Ring1b Regulates ES Cell Fate Figure 6. Deregulation of genes bivalent or H3K4me3 marked genes with HCPs in Ring1b-deficient ES cells. (A–D) Circle graphs show that Ring1b primarily occupies (C) and regulates (D) transcription of genes with high CpG content promoters (HCPs) rather than intermediate (ICP) or ‘low’ (LCP) CpG content promoters, compared to the distribution of HCPs in all genes (A) or only the deregulated genes (B) at the microarray (‘genome-wide’). (E) Bar graph showing the distribution (percentages) of the chromatin state of HCPs for all genes on the whole microarray (‘genome- wide’); only the genes deregulated in absence of Ring1b; Ring1b bound genes; only deregulated Ring1b bound genes in Ring1b-deficient ES cells. Graph shows that Ring1b binds and regulates transcription of bivalent, and to a lesser extend H3K4me3 marked genes (final two columns), in contrast to the genome-wide distribution of these marks on promoters (first two columns). (F) as (E), but for genes with ICPs. Graph shows that bivalent Ring1b bound genes with ICPs are not deregulated in Ring1b-deficient ES cells. (G) Blotplot representing Affimetrix array data of RNA expression levels in wild type ES cells [30], for the genes represented in (E). The blotplot shows that the median expression levels of H3K4me3 marked genes on the whole microarray (‘genome-wide’ ) are higher compared to the median RNA expression levels of H3K4me3+H3K27me3 marked genes (first two columns). This is similar for genes that are bound by Ring1b (middle two columns), and for Ring1b bound genes that are deregulated in Ring1b-deficient ES cells (last two columns). Black diamond represents median RNA expression level. (H–K) Line graph showing changes (log2 ratio) in gene expression of bivalently (H–I) or H3K4me3 (J–K) marked genes bound by Ring1b following deletion of Ring1b in Ring1b2/Lox;CreERT2 ES cells. Time is indicated in number of days of 4-OHT treatment. doi:10.1371/journal.pone.0002235.g006 sponding transcriptional state, according to the chromatin-state maps generated for these cell types [30] (Figure S3). In contrast, of the bivalently marked genes only 11% and 51% retain both histone marks in NPCs and MEFs, respectively [30] (Figure S3), indicating that Ring1b does not act to silence the H3K4me3 marked genes in ES cells or during development. Although we cannot exclude that differentiation-associated changes affect transcription of the Ring1b bound genes, these data suggest that Ring1b predominantly represses bivalent genes, and is also involved in modulating, rather than silencing, the transcriptional activity of genes with the active H3K4me3 histone mark in ES cells. Strikingly, a specific subset of Ring1b bound genes was not affected following Ring1b deletion. This subset contains LCPs, which were all without H3K27me3 or H3K4me3 marks, and bivalently marked genes with ICPs (Figure 6D, 6F). This implies that other mechanisms are involved in regulation of these genes in ES cells. Summarizing, these data suggest that Ring1b is involved in direct regulation of RNAPII controlled promoters with ‘high’ CpG content and epigenetically distinct histone marks, namely bivalent marks or the active H3K4me3 mark, in mouse ES cells. Discussion To investigate the role of Ring1b in mouse ES cells, we employed a conditional knockout system. By performing micro- array analysis, we studied the genome-wide changes in gene transcription that occur following Ring1b deletion in ES cells. We PLoS ONE | www.plosone.org 9 May 2008 | Volume 3 | Issue 5 | e2235