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H3k27me2

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H3K27me2 is a histone modification that is associated with gene silencing. It is a specific antibody that can be used to detect the presence of dimethylation on lysine 27 of histone H3.

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Lab products found in correlation

H3k4me2, by Abcam (5 mentions) H3k9me3, by Abcam (4 mentions) H3k27me3, by Merck Group (4 mentions) H3k9me2, by Merck Group (2 mentions) Suv39h1, by Merck Group (2 mentions) H3k9ac, by Active Motif (2 mentions) Chip isolation kit, by Merck Group (2 mentions) H3k4me, by Merck Group (2 mentions) Eset setdb1, by Merck Group (2 mentions) G9a ehmt2 c6h3, by Abcam (2 mentions) H3k4me3, by Merck Group (2 mentions) Ubiquitin, by Abcam (2 mentions) Rna pol 2, by Abcam (2 mentions) Pgc 1α, by Abcam (2 mentions) Atg7 sirna, by Thermo Fisher Scientific (2 mentions) P s6 kinase, by Cell Signaling Technology (2 mentions) Mouse creb sirna, by Thermo Fisher Scientific (2 mentions) Fxr sirna and control sirna, by Horizon Discovery (2 mentions) β tubulin, by Santa Cruz Biotechnology (2 mentions) H3k27me3, by Abcam (2 mentions)

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Anti-H3K27me2 (4 citations)

13 protocols using h3k27me2

1

Chromatin Immunoprecipitation for Studying Histone Modifications

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Antibodies used for immunoprecipitation were purchased from: histone H3 di methyl K27 (H3K27me2, Abcam) and Histone H3 trimethyl K27 (H3K27me3, Diagenode). 10 µg of each antibody or appropriate irrelevant antibody control were used in each ChIP reaction as described previously [18] (link). Primers used for detection of relevant rat genomic sequences were: IFNγ −43 kb sense 5′- aaggtcaagccataacattc-3′ and antisense 5′- cagggatgaacaaggaccag-3′; IFNγ −0.5 kb sense 5′- cttttgtaaccgaacgccttc-3′ and antisense 5′- cttttacttcacaccatttg-3′; IFNγ 0.4 kb sense 5′- tcggtgaggtgttcgttgac-3′ and antisense 5′- aagaatgaaaaccatgaagg-3′ and IFNγ 1.1 kb sense 5′- gagttgagtttatttgtgg-3′ and antisense 5′- ctgtggagttttgttgaatg-3′. Each PCR reaction was performed in triplicate and the analysis was repeated three times from independent ChIP experiments. A signal intensity value for each sample was calculated from the average of the experiments. Average values of eluates were normalized to average values of control antibody sample and expressed as fold enrichment above background (i.e. control antibody) [18] (link).
Wilson C.L., Mann J., Walsh M., Perrugoria M.J., Oakley F., Wright M.C., Brignole C., Di Paolo D., Perri P., Ponzoni M., Karin M, & Mann D.A. (2014). Quiescent Hepatic Stellate Cells Functionally Contribute to the Hepatic Innate Immune Response via TLR3. PLoS ONE, 9(1), e83391.
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2

Chromatin Immunoprecipitation Assay Protocol

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The ChIP assays were performed using a ChIP isolation kit (Millipore, Billerica, MA). We treated 1 × 105 to 5 × 105 murine naïve T cells with 1% formaldehyde to cross-link histones to DNA. The fixed cells were sonicated to yield chromatin fragments of 200–500 base pairs (bp). The antibodies used in the ChIP assays included: H3Ac (Millipore), H4Ac (Upstate Biotechnology, Lake Placid, NY), H3K9Ac (Active Motif, Carlsbad, CA), H3K4Me3 (Millipore), H3K4Me2 (Abcam), H3K4Me (Millipore), H3K9Me2 (Millipore), H3K9Me3 (Abcam) H3K27me2 (Abcam) H3K27me3 (Millipore), p300 (Abcam), PCAF (Abcam), SUV39H1 (Millipore), CBP (Abcam), ESET/SetDB1(Millipore), Ezh2 (Cell Signaling Technology, Beverly, MA), G9a/EHMT2 (C6H3) (Abcam), HP1α (Millipore), HP1β (Active Motif), and HP1γ (Millipore). DNA was recovered by a Chelex 10% slurry and sample boiling method,10 (link) or using the IP-STAR (Diagenode, Denville, NJ) direct chip protocol followed by the IPPURE DNA purification. 1% Pre-enriched chromatin (input) served as the percentage input for sample quantification, confirmed by fold over IgG changes. The UV-exposed 4% agarose gels were imaged, and the digitized images were analyzed using the software VisionWorks (UVP). Protein signals were indexed by measuring their relative mean grey value per area.
Sarmento O.F., Svingen P.A., Xiong Y., Xavier R.J., McGovern D., Smyrk T.C., Papadakis K.A., Urrutia R.A, & Faubion W.A. (2014). A Novel Role for Kruppel-like Factor 14 (KLF14) in T-Regulatory Cell Differentiation. Cellular and Molecular Gastroenterology and Hepatology, 1(2), 188-202.e4.
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3

Transcriptional Regulation of Cellular Pathways

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Antibodies were purchased for FXR (sc-13063), CREB (sc-186), CRTC2 (sc-46272, sc-271912), RNA pol II (sc-9001), NcoR (sc-8994), SMRT (sc-1612), p300 (sc-584), PGC-1α (sc-13067), H3K27-me2 (Abcam 050851), H3K4-me2 (Abcam 8580), ubiquitin (sc-9133), lamin A (sc-20680), β-tubulin (sc-5274), and actin (sc-1616) from Santa Cruz Biotechnology; for p(S133)-CREB (#9198S), LC3 (#4108), p62 (#5114), S6 kinase (#9202), p-S6 kinase (#9208), and ATG7 (#2631) from Cell Signaling; and for p(S171)-CRTC2 (bs-3415R) from Bioss USA. Mouse CREB siRNA was purchased from Thermo Scientific, FXR siRNA and control siRNA from Dharmacon, and Atg7 siRNA from Life Technologies (AM16708).
Seok S., Fu T., Choi S.E., Li Y., Zhu R., Kumar S., Sun X., Yoon G., Kang Y., Zhong W., Ma J., Kemper B, & Kemper J.K. (2014). Transcriptional regulation of autophagy by an FXR/CREB axis. Nature, 516(7529), 108-111.
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4

Western Blot Analysis of Histone Modifications

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Whole-cell extracts were prepared, and the protein concentration of samples was determined by using a Bradford DC assay kit (Bio-Rad). Whole-cell extracts from samples of interest were then fractionated by electrophoresis through a 9% sodium dodecyl sulfate-polyacrylamide gel. Gels were run at 100 V for 1.5 hr before transfer onto nitrocellulose. After thr blockade of nonspecific protein binding, nitrocellulose blots were incubated for 1 hr with primary antibodies diluted in Tris-buffered saline (TBS)/Tween 20 (0.075%) containing 5% bovine serum albumin. Rabbit polyclonal antibody-recognizing EZH2 was used at 1/500 dilution (Active Motif, catalog no. 39103), H3K27me2 (Abcam ab24684), H3K27me3 (Abcam, ab6002), H3K4me3 (Abcam, ab8580), and β-actin at 1/1000 dilution (Sigma). After incubation with primary antibodies, blots were washed three times in TBS/Tween 20 before incubation for 1 hr in appropriate horseradish peroxidase-conjugated secondary antibody. After extensive washing in TBS/Tween 20, the blots were processed with distilled water for detection of antigen by using the enhanced chemiluminescence system (Amersham Biosciences).
Zeybel M., Luli S., Sabater L., Hardy T., Oakley F., Leslie J., Page A., Moran Salvador E., Sharkey V., Tsukamoto H., Chu D.C., Singh U.S., Ponzoni M., Perri P., Di Paolo D., Mendivil E.J., Mann J, & Mann D.A. (2017). A Proof-of-Concept for Epigenetic Therapy of Tissue Fibrosis: Inhibition of Liver Fibrosis Progression by 3-Deazaneplanocin A. Molecular Therapy, 25(1), 218-231.
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5

Immunohistochemistry of Epigenetic Markers

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IHC was performed using a Vectastain Elite kit (AK-5001, Vector Laboratories, Burlingame, CA) as described previously34 (link),42 (link),43 (link). Primary antibodies included the human-specific Ki67 (1:50) (Thermo Fisher, Waltham, MA), and rabbit anti-EZH2 (1:50) (Thermo Fisher, Waltham, MA) and H3K27me2 (1:100) (ABCAM INC, Cambridge, CA), and H3K27me3(1:50) (Thermo Fisher, Waltham, MA).
Mackenzie A., Hallam N., Mitchell E, & Beattie T. (1999). Near patient testing for respiratory syncytial virus in paediatric accident and emergency: prospective pilot study. BMJ : British Medical Journal, 319(7205), 289-290.
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6

Comprehensive Transcriptome and Epigenome Analysis

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CDNA synthesis, QRT-PCR, and data analysis was performed as described previously [39 (link)]. QRT-PCR primers were designed and selected for optimal efficiency based on their performance with a standard curve of cDNA template. QRT-PCR was performed with at least three replicates. Primer sequences are listed in Supplementary Table 1. Antibodies used for western blotting were: H3K4me3 (Active Motif 39159), H3K4me2 (Millipore 07-030), H3K4me1 (Abcam ab8895), Histone H3 (Abcam ab1791), WDR5 (Bethyl A302-430A), KU70 (Santa Cruz sc9033), H3K27me3 (Millipore 07-449), H3K27me2 (Abcam ab24684), H3K27me1 (Millipore 07-448), H2AK119ub (Millipore 05-678), RNF2 (a gift from H. Koseki), CBX4 (Santa Cruz sc19299), and PCNA (Santa Cruz sc56).
Putiri E.L., Tiedemann R.L., Liu C., Choi J.H, & Robertson K.D. (2014). Impact of human MLL/COMPASS and polycomb complexes on the DNA methylome. Oncotarget, 5(15), 6338-6352.
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7

Chromatin Immunoprecipitation (ChIP) Assay

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ChIP assays were performed using a ChIP isolation kit (Millipore). 1 × 105 to 5 × 105 murine naïve T cells were treated with 1% formaldehyde to cross-link histones to DNA. Fixed cells were sonicated to yield chromatin fragments of 200–500 bp. Antibodies used in the ChIP assays included: H3Ac (Millipore), H4Ac (upstate), H3K9Ac (Active Motif), H3K4Me3 (Millipore), H3K4Me2 (Abcam), H3K4Me (Millipore), H3K9Me2 (Millipore), H3K9Me3 (Abcam) H3K27me2 (Abcam) H3K27me3 (Millipore), p300 (Abcam), PCAF (Abcam), SUV39H1(Millipore) , CBP (Abcam), ESET/SetDB1(Millipore), Ezh2 (Cell Signalling), G9a/EHMT2 (C6H3) (Abcam), HP1α (millipore), HP1β (Active Motif), and HP1γ (Millipore ). DNA was recovered by Chelex 10% slurry and sample boiling method (Nature protocols), or utilizing the IP-STAR (diagenode) direct chip protocol followed by the IPPURE DNA purification. 1% Pre-enriched chromatin (Input) served as percent Input for sample quantification, confirmed by fold over IgG changes. UV exposed 4% agarose gels were imagined and digitized images were analyzed using the software visionworks (UVP). Protein signals were indexed by measuring their relative mean grey value per area.
Sarmento O.F., Svingen P.A., Xiong Y., Xavier R.J., McGovern D., Smyrk T.C., Papadakis K.A., Urrutia R.A, & Faubion W.A. (2014). A Novel Role for Kruppel-like Factor 14 (KLF14) in T-Regulatory Cell Differentiation. Cellular and Molecular Gastroenterology and Hepatology, 1(2), 188-202.e4.
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8

Antibody-based Epigenetic Profiling Protocol

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Antibodies against D2HGDH (Proteintech, 13895–1-AP), L2HGDH (Proteintech, 15707–1-AP), Flag (Sigma-Aldrich, F1804), Tubulin (Neomarker, MS-581-P1), β-actin (Genescript, A00702), IDH1 (Epitomics, 8057–1), H3 (CST, 4499), H3K4me2 (Millipore, 07–030), H3K9me2 (Abcam, ab1220), H3K9me3 (Abcam, ab8898), H3K27me2 (Abcam, ab1298), H3K27me3 (Millipore, 17–622), H3K79me2 (Abcam, ab3594), 5 hmC (Active Motif, 39769), Collagen Type IV (Rockland 600–401-106), HIF-1α (BD, 610958) and Endostatin (Abcam, ab64569) were purchased commercially.
Ma S., Jiang B., Deng W., Gu Z.K., Wu F.Z., Li T., Xia Y., Yang H., Ye D., Xiong Y, & Guan K.L. (2015). D-2-hydroxyglutarate is essential for maintaining oncogenic property of mutant IDH-containing cancer cells but dispensable for cell growth. Oncotarget, 6(11), 8606-8620.
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9

ChIP Analysis of Histone Modifications and Transcription Factors

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The antibodies utilized in ChIP were HitoneH3Ac (Millipore, 06-599), H3K27me2 (Abcam, ab24684), GR (CST, 3660), Sp1 (Santa Cruz, SC-420), and p53 (Santa Cruz, SC-126). ChIP was carried out using a ChIP kit (Active Motif) according to the manufacturer’s specifications, and then the precipitated material was used in quantitative analysis. ChIP was performed with the following CpG island-specific primer pairs: ChIP primer set for human cells (forward 5′-GCAAAGAAAGCTCAGCATAGTATC-3′, reverse 5′-GAGCAGAGAGTGCTAAGTGGAC-3′), ChIP-TF primer set for human cells (forward 5′-CTGTGCACGGGAGAGAGAG-3′, reverse 5′-CTATTAGGTCCTCTGCCGGG-3′), and the ChIP primer set for mouse cells (forward 5′-GATGGGGTACAGATGGGCAT-3′, reverse 5′-GTCCCCGCTCTTCACTCTG -3′).
Kim H.K., Jeong Y.J., Song I.S., Noh Y.H., Seo K.W., Kim M, & Han J. (2017). Glucocorticoid receptor positively regulates transcription of FNDC5 in the liver. Scientific Reports, 7, 43296.
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10

Histone Demethylase Activity Assay

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For in vitro demethylation assays, SlJMJ4-GST fusion proteins were purified using glutathione sepharose 4B (GE Healthcare). Afterwards, histone demethylase activity was analyzed as previously described [18 (link)]. In brief, the purified GST-tagged SlJMJ14 (4.0 μg) was incubated with calf thymus histones (Sigma) in a reaction buffer containing 150 mM NaCl, 80 μM Fe(NH4)2(SO4)2, 50 mM Tris–HCl (pH 7.0), 1 mM α-KG, and 2 mM ascorbic acid for 6 h at 37°C. The reaction was terminated with 10 μM EDTA and subjected to western blotting analysis. For in vitro demethylation assays, histone proteins were extracted from 2-month-old leaves of SlJMJ4-OE and WT plants with the EpiQuik Total Histone Extraction Kit (Epigentek, Farmingdale, NY, USA) and analyzed by western blotting. The antibodies used in this experiment were from Abcam: H3K4me1 (ab176877, 1:3000 dilution), H3K4me2 (ab11946, 1:3000 dilution), H3K4me3 (ab8580, 1:3000 dilution), H3K9me1 (ab9045, 1:3000 dilution), H3K9me2 (ab1220, 1:1000 dilution), H3K9me3 (ab8898, 1:1000 dilution), H3K27me1 (ab115068, 1:3000 dilution), H3K27me2 (ab24684, 1:3000 dilution), H3K27me3 (ab6002, 1:3000 dilution), H3K36me1 (ab176920, 1:3000 dilution), H3K36me2 (ab176921, 1:3000 dilution), H3K36me3 (ab9050, 1:3000 dilution), and H3 (ab1791, 1:5000). H3 was used as a loading control.
Ding X., Zhang D., Gu D., Li Z., Liang H., Zhu H., Jiang Y, & Duan X. (2022). The histone H3K27 demethylase SlJMJ4 promotes dark- and ABA-induced leaf senescence in tomato. Horticulture Research, 9, uhab077.
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