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Title: Genome-Wide uH2A Localization Analysis Highlights Bmi1-Dependent Deposition of the Mark at Repressed Genes}
Article:
Abstract:
Polycomb group( PcG) proteins control organism development by regulating the expression of developmental genes. Transcriptional regulation by PcG proteins is achieved, at least partly, through the PRC2-mediated methylation on lysine 27 of histone H3( H3K27) and PRC1-mediated ubiquitylation on lysine 119 of histone H2A( uH2A). As an integral component of PRC1, Bmi1 has been demonstrated to be critical for H2A ubiquitylation. Although recent studies have revealed the genome-wide binding patterns of some of the PRC1 and PRC2 components, as well as the H3K27me3 mark, there have been no reports describing genome-wide localization of uH2A. Using the recently developed ChIP-Seq technology, here, we report genome-wide localization of the Bmi1-dependent uH2A mark in MEF cells. Gene promoter averaging analysis indicates a peak of uH2A just inside the transcription start site( TSS) of well-annotated genes. This peak is enriched at promoters containing the H3K27me3 mark and represents the least expressed genes in WT MEF cells. In addition, peak finding reveals regions of local uH2A enrichment throughout the mouse genome, including almost 700 gene promoters. Genes with promoter peaks of uH2A exhibit lower-level expression when compared to genes that do not contain promoter peaks of uH2A. Moreover, we demonstrate that genes with uH2A peaks have increased expression upon Bmi1 knockout. Importantly, local enrichment of uH2A is not limited to regions containing the H3K27me3 mark. We describe the enrichment of H2A ubiquitylation at high-density CpG promoters and provide evidence to suggest that DNA methylation may be linked to uH2A at these regions. Thus, our work not only reveals Bmi1-dependent H2A ubiquitylation, but also suggests that uH2A targeting in differentiated cells may employ a different mechanism from that in ES cells.
Introduction:
In higher eukaryotes, DNA is organized in the form of chromatin. The basic repeating unit of chromatin is called the nucleosome, which consists of 146 bp of DNA wrapped around a core histone octamer. One unique feature of core histones is their proclivity for covalent modification including acetylation, methylation, ubiquitylation, and phosphorylation[1]. In addition, DNA can be modified directly through methylation. These covalent modifications can affect gene transcription directly or indirectly through the recruitment of additional modulatory factors[2]. Therefore, different combinations of modifications on chromatin may ultimately determine distinct cellular states through regulating the transcriptional programs that cells adopt. Thus, identification and characterization of the proteins that are responsible for the placement and maintenance of these epigenetic marks is of great importance in understanding cellular proliferation and differentiation. The addition of a single ubiquitin molecule to histone H2A at lysine 119 was first discovered over thirty years ago[3]. Classic experiments demonstrated that uH2A accounts for about 10% of total H2A[4]. Despite the knowledge of its existence, the identity of the responsible enzymes and the function of this modification have only recently begun to be elucidated. The first H2A ubiquitin E3 ligase was identified as the core components of the Polycomb repressive complex 1( PRC1) composed of RING1/2, BMI1, and HPH2[5]. Biochemical and functional analysis of the PRC1 complex has revealed RING2/Ring1b as the catalytic subunit, which can be greatly stimulated by Bmi1 and Ring1a, as loss function on any of these two proteins resulted in drastic genome-wide reduction of uH2A[6],[7]. Genome-wide location studies revealed that PRC1 occupies the promoters of a subset of Polycomb repressive complex 2( PRC2) targets and both PRC1 and PRC2 are enriched at genes involved in developmental processes[8]–[10]. Recent studies have uncovered that Bmi1 homologs, such as Mel18 and NSPc1, can target the PRC1 complex in various cell types[11],[12]. In addition, a new E3 ligase for H2A, 2A-HUB, has also been reported[13] highlighting the fact that there must be Bmi1-dependent and Bmi1-independent pools of uH2A in the genome. Unlike PRC1, PRC2 possesses H3K27-specific histone methyltransferase activity[14]. The discovery that a component of PRC1, Pc, can specifically recognize and bind to H3K27me3[15]–[17] has prompted researchers to embrace a sequential recruitment model whereby PRC2-mediated H3K27 methylation contributes to PRC1 recruitment and subsequent ubiquitylation of histone H2A. This model is supported by three pieces of evidence. First, studies on Hox and Ink4a/Arf loci indicate that PRC1 knockdown reduced local uH2A levels which correlate with upregulation of gene expression[7],[18],[19]. Second, knockdown of the H3K27me3 demethylase, Utx, results in enrichment of both PRC1 and uH2A at PRC2 target genes[20]. Third, the majority of genome-wide Ring1b enriched regions in mouse embryonic stem cells( mES) co-localize with peaks of H3K27me3[21]. In addition to the relationship between H3K27 methylation and H2A ubiquitylation, several studies also suggest a potential link between H3K27 methylation and DNA methylation. For example, H3K27 methylation has been demonstrated to play an important role in imprinted gene silencing[22],[23]. Components of PRC2, such as Ezh2, have been reported to interact with Dnmt1/3a/3b and are required for efficient DNA methylation at several target genes[24]. On the other hand, Dnmt1 may contribute to the recruitment of PRC1 as knockdown of Dnmt1 abrogates localization of PRC1 components to Polycomb bodies in cultured cells[25]. Consistent with this notion, recent studies have demonstrated that components of PRC1 can interact with a methyl-DNA binding protein, Mbd1[26], and the Dnmt1-associated protein, Dmap1[27]. Despite these reports, a general correlation between H3K27 methylation and DNA methylation may not exist as genome-wide epigenetic profiling revealed only a small subset of H3K27me3 positive promoters were found to be hypermethylated[28],[29]. Whether there exists a genome-wide link between PRC1 mediated H2A ubiquitylation and DNA methylation remains to be determined. The advent of chromatin immunoprecipitation coupled to genomic tiling arrays( ChIP-chip) has provided scores of reports highlighting genome-wide maps of histone modifications[30], histone modifying enzyme binding profiles[9], and transcription modulators[31],[32]. Recent advances in ChIP-coupled deep sequencing have greatly expedited the tedious task of dissecting the interplay between epigenetic modifications and complex transcriptional output[33]. Although genome-wide analysis of most epigenetic marks[34]–[36], as well as transcription factors[37], has been reported for various cell lines, uH2A distribution remains a mystery. In addition, the recent discovery that the majority of uH2A in the fly genome is placed by a complex containing the Bmi1 homolog but lacking Pc[38] calls the generality of the sequential recruitment model into question. To understand how uH2A fits into the complex epigenetic architecture associated with mammalian chromatin, we describe the genome-wide profile of Bmi1-dependent uH2A by comparing the enrichment of this mark in Bmi1 wild-type and null MEF cells. This analysis provides evidence that while Bmi1 dependent uH2A is enriched at genes containing the H3K27me3 mark, it is not limited to these regions. In addition, analysis of genome-wide DNA methylation patterns reveals a link between uH2A and DNA methylation in high-density CpG promoters. Transcription profiling of wild-type MEF cells indicates that Bmi1-dependent uH2A is enriched at genes with low levels of de novo expression. Finally, genes containing the highest levels of Bmi1 dependent uH2A at their promoters are expressed higher upon Bmi1 loss of function than genes harboring low levels of uH2A. Thus, our study uncovers some previously unrecognized features of uH2A.
Discussion:
By combining genome averaging and peak localization analyses, this study reveals the first picture of the genome-wide localization of the uH2A mark and identifies Bmi1-dependent enrichment within both genic and non-genic regions of the mouse genome. On average, uH2A tag density is enriched at gene promoter regions( Figure 1A) with further enrichment at genes marked by H3K27me3( Figure 1B). Peak analysis reveals that Bmi1-dependent uH2A enriched regions coincide with gene promoters at a much higher rate than can be expected by chance( Table 1), and these peaks encompass significantly higher tag values when compared with non-promoter peaks( Figure 3B). In addition, promoter peak tag values are enriched at genes also marked by H3K27me3( Figure 3C). Interestingly, the gross distribution of genic uH2A peaks is skewed towards the 3′ end of genes, indicating that more regions of uH2A enrichment are found within these genic regions( Figure S1A). However, these peaks represent lower enrichment regions when compared to promoter peaks( Figure S1B). Together, these data support the notion that H3K27me3 can contribute to the recruitment of PRC2 and ubiquitylation of H2A in promoters[16],[17]. However, even though the highest regions of Bmi- dependent uH2A enrichment are at H3K27me3 genes, clear peaks also exist at genomic regions( both genic and non-genic) that do not contain this mark. This uH2A distribution pattern is very different from what would be expected given a recent study of Ring1b and Bmi1 binding profiles in mES cells[21] but are more consistent with a previous study which described a proportion of gene promoters positive for Ring1b/Bmi1 but negative for PRC2 binding[8]. Although this discrepancy may reflect the differences of uH2A distribution in ES cells and MEF cells, it is possible that similar to the observations in Drosophila[38], a Pc independent mechanism of Ring1b/Bmi1 recruitment may exist in MEF cells. Along these lines, about half of all the most enriched Bmi1-dependent uH2A regions lie outside of both promoter and transcribed regions of the genome( Table 1). Interestingly, these regions do not overlap significantly with peaks of H3K27me3. Since the initial finding linking Polycomb silencing to DNA methylation[24], genome-wide studies have called the generality of this association into question as very little overlap exists between genes methylated at H3K27 and genes that contain high levels of CpG island methylation associated with their promoters[28],[29],[49]. Recently, several reports have demonstrated a role for PRC1 in recognizing methylated DNA at specific loci or heterochromatic regions[25] either through Bmi1 interaction with Dmap1[27] or Ring1b interaction with Mbd1[26]. However, whether these specific instances of convergence of silencing pathways are linked to global gene regulation has not been determined. Here we provide evidence to support a functional link between DNA methylation and histone ubiquitylation through Bmi1-dependent mechanisms in a group of high CpG-containing genes. Not only does the level of DNA methylation increase at HCP promoters marked by uH2A( Figure 4B), a result that may be explained by an increase in H3K27 methylation on these genes, but knock-out of Dnmt1, which results in the ablation of CpG methylation genome wide, causes a global decrease in the uH2A levels( Figure 4C). In contrast, Bmi1 knock-out results in only a small, statistically insignificant, increase in global DNA methylation( Figure 4D). Taken together, these data suggest that DNA methylation may be upstream of Bmi1-dependent H2A ubiquitylation. Future work will reveal how DNA methylation contributes to H2A ubiquitylation. Recent studies in a limited gene set indicate that uH2A is mostly linked to transcriptional repression[7],[40],[48],[50]. Consistent with these studies, our genome-wide analysis indicate that Bmi1-dependent uH2A exhibits a broad enrichment at the most repressed genes in the mouse genome( Figure 5A and 5B). Peak centered analysis of promoter uH2A confirms this result( Figure 5C) and reveals that this enrichment is most likely a consequence of over-representation of the H3K27me3 mark( Figure 5C, 5D, and 5E). These results indicate that unlike H3K27me3[34],[35], uH2A by itself is not an accurate predictor of de novo expression levels and could serve to explain earlier studies which have reported the presence of uH2A at actively transcribed genes[51],[52]. Instead, our results suggest that uH2A plays a much more refined role in the control of gene expression. Analysis of expression changes in Bmi1 knockout MEFs revealed that, on average, genes marked by promoter peaks of uH2A are upregulated upon Bmi1 knockout. In addition, the level of enrichment of these promoter uH2A peaks, as indicated by sequence tag density, reveal a clear increase in average fold change when higher density peaks are compared with lower density peaks( Figure 5F). Thus, our work extends gene specific studies and confirms the existence of a genome-wide link between uH2A and gene silencing.
A wealth of recent studies has demonstrated the role of Bmi1-stimulated histone ubiquitylation in the repression of transcription at targeted genetic loci . However , the repressive function of this mark has never been extrapolated genome-wide . We have used deep sequencing technology to explore the global deposition of Bmi1-dependent H2A ubiquitylation ( uH2A ) in mouse embryonic fibroblast cells . Our study confirms the gene-specific repressive function of the uH2A mark on a genome-wide scale . In addition , we also analyzed the general trends of uH2A distribution with respect to genomic elements , such as various classes of gene promoters and transcribed regions . Our work implies that the mechanism of uH2A distribution in differentiated cells may vary from that in embryonic stem cells . Given the importance of the uH2A modification in fundamental biological processes and cancer , insight into the distribution of this modification has reaching implications in understanding the contribution of epigenetic silencing to cellular physiology .