ESCs represent a unique type of stem cell that can undergo indefinite cycles of self-renewal while maintaining pluripotency. Previous studies have identified many crucial gene transcription factors and regulatory networks that are required for maintaining the “stemness” of ESCs (Avilion et al., 2003
; Chambers et al., 2003
; Ivanova et al.,2006
; Mitsui et al., 2003
; Nichols et al., 1998
; Walker et al, 2007
). One of the insightful conclusions is that Oct4, Nanog, and Sox2 form transcriptional circuitry to activate their own expression in a forward feedback manner; furthermore, the Oct4/Nanog/Sox2 complex promotes expression of those genes required for the self-renewal of ESCs, but represses the developmental genes that will only be activated upon cell differentiation (Boyer et al., 2006
; Loh et al., 2006
). In addition, along with Oct4/Nanog/Sox2, Trithorax- and Polycomb-complex mediated histone modifications are also involved in the activation or repression genes, as well as in maintaining the “poised” nature of some genes (Mikkelsen et al., 2007
; Walker et al., 2007
; Pan et al., 2007
; Zhao et al., 2007
). In this report, we have comprehensively mapped CpG methylation in the proximal gene promoter regions and identified 6,127 methylated and 5,074 unmethylated proximal gene promoters. Our data help refine the emerging epigenetic landscape of mESCs. By comparing promoter DNA methylation with histone modifications, we can gain insights into how overlapping or independent epigenetic regulators regulate particular sets of genes (). One of the novel findings in this study is that DNA methylation occurs in approximately 87% of the genes in ESCs that lack either H3K4me3 and H3K27me3 (). This population of methylated genes could potentially constitute close to one-third of all annotated genes in mouse and human ESCs (Mikkelsen et al., 2007
; Pan et al., 2007
; Zhao et al., 2007
). Therefore, we conclude that DNA methylation in proximal gene promoter regions represents another major epigenetic marker that can distinguish different classes of genes in undifferentiated ESCs ().
A Schematic Summary of Epigenetic and Transcriptional Regulation in mESCs
In this study, we provided evidence that DNA methylation is causally linked to the silencing of a cluster of X-linked genes and a subset of developmentally regulated genes. It should be noted that the mESCs used in these experiments are male (XY) and lack X-inactivation; therefore, up-regulation of X-linked genes in TKO mESCs cannot be related to de-regulation of X-inactivation. However, it is known that genes involved in germ cell differentiation and sex development are over-represented on the X-chromosome (Wang et al., 2001
), and many of these genes tend to be duplicated on the X-chromosome. Furthermore, DNA methylation is proposed to directly silence many genes involved in germ cell development (Maatouk et al., 2006
). Indeed, Rhox
family genes which have been shown to be duplicated extensively on the X chromosome, are repressed by DNA methylation in somatic cells and ESCs (Maclean et al., 2005
; MacLean et al., 2006
; Oda et al., 2006
). Similarly, DNA methylation represses the Mage
gene family and the Dazl
gene that are related to germ cell development (Chuang et al., 2005
; De Smet et al., 1999
; Maatouk et al., 2006
). Finally, demethylation-induced over-expression of Mage
family genes is also observed in somatic cells treated with 5’azacytodine to inhibit DNMTs (Chuang et al., 2005
). It is worth noting that the up-regulation of a subset of X-linked and development genes apparently does not interfere with the self-renewal of demethylated ESCs. Thus, ESCs can tolerate the over-expression of a subset of genes that serve a specialized function in germ cell or other types of somatic cells.
Although our study provides direct evidence that DNA demethylation can induce gene activation for a number of cell differentiation genes, we also found many genes are not up-regulated in the absence of DNA methylation. This result is in contrast to the result observed in demethylated primary fibroblasts, in which up to 10% of expressed genes can be up-regulated compared to wildtype primary fibroblasts (Jackson-Grusby, et al. 2001
). Genome-wide mapping of histone modifications indicated that bivalent H3K4/K27me3 is more widespread than the association of Polycomb proteins (Boyer, et al., 2006
; Mikkelsen, et al., 2007
; ). Moreover, many methylated genes also contain H3K27me3 or bivalent H3K4/K27me3 markers in mESCs, raising the possibility that repressive histone marks can to some extent compensate for loss of DNA methylation in gene repression. It is also possible that compensatory repressive mechanisms have become activated during generation and culture of the TKO cell line. In addition, a lack of proper gene transcription activators in mESCs for those demethylated genes may also account for the continued inactive status of a majority of demethylated genes in TKO mESCs. Our results are consistent with the notion that DNA demethylation is necessary but not sufficient for gene activation. Conversely, methylation of a promoter is not always sufficient for gene repression. Using previously published gene expression data for mESCs, we found that up to 36% of genes are still expressed even if methylated in the proximal promoter. However, 80% of the expressed genes that exhibit promoter methylation are marked by the active histone H3K4me3 mark and are HCP genes. These observations are consistent with the suggestion by Weber et al. (2007)
that a low density of DNA methylation in a gene promoter may not be sufficient to silence gene transcription by itself.
The mapping of promoter methylation patterns in mESCs could provide insights into why mESCs are a good cell source for somatic nuclear transfer experiments when compared to differentiated somatic cells (Yamanaka, 2007
). One of the major hurdles in somatic nuclear transfer experiments is the efficiency of epigenetic reprogramming. It has been shown that a panel of genes including Oct4 and Dppa4 are only partially reactivated in somatic nuclear reconstituted blastocyst embryos, which could be attributed to the incomplete demethylation of Oct4 and Dppa family genes in somatic nuclear during reprogramming (Bortvin et al., 2003
). Our methylation mapping indicated that both Oct4 and Dppa4 genes are demethylated in mESCs, supporting the notion that ESCs are more easily reprogrammed than somatic nuclei.
In summary, our comprehensive mapping of DNA methylation of gene promoters in mESCs provides a valuable resource for understanding the function of DNA methylation in the maintenance of self-renewal and pluripotency. Furthermore, the methylation patterns in gene promoters may also represent an epigenetic code that underlies the program of lineage specific differentiation.