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Mapping polycomb complexes in human and mouse embryonic stem cells (human)
External Reference: GSE13084    
Measurement Type: Transcription Factor Binding (ChIP-Seq) Factors: Immunoprecipitation Antibody
Contact(s): Tarjei Mikkelsen, Bradley E. Bernstein
Curator(s): Emily Merrill
Submitted Date: 12/30/2014

summary

In embryonic stem (ES) cells, bivalent chromatin domains with overlapping repressive (H3 lysine 27 tri-methylation) and activating (H3 lysine 4 tri-methylation) histone modifications mark the promoters of more than 2000 genes. To gain insight into the structure and function of bivalent domains, we mapped key histone modifications and subunits of Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) genomewide in human and mouse ES cells by chromatin immunoprecipitation followed by ultra high-throughput sequencing. We find that bivalent domains can be segregated into two classes: the first occupied by both PRC2 and PRC1 (PRC1-positive) and the second specifically bound by PRC2 (PRC2-only). PRC1-positive bivalent domains appear functionally distinct as they more efficiently retain lysine 27 tri-methylation upon differentiation, show stringent conservation of chromatin state, and associate with an overwhelming number of developmental regulator gene promoters. We also used computational genomics to search for sequence determinants of Polycomb binding. This analysis revealed that the genomewide locations of PRC2 and PRC1 can be largely predicted from the locations, sizes and underlying motif contents of CpG islands. We propose that large CpG islands depleted of activating motifs confer epigenetic memory by recruiting the full repertoire of Polycomb complexes. H3K4me3, H3K27me3, H3K36me3, Ezh2 and Ring1b ChIP-Seq in singlicate from human embyonic stem cells (hES; H9).


Growth Protocol

Embryonic stem cells were cultured as described in (Mikkelsen et al. 2007, PMID 17603471) and (Thomson et al. 1998, PMID 9804556 ). Mouse v6.5 (genotype 129SvJae×C57BL6, male, passages 10–15) ES cells were cultured on fibroblast feeders in DMEM (Sigma) with 15% fetal bovine serum (Hyclone), GlutaMax (Invitrogen), MEM non-essential amino acids (Invitrogen), pen/strep (Invitrogen), ESGRO (Chemicon) and 2-mercaptoethanol (Sigma), incubating at 37°C, 5% CO2 [16]. Prior to harvest, these cells were passaged 2–3 times on feeder-free gelatinized tissue culture plates. Human H9 (female, passage 45) ES cells were cultured as described [58] and at http:/www.WiCell.org. Briefly, the human ES cells were cultivated on irradiated MEFs (strain DR4) in Knockout DMEM (Invitrogen) containing 10% Knockout Serum Replacement (Invitrogen), 10% Plasmanate (Bayer Healthcare), GlutaMax (2 mM), pen/strep, MEM non-essential amino acids (0.1 mM), 10 ng/ml β-FGF (Invitrogen) and 2-mercaptoethanol. Cells were incubated at 37°C, 5% CO2. MEF-free ES cells were used for analysis. MEF-free culture was prepared in the following manner: First, MEFs were depleted at the time of trypsin passaging through brief transfer (thirty minutes) of hES cells onto gelatin-coated plates. MEF-subtracted ES cells were then propagated on plates coated with Matrigel (Invitrogen). ES cells grown on Matrigel were supported with the aforementioned human ES cell medium that had first been conditioned on MEFs for 24 hours. Fresh β-FGF was added to the conditioned medium immediately prior to use.

Treatment Protocol


Extraction Protocol

All ChIP experiments were performed as described in (Mikkelsen et al. 2007, PMID 17603471), except for Suz12 and Ezh2, for which an additional nucleus isolation step was included, as described in (Weinmann et al. 2001, PMID 11564866). ES cells were crosslinked in 1% formaldehyde, lysed and sonicated with either a Branson 250 Sonifier (mouse ES cells) or a Diagenode bioruptor (human ES cells) to obtain chromatin fragments in a size range between 200 and 700 bp. Solubilized chromatin (whole cell lysate or ‘WCE’) was diluted in ChIP dilution buffer (1[ratio]10) and incubated with antibody overnight at 4°C. Protein A sepharose beads (Sigma) were used to capture the antibody-chromatin complex and washed with low salt, LiCl, as well as TE (pH 8.0) wash buffers. Enriched chromatin fragments were eluted at 65°C for 10 min, subjected to crosslink reversal at 65°C for 5 hrs, and treated with Proteinase K (1 mg/ml), before being extracted by phenol-chloroform-isoamyl alcohol, and ethanol precipitated. ChIP DNA was then quantified by Quant-iT Picogreen dsDNA Assay kit (Invitrogen). ChIP experiments for Ezh2 and Suz12 were carried out on nuclear preps. Crosslinked ES cells were incubated in swelling buffer (0.1 M Tris pH 7.6, 10 mM KOAc, 15 mM MgOAc, 1% NP40), on ice for twenty minutes, passed through a 16G needle 20 times and centrifuged to collect nuclei [60]. Isolated nuclei were then lysed, sonicated and immunoprecipitated as described above.

Name

hES_RING1B_ChIPSeq


Organism

Homo sapiens


Cell type

H9


Disease state


Platform

Illumina Genome Analyzer



citations

Genomewide analysis of PRC1 and PRC2 occupancy identifies two classes of bivalent domains. Ku M, Koche RP, Rheinbay E, Mendenhall EM, Endoh M, Mikkelsen TS, Presser A, Nusbaum C, Xie X, Chi AS, et al. PLoS Genet. 2008 Oct; 4(10):e1000242. PMID: 18974828.