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
]. This dynamic exchange is thought to maintain an accessible and transcriptionally competent state [4
]. 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 [2
]. In fact, some of these loci may already be engaged by initiating RNA polymerase II (RNAPII) [6
]. 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 [7
Replication-independent histone deposition is of particular interest as it is targeted to DNA sequences under active regulation [8
]. Rapid nucleosome turnover is a general feature of promoters and epigenetic regulatory elements in yeast [10
] and in fly [11
]. 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 [12
]. 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 [13
]. 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
]. 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
]. H2A.Z-containing nucleosomes are unusually susceptible to nuclease digestion and stringent ionic conditions [16
], and it has been speculated that this structural instability is because of amino acid substitutions at the interface between H2A.Z and H3/H4 [18
]. 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
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 [21
]. 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 [15
], and antibody reagents that may not account for potential modifications [22
]. 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.