant blue staining ( left , bottom) of GST-HDAC4 fusion protein
    Hdac Phosphorylation, supplied by Active Motif, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Active Motif histone h3k4me3
    Calix[4]arenes mimic the aromatic cage of PHD fingers. a , schematic showing the disruption of the association of a PHD finger ( orange ) with the histone <t>H3K4me3</t> tail by calixarene ( yellow ). b , distances between the aromatic groups in the methyllysine-binding
    Histone H3k4me3, supplied by Active Motif, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Knockout of EZH2 gene by CRISPR/Cas9 and stable infection of EBV. (A) Loss of EZH2 expression in the EZH2-KO Akata(−) cells. Two independent clones were used (named cl1 and cl2) for both WT and KO. Levels of EZH2, H3K27me3, histone H3, and β-actin in the samples from WT and EZH2-KO cells were examined by Western blotting. (B) Growth kinetics of EZH2-KO Akata(−) cells. A quantity of 1.0 × 10 5 cells/ml was seeded and cultured. Cell numbers were counted after the indicated number of days. (C) Reduced growth speed in the EZH2-KO cells. The WT and EZH2-KO Akata cells were infected with EBV and cultured in the presence of G418. At 0, 4, 8, and 12 weeks postinfection, cells were seeded at 1.0 × 10 5 cells/ml, and cell numbers were counted after 3 days. The average and SD from 2 independent replicates are shown as a ratio relative to the value of the starting point (0 week). Student’s t test was performed, and asterisks indicate statistical significance (*, P

    Journal: mSphere

    Article Title: Regulation of Epstein-Barr Virus Life Cycle and Cell Proliferation by Histone H3K27 Methyltransferase EZH2 in Akata Cells

    doi: 10.1128/mSphere.00478-18

    Figure Lengend Snippet: Knockout of EZH2 gene by CRISPR/Cas9 and stable infection of EBV. (A) Loss of EZH2 expression in the EZH2-KO Akata(−) cells. Two independent clones were used (named cl1 and cl2) for both WT and KO. Levels of EZH2, H3K27me3, histone H3, and β-actin in the samples from WT and EZH2-KO cells were examined by Western blotting. (B) Growth kinetics of EZH2-KO Akata(−) cells. A quantity of 1.0 × 10 5 cells/ml was seeded and cultured. Cell numbers were counted after the indicated number of days. (C) Reduced growth speed in the EZH2-KO cells. The WT and EZH2-KO Akata cells were infected with EBV and cultured in the presence of G418. At 0, 4, 8, and 12 weeks postinfection, cells were seeded at 1.0 × 10 5 cells/ml, and cell numbers were counted after 3 days. The average and SD from 2 independent replicates are shown as a ratio relative to the value of the starting point (0 week). Student’s t test was performed, and asterisks indicate statistical significance (*, P

    Article Snippet: Antibodies against EZH2, histone H3K27me3, H4K20me3, and H3K9ac were from Active Motif.

    Techniques: Knock-Out, CRISPR, Infection, Expressing, Clone Assay, Western Blot, Cell Culture

    Effect of GSK343, an EZH2 inhibitor, on EBV gene expression in Akata cells. (A) EBV-positive Akata cells were pretreated daily with DMSO (control) or 1 μM GSK343 for 3 days and harvested for Western blotting. (B) EBV-positive Akata cells were treated daily with DMSO (Cont) or 1 μM GSK343 for 3 days and subjected to ChIP assays using anti-histone H3 and -histone H3K27me3 antibodies. The data for H3K27me3 are shown after normalization to the value of histone H3. Qp, Q promoter; Cp, C promoter; Zp, BZLF1 promoter; Ap, BALF2 promoter; Globinp, globin promoter. Student’s t test was performed. *, P

    Journal: mSphere

    Article Title: Regulation of Epstein-Barr Virus Life Cycle and Cell Proliferation by Histone H3K27 Methyltransferase EZH2 in Akata Cells

    doi: 10.1128/mSphere.00478-18

    Figure Lengend Snippet: Effect of GSK343, an EZH2 inhibitor, on EBV gene expression in Akata cells. (A) EBV-positive Akata cells were pretreated daily with DMSO (control) or 1 μM GSK343 for 3 days and harvested for Western blotting. (B) EBV-positive Akata cells were treated daily with DMSO (Cont) or 1 μM GSK343 for 3 days and subjected to ChIP assays using anti-histone H3 and -histone H3K27me3 antibodies. The data for H3K27me3 are shown after normalization to the value of histone H3. Qp, Q promoter; Cp, C promoter; Zp, BZLF1 promoter; Ap, BALF2 promoter; Globinp, globin promoter. Student’s t test was performed. *, P

    Article Snippet: Antibodies against EZH2, histone H3K27me3, H4K20me3, and H3K9ac were from Active Motif.

    Techniques: Expressing, Western Blot, Chromatin Immunoprecipitation

    Low H3K27me3 modification in the EZH2-KO cells during reactivation from latency. (A to E) WT and EZH2-KO cells latently infected with EBV (cl2) were treated with anti-IgG. Cells were harvested at 0 (latency) and 10 hpi and analyzed by ChIP using anti-histone H3 (A), anti-histone H3K27me3 (B), anti-histone H3K9me3 (C), anti-histone H3K4me3 (D), or anti-histone H3ac (E) antibodies. Data for histone H3 are shown as the percentage of the input sample (A). The data for other markers (B to E) are shown after normalization to the value of histone H3. Lp, LMP1 promoter; Qp, Q promoter; Cp, C promoter; Zp, BZLF1 promoter; Mp, BMRF1 promoter; Ap, BALF2 promoter; Globinp, Globin promoter; GAPDHp, GAPDH promoter. Student’s t test was performed. *, P

    Journal: mSphere

    Article Title: Regulation of Epstein-Barr Virus Life Cycle and Cell Proliferation by Histone H3K27 Methyltransferase EZH2 in Akata Cells

    doi: 10.1128/mSphere.00478-18

    Figure Lengend Snippet: Low H3K27me3 modification in the EZH2-KO cells during reactivation from latency. (A to E) WT and EZH2-KO cells latently infected with EBV (cl2) were treated with anti-IgG. Cells were harvested at 0 (latency) and 10 hpi and analyzed by ChIP using anti-histone H3 (A), anti-histone H3K27me3 (B), anti-histone H3K9me3 (C), anti-histone H3K4me3 (D), or anti-histone H3ac (E) antibodies. Data for histone H3 are shown as the percentage of the input sample (A). The data for other markers (B to E) are shown after normalization to the value of histone H3. Lp, LMP1 promoter; Qp, Q promoter; Cp, C promoter; Zp, BZLF1 promoter; Mp, BMRF1 promoter; Ap, BALF2 promoter; Globinp, Globin promoter; GAPDHp, GAPDH promoter. Student’s t test was performed. *, P

    Article Snippet: Antibodies against EZH2, histone H3K27me3, H4K20me3, and H3K9ac were from Active Motif.

    Techniques: Modification, Infection, Chromatin Immunoprecipitation

    Effect of GSK343 on EBV gene expression in LCLs. (A) LCLs were pretreated daily with DMSO (control) or 1 μM GSK343 for 3 days and harvested for Western blotting. (B) LCLs were treated daily with DMSO (Cont) or 1 μM GSK343 for 3 days and subjected to ChIP assays using anti-histone H3 and -histone H3K27me3 antibodies. The data for H3K27me3 are shown after normalization to the value of histone H3. Qp, Q promoter; Cp, C promoter; Zp, BZLF1 promoter; Ap, BALF2 promoter; Globinp, globin promoter. Student’s t test was performed. *, P

    Journal: mSphere

    Article Title: Regulation of Epstein-Barr Virus Life Cycle and Cell Proliferation by Histone H3K27 Methyltransferase EZH2 in Akata Cells

    doi: 10.1128/mSphere.00478-18

    Figure Lengend Snippet: Effect of GSK343 on EBV gene expression in LCLs. (A) LCLs were pretreated daily with DMSO (control) or 1 μM GSK343 for 3 days and harvested for Western blotting. (B) LCLs were treated daily with DMSO (Cont) or 1 μM GSK343 for 3 days and subjected to ChIP assays using anti-histone H3 and -histone H3K27me3 antibodies. The data for H3K27me3 are shown after normalization to the value of histone H3. Qp, Q promoter; Cp, C promoter; Zp, BZLF1 promoter; Ap, BALF2 promoter; Globinp, globin promoter. Student’s t test was performed. *, P

    Article Snippet: Antibodies against EZH2, histone H3K27me3, H4K20me3, and H3K9ac were from Active Motif.

    Techniques: Expressing, Western Blot, Chromatin Immunoprecipitation

    Prominent decrease in H3K27me3 modification in EZH2-KO cells. (A to C) WT and EZH2-KO Akata cells latently infected with EBV (cl1) were subjected to chromatin immunoprecipitation (ChIP) analysis using normal IgG (A), anti-histone H3 (B), or anti-histone H3K27me3 (C) antibodies. Data for normal IgG and histone H3 are shown as the percentage of the input sample. Data for H3K27me3 (C) are shown after normalization to the value of histone H3. Lp, LMP1 promoter; Qp, Q promoter; Cp, C promoter; Zp, BZLF1 promoter; Mp, BMRF1 promoter; Ap, BALF2 promoter; Globinp, globin promoter; GAPDHp, GAPDH promoter. Student’s t test was performed. *, P

    Journal: mSphere

    Article Title: Regulation of Epstein-Barr Virus Life Cycle and Cell Proliferation by Histone H3K27 Methyltransferase EZH2 in Akata Cells

    doi: 10.1128/mSphere.00478-18

    Figure Lengend Snippet: Prominent decrease in H3K27me3 modification in EZH2-KO cells. (A to C) WT and EZH2-KO Akata cells latently infected with EBV (cl1) were subjected to chromatin immunoprecipitation (ChIP) analysis using normal IgG (A), anti-histone H3 (B), or anti-histone H3K27me3 (C) antibodies. Data for normal IgG and histone H3 are shown as the percentage of the input sample. Data for H3K27me3 (C) are shown after normalization to the value of histone H3. Lp, LMP1 promoter; Qp, Q promoter; Cp, C promoter; Zp, BZLF1 promoter; Mp, BMRF1 promoter; Ap, BALF2 promoter; Globinp, globin promoter; GAPDHp, GAPDH promoter. Student’s t test was performed. *, P

    Article Snippet: Antibodies against EZH2, histone H3K27me3, H4K20me3, and H3K9ac were from Active Motif.

    Techniques: Modification, Infection, Chromatin Immunoprecipitation

    Complementary mechanisms for thermogenic gene induction in acute and chronic cold stress via overlapping, but distinct mechanisms of JMJD1A. Brown fat cells mediate acute and robust thermogenic activation of Ucp1 , while scWAT-derived beige fat cells contribute to an adaptive response against chronic cold exposure (top). The acute response in BAT requires a BAR-dependent phosphorylation of JMJD1A that facilitates long-range enhancer-promoter interactions and stimulate thermogenic gene expressions, but this does not require the intrinsic H3K9me2 demethylation activity of JMJD1A (left bottom). The chronic adaptation in beigeing requires both phosphorylation-dependent chromatin recruitment and H3K9me2 demethylation activity of JMJD1A (right bottom). These histone demethylation-independent acute Ucp1 induction in BAT and demethylation-dependent chronic Ucp1 expression in beige scWAT ensure an ordered transition between acute and chronic adaptation to cold stress. TXN transcription

    Journal: Nature Communications

    Article Title: Histone demethylase JMJD1A coordinates acute and chronic adaptation to cold stress via thermogenic phospho-switch

    doi: 10.1038/s41467-018-03868-8

    Figure Lengend Snippet: Complementary mechanisms for thermogenic gene induction in acute and chronic cold stress via overlapping, but distinct mechanisms of JMJD1A. Brown fat cells mediate acute and robust thermogenic activation of Ucp1 , while scWAT-derived beige fat cells contribute to an adaptive response against chronic cold exposure (top). The acute response in BAT requires a BAR-dependent phosphorylation of JMJD1A that facilitates long-range enhancer-promoter interactions and stimulate thermogenic gene expressions, but this does not require the intrinsic H3K9me2 demethylation activity of JMJD1A (left bottom). The chronic adaptation in beigeing requires both phosphorylation-dependent chromatin recruitment and H3K9me2 demethylation activity of JMJD1A (right bottom). These histone demethylation-independent acute Ucp1 induction in BAT and demethylation-dependent chronic Ucp1 expression in beige scWAT ensure an ordered transition between acute and chronic adaptation to cold stress. TXN transcription

    Article Snippet: For immunoblot analysis of histones, histones were acid extracted from BAT or scWAT of C57BL/6J mice using a Histone Purification Mini Kit (Active Motif), following the manufacturer’s instructions as described .

    Techniques: Activation Assay, Derivative Assay, Activity Assay, Expressing

    Insulin resistance phenotype of Jmjd1a -S265A KI/KI mice. a Body weight changes. WT (+/+) and Jmjd1a -S265A KI/KI mice ( WT : n = 6; Jmjd1a -S265A KI/KI : n = 8) were fed on HFD for 4 weeks at 30 °C, and then switched to 4 °C for 4 weeks. b , c Glucose tolerance test (GTT) ( b ) and insulin tolerance test (ITT) ( c ) in each genotype group mice fed on HFD after cold acclimation in a ( WT : n = 6; Jmjd1a -S265A KI/KI : n = 7). d Assessment of insulin signaling as quantified by the phosphorylation of AKT-S473 in scWAT, BAT, or soleus from each genotype mice fed on HFD after cold acclimation presented in a . Uncropped images of the blots are shown in Supplementary Fig. 8 . Data are mean ± s.e.m. ( a – c ) Student’s t test was performed for comparisons in a – c . * P

    Journal: Nature Communications

    Article Title: Histone demethylase JMJD1A coordinates acute and chronic adaptation to cold stress via thermogenic phospho-switch

    doi: 10.1038/s41467-018-03868-8

    Figure Lengend Snippet: Insulin resistance phenotype of Jmjd1a -S265A KI/KI mice. a Body weight changes. WT (+/+) and Jmjd1a -S265A KI/KI mice ( WT : n = 6; Jmjd1a -S265A KI/KI : n = 8) were fed on HFD for 4 weeks at 30 °C, and then switched to 4 °C for 4 weeks. b , c Glucose tolerance test (GTT) ( b ) and insulin tolerance test (ITT) ( c ) in each genotype group mice fed on HFD after cold acclimation in a ( WT : n = 6; Jmjd1a -S265A KI/KI : n = 7). d Assessment of insulin signaling as quantified by the phosphorylation of AKT-S473 in scWAT, BAT, or soleus from each genotype mice fed on HFD after cold acclimation presented in a . Uncropped images of the blots are shown in Supplementary Fig. 8 . Data are mean ± s.e.m. ( a – c ) Student’s t test was performed for comparisons in a – c . * P

    Article Snippet: For immunoblot analysis of histones, histones were acid extracted from BAT or scWAT of C57BL/6J mice using a Histone Purification Mini Kit (Active Motif), following the manufacturer’s instructions as described .

    Techniques: Mouse Assay

    Ucp1 expression and H3K9me2 levels in adipose tissues and BAT thermogenic function in Jmjd1a -S265A KI/KI mice . a Ucp1 mRNA ( n = 3) (left) and protein levels detected by immunoblotting (IB) (right) in BAT, and scWAT of mice exposed to 4 °C for 6 h. b H3K9me2 immunoblotting using purified histones from adipose tissues of mice housed at RT. c H3K9me2 ChIP-qPCR in BAT, and scWAT of mice exposed to 4 °C for 6 h ( n = 3). d NE-induced Ucp1 mRNA levels in im-BATs stably expressing WT or indicated mutants of hJMJD1A. The relative quantity in im-BATs expressing WT-JMJD1A on day 8 before NE treatment (0 h) is defined as 1. e Ucp1 mRNA expressions ( n = 3) (left) and proteins (right) in BAT, and scWAT from mice exposed to 30 °C or 4 °C for 1 week. Uncropped images of the blots ( a , b , e ) are shown in Supplementary Fig. 8 . f , g H3K9me2 ChIP-qPCR in scWAT from mice placed at 4 °C for 1 week ( n = 4) ( f ) and in im-scWATs during beige adipogenesis (mean ± s.e.m. of three technical replicates) ( g ). h Bi-phasic Ucp1 expressions during cold exposure in BAT and scWAT. Mice were exposed to 4 °C for the indicated time, and Ucp1 mRNAs in BAT and scWATs were quantified by qPCR ( n = 3). The data were converted to copy number per ng of total RNA. i In BAT, Ucp 1 locus is in euchromatin (left), while in scWAT, it is in heterochromatin with H3K9me2 (right). In BAT, cold exposure leads to acute induction of Ucp1 mRNA through the mechanisms independent of H3K9me2 demethylation (left). In scWAT, H3K9me2 at Ucp1 gene locus needs to be removed for beige adipogenesis (right bottom). j Cold intolerance in Jmjd1a -S265A KI/KI mice ( n = 6 per genotype group). Shown is the body temperature of 9-week-old mice at different times after cold exposure (4 °C). k Impaired NE-induced activation of selective genes in BAT ( n = 6 per genotype group). l , m Reduced NE-induced mitochondrial respiration ( l ) and NE-induced glycerol release ( m ) in primary brown adipocytes from Jmjd1a -S265A KI/KI mice. Data are mean ± s.e.m. of five technical replicates ( l ) and three independent experiments ( m ). Data are mean ± s.e.m. a , c , e , f , j , k Student’s t test ( a , e , f , j , l , m ) or analysis of variance, followed by Tukey’s post hoc comparison ( k ) were performed for comparisons. * P

    Journal: Nature Communications

    Article Title: Histone demethylase JMJD1A coordinates acute and chronic adaptation to cold stress via thermogenic phospho-switch

    doi: 10.1038/s41467-018-03868-8

    Figure Lengend Snippet: Ucp1 expression and H3K9me2 levels in adipose tissues and BAT thermogenic function in Jmjd1a -S265A KI/KI mice . a Ucp1 mRNA ( n = 3) (left) and protein levels detected by immunoblotting (IB) (right) in BAT, and scWAT of mice exposed to 4 °C for 6 h. b H3K9me2 immunoblotting using purified histones from adipose tissues of mice housed at RT. c H3K9me2 ChIP-qPCR in BAT, and scWAT of mice exposed to 4 °C for 6 h ( n = 3). d NE-induced Ucp1 mRNA levels in im-BATs stably expressing WT or indicated mutants of hJMJD1A. The relative quantity in im-BATs expressing WT-JMJD1A on day 8 before NE treatment (0 h) is defined as 1. e Ucp1 mRNA expressions ( n = 3) (left) and proteins (right) in BAT, and scWAT from mice exposed to 30 °C or 4 °C for 1 week. Uncropped images of the blots ( a , b , e ) are shown in Supplementary Fig. 8 . f , g H3K9me2 ChIP-qPCR in scWAT from mice placed at 4 °C for 1 week ( n = 4) ( f ) and in im-scWATs during beige adipogenesis (mean ± s.e.m. of three technical replicates) ( g ). h Bi-phasic Ucp1 expressions during cold exposure in BAT and scWAT. Mice were exposed to 4 °C for the indicated time, and Ucp1 mRNAs in BAT and scWATs were quantified by qPCR ( n = 3). The data were converted to copy number per ng of total RNA. i In BAT, Ucp 1 locus is in euchromatin (left), while in scWAT, it is in heterochromatin with H3K9me2 (right). In BAT, cold exposure leads to acute induction of Ucp1 mRNA through the mechanisms independent of H3K9me2 demethylation (left). In scWAT, H3K9me2 at Ucp1 gene locus needs to be removed for beige adipogenesis (right bottom). j Cold intolerance in Jmjd1a -S265A KI/KI mice ( n = 6 per genotype group). Shown is the body temperature of 9-week-old mice at different times after cold exposure (4 °C). k Impaired NE-induced activation of selective genes in BAT ( n = 6 per genotype group). l , m Reduced NE-induced mitochondrial respiration ( l ) and NE-induced glycerol release ( m ) in primary brown adipocytes from Jmjd1a -S265A KI/KI mice. Data are mean ± s.e.m. of five technical replicates ( l ) and three independent experiments ( m ). Data are mean ± s.e.m. a , c , e , f , j , k Student’s t test ( a , e , f , j , l , m ) or analysis of variance, followed by Tukey’s post hoc comparison ( k ) were performed for comparisons. * P

    Article Snippet: For immunoblot analysis of histones, histones were acid extracted from BAT or scWAT of C57BL/6J mice using a Histone Purification Mini Kit (Active Motif), following the manufacturer’s instructions as described .

    Techniques: Expressing, Mouse Assay, Purification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Stable Transfection, Activation Assay

    Phospho-S265 JMJD1A induces beige biogenesis. a qPCR analysis demonstrates decreased expression of beige-selective genes in scWAT from Jmjd1a -S265A KI/KI mice exposed to 30 °C or 4 °C for 1 week ( n = 5 per genotype group). b Immunoblot analysis of UCP1 and PPARγ in tissue homogenates of scWAT from mice presented in a . Uncropped images of the blots are shown in Supplementary Fig. 8 . c Haematoxylin and eosin (H E) and UCP1 staining sections of scWAT from WT and Jmjd1a -S265A KI/KI mice exposed to chronic cold exposure (4 °C for 1 week) (scale bar, 100 μm). d NE-induced oxygen consumption rate (OCR) in mice exposed to chronic cold exposure (4 °C for 4 weeks) ( n = 7 per genotype group) (left). OCR before and 30 min after NE treatment are analyzed (right) ( n = 7). e OCR of scWAT from mice exposed to 30 °C or 4 °C for 1 week ( WT : n = 3; Jmjd1a -S265A KI/KI : n = 4). Data are mean ± s.e.m. a , d , e Analysis of variance were performed followed by Tukey’s post hoc comparison in a . Student’s t test was performed for comparisons in d , e . * P

    Journal: Nature Communications

    Article Title: Histone demethylase JMJD1A coordinates acute and chronic adaptation to cold stress via thermogenic phospho-switch

    doi: 10.1038/s41467-018-03868-8

    Figure Lengend Snippet: Phospho-S265 JMJD1A induces beige biogenesis. a qPCR analysis demonstrates decreased expression of beige-selective genes in scWAT from Jmjd1a -S265A KI/KI mice exposed to 30 °C or 4 °C for 1 week ( n = 5 per genotype group). b Immunoblot analysis of UCP1 and PPARγ in tissue homogenates of scWAT from mice presented in a . Uncropped images of the blots are shown in Supplementary Fig. 8 . c Haematoxylin and eosin (H E) and UCP1 staining sections of scWAT from WT and Jmjd1a -S265A KI/KI mice exposed to chronic cold exposure (4 °C for 1 week) (scale bar, 100 μm). d NE-induced oxygen consumption rate (OCR) in mice exposed to chronic cold exposure (4 °C for 4 weeks) ( n = 7 per genotype group) (left). OCR before and 30 min after NE treatment are analyzed (right) ( n = 7). e OCR of scWAT from mice exposed to 30 °C or 4 °C for 1 week ( WT : n = 3; Jmjd1a -S265A KI/KI : n = 4). Data are mean ± s.e.m. a , d , e Analysis of variance were performed followed by Tukey’s post hoc comparison in a . Student’s t test was performed for comparisons in d , e . * P

    Article Snippet: For immunoblot analysis of histones, histones were acid extracted from BAT or scWAT of C57BL/6J mice using a Histone Purification Mini Kit (Active Motif), following the manufacturer’s instructions as described .

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Mouse Assay, Staining

    P-JMJD1A cell autonomously induces beige adipogenesis. a pSer265-JMJD1A protein levels in WT (+/+) and S265A knock-in whole-cell lysates (WCL) from scWAT cultures treated with NE or vehicle for 1 h. b Decreased beige-selective gene expressions in S265A knock-in scWAT cultures treated with NE (10 μM) for 2 h (mean ± s.e.m. of three technical replicates). ORO staining of indicated genotype of scWAT cultures (inset). c Immunoblot analysis using anti-UCP1, anti-PGC1α, anti-PRDM16, anti-PPARγ, or anti-total OXPHOS antibodies cocktail, using WCL from WT and S265A knock-in scWAT cultures. d MitoTracker staining in indicated genotype scWAT cultures (scale bar, 100 μm). e Mitochondrial DNA (mt-DNA) contents measured by qPCR in indicated scWAT cultures (mean ± s.e.m. of three independent experiments). f Electron micrographs of indicated genotype of scWAT cultures (bar, 1 μm). Mitochondria (M) and lipid droplets (L) are indicated. g The OCR of indicated scWAT cultures (left). The arrows indicate the time of addition for oligomycin (Oligo), FCCP, and rotenone/antimycin A (Rot/Anti). Basal, maximum, and uncoupled respiration were calculated (mean ± s.e.m. of five technical replicates) (right). h Glycerol release from indicated scWAT cultures after the treatment with NE for 3 h (mean ± s.e.m. of three independent experiments). i Increased expressions of beige-selective genes in S265D-hJMJD1A-transduced im-scWATs (mean ± s.e.m. of three technical replicates). ORO staining and MitoTracker staining (inset) (scale bar, 50 μm). j Immunoblotting with anti-UCP1, anti-PGC1α, anti-PPARγ, or anti-total OXPHOS antibodies cocktail using WCL from indicated im-scWATs. Uncropped images of the blots ( a , c , j ) are shown in Supplementary Fig. 8 . k Mitochondrial DNA content measured by qPCR in indicated im-scWATs (mean ± s.e.m. of three technical replicates). Student’s t test was performed for comparisons in b , g , h . * P

    Journal: Nature Communications

    Article Title: Histone demethylase JMJD1A coordinates acute and chronic adaptation to cold stress via thermogenic phospho-switch

    doi: 10.1038/s41467-018-03868-8

    Figure Lengend Snippet: P-JMJD1A cell autonomously induces beige adipogenesis. a pSer265-JMJD1A protein levels in WT (+/+) and S265A knock-in whole-cell lysates (WCL) from scWAT cultures treated with NE or vehicle for 1 h. b Decreased beige-selective gene expressions in S265A knock-in scWAT cultures treated with NE (10 μM) for 2 h (mean ± s.e.m. of three technical replicates). ORO staining of indicated genotype of scWAT cultures (inset). c Immunoblot analysis using anti-UCP1, anti-PGC1α, anti-PRDM16, anti-PPARγ, or anti-total OXPHOS antibodies cocktail, using WCL from WT and S265A knock-in scWAT cultures. d MitoTracker staining in indicated genotype scWAT cultures (scale bar, 100 μm). e Mitochondrial DNA (mt-DNA) contents measured by qPCR in indicated scWAT cultures (mean ± s.e.m. of three independent experiments). f Electron micrographs of indicated genotype of scWAT cultures (bar, 1 μm). Mitochondria (M) and lipid droplets (L) are indicated. g The OCR of indicated scWAT cultures (left). The arrows indicate the time of addition for oligomycin (Oligo), FCCP, and rotenone/antimycin A (Rot/Anti). Basal, maximum, and uncoupled respiration were calculated (mean ± s.e.m. of five technical replicates) (right). h Glycerol release from indicated scWAT cultures after the treatment with NE for 3 h (mean ± s.e.m. of three independent experiments). i Increased expressions of beige-selective genes in S265D-hJMJD1A-transduced im-scWATs (mean ± s.e.m. of three technical replicates). ORO staining and MitoTracker staining (inset) (scale bar, 50 μm). j Immunoblotting with anti-UCP1, anti-PGC1α, anti-PPARγ, or anti-total OXPHOS antibodies cocktail using WCL from indicated im-scWATs. Uncropped images of the blots ( a , c , j ) are shown in Supplementary Fig. 8 . k Mitochondrial DNA content measured by qPCR in indicated im-scWATs (mean ± s.e.m. of three technical replicates). Student’s t test was performed for comparisons in b , g , h . * P

    Article Snippet: For immunoblot analysis of histones, histones were acid extracted from BAT or scWAT of C57BL/6J mice using a Histone Purification Mini Kit (Active Motif), following the manufacturer’s instructions as described .

    Techniques: Knock-In, Staining, Real-time Polymerase Chain Reaction

    Histone deacetylase (HDAC) phosphorylation and myocyte enhancer factor 2 (MEF2) activities in hearts of E16.5 Ryr2 +/+ and Ryr2 ADA/ADA mice. A : [ 32 P] incorporation ( left , top) and Coomassie brilliant blue staining ( left , bottom) of GST-HDAC4 fusion protein

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Dysfunctional ryanodine receptor and cardiac hypertrophy: role of signaling molecules

    doi: 10.1152/ajpheart.00719.2010

    Figure Lengend Snippet: Histone deacetylase (HDAC) phosphorylation and myocyte enhancer factor 2 (MEF2) activities in hearts of E16.5 Ryr2 +/+ and Ryr2 ADA/ADA mice. A : [ 32 P] incorporation ( left , top) and Coomassie brilliant blue staining ( left , bottom) of GST-HDAC4 fusion protein

    Article Snippet: HDAC phosphorylation and MEF2 activity were determined in E16.5 heart (without atria) lysates as described ( ) using GST-HDAC4 fusion protein (amino acids 419–670) ( ) and Active Motif TransAM MEF2 kit (Carlsbad, CA), respectively.

    Techniques: Histone Deacetylase Assay, Mouse Assay, Staining

    Calix[4]arenes mimic the aromatic cage of PHD fingers. a , schematic showing the disruption of the association of a PHD finger ( orange ) with the histone H3K4me3 tail by calixarene ( yellow ). b , distances between the aromatic groups in the methyllysine-binding

    Journal: The Journal of Biological Chemistry

    Article Title: Molecular Insights into Inhibition of the Methylated Histone-Plant Homeodomain Complexes by Calixarenes *

    doi: 10.1074/jbc.M115.669333

    Figure Lengend Snippet: Calix[4]arenes mimic the aromatic cage of PHD fingers. a , schematic showing the disruption of the association of a PHD finger ( orange ) with the histone H3K4me3 tail by calixarene ( yellow ). b , distances between the aromatic groups in the methyllysine-binding

    Article Snippet: FLAG-MLL5 was labeled with a mouse monoclonal anti-FLAG antibody (Sigma-Aldrich, catalog no. F1804), and histone H3K4me3 was labeled with a rabbit polyclonal antibody (Active Motif, catalog no. 39159) diluted 1:2000 and 1:500, respectively, in the diluent provided with the kit.

    Techniques: Binding Assay

    Calixarene (1) impairs the interaction of the ING2 PHD finger with H3K4me3 but does not disturb the interaction with H3K4me2. a , binding of the GST-fusion ING2 PHD finger to the indicated biotinylated histone peptides in the absence or presence of the

    Journal: The Journal of Biological Chemistry

    Article Title: Molecular Insights into Inhibition of the Methylated Histone-Plant Homeodomain Complexes by Calixarenes *

    doi: 10.1074/jbc.M115.669333

    Figure Lengend Snippet: Calixarene (1) impairs the interaction of the ING2 PHD finger with H3K4me3 but does not disturb the interaction with H3K4me2. a , binding of the GST-fusion ING2 PHD finger to the indicated biotinylated histone peptides in the absence or presence of the

    Article Snippet: FLAG-MLL5 was labeled with a mouse monoclonal anti-FLAG antibody (Sigma-Aldrich, catalog no. F1804), and histone H3K4me3 was labeled with a rabbit polyclonal antibody (Active Motif, catalog no. 39159) diluted 1:2000 and 1:500, respectively, in the diluent provided with the kit.

    Techniques: Binding Assay

    Calixarenes display a selectivity for methylated histone peptides. a , representative FDA binding curves for the indicated peptides and calixarenes. b , dissociation constants measured by FDA. Selectivity for H3K4me3 determined by the ratio of H3K4me3

    Journal: The Journal of Biological Chemistry

    Article Title: Molecular Insights into Inhibition of the Methylated Histone-Plant Homeodomain Complexes by Calixarenes *

    doi: 10.1074/jbc.M115.669333

    Figure Lengend Snippet: Calixarenes display a selectivity for methylated histone peptides. a , representative FDA binding curves for the indicated peptides and calixarenes. b , dissociation constants measured by FDA. Selectivity for H3K4me3 determined by the ratio of H3K4me3

    Article Snippet: FLAG-MLL5 was labeled with a mouse monoclonal anti-FLAG antibody (Sigma-Aldrich, catalog no. F1804), and histone H3K4me3 was labeled with a rabbit polyclonal antibody (Active Motif, catalog no. 39159) diluted 1:2000 and 1:500, respectively, in the diluent provided with the kit.

    Techniques: Methylation, Binding Assay

    Calixarenes block the histone-binding activity of the MLL5 PHD finger in vitro and in vivo . a , crystal structure of the MLL5 PHD finger in complex with H3K4me3 peptide (PDB code 2LV9 ). The aromatic cage residues are colored green. b , superimposed 1 H,

    Journal: The Journal of Biological Chemistry

    Article Title: Molecular Insights into Inhibition of the Methylated Histone-Plant Homeodomain Complexes by Calixarenes *

    doi: 10.1074/jbc.M115.669333

    Figure Lengend Snippet: Calixarenes block the histone-binding activity of the MLL5 PHD finger in vitro and in vivo . a , crystal structure of the MLL5 PHD finger in complex with H3K4me3 peptide (PDB code 2LV9 ). The aromatic cage residues are colored green. b , superimposed 1 H,

    Article Snippet: FLAG-MLL5 was labeled with a mouse monoclonal anti-FLAG antibody (Sigma-Aldrich, catalog no. F1804), and histone H3K4me3 was labeled with a rabbit polyclonal antibody (Active Motif, catalog no. 39159) diluted 1:2000 and 1:500, respectively, in the diluent provided with the kit.

    Techniques: Blocking Assay, Binding Assay, Activity Assay, In Vitro, In Vivo

    Calixarene (1) disrupts binding of MLL5 to H3K4me3 in vivo . a , C2C12 cells expressing FLAG-MLL5 were fixed and stained with anti-FLAG and anti-H3K4me3 antibodies and with secondary antibodies conjugated with Duolink PLA probes and Duolink PLA detection

    Journal: The Journal of Biological Chemistry

    Article Title: Molecular Insights into Inhibition of the Methylated Histone-Plant Homeodomain Complexes by Calixarenes *

    doi: 10.1074/jbc.M115.669333

    Figure Lengend Snippet: Calixarene (1) disrupts binding of MLL5 to H3K4me3 in vivo . a , C2C12 cells expressing FLAG-MLL5 were fixed and stained with anti-FLAG and anti-H3K4me3 antibodies and with secondary antibodies conjugated with Duolink PLA probes and Duolink PLA detection

    Article Snippet: FLAG-MLL5 was labeled with a mouse monoclonal anti-FLAG antibody (Sigma-Aldrich, catalog no. F1804), and histone H3K4me3 was labeled with a rabbit polyclonal antibody (Active Motif, catalog no. 39159) diluted 1:2000 and 1:500, respectively, in the diluent provided with the kit.

    Techniques: Binding Assay, In Vivo, Expressing, Staining, Proximity Ligation Assay

    Calixarenes exhibit selectivity for the PHD fingers of JARID1A and MLL1. a , the crystal structure of the JARID1A PHD3 finger in complex with H3K4me3 peptide (PDB code 3GL6 ). The aromatic cage residues are colored yellow. b , superimposed 1 H, 15 N HSQC spectra

    Journal: The Journal of Biological Chemistry

    Article Title: Molecular Insights into Inhibition of the Methylated Histone-Plant Homeodomain Complexes by Calixarenes *

    doi: 10.1074/jbc.M115.669333

    Figure Lengend Snippet: Calixarenes exhibit selectivity for the PHD fingers of JARID1A and MLL1. a , the crystal structure of the JARID1A PHD3 finger in complex with H3K4me3 peptide (PDB code 3GL6 ). The aromatic cage residues are colored yellow. b , superimposed 1 H, 15 N HSQC spectra

    Article Snippet: FLAG-MLL5 was labeled with a mouse monoclonal anti-FLAG antibody (Sigma-Aldrich, catalog no. F1804), and histone H3K4me3 was labeled with a rabbit polyclonal antibody (Active Motif, catalog no. 39159) diluted 1:2000 and 1:500, respectively, in the diluent provided with the kit.

    Techniques:

    Calixarene (1) inhibits binding of the ING2 PHD finger to H3K4me3. a , molecular model depicting disassociation of the ING2 PHD-H3K4me3 complex because of (1). The crystal structure of the H3K4me3-bound ING2 PHD finger (PDB code 2G6Q ) is shown as a ribbon

    Journal: The Journal of Biological Chemistry

    Article Title: Molecular Insights into Inhibition of the Methylated Histone-Plant Homeodomain Complexes by Calixarenes *

    doi: 10.1074/jbc.M115.669333

    Figure Lengend Snippet: Calixarene (1) inhibits binding of the ING2 PHD finger to H3K4me3. a , molecular model depicting disassociation of the ING2 PHD-H3K4me3 complex because of (1). The crystal structure of the H3K4me3-bound ING2 PHD finger (PDB code 2G6Q ) is shown as a ribbon

    Article Snippet: FLAG-MLL5 was labeled with a mouse monoclonal anti-FLAG antibody (Sigma-Aldrich, catalog no. F1804), and histone H3K4me3 was labeled with a rabbit polyclonal antibody (Active Motif, catalog no. 39159) diluted 1:2000 and 1:500, respectively, in the diluent provided with the kit.

    Techniques: Binding Assay