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 
. 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 
. 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 
. Classic experiments demonstrated that uH2A accounts for about 10% of total H2A 
. 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 
. 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 
. 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 
. Recent studies have uncovered that Bmi1 homologs, such as Mel18 and NSPc1, can target the PRC1 complex in various cell types 
. In addition, a new E3 ligase for H2A, 2A-HUB, has also been reported 
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 
. The discovery that a component of PRC1, Pc, can specifically recognize and bind to H3K27me3 
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 
. Second, knockdown of the H3K27me3 demethylase, Utx, results in enrichment of both PRC1 and uH2A at PRC2 target genes 
. Third, the majority of genome-wide Ring1b enriched regions in mouse embryonic stem cells (mES) co-localize with peaks of H3K27me3 
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 
. 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 
. 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 
. Consistent with this notion, recent studies have demonstrated that components of PRC1 can interact with a methyl-DNA binding protein, Mbd1 
, and the Dnmt1-associated protein, Dmap1 
. 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 
. 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 
, histone modifying enzyme binding profiles 
, and transcription modulators 
. Recent advances in ChIP-coupled deep sequencing have greatly expedited the tedious task of dissecting the interplay between epigenetic modifications and complex transcriptional output 
. Although genome-wide analysis of most epigenetic marks 
, as well as transcription factors 
, 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 
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.