In this study, we examine H3 variant localization in mammalian ES cells and differentiated NPCs. Genome-wide patterns of H3.3 are dependent on H3.3-specific amino acid sequence; the enrichment of H3.3 at cell-type specific genes and TFBS is dependent on cellular state. We describe three general categories of H3.3 enrichment in mammalian cells: 1) genes and other transcribed non-repetitive sequences, 2) TFBS, and 3) telomeres. Remarkably, we find that each of these general categories of H3.3 enrichment in ES cells is mediated by distinct mechanisms. As expected, Hira is required for genic enrichment of H3.3. Unexpectedly, localization of H3.3 at specific TFBS and telomeres is Hira-independent, and we have identified Atrx as required for H3.3 localization at telomeres. Our results demonstrate that distinct factors control H3.3 localization at specific genomic locations in mammalian cells.
We find that H3.3 is constitutively enriched around the TSS of active and repressed HCP genes in mammalian ES cells and NPCs, including the TSS of repressed bivalent genes in ES cells. Although a recent genome-wide study found that H3.3 is unenriched at the TSS of repressed genes in HeLa cells (Jin et al., 2009
), these results are not necessarily in conflict with our findings. Low CpG content promoter (LCP) and HCP genes have been described to display distinct modes of regulation (Mikkelsen et al., 2007
; Ramirez-Carrozzi et al., 2009
; Saxonov et al., 2006
). Most HCP genes show evidence of transcriptional initiation, assemble unstable nucleosomes, and do not require SWI/SNF nucleosome remodeling complexes for gene induction, while LCP genes assemble stable nucleosomes and require SWI/SNF (Guenther et al., 2007
; Ramirez-Carrozzi et al., 2009
). Less differentiated cells such as ES cells contain large numbers of HCP genes with characteristics of transcriptional initiation (Guenther et al., 2007
; Mikkelsen et al., 2007
). Indeed, nearly all (99%) of HCP genes are marked by H3K4me3 in mouse ES cells, whether they are transcriptionally active or repressed (Mikkelsen et al., 2007
). In contrast to HCP genes, we do not observe any significant pattern of H3.3 enrichment at LCP genes in ES cells and NPCs (data not shown). Our results are therefore consistent with a model in which H3K4 methylation and H3.3 localization at HCP TSS are coupled to transcriptional initiation.
We find that enrichment of H3.3 in the gene body and after the TES is proportional to transcriptional activity. As with previous studies in Drosophila
and human cells (Henikoff et al., 2009
; Jin et al., 2009
; Mito et al., 2005
), we find peaks of H3.3 after the TES of highly active genes, and we observe that these peaks are closely paralleled by peaks of Ser-5 phosphorylated RNAPII itself. We demonstrate that chromatin-based “transcriptional punctuation” (Siegel et al., 2009
; Talbert and Henikoff, 2009
) by H3.3 and phosphorylated RNAPII marks the boundaries of highly expressed genes in both undifferentiated and differentiated mammalian cells, calling attention to a potentially more universal mechanism for histone variant utilization as a genomic “boundary marker.”
To our knowledge, our report is the first genome-wide study to compare chromatin in the presence and absence of a mammalian histone chaperone. We find that H3.3 enrichment at active and repressed genes is dependent on the histone chaperone Hira. Previous studies suggest that H3.3 deposition in actively transcribed gene bodies may be coupled to transcription, potentially mediated by factors associated with elongating polymerase (Daury et al., 2006
; Janicki et al., 2004
; Schwartz and Ahmad, 2005
). Our data are consistent with Hira-dependent transcription-coupled deposition of H3.3 at transcribed non-repetitive sequences.
Intriguingly, we do not observe significant abnormalities in Hira −/− ES cells, despite a global lack of H3.3 enrichment at active and repressed genes, and despite the requirement of Hira for early embryonic development (Roberts et al., 2002
). We speculate that Hira −/− ES cells may be rescued by the replication-coupled deposition of histones during the frequent S-phases of rapidly dividing ES cells (Burdon et al., 2002
). Hira −/− ES cells divide as rapidly as wild-type ES cells, and show a similar preponderance of cells in S phase (A.C. and P.J.S., unpublished data). Overall, our data are consistent with a role for Hira in genic deposition of H3.3.
H3.3 enrichment has recently been shown at TFBS in Drosophila
and human cells (Jin et al., 2009
; Mito et al., 2007
). Deposition of H3.3 at TFBS may serve as a mechanism for the maintenance of regulatory elements in a more accessible chromatin conformation (Henikoff, 2008
). Close comparison of our data to a recent dataset of 13 different TFs in mouse ES cells (Chen et al., 2008
) shows H3.3 enriched in ES cells at all known types of TFBS genome-wide, whether in gene bodies, promoters, or intergenic regions. We also find a strong positive correlation between MTL and H3.3 localization, indicating particular enrichment of H3.3 at enhancer elements. Our data demonstrate that Hira is involved in H3.3 localization at some genic and intergenic TFBS. However, we also find that genome-wide H3.3 enrichment at many regulatory elements is Hira-independent and Atrx-independent. Our data therefore suggest that H3.3 localization at TFBS may be mediated by multiple and distinct factors, including Hira, with as yet unidentified factors mediating H3.3 localization at specific regulatory elements.
We find that H3.3 is specifically enriched in the canonical (TTAGGG)n repeat that is the hallmark of telomeres in vertebrates (Meyne et al., 1989
). Previous immunofluorescence studies localizing GFP-tagged Hira to telomeres suggested that Hira facilitates H3.3 deposition at this location (Wong et al., 2009
). However, using genome-wide ChIP-seq and cell imaging analyses in Hira −/− ES cells, we show that localization of H3.3 at telomeres in ES cells is Hira-independent. Further, we identify Atrx and Daxx as proteins that associate with H3.3 nucleosomes in the presence and absence of Hira.
Recent studies have shown that the Drosophila
homolog of Atrx, XNP, co-localizes with H3.3 at sites of nucleosome replacement on polytene chromosomes, but is not required for H3.3 localization at these sites (Schneiderman et al., 2009
). We find that Atrx is required for enrichment of H3.3 at mammalian ES cell telomeres, suggesting a divergence of homolog function. Moreover, we demonstrate that in the absence of Atrx, ES cells show upregulation of TERRA. Could ATRX and Daxx serve as specific H3.3 variant deposition machinery for specialized regions of heterochromatin? The ATRX/Daxx complex has previously been shown to have chromatin remodeling activity (Tang et al., 2004
; Xue et al., 2003
), and we show that Atrx is physically associated with ES cell telomeres. In addition to telomeres, our preliminary studies indicate that Atrx is also required for H3.3 enrichment at ribosomal DNA (data not shown), another transcribed repetitive element with characteristics of heterochromatin (McStay and Grummt, 2008
Our findings raise multiple questions. What is the function of H3.3 at genes, TFBS, and telomeres? Do cellular requirements for H3.3 differ in dividing versus post-mitotic cells, where replication-independent deposition might play a larger role? Is Atrx-mediated localization of H3.3 also replication-independent, like Hira, or does it occur during replication? Broadly, our study raises the prospect that distinct, region-specific chaperone and remodeling complexes may mediate the localization of a single histone variant (H3.3) to particular genomic regions. Although key factors required for region-specific H3.3 localization have now been identified, the exact deposition mechanisms at play remain an important challenge for future work.