BindDB: Bivalent genes Case Study
Validate and Expand Established Epigenetic Profiles In order to validate the ability of BindDB to detect an already well-established epigenetic profile, we began with the large group of 'bivalent' genes coined on the premise of the epigenetic characteristics of their promoters. These 3913 genes in human and 2984 genes in mouse(Azuara et al., 2006; Bernstein et al., 2006; Li et al., 2013) have the H3K4me3 active promoter hallmark as well as the H3K27me3 repressive histone modification in close proximity to their transcriptional start sites and are lowly expressed in ESCs(Bernstein et al., 2006). The repressed nature of the bivalent promoters in ESCs is conferred by the Polycomb repressive complexes (PRC), which interact with the H3K27me3 mark. We uploaded the list of bivalent genes by gene name to BindDB, selected the 'proximal promoter (-1000, +1000) as the gene portion to query and within less than 5 minutes, could confirm all of the above. Enrichment score analysis (the ratio of factor binding to the queried gene promoters divided by the ratio of factor binding to all gene promoters) clearly indicates an enrichment in the characteristic "K4-K27" (Figure S1A, blue arrows) histone marking at bivalent gene promoters. In addition, the depletion of H3K36me3 indicates that these genes, although exhibiting an active chromatin mark, are not expressed at high levels. H3K36me3 is also depleted across the bivalent gene bodies when compared to all gene bodies (data not shown).This finding complies with the hypothesis that PHF19, a component of the Polycomb complex, recruits histone-lysine demethylase NO66 in order to reduce levels of H3K36me3 at bivalent genes(Brien et al., 2012) and is strengthened by the enrichment of H3K36me2 instead, and PHF19 itself at these promoters. In addition to PHF19, many other components of the Polycomb complexes can be found as well, including EZH1 and EZH2, SUZ12, JARID2, and RING1 proteins. The presence of KDM2A and KDM2B may imply that these proteins are involved in the demethylation of either lysine 4 or lysine 27 during cell fate determination. Alternatively, KDM2B (FBXL10) along with CBX7, which also shows enrichment in the BindDB analysis (Figure S1A, left), have been shown to be involved in the recruitment of the PRC1 complex to the H3K27me3 histone mark (He et al., 2013; Morey et al., 2012). Strikingly, hardly any significant enrichment of transcription factors could be detected at bivalent genes in ESCs (Figure S1B, filtered heatmap), except for OCT4 and SOX2, in line with previous findings that these key ESC transcription factors bind approximately one third of PRC2-occupied genes that also encode developmental transcription factors (Boyer et al., 2006; Lee et al., 2006). This phenomenon is unique to this group of genes (see subsequent examples) and signifies the important role of epigenetic regulation on these 'poised', yet inactive genes in ESCs.