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H3k27me3

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H3K27me3 is a histone modification specific to the trimethylation of lysine 27 on histone H3. It is a widely studied epigenetic mark associated with transcriptional repression and heterochromatin formation. This product provides a tool for detecting and analyzing this histone modification.

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Lab products found in correlation

H3k4me3, by Diagenode (13 mentions) H3k27ac, by Diagenode (10 mentions) H3k4me1, by Diagenode (8 mentions) H3k9me3, by Abcam (6 mentions) H3k9me3, by Diagenode (4 mentions) Paxillin, by Merck Group (4 mentions) H3k4me3, by Abcam (4 mentions) Hiseq 4000, by Illumina (3 mentions) Thinprep, by Hologic (2 mentions) Cc1 basic buffer, by Roche (2 mentions) H3k36me3, by Diagenode (2 mentions) Chip it high sensitivity kit, by Active Motif (2 mentions) H3k27me3, by Active Motif (2 mentions) Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions) Nitrocellulose membrane, by Cytiva (2 mentions) Irdye secondary antibody, by LI COR (2 mentions) Nupage sample reducing agent, by Thermo Fisher Scientific (2 mentions) Nupage mops sds running buffer, by Thermo Fisher Scientific (2 mentions) E cadherin, by BD (2 mentions) Lamin b1, by Abcam (2 mentions)

Close spelling variants of this product

Anti-H3K27me3 (23 citations) Anti-H3K27me3: C15410195 (3 citations) Anti-H3K27me3 antibody (3 citations) α-H3K27me3 (2 citations) H3K27me3 antibody (2 citations) H3K27me3 C15410195 (2 citations)

40 protocols using h3k27me3

1

ChIP Assay for Histone Modifications

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Chromatin immunoprecipitation (ChIP) was done as described in Lindeman et al. [49 (link)]. Antibodies, H3K4me3 (cat #. C15410003), H3K27me3 (cat # C15410069) and H3-pan (cat# C15310135) were purchased from Diagenode, Belgium. ChIP-primers for cebpa locus were designed in Primer3 v4.0.0 [50 (link), 51 (link)] and purchased from ThermoFisher Scientific (Additional file 1: Table S1). The precipitated DNA was quantified with qPCR (SYBR green, Roche) using 2.5 µL ChIP DNA as input template. Both ChIP and the qPCR experiments were performed in duplicates.
den Broeder M.J., Ballangby J., Kamminga L.M., Aleström P., Legler J., Lindeman L.C, & Kamstra J.H. (2020). Inhibition of methyltransferase activity of enhancer of zeste 2 leads to enhanced lipid accumulation and altered chromatin status in zebrafish. Epigenetics & Chromatin, 13, 5.
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2

Histone H3 Lysine 27 Trimethylation Immunohistochemistry

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Cells were centrifugated during 10 min at 377 g and fixed in an alcohol based fixative solution Thinprep® (Hologic) during 15 min. After centrifugated during 5 min at 377 g, the cells were resuspended in 10 ml of Epredia™ Gel (Richard-Allan Scientific™ HistoGel™). The gel was hardened during 15 min at 4 °C and the corresponding blocks were dehydrated and embedded in paraffin. 3 µm-sections were immunostained using an antibody anti-histone H3 containing the trimethylated lysine 27 (H3K27me3) (Diagenode). Heat induced antigen retrieval was done using CC1 basic buffer (Ventana). Staining was performed using DAB Ultraview detection system (Ventana).
Durand S., Bruelle M., Bourdelais F., Bennychen B., Blin-Gonthier J., Isaac C., Huyghe A., Martel S., Seyve A., Vanbelle C., Adrait A., Couté Y., Meyronet D., Catez F., Diaz J.J., Lavial F., Ricci E.P., Ducray F, & Gabut M. (2023). RSL24D1 sustains steady-state ribosome biogenesis and pluripotency translational programs in embryonic stem cells. Nature Communications, 14, 356.
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3

ChIP-seq of Primary Human Tumors

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ChIP-seq for three primary human tumors was done as described previously11 (link). Chromatin was prepared from 20 to 50 sections (25 µm each) of snap-frozen tumors obtained by microtome sectioning. The following antibodies were used: H3K4me3 (1 µg/ChIP; Diagenode, C15410003-50), H3K27me3 (1 µg/ChIP; Diagenode, C15410195), H3K4me1 (1 µg/ChIP; Diagenode, C15410194), H3K27ac (1 µg/ChIP; Diagenode, C15410196), H3K56ac (4 µl/ChIP; Active Motif, 39281), H3K9me3 (1 µg/ChIP; Diagenode, C15410193), and H3K36me3 (1 µg/ChIP; Diagenode, C15410192). Library preparation for ChIP DNA and input control DNA was performed using the NEBNext Ultra kit (New England Biolabs, E7370S/L) following the manufacturer’s instructions. Quality control for the final libraries was done by measuring the DNA concentration with the Qubit dsDNA HS assay (Life Technologies, Q32851) on Qubit 2.0 Fluorometer (Life Technologies, Q32866) and by determining library fragment sizes with the Experion DNA 1K Analysis kit (Bio-Rad, 700-7107) on the Experion Automated Electrophoresis Station (Bio-Rad, 701-7000). Libraries were sequenced on Illumina HiSeq 2000/2500 machines.
Sheffield N.C., Pierron G., Klughammer J., Datlinger P., Schönegger A., Schuster M., Hadler J., Surdez D., Guillemot D., Lapouble E., Freneaux P., Champigneulle J., Bouvier R., Walder D., Ambros I.M., Hutter C., Sorz E., Amaral A.T., de Álava E., Schallmoser K., Strunk D., Rinner B., Liegl-Atzwanger B., Huppertz B., Leithner A., de Pinieux G., Terrier P., Laurence V., Michon J., Ladenstein R., Holter W., Windhager R., Dirksen U., Ambros P.F., Delattre O., Kovar H., Bock C, & Tomazou E.M. (2017). DNA methylation heterogeneity defines a disease spectrum in Ewing sarcoma. Nature medicine, 23(3), 386-395.
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4

CUT&RUN Profiling of Epigenetic Marks

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CUT&RUN experiments were performed as previously described [20 (link)]. For each condition, 100,000 cells were incubated overnight with (1:100) dilution of the following antibodies, H3K27ac (#C15410174, Diagenode), H3K4me1 (#C15410194, Diagenode), H3K4me3 (#C15410003, Diagenode), H3K27me3 (#C15410069, Diagenode), and (1:50) delusion of RUNX1/ETO (#C15310197, Diagenode), RUNX1 (#ab35962; Abcam), CBFB (#C15310002, Diagenode), and Rabbit IgG (#C15410206, Diagenode). The nuclease pAG/MNase (addgene #123461) was produced and purified in-house. Libraries were constructed from released DNA and subjected to paired-end Illumina sequencing (2 × 150 cycle).
Issa H., Swart L.E., Rasouli M., Ashtiani M., Nakjang S., Jyotsana N., Schuschel K., Heuser M., Blair H, & Heidenreich O. (2023). Nanoparticle-mediated targeting of the fusion gene RUNX1/ETO in t(8;21)-positive acute myeloid leukaemia. Leukemia, 37(4), 820-834.
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5

Chromatin Immunoprecipitation Sequencing

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BMI1, H3K4me3 and H3K27me3 ChIPs were performed using ChIP-IT High Sensitivity kit (Active Motif) following the manufacturer’s instructions. Briefly, 1 × 106 cells were fixed with the formaldehyde-based fixing solution for 15 min at room temperature and lysed with provided lysis solution supplemented with protease inhibitors. Next, nuclei pellets were lysed and chromatin sonicated with Bioruptor Plus sonication device (Diagenode) to obtain DNA fragments within the recommended 200–1200-bp range. In total, 25 µg or 10 µg of sheared chromatin was then incubated with 4 µg of antibody against BMI1 (BMI1, clone AF27, Active Motif), H3K27me3 (Diagenode) or H3K4me3 (Diagenode) overnight at 4 °C with rotation. Following incubation with Protein G agarose beads, bound chromatin was washed, eluted and purified following the manufacturer’s protocols. Validation by qPCR-ChIP on target genes was done before proceeding to sequencing. ChIPed DNA was end-repaired, A-tailed and adapter-ligated before size selection and amplification. The obtained libraries were QC’ed and multiplexed before 75-bp paired-end sequencing on HiSeq4000 (Illumina).
Badodi S., Pomella N., Zhang X., Rosser G., Whittingham J., Niklison-Chirou M.V., Lim Y.M., Brandner S., Morrison G., Pollard S.M., Bennett C.D., Clifford S.C., Peet A., Basson M.A, & Marino S. (2021). Inositol treatment inhibits medulloblastoma through suppression of epigenetic-driven metabolic adaptation. Nature Communications, 12, 2148.
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6

Western Blot Analysis of Cellular Markers

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Cell lysis was carried out with RIPA lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS) supplemented with FPIC (Fast Protease Inhibitors cocktail; Sigma-Aldrich; Cat#: S8830-20TAB). The lysates were reduced in NuPAGE LDS Sample Buffer (Thermo Fisher; Cat#: NP0007) supplemented with NuPAGE Sample Reducing Agent (Thermo Fisher; Cat#: NP0009), separated by polyacrylamide gel in NuPAGE MOPS SDS Running Buffer (Thermo Fisher; Cat#: NP0001) and transferred into nitrocellulose membrane (Amersham; Cat#: 10600007). Membranes were blocked in 5% milk powder in PBST Buffer (1× PBS, 0.2% Tween 20) and incubated overnight at 4°C with the primary antibodies against SETDB1 (Abcam; Cat#: ab107225), H3 (Santa Cruz Biotechnology; Cat#: sc-8654), H3K9me3 (Abcam; Cat#: ab8898), H3K27me3 (Diagenode; Cat#: C15410069), Lamin A/C (Proteintech; Cat#: 10298-1-AP), Lamin B1 (Abcam; Cat#: ab16048), E-cadherin (BD Transduction Laboratory; Cat#: 610181), Paxillin (Millipore; Cat#: 05-417), β-actin (Sigma-Aldrich; Cat#: A5441). Membranes were incubated with the appropriate LI-COR IRDye secondary antibody (LI-COR Biosciences GmbH) and revealed by Odyssey Fc imaging system (LI-COR Biosciences GmbH). The images obtained from the slot blot assay were analyzed with the Image Studio Lite 5.2.5 software.
Zakharova V.V., Magnitov M.D., Del Maestro L., Ulianov S.V., Glentis A., Uyanik B., Williart A., Karpukhina A., Demidov O., Joliot V., Vassetzky Y.S., Mège R.M., Piel M., Razin S.V, & Ait-Si-Ali S. (2022). SETDB1 fuels the lung cancer phenotype by modulating epigenome, 3D genome organization and chromatin mechanical properties. Nucleic Acids Research, 50(8), 4389-4413.
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7

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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8

Western Blot Analysis of Chromatin Proteins

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Loading buffer (Lane Marker Reducing Sample Buffer
Thermo-Scientific) was added to 5 μg or 1 μg of nuclear or
chromatin protein respectively and the sample denatured at 95° C for
5 min, spun briefly and kept on ice. The samples and a molecular weight
marker (Spectra Multicolor Broad Range Protein Ladder, Invitrogen) were
loaded in a 4–20 % polyacrylamide Amersham ECL Gel (GE Healthcare,
UK). The run was performed at a constant 160 mV in Tris – Glycine
Running Buffer. The electrophoresed proteins were transferred to a
nitrocellulose membrane (Pierce) in a Tris-Glycine-Ethanol Transfer Buffer,
by application of an electrical field of 100 mV for 45 min. Then they were
incubated in blocking solution.
The membrane was incubated in a solution composed of primary
antibodies in T-TBS (BAF47 1:1000, 612110 BD Biosciences; SMARCA4 1:1000,
sc-10760 SantaCruz, H3K27me3 1:20000, C15410196 Diagenode; H3K27ac 1:20000,
C15410196 Diagenode; H3 1:20000, ab1791 Abcam; B- actin:20000, ab49900
Abcam; Anti-Mouse IgG 1:10000, ab6728 Abcam; Anti-Rabbit IgG 1:10000,
ab205718 Abcam; ) for 1 hour at room temperature. It was washed three times
in a solution of T-TBS then incubated in a diluted specific secondary
antibody (1:10000) for 1 hour at room temperature and washed in T-TBS as
before. The blot was developed using SuperSignal West Pico Chemiluminescent
Substrate (Pierce) and imaged using ChemiDoc XRS+ (Biorad).
Erkek S., Johann P.D., Finetti M.A., Drosos Y., Chou H.C., Zapatka M., Sturm D., Jones D.T., Korshunov A., Rhyzova M., Wolf S., Mallm J.P., Beck K., Witt O., Kulozik A.E., Frühwald M.C., Northcott P.A., Korbel J.O., Lichter P., Eils R., Gajjar A., Roberts C.W., Williamson D., Hasselblatt M., Chavez L., Pfister S.M, & Kool M. (2018). Comprehensive analysis of chromatin states in atypical teratoid/rhabdoid tumor identifies diverging roles for SWI/SNF and Polycomb in gene regulation. Cancer cell, 35(1), 95-110.e8.
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9

Immunofluorescence Staining of Stem Cells

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ESCs or MEFs were split onto slides 16 hr before staining at low density (without feeders). Slides were then washed in PBS, fixed with 2% formaldehyde in PBS for 15 min, and permeabilized with 0.4% Triton X-100 in PBS for 5 min. After washing with PBS, the slides were blocked for 30 min in 0.2% fish gelatin (Sigma) in PBS and incubated for 2 hr with primary antibody (diluted in 0.2% fish gelatin and 5% normal goat serum). Slides were washed three times in 0.2% fish gelatin and incubated for 2 hr with Alexa Fluor conjugated secondary antibody (Life Technologies). After two washes in fish gelatin and two washes in PBS, the slides were stained with DAPI (1 μg/ml) and mounted using mounting media (Dako).
The following primary antibodies were used for IF: H2AK119u1 (1:500, rabbit monoclonal, 8240; Cell Signaling Technology), H3K27me3 (1:500, rabbit polyclonal, pAB-069-050; Diagenode), H3K27me3 (1:1,000, mouse monoclonal, 61017; Active Motif), Flag (1:500, mouse M2 monoclonal; Sigma), H3K9me3 (1:500, rabbit polyclonal 39161; Active Motif), H3K4me3 (1:500, rabbit polyclonal, ab8580; Abcam), HP1α (1:500, mouse monoclonal MAB3584; Millipore), and GFP (1:100, mouse monoclonal, sc-9996; Santa Cruz).
Cooper S., Dienstbier M., Hassan R., Schermelleh L., Sharif J., Blackledge N.P., De Marco V., Elderkin S., Koseki H., Klose R., Heger A, & Brockdorff N. (2014). Targeting Polycomb to Pericentric Heterochromatin in Embryonic Stem Cells Reveals a Role for H2AK119u1 in PRC2 Recruitment. Cell Reports, 7(5), 1456-1470.
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10

Chromatin Immunoprecipitation Assay

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ChIP was performed with 10,000 FACS-sorted cells per IP using Diagenode Low Cell ChIP kit (cat# C01010072) according to manufacturer’s protocol. 1 µg of corresponding antibody from Diagenode was used per IP: H3K4me3 (cat# C15410003-50, lot# A5051-001P), H3K4me1 (cat# C15410194, lot# A1862D), H3K27ac (cat# C15410196, lot# A1723-0041D), H3K27me3 (cat# C15410195, lot# A1811-001P).
Ferguson G.B., Van Handel B., Bay M., Fiziev P., Org T., Lee S., Shkhyan R., Banks N.W., Scheinberg M., Wu L., Saitta B., Elphingstone J., Larson A.N., Riester S.M., Pyle A.D., Bernthal N.M., Mikkola H.K., Ernst J., van Wijnen A.J., Bonaguidi M, & Evseenko D. (2018). Mapping molecular landmarks of human skeletal ontogeny and pluripotent stem cell-derived articular chondrocytes. Nature Communications, 9, 3634.
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