How chromatin organization in ES cells contributes to the preservation of cell identity while maintaining the genome in a flexible state that allows for differentiation into multiple lineages remains an open question. In ES cells, the promoters of a subset of silent genes with roles in development are marked with “bivalent” histone modifications that correlate with both transcription initiation (H3K4me3) and gene silencing (H3K27me3) (Azuara et al., 2006
; Bernstein et al., 2006
; Mikkelsen et al., 2007
). These chromatin marks are a result of the activity of Trithorax- and Polycomb-group (Trx and PcG) proteins, respectively. Core components of Polycomb Repressive Complex PRC2 (Suz12 and Eed, as well as the modification it catalyzes, H3K27me3) and PRC1 (Rnf2 and Phc1) have been found to significantly overlap in ES cells (Boyer et al., 2006
; Lee et al., 2006
). Our finding that H2AZ and Suz12 occupy the promoter regions of the same genes in ES cells suggests that this variant is an additional regulatory component at bivalent promoters and links H2AZ to a large class of genes with known roles in development. Consistent with a role for H2AZ in gene regulation, target genes are de-repressed and PcG proteins are lost from promoters upon RNAi-mediated H2AZ depletion in ES cells. It remains unclear, however, whether H2AZ incorporation is necessary for gene repression similarly to PcG proteins or whether it may be required for subsequent gene activation as has been observed in yeast (Li et al., 2005
; Zhang et al., 2005
Prior studies in metazoans have shown that H2AZ enrichment is directly proportional to gene activation (Barski et al., 2007
; Mavrich et al., 2008
; Schones et al., 2008
), so it was surprising that H2AZ was enriched at a large subset of silent genes in ES cells. Recent studies have revealed that bivalent promoters experience transcription initiation but show no evidence of elongation, suggesting that they may be regulated at post-initiation steps (Guenther et al., 2007
). An additional feature of these promoters is the presence of a paused RNA Polymerase II (Pol II) complex (Guenther et al., 2007
; Stock et al., 2007
). Interestingly, Polycomb-mediated ubiquitination of histone H2A, as catalyzed by the PRC1 component Rnf2/Ring1b (de Napoles et al., 2004
), is necessary to maintain this configuration because its loss results in release of the paused polymerase and in gene de-repression (Stock et al., 2007
). The observed loss of gene silencing and of Rnf2/Ring1b from target promoters in H2AZ-depleted cells is consistent with the idea that H2AZ is a target for Polycomb-mediated ubiquitination. It has been proposed that the incorporation of H2AZ favors nucleosome eviction (Mavrich et al., 2008
), which would promote histone turnover and chromatin accessibility. As such, it will also be of interest to investigate whether H2AZ loss or de-ubiquitination is required for release of a paused polymerase and transcriptional elongation. Thus, H2AZ incorporation may be a key mechanism to allow developmental genes to remain silent, yet poised for activation in ES cells.
While the majority of H2AZ bound regions encompass narrow intervals (< 2 kb) at gene promoters, a small proportion of these regions extended from the promoter into the coding region and in some cases included contiguous genes such as those located in the HOX gene clusters (Lee et al., 2006
). Biophysical analyses of H2AZ-containing nucleosomal arrays suggest that H2AZ inhibits the formation of highly compacted chromatin fibers (Fan et al., 2004
). In this scenario, H2AZ incorporation into large regions with PcG proteins may allow these genes to remain primed for activity by contending with Polycomb-mediated chromatin compaction (Francis et al., 2004
). Together, these observations suggest that H2AZ and PcG proteins together may establish a specialized, structurally distinct chromatin conformation that has important consequences on gene regulation.
The promoter configuration at developmental regulators where both H2AZ and PcG proteins are necessary to maintain proper gene control appears to be unique to pluripotent cells. We find that H2AZ and H3K27me3 are co-enriched at genes in ES cells whereas this association is not maintained upon differentiation into multi-potent neural precursors. Rather, in NPs, H2AZ occupies active genes with a wide range of roles in metabolic processes. This observation is in accordance with previous studies in human T cells in that H2AZ localized to discrete regions at the promoters of active genes (Barski et al., 2007
; Schones et al., 2008
). How can H2AZ mediate seemingly opposing functions? H2AZ can be post-translationally modified and its incorporation correlates with particular histone modification patterns such as acetylation (Guillemette and Gadreau, 2006
). Interestingly, the acetylated form of H2AZ correlates with its localization to the 5’ regions of active genes in yeast and vertebrates (Bruce et al., 2005
; Millar et al., 2006
) suggesting that post-translational modification of H2AZ may underlay its differential distribution in ES cells compared to lineage-committed cells. Alternatively, H2AZ and the histone variant H3.3 can co-occupy the same nucleosome and this has been shown to impact nucleosome stability (Jin and Felsenfeld, 2007
). Thus, the function of H2AZ may depend on the nucleosome into which it is incorporated. As such, it will be of tremendous interest to determine how H3.3 is distributed in pluripotent and lineage-committed cells. Together, these studies suggest that H2AZ distribution changes dramatically during development and that its developmental or stage-specific localization and function likely depends on additional modifiers.
We find that the association between H2AZ and PRC1 and PRC2 is mutually interdependent at promoters, as loss of one leads to eviction of the other. This provides strong evidence for an obligate relationship between these regulators and their effect on chromatin structure and target gene regulation in ES cells. Their highly defined distribution patterns suggest that these regulators are specifically recruited to genomic sites. While considerable evidence exists that PcG proteins are targeted by DNA binding factors in Drosophila (Ringrose and Paro, 2007
), it is currently unknown how these repressors are localized to target genes in mammals. It is intriguing to speculate that H2AZ may play such a role in ES cells. However, we failed to detect an interaction between these proteins by co-immunoprecipitation assay suggesting that recruitment may occur through a different mechanism. Interestingly, a recent report suggests that long non-coding RNA may facilitate PcG protein localization to HOX gene clusters in human cells (Rinn et al., 2007
), highlighting a potential role for ongoing transcription of non-coding RNA in this process. Given that H2AZ is also redistributed during ES cell differentiation, how H2AZ is conscripted at target promoters is an equally important question. H2AZ deposition is catalyzed by the ATP-dependent activity of the SRCAP/SWR1 complex in mammals (Wong et al., 2007
). Thus, it is possible that H2AZ incorporation is mediated by recruitment of this complex independently of PcG proteins. Given that both H2AZ and PcG proteins have been linked to cancer progression (Hua et al., 2008
; Sparmann and van Lohuizen, 2006
), it will be of extreme interest to elucidate the mechanisms by which these regulators are recruited to genomic sequences.
This study demonstrates a requirement for H2AZ to initiate developmental programs, but not for maintenance of the ES cell state suggesting an important role for H2AZ in mediating cell fate transitions. This phenotype is similar to loss of Suz12 where null ES cells display de-repression of target genes and failure to differentiate into multiple lineages (Pasini et al., 2007
). In contrast, although the PRC2 component Eed is essential for early development, null ES cells can contribute to tissues of all three germ layers in chimeric assays (Chamberlain et al., 2008
) so it will be important to further explore its relationship between H2AZ and PRC2 in ES cells. Our findings demonstrate an important functional interaction between these two chromatin regulatory pathways in ES that is necessary for the control of developmental gene expression programs. Genetic interactions between H2AZ or its deposition complex and PcG proteins have been reported in Drosophila (Ruhf et al., 2001
; Swaminathan et al., 2005
). It will be important to explore this relationship in mammals to fully understand the nature of this interaction. Nonetheless, our data suggest that the association between H2AZ and PcG proteins provides an important functional switch to control the initial stages of lineage commitment. Moreover, this work provides a model for understanding the role of chromatin states in cell fate specification as well as in the progression from a normal to disease state.