H2A.Z landscapes and dual modifications in pluripotent and multipotent stem cells underlie complex genome regulatory functions
© Ku et al.; licensee BioMed Central Ltd. 2012
Received: 30 April 2012
Accepted: 3 October 2012
Published: 3 October 2012
The histone variant H2A.Z has been implicated in nucleosome exchange, transcriptional activation and Polycomb repression. However, the relationships among these seemingly disparate functions remain obscure.
We mapped H2A.Z genome-wide in mammalian ES cells and neural progenitors. H2A.Z is deposited promiscuously at promoters and enhancers, and correlates strongly with H3K4 methylation. Accordingly, H2A.Z is present at poised promoters with bivalent chromatin and at active promoters with H3K4 methylation, but is absent from stably repressed promoters that are specifically enriched for H3K27 trimethylation. We also characterized post-translational modification states of H2A.Z, including a novel species dually-modified by ubiquitination and acetylation that is enriched at bivalent chromatin.
Our findings associate H2A.Z with functionally distinct genomic elements, and suggest that post-translational modifications may reconcile its contrasting locations and roles.
KeywordsAcetylation bivalent ChIP-Seq H2A.Z mass spectrometry; ubiquitination
Pluripotent embryonic stem (ES) cells are characterized by a plastic epigenome conducive to self-renewal and broad differentiation potential. Histones and chromatin proteins in ES cells are subject to relatively rapid turnover [1–3]. This dynamic exchange is thought to maintain an accessible and transcriptionally competent state [4, 5]. During development, this initially permissive chromatin configuration becomes restricted as cells progressively commit to specific lineages.
Pluripotent chromatin is distinguished by characteristic post-translational histone modifications. Bivalent domains that contain 'active' H3 lysine 4 trimethylation (H3K4me3) and 'repressive' H3 lysine 27 trimethylation (H3K27me3) are prevalent in ES cells. Bivalent domains and associated Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) silence developmental loci while maintaining their potential for future activation . In fact, some of these loci may already be engaged by initiating RNA polymerase II (RNAPII) . During lineage specification, bivalent domains often resolve into monovalent domains enriched for either modification in accordance with gene expression. Developmental genes that are not expressed within the relevant lineage often retain H3K27me3 domains .
Replication-independent histone deposition is of particular interest as it is targeted to DNA sequences under active regulation [8, 9]. Rapid nucleosome turnover is a general feature of promoters and epigenetic regulatory elements in yeast  and in fly . In flies and mammals, nucleosome-exchange hotspots, including promoters, sites of transcriptional initiation and transcription factor (TF) binding sites, are also enriched for the histone variant H3.3 . In mammals, H3.3 can coexist with H2A.Z in the same nucleosome, and these double-variant-containing nucleosomes represent the most labile fraction of the accessible active promoters, enhancers and putative insulators . H2A.Z, an evolutionarily conserved H2A variant, has been implicated in multiple functions. H2A.Z localizes to transcription start sites (TSSs) where it frequently flanks nucleosome-deficient regions [14, 15]. This variant is also associated with other genomic sites undergoing histone exchange, including intergenic CCCTC-binding factor (CTCF) binding sites in mammals and boundary elements in yeast [8, 13, 15]. H2A.Z-containing nucleosomes are unusually susceptible to nuclease digestion and stringent ionic conditions [16, 17], and it has been speculated that this structural instability is because of amino acid substitutions at the interface between H2A.Z and H3/H4 . Overall, these findings suggest that H2A.Z indexes genomic regions of specific regulatory functions for rigorous nucleosome disassembly and reassembly. That this variant is also essential for mammalian development reinforces the significance of chromatin dynamics to genome regulation [19, 20].
In addition to its pervasive roles at TSSs and active regulatory elements, H2A.Z has also been linked to Polycomb regulation. A microarray-based chromatin immunoprecipitation (ChIP-chip) analysis in ES cells found that H2A.Z associates exclusively with silent promoters bound by PRC2 . Upon differentiation, H2A.Z was found to relocate to active TSSs. These findings suggested that H2A.Z plays a distinct role in ES cells that is tightly linked to Polycomb repression. However, this study relied primarily on promoter microarrays that are not comprehensive , and antibody reagents that may not account for potential modifications . Moreover, the findings are not entirely consistent with those of H2A.Z studies carried out in other cell models and in other organisms.
To clarify the distribution and potential functions of H2A.Z in ES cells, we used ChIP coupled with high-throughput sequencing (ChIP-Seq) to query the localization of this variant in mouse and human ES cells, and in lineage-restricted progenitors. We found that H2A.Z is ubiquitously deposited at promoters, putative enhancers and other intergenic regulatory elements marked by H3K4 methylation. H2A.Z is also deposited at K27me3 regions/PRC2 binding sites, but it is restricted to those sites that have coexisting H3K4 methylation, and thus constitute bivalent domains. Notably, we found that bivalent chromatin is enriched for a novel population of H2A.Z simultaneously modified by N-terminal acetylation and C-terminal ubiquitination. We propose that distinct modification states enable H2A.Z to facilitate regulation of bivalent PRC2 targets as well as to act at a diversity of other histone exchanging elements in mammalian genomes.
Results and discussion
Genomewide H2A.Z enrichment at active and bivalent promoters
Principles of H2A.Z occupancy conserved between species and cell states
We used the same ChIP-Seq procedures and antibody to profile H2A.Z in human ES (hES) cells (Figure 1b,d, hES cells). We again compared bivalent, H3K4me3-only and no-mark promoters, from previously described hES cell ChIP-Seq data . The H2A.Z signal in hES cells shows a strong bimodal pattern of intensity at H3K4me3-only and bivalent TSSs (Figure 1b,d, hES cells; Figure 2b); an identical promoter architecture to that observed in mES cells (Figure 1a). Next, we examined mouse neural progenitors (mNPs), where H2A.Z again localizes to active promoters as previously reported for differentiated cell types (Figure 1c,e). H2A.Z is also enriched at some PRC2 target promoters in mNPs, but is limited to the subset of Polycomb targets that also carries H3K4me3. Together, these findings suggest that H2A.Z patterns are conserved among species and between cell types, and that co-localization of H2A.Z with PRC2 binding sites reflects coexisting active chromatin modifications (Figure 1c). Furthermore, the data suggest that progression of certain PRC2 target loci from bivalent to H3K27me3-only during differentiation is accompanied by marked reductions in the accessibility of chromatin, which might underlie a more stably repressed chromatin state.
H2A.Z correlates with alternate H3K4 methylation states at promoters and enhancers
Further evidence for correlation between H2A.Z and H3K4 methylation emerges from genome-wide analyses of H3K4 monomethylation (me1), dimethylation (me2) and trimethylation (me3). Respectively, 51% of H3K4me1-, 74% of H3K4me2- and 78% of H3K4me3-enriched regions overlap with H2A.Z sites (Figure 2a). H2A.Z enriches at similar level in promoters, as it does in identified intergenic regions (Figure 2b). H3K4me1, which is enriched at enhancers, strongly associates with intergenic H2A.Z (Figure 2c) . As previously described, H3K4me2 is enriched at both promoters and enhancers (Figure 2d), while H3K4me3 predominantly marks promoters (Figure 2e) . In all H3K4 methylation contexts, H2A.Z was suitably enriched. The localization of H2A.Z in ES cells is also associated with the presence of histone H3 variant, H3.3, which has been linked to replication-independent deposition . Indeed, we observed relative enrichment of H3.3 at H2A.Z-positive enhancers and promoters (Figure 2f, Additional file 1). H2A.Z also occupies between 20% and 30% of multiple TF binding loci (MTLs) (Figure 2g), supporting its localization at accessible chromatin [26–29]. Together, the data suggest a high degree of correspondence between H2A.Z and sites of euchromatin in ES cells.
Promoter H2A.Z sites flank nucleosome-deficient regions enriched for transcriptional machinery
These observations led us to consider the basis for the presence of H2A.Z and associated nucleosome replacement with histone variants at bivalent PRC2 target sites in ES cells. We therefore acquired and analyzed ChIP-Seq maps for the pre-initiation complex component TFIID (transcription Factor II D), hypo-phosphorylated RNAPII and pan-histone H3. We also incorporated published RNA-Seq data for mES cells [7, 30]. Integrative analyses across different classes of TSSs reveal strong enrichment for RNAPII and TFIID over the nucleosome-deficient regions of H3K4me3-only TSSs (Figure 1a, TFIID, RNAPII). As expected, RNA transcripts are also strongly represented across this class of TSSs (Figure 1a, mRNA). By contrast, promoters lacking H3K4me3 (no-mark) show essentially no signal for RNAPII, TFIID or RNA transcripts (Figure 1a, mRNA). PRC2-bound bivalent genes exhibit subtle enrichments for TFIID and, to a lesser extent, for RNAPII, but no productive mRNA is observed. These data suggest that bivalent TSSs are accessible to the transcriptional apparatus, although the associations are likely transient. ChIP-Seq analysis of pan-histone H3 shows that clearly defined nucleosome-deficient regions are present at K4-only and bivalent promoters, but not at no-mark promoters (Additional file 2). These activities might be sufficient to promote and maintain nucleosome-deficient regions and facilitate H2A.Z turnover at bivalent TSSs to retain their transcriptional competence. Alternatively, the chromatin patterns could reflect yet undefined sequence determinants that directly recruit chromatin regulators and RNAs, or innately destabilize nucleosomes [31, 32].
Monoubiquitination of H2A.Z is downstream of Ring1B
H2A.Z has been shown to be subject to C-terminal ubiquitination by the PRC1 component Ring1B . Western blots performed on acid-extracted histones from mES cells with antibody against H2A.Z revealed the presence of the native variant as well as an additional species (approximately 22 kDa) whose molecular weight is consistent with the addition of one ubiquitin moiety (Figure 3b, anti-H2A.Z: Ub0 and Ub1 band). Mass spectrometry (MS) analysis confirmed that the majority of H2A.Z in the higher molecular weight species contains the residual adduct of a ubiquitin distributed amongst K120 (>60%), K121 (approximately 30%) and K125 (approximately 10%) (Figure 3c,d,e). The observation of multiple ubiquitin acceptor sites is consistent with prior reports, and may reflect promiscuity of the E3 ligase . We then tested whether all sites depend on the PRC1 component Ring1B. The monoubiquitination levels of all three lysines are dramatically reduced in Ring1B knockout mES cells according to western blot and MS analyses (Figure 3b, right panel; 3f). While some redundancy or slow turnover of non-ubiquitinated species may account for residual ubiquitinylation in our system, our MS data clearly confirm Ring1B as the principal ubiquitin E3 ligase for all sites on the H2A.Z C-terminus.
A dually modified H2A.Z species with ubiquitination and acetylation
We next explored the relationship between C-terminal ubiquitination and N-terminal acetylation on H2A.Z. Although prior studies have linked H2A.Z acetylation to transcriptional activity [34–37], western blot analysis showed that the anti-acetylated-H2A.Z antibody also recognizes the '+1 ubiquitin' species (Figure 3b, anti-acH2A.Z, Ub1 band). MS analysis further supports the co-occurrence of the two types of H2A.Z modifications on the same molecule. Because the +Ub1 shifts H2A.Z significantly on SDS-PAGE analysis, we were able to isolate individual H2A.Zub0 and H2A.Zub1 bands respectively. We performed MS analyses on these isolated bands and confirmed that the H2A.Zub1 band is essentially all ubiquitinated (Figure 3d). Furthermore, within the H2A.Zub1 fraction, MS analysis specifically shows that approximately 21% of H2A.Zub1 is acetylated, indicating that about one fifth of the H2A.Zub1 population carries ubiquitination and acetylation concurrently (Figure 3g). Remarkably, quantitative MS analysis also indicates that H2A.Zub1 species have higher levels of N-terminal acetylation relative to their non-ubiquitinated counterpart, but possess differential acetylation profiles (Figure 3g).
Dually modified H2A.Z enriched within bivalent chromatin in mouse embryonic stem cells
We sought to confirm that the ubiquitinated and dually modified species also localize to bivalent domains. However, we were unable to acquire high-quality ChIP-Seq maps using an antibody against H2A.Zub1. We therefore implemented an alternative approach in which we immunoprecipitated mononucleosomes from mES cells using an antibody against H3K27me3, and then used western blots to evaluate H2A.Z. We found that both H2A.Zub0 and H2A.Zub1 are enriched, and that the level of H2A.Zub1 relative to H2A.Zub0 is significantly higher in the H3K27me3 pull-down relative to control input fractions (Figure 4c). We also performed the same experiment for the ubiquitinated and non-ubiquitinated levels in acetylated H2A.Z (acH2A.Z). We found that the ratio between acH2A.Zub1 and acH2A.Zub0 is significantly higher in H3K27me3-enriched mononucleosomal fractions, normalized by input chromatin. Given that the vast majority of sites with H3K27me3 in mES cells also carry H3K4me3 and are thus bivalent, these results suggest that both ubiquitinated (H2A.Zub1) and dually modified H2A.Z (acH2A.Zub1) are enriched at bivalent chromatin.
H2A.Z acetylation patterns related to transcriptional status
Acetylated histones have long been known to be a marker of active transcription and are thought to act in part by neutralizing charge interactions to open chromatin and allow access for transcription machinery [34–37]. To evaluate the correspondence between acH2A.Z levels and transcriptional output, we divided all genes marked by H3K4me3 but without H3K27me3 (H3K4me3-only) into categories according to their expression level. H2A.Z occupancy at the 5' end of transcripts is inversely related to transcriptional activity, similar to published reports (Figure 5d) [13, 15]. We specifically found that the most highly active genes in the top expression quartile show lower H2A.Z levels at their 5' ends, possibly due to eviction as a consequence of transcriptional elongation (Figure 5d). Interestingly, acH2A.Z level at the 5' end of the transcript is preserved as transcription level increases (Figure 5e). These data suggest that, as transcription activity escalates, total H2A.Z decreases at the 5' end of the transcript but an increasing proportion of the variant becomes acetylated. The asymmetric distribution of H2A.Z and acH2AZ at active promoters suggests that this histone variant and/or associated chromatin structures may help direct transcription by favoring procession of the transcription machinery towards the 3' direction from TSSs .
Through comprehensive analyses of ChIP-Seq, MS and biochemical data, we document the existence of a novel, dually modified H2A.Z species that preferentially localize to bivalent chromatin domains in ES cells. Our results also clarify that, in both mouse and human ES cells, H2A.Z promiscuously co-localizes to genomic loci enriched for H3K4 methylation, including both bivalent PRC2 targets and as active TSSs as well as at distal enhancer elements. We demonstrate a strong association between H2A.Z acetylation and transcriptional activity. The co-occurrence on the same histone molecule of acetylation, previously linked to gene activity and induction, and ubiquitination events, downstream of Polycomb repressors, echoes the duality of the bivalent H3K4me3 and H3K27me3 combination. We propose that the modified variant contributes to the transcriptional dynamics and epigenomic plasticity of pluripotent ES cells by maintaining dynamic chromatin at key loci poised for alternate developmental fates. This study provides a framework for future studies into the nature of bivalent chromatin functions, and opens up new avenues for decoding the interplay between chromatin-modifying enzymes and modifications of variant histones.
Materials and methods
mES cells v6.5 (male, strain 129SVJae × C57BL6) were cultured using standard procedures and reagents . Ring1B KO (Ring1A -/- ;Ring1B fl/fl ; Rosa26::CreERT2) mES cells have been described previously . mNP cells were in vitro differentiated from mES cells as previously described . hES cells (H1) were cultured on Matrigel (BD Biosciences, San Jose, CA, USA) in feeder-free, serum-free modified mTeSR1 media and passaged by dispase digestion (Cellular Dynamics International, Madison, WI, USA) .
ChIP experiments for H2A.Z, acH2AZ and other histone modifications were performed in whole cell extract preparation as described previously . The antibody against H2A.Z recognizes both H2A.Z.1 and H2A.Z.2.1. TFIID ChIP was performed by immunoprecipitation of the TFIID subunit TBP (TATA binding protein) in nuclear preparations as detailed previously . Notably, ChIP procedures performed in nuclei preparations were identical to that of whole cell extract, except nuclei were isolated prior to cell lysis and sonication. Crosslinked ES cells were incubated in swelling buffer (0.1 M Tris pH 7.6, 10 mM potassium acetate, 15 mM magnesium acetate, 1% nonyl phenoxypolyethoxylethanol) on ice, and then passed through 16 G needles to disrupt plasma membranes. Nuclei were collected by centrifugation. A summary of ChIP-Seq data sets is listed in Additional file 3 and antibody information is listed in Additional file 4.
Histones were purified from mES cells as described except that a C5 HPLC column was employed (Phenomenex, Torrance, CA, USA) . Each one-minute fraction collected from the HPLC separation of the histones was subjected to SDS-PAGE. Subsequent LC-MS/MS experiments were performed on an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) fed by an Agilent 1100 nano-HPLC system following procedures previously described .
Coomassie-stained visible bands from PAGE gels were interrogated by tryptic and chymotryptic digestion. Peptides unique to H2A.Z (H2AFZ or H2A.Z.1, and not derived from other H2A variants) were detected in bands of approximately 14 kDa and approximately 20 kDa that co-purified with the core H4 protein (approximately 12 kDa, see Figure 3c). Next, these bands (from a parallel preparation) were subject to in-gel propionylation using d 10 -proprionic anhydride according to . To study the C-terminal ubiquitination of H2A.Z, chymotryptic peptides were analyzed. To study the N-terminal acetylation of H2A.Z, tryptic peptides were analyzed. M/z values corresponding to the various acetylated and ubiquitin-residual peptides (recall that the proteases will cleave ubiquitin as well as H2A.Z, leaving a branched peptide residual) were calculated . Separate acquisition methods were designed for the study of acetylation or ubiquitination. Selective-ion monitoring windows were designed around these m/zs as appropriate and data-independent MS/MS scans were acquired at these m/zs as dictated by each experiment. The sample was introduced to the mass spectrometer via liquid chromatography with conditions identical to those previously described .
C-terminal ubiquitinated positional isomers were assigned from collisional MS/MS spectra (Figure 3e). The percentage of each positional isomer was determined using integrated chromatographic peak area of extracted ion chromatograms (Figure 3c, right panel).
N-terminal acetyl positional isomers were also assigned from collisional MS/MS spectra (Figure 3e and data not shown). As positional isomers did not cleanly resolve during chromatographic separation, the percentage of ion current corresponding to d 5 -propionylated (non-acetylated) or acetylated b-series fragment ions at each potentially modified residue (K4, K7, K11, K13, K15) in a composite spectrum averaged across the entire elution frame was taken as a proxy for acetylation at that residue. In the case of diacetyl precursors, mixture modeling was conducted to deconvolute percentage occupancy at each individual residue. The total percentage of acetylation was derived from extracted ion chromatograms corresponding to the precursors of the H2A.Z tryptic N-terminus bearing zero to four acetyls (where the balance of primary amines are d 5 -propionylated). MS data are available at the Broad Institute Proteomics Platform public data portal .
Histones were extracted from mES cells using a standard acid extraction protocol described previously . For the anti-Ring1B western blot, mES cells were incubated with radioimmunoprecipitation assay buffer (BP-115; Boston BioProducts Inc., Ashland MA, USA). Protein concentration was measured by Quant-iT Protein Assay Kit (Q33210; Invitrogen, Carlsbad, CA, USA) to ensure equal loading. Lysate was boiled with NuPage lithium dodecyl sulfate sample buffer (Invitrogen NP0008) and 1% β-mercaptoethanol for 8 minutes before loading on the NuPAGE Novex 4-12% gradient Bis-Tris Gel (Invitrogen NP0322BOX) for electrophoresis. Proteins were transferred using the iBlot system (Invitrogen IB1001) according to manufacturer's instructions. Transferred nitrocellulose membrane was incubated with blocking buffer (Odyssey 927-40000; LI-COR Biosciences, Lincoln, NE, USA), and blotted with respective antibodies overnight. Secondary antibodies conjugated with infrared dye were incubated for 30 minutes in the dark and imaged on the Odyssey® Infrared imaging system (LI-COR Biosciences). For mononucleosome immunoprecipitation, western blot was carried out as described above. Ubiquitinated and non-ubiquitinated bands of H2A.Z and acH2A.Z blots were quantified by using ImageJ software .
Chromatin preparation and mononucleosome immunoprecipitation of mES cells were performed as described with modifications [22, 50]. mES cells were incubated with 0.5% Triton X-100/buffer 1 (10 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.5, 10 mM sodium butyrate, 60 mM potassium chloride (KCl), 15mM sodium chloride (NaCl), 5 mM magnesium chloride (MgCl2), 0.25 M sucrose) on ice. Nuclei were then layered onto 2.5× volume of 30% sucrose/buffer 1 and centrifuged at 4,000 rpm. Purified nuclei were resuspended into buffer 2 (20 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.8, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 2 mM calcium chloride, 1 mM dithiothreitol) with 200 U of micrococcal nuclease (10107921001; Roche Diagnostics, Indianapolis, IN, USA) at room temperature until mainly mononucleosomes was achieved, then digestion was stopped by adding a final 1 mM of ethylene glycol tetraacetic acid (EGTA). Digested chromatin was resuspended in buffer 3 (20 mM HEPES, pH 7.8, 1.5 mM MgCl2, 0.42 M NaCl and 0.2 mM EGTA), incubated on ice for 1 hour and centrifuged at 1000 g. Finally, 2.8× volume of buffer 4 (20 mM HEPES, pH 7.8, 1.5 mM MgCl2, 25% glycerol, 0.2 mM EGTA) was added drop-wise to the supernatant while vortexing.
The supernatant was used for subsequent immunoprecipitation by adding antibodies and incubated at 4°C. Protein-A or -G Sepharose beads (Sigma-Aldrich, St. Louis, MO, USA) were added and incubated for 2 hours then subjected to eight washes of buffer 5 (20 mM HEPES, pH 7.8, 1.5 mM MgCl2, 10% glycerol, 0.2% Triton X-100, 150 mM NaCl, 0.2 mM EGTA). Buffers 1 to 5 were supplemented by phenylmethylsulfonyl fluoride and benzamidine (0.1 mM each). Histones were eluted by boiling the washed beads in lithium dodecyl sulfate sample buffer, and 1% β-mercaptoethanol.
Illumina sequencing libraries were prepared from ChIP-enriched DNA as described previously [7, 25], and sequenced on the Illumina Genome Analyzer IIx and HiSeq2000 (Illumina, San Diego, CA) according to the manufacturer's specifications. ChIP-Seq data was compiled, processed and aligned as published . All ChIPs performed in mouse cells were aligned to mm8, and ChIPs in human cells were aligned to hg18 reference genomes.
ChIP-Seq data was processed and aligned to the reference genomes (mm8 for mES, mNP and hg18 for hES) as described in a previous study . Promoter classification and ChIP-Seq enriched intervals were carried out as described [7, 25]. Promoters were defined as 0.5 kb upstream and 2 kb downstream of all annotated TSSs, generating 17,760 mouse and 18,522 human promoters respectively. Genomewide-enriched windows were calculated in sliding one-kilobase windows and are merged if distance between the two is less than 2 kb. ChIP-Seq enriched intergenic regions are defined as enriched windows that are least ±4 kb from known gene promoters and gene bodies to prevent contamination of proximal or alternate promoters. Heatmaps were generated by measuring ChIP-Seq signals in 200 bp sliding windows spanning ±5 kb of the TSS. Composite plots were generated by averaging values in each of the 200 bp windows. Statistical significance of enrichment (P < 10-4) was determined based on background distribution of randomized reads specific for each independent genomewide ChIP analysis. MTLs were defined previously as described . Chromosome positions for MTLs were extended ±2 kb. MTLs that are located in ±4 kb of TSSs and transcription end sites, or gene bodies were removed to yield intergenic MTLs, and query the overlap with H2A.Z defined genomewide-enriched windows (mentioned above). mES cell mRNA enrichment analysis was generated using published RNA-Seq data , and a heatmap was generated as described above. ChIP-seq data are available at the Broad Institute Epigenomics Public Data Portal .
The data sets are available in the Gene Expression Omnibus (GEO) database under the accession number GSE:.
acetylated and non-ubiquitinated H2A.Z
acetylated and monoubiquitinated H2A.Z
chromatin immunoprecipitation coupled with microarray
chromatin immunoprecipitation coupled with high-throughput sequencing
ethylene glycol tetraacetic acid
histone 3 lysine 4 trimethylation
histone 3 lysine 27 trimethylation
human embryonic stem
high performance liquid chromatography
mouse embryonic stem
mouse neural progenitors
multiple transcription factor binding loci
Polycomb repressive complexes
RNA polymerase II
transcription Factor II D
transcription start sites.
We thank Shawn Gillespie, Kaylyn Williamson, Tim Durham, Robbyn Issner, Xiaolan Zhang, Jing Gao, Chuck Epstein, Noam Shoresh, Michael Coyne and the Broad Institute Genome Sequencing Platform for providing reagents and assistance in generating, processing, displaying and submitting ChIP-Seq data. We are grateful for Dr Kevin Haigis and Krystle Kendall for the permission to use their Odyssey® Infrared Imaging system. We thank Dr Joseph A Goldman for helpful discussions on micrococcal nuclease digestion protocols. MK is supported in part by a Croucher postdoctoral fellowship.
- Hager GL, McNally JG, Misteli T: Transcription dynamics. Mol Cell. 2009, 35: 741-753. 10.1016/j.molcel.2009.09.005.PubMedView ArticleGoogle Scholar
- Mendenhall EM, Bernstein BE: Chromatin state maps: new technologies, new insights. Curr Opin Genet Dev. 2008, 18: 109-115. 10.1016/j.gde.2008.01.010.PubMedPubMed CentralView ArticleGoogle Scholar
- Jaenisch R, Young R: Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell. 2008, 132: 567-582. 10.1016/j.cell.2008.01.015.PubMedPubMed CentralView ArticleGoogle Scholar
- Mattout A, Meshorer E: Chromatin plasticity and genome organization in pluripotent embryonic stem cells. Curr Opin Cell Biol. 2010, 22: 334-341. 10.1016/j.ceb.2010.02.001.PubMedView ArticleGoogle Scholar
- Xu J, Watts JA, Pope SD, Gadue P, Kamps M, Plath K, Zaret KS, Smale ST: Transcriptional competence and the active marking of tissue-specific enhancers by defined transcription factors in embryonic and induced pluripotent stem cells. Genes Dev. 2009, 23: 2824-2838. 10.1101/gad.1861209.PubMedPubMed CentralView ArticleGoogle Scholar
- Stock JK, Giadrossi S, Casanova M, Brookes E, Vidal M, Koseki H, Brockdorff N, Fisher AG, Pombo A: Ring1-mediated ubiquitination of H2A restrains poised RNA polymerase II at bivalent genes in mouse ES cells. Nat Cell Biol. 2007, 9: 1428-1435. 10.1038/ncb1663.PubMedView ArticleGoogle Scholar
- Mikkelsen TS, Ku M, Jaffe DB, Issac B, Lieberman E, Giannoukos G, Alvarez P, Brockman W, Kim TK, Koche RP, Lee W, Mendenhall E, O'Donovan A, Presser A, Russ C, Xie X, Meissner A, Wernig M, Jaenisch R, Nusbaum C, Lander ES, Bernstein BE: Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature. 2007, 448: 553-560. 10.1038/nature06008.PubMedPubMed CentralView ArticleGoogle Scholar
- Zlatanova J, Thakar A: H2A.Z: view from the top. Structure. 2008, 16: 166-179. 10.1016/j.str.2007.12.008.PubMedView ArticleGoogle Scholar
- Deal RB, Henikoff S: Gene regulation: a chromatin thermostat. Nature. 2010, 463: 887-888. 10.1038/463887a.PubMedPubMed CentralView ArticleGoogle Scholar
- Dion MF, Kaplan T, Kim M, Buratowski S, Friedman N, Rando OJ: Dynamics of replication-independent histone turnover in budding yeast. Science. 2007, 315: 1405-1408. 10.1126/science.1134053.PubMedView ArticleGoogle Scholar
- Deal RB, Henikoff JG, Henikoff S: Genome-wide kinetics of nucleosome turnover determined by metabolic labeling of histones. Science. 2010, 328: 1161-1164. 10.1126/science.1186777.PubMedPubMed CentralView ArticleGoogle Scholar
- Goldberg AD, Banaszynski LA, Noh KM, Lewis PW, Elsaesser SJ, Stadler S, Dewell S, Law M, Guo X, Li X, Wen D, Chapgier A, DeKelver RC, Miller JC, Lee YL, Boydston EA, Holmes MC, Gregory PD, Greally JM, Rafii S, Yang C, Scambler PJ, Garrick D, Gibbons RJ, Higgs DR, Cristea IM, Urnov FD, Zheng D, Allis CD: Distinct factors control histone variant H3.3 localization at specific genomic regions. Cell. 2010, 140: 678-691. 10.1016/j.cell.2010.01.003.PubMedPubMed CentralView ArticleGoogle Scholar
- Jin C, Zang C, Wei G, Cui K, Peng W, Zhao K, Felsenfeld G: H3.3/H2A.Z double variant-containing nucleosomes mark 'nucleosome-free regions' of active promoters and other regulatory regions. Nat Genet. 2009, 41: 941-945. 10.1038/ng.409.PubMedPubMed CentralView ArticleGoogle Scholar
- Talbert PB, Henikoff S: Histone variants--ancient wrap artists of the epigenome. Nat Rev Mol Cell Biol. 2010, 11: 264-275.PubMedView ArticleGoogle Scholar
- Barski A, Cuddapah S, Cui K, Roh TY, Schones DE, Wang Z, Wei G, Chepelev I, Zhao K: High-resolution profiling of histone methylations in the human genome. Cell. 2007, 129: 823-837. 10.1016/j.cell.2007.05.009.PubMedView ArticleGoogle Scholar
- Tolstorukov MY, Kharchenko PV, Goldman JA, Kingston RE, Park PJ: Comparative analysis of H2A.Z nucleosome organization in the human and yeast genomes. Genome Res. 2009, 19: 967-977. 10.1101/gr.084830.108.PubMedPubMed CentralView ArticleGoogle Scholar
- Henikoff S, Henikoff JG, Sakai A, Loeb GB, Ahmad K: Genome-wide profiling of salt fractions maps physical properties of chromatin. Genome Res. 2009, 19: 460-469.PubMedPubMed CentralView ArticleGoogle Scholar
- Suto RK, Clarkson MJ, Tremethick DJ, Luger K: Crystal structure of a nucleosome core particle containing the variant histone H2A.Z. Nat Struct Biol. 2000, 7: 1121-1124. 10.1038/81971.PubMedView ArticleGoogle Scholar
- Bernstein BE, Meissner A, Lander ES: The mammalian epigenome. Cell. 2007, 128: 669-681. 10.1016/j.cell.2007.01.033.PubMedView ArticleGoogle Scholar
- Margueron R, Reinberg D: Chromatin structure and the inheritance of epigenetic information. Nat Rev Genet. 2010, 11: 285-296. 10.1038/nrg2752.PubMedPubMed CentralView ArticleGoogle Scholar
- Creyghton MP, Markoulaki S, Levine SS, Hanna J, Lodato MA, Sha K, Young RA, Jaenisch R, Boyer LA: H2AZ is enriched at polycomb complex target genes in ES cells and is necessary for lineage commitment. Cell. 2008, 135: 649-661. 10.1016/j.cell.2008.09.056.PubMedPubMed CentralView ArticleGoogle Scholar
- Sarcinella E, Zuzarte PC, Lau PN, Draker R, Cheung P: Monoubiquitylation of H2A.Z distinguishes its association with euchromatin or facultative heterochromatin. Mol Cell Biol. 2007, 27: 6457-6468. 10.1128/MCB.00241-07.PubMedPubMed CentralView ArticleGoogle Scholar
- Xiao S, Xie D, Cao X, Yu P, Xing X, Chen CC, Musselman M, Xie M, West FD, Lewin HA, Wang T, Zhong S: Comparative epigenomic annotation of regulatory DNA. Cell. 2012, 149: 1381-1392. 10.1016/j.cell.2012.04.029.PubMedPubMed CentralView ArticleGoogle Scholar
- Illingworth RS, Botting CH, Grimes GR, Bickmore WA, Eskeland R: PRC1 and PRC2 are not required for targeting of H2A.Z to developmental genes in embryonic stem cells. PLoS One. 2012, 7: e34848-10.1371/journal.pone.0034848.PubMedPubMed CentralView ArticleGoogle Scholar
- Ku M, Koche RP, Rheinbay E, Mendenhall EM, Endoh M, Mikkelsen TS, Presser A, Nusbaum C, Xie X, Chi AS, Adli M, Kasif S, Ptaszek LM, Cowan CA, Lander ES, Koseki H, Bernstein BE: Genomewide analysis of PRC1 and PRC2 occupancy identifies two classes of bivalent domains. PLoS Genet. 2008, 4: e1000242-10.1371/journal.pgen.1000242.PubMedPubMed CentralView ArticleGoogle Scholar
- Heintzman ND, Stuart RK, Hon G, Fu Y, Ching CW, Hawkins RD, Barrera LO, van Calcar S, Qu C, Ching KA, Wang W, Weng Z, Green RD, Crawford GE, Ren B: Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat Genet. 2007, 39: 311-318. 10.1038/ng1966.PubMedView ArticleGoogle Scholar
- Mito Y, Henikoff JG, Henikoff S: Histone replacement marks the boundaries of cis-regulatory domains. Science. 2007, 315: 1408-1411. 10.1126/science.1134004.PubMedView ArticleGoogle Scholar
- Chen X, Xu H, Yuan P, Fang F, Huss M, Vega VB, Wong E, Orlov YL, Zhang W, Jiang J, Loh YH, Yeo HC, Yeo ZX, Narang V, Govindarajan KR, Leong B, Shahab A, Ruan Y, Bourque G, Sung WK, Clarke ND, Wei CL, Ng HH: Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell. 2008, 133: 1106-1117. 10.1016/j.cell.2008.04.043.PubMedView ArticleGoogle Scholar
- Hardy S, Jacques PE, Gevry N, Forest A, Fortin ME, Laflamme L, Gaudreau L, Robert F: The euchromatic and heterochromatic landscapes are shaped by antagonizing effects of transcription on H2A.Z deposition. PLoS Genet. 2009, 5: e1000687-10.1371/journal.pgen.1000687.PubMedPubMed CentralView ArticleGoogle Scholar
- Cloonan N, Forrest AR, Kolle G, Gardiner BB, Faulkner GJ, Brown MK, Taylor DF, Steptoe AL, Wani S, Bethel G, Robertson AJ, Perkins AC, Bruce SJ, Lee CC, Ranade SS, Peckham HE, Manning JM, McKernan KJ, Grimmond SM: Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nat Methods. 2008, 5: 613-619. 10.1038/nmeth.1223.PubMedView ArticleGoogle Scholar
- Ramirez-Carrozzi VR, Braas D, Bhatt DM, Cheng CS, Hong C, Doty KR, Black JC, Hoffmann A, Carey M, Smale ST: A unifying model for the selective regulation of inducible transcription by CpG islands and nucleosome remodeling. Cell. 2009, 138: 14-128. 10.1016/j.cell.2009.06.028.View ArticleGoogle Scholar
- Kanhere A, Viiri K, Araujo CC, Rasaiyaah J, Bouwman RD, Whyte WA, Pereira CF, Brookes E, Walker K, Bell GW, Pombo A, Fisher AG, Young RA, Jenner RG: Short RNAs are transcribed from repressed polycomb target genes and interact with polycomb repressive complex-2. Mol Cell. 2010, 38: 675-688. 10.1016/j.molcel.2010.03.019.PubMedPubMed CentralView ArticleGoogle Scholar
- Zilberman D, Coleman-Derr D, Ballinger T, Henikoff S: Histone H2A.Z and DNA methylation are mutually antagonistic chromatin marks. Nature. 2008, 456: 125-129. 10.1038/nature07324.PubMedPubMed CentralView ArticleGoogle Scholar
- Thambirajah AA, Li A, Ishibashi T, Ausio J: New developments in post-translational modifications and functions of histone H2A variants. Biochem Cell Biol. 2009, 87: 7-17. 10.1139/O08-103.PubMedView ArticleGoogle Scholar
- Bruce K, Myers FA, Mantouvalou E, Lefevre P, Greaves I, Bonifer C, Tremethick DJ, Thorne AW, Crane-Robinson C: The replacement histone H2A.Z in a hyperacetylated form is a feature of active genes in the chicken. Nucleic Acids Res. 2005, 33: 5633-5639. 10.1093/nar/gki874.PubMedPubMed CentralView ArticleGoogle Scholar
- Valdes-Mora F, Song JZ, Statham AL, Strbenac D, Robinson MD, Nair SS, Patterson KI, Tremethick DJ, Stirzaker C, Clark SJ: Acetylation of H2A.Z is a key epigenetic modification associated with gene deregulation and epigenetic remodeling in cancer. Genome Res. 2012, 22: 307-321. 10.1101/gr.118919.110.PubMedPubMed CentralView ArticleGoogle Scholar
- Ishibashi T, Dryhurst D, Rose KL, Shabanowitz J, Hunt DF, Ausio J: Acetylation of vertebrate H2A.Z and its effect on the structure of the nucleosome. Biochemistry. 2009, 48: 5007-5017. 10.1021/bi900196c.PubMedPubMed CentralView ArticleGoogle Scholar
- Hardy S, Robert F: Random deposition of histone variants: a cellular mistake or a novel regulatory mechanism?. Epigenetics. 2010, 5: 368-372. 10.4161/epi.5.5.11787.PubMedView ArticleGoogle Scholar
- Endoh M, Endo TA, Endoh T, Fujimura Y, Ohara O, Toyoda T, Otte AP, Okano M, Brockdorff N, Vidal M, Koseki H: Polycomb group proteins Ring1A/B are functionally linked to the core transcriptional regulatory circuitry to maintain ES cell identity. Development. 2008, 135: 1513-1524. 10.1242/dev.014340.PubMedView ArticleGoogle Scholar
- Conti L, Pollard SM, Gorba T, Reitano E, Toselli M, Biella G, Sun Y, Sanzone S, Ying QL, Cattaneo E, Smith A: Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. PLoS Biol. 2005, 3: e283-10.1371/journal.pbio.0030283.PubMedPubMed CentralView ArticleGoogle Scholar
- Ludwig TE, Bergendahl V, Levenstein ME, Yu J, Probasco MD, Thomson JA: Feeder-independent culture of human embryonic stem cells. Nat Methods. 2006, 3: 637-646. 10.1038/nmeth902.PubMedView ArticleGoogle Scholar
- Thomas CE, Kelleher NL, Mizzen CA: Mass spectrometric characterization of human histone H3: a bird's eye view. J Proteome Res. 2006, 5: 240-247. 10.1021/pr050266a.PubMedView ArticleGoogle Scholar
- Kinter M, Sherman NE: Protein Sequencing and Identification using Tandem Mass Spectrometry. 2000, New York, NY: Wiley-InterscienceView ArticleGoogle Scholar
- Garcia BA, Mollah S, Ueberheide BM, Busby SA, Muratore TL, Shabanowitz J, Hunt DF: Chemical derivatization of histones for facilitated analysis by mass spectrometry. Nat Protoc. 2007, 2: 933-938. 10.1038/nprot.2007.106.PubMedPubMed CentralView ArticleGoogle Scholar
- Peng J, Schwartz D, Elias JE, Thoreen CC, Cheng D, Marsischky G, Roelofs J, Finley D, Gygi SP: A proteomics approach to understanding protein ubiquitination. Nat Biotechnol. 2003, 21: 921-926. 10.1038/nbt849.PubMedView ArticleGoogle Scholar
- Jaffe JD, Keshishian H, Chang B, Addona TA, Gillette MA, Carr SA: Accurate inclusion mass screening: a bridge from unbiased discovery to targeted assay development for biomarker verification. Mol Cell Proteomics. 2008, 7: 1952-1962. 10.1074/mcp.M800218-MCP200.PubMedPubMed CentralView ArticleGoogle Scholar
- Broad Institute Proteomics Data on H2A.Z Acetylation and Ubiquitination Status in mES Cells. [ftp://ftp.broadinstitute.org/pub/proteomics/public_datasets/Ku_GenomeBiol_2012/]
- Abcam Histone Extraction Protocol. [http://www.abcam.com/ps/pdf/protocols/Histone%20extraction%20protocol.pdf]
- Rasband WW: ImageJ. 1997, Bethesda, MD: US NIH, [http://rsb.info.nih.gov/ij]Google Scholar
- Hebbes TR, Clayton AL, Thorne AW, Crane-Robinson C: Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken beta-globin chromosomal domain. EMBO J. 1994, 13: 1823-1830.PubMedPubMed CentralGoogle Scholar
- Broad Institute H2A.Z ChIP-Seq Data in mES, hES and mNP cells. [http://www.broadinstitute.org/cgi-bin/epigenomics/public/prod/cloneportal.cgi?data=ku_h2az_portal]
- Matsui SI, Seon BK, Sandberg AA: Disappearance of a structural chromatin protein A24 in mitosis: implications for molecular basis of chromatin condensation. Proc Natl Acad Sci USA. 1979, 76: 6386-6390. 10.1073/pnas.76.12.6386.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is cited.