Ctional domains. Considerable evidence suggests that posttranslational modifications to DNA and histones define a `chromatin state’ that dictates a distinct cellular state and thus a particular transcriptional program (Reviewed in [1?]). Genome-wide maps of chromatin state have been made for numerous modifications in a variety of cell types. The resulting maps show that modifications often exist in specific combinations corresponding to unique functional genomic features. For example, trimethylation of histone H3 at MedChemExpress Madrasin lysine 4 (H3K4me3) and lysine 27 (H3K27me3) exists at the promoters of a subset of genes in ES cells [4,5]. Such `bivalent’ genes tend to be associated with developmental functions and are repressed in ES cells, but poised for activation upon differentiation. A more recent study examined nine histone modifications in nine human cell types and found 15 chromatin states with distinct profiles of chromatin marks and functional enrichments [6]. Epigenetic modifications may also be antagonistic. In Arabidopsis thaliana the histone Hvariant H2A.Z and DNA methylation (DNAme) are mutually antagonistic [7]. DNA methylation is associated with repression while H2A.Z promotes transcriptional competence. Mutation of the PIE1 subunit of the Swr1 complex that deposits H2A.Z leads to genome-wide hypermethylation, while mutation of the MET1 DNA methyltransferase engenders opposite changes in DNA methylation and H2A.Z deposition. In addition to the examples described, coordinate regulation of epigenetic modifications has been demonstrated in a number of studies, consistent with the hypothesis of a histone code [8?1]. DNA methylation and H3K27me3 are both involved in the establishment and maintenance of epigenetic gene silencing. There are data showing coordinate regulation between the marks. Some evidence points toward a cooperative relationship. For example, the polycomb group protein EZH2 has been shown to positively regulate DNA methylation [12]. In these studies, EZH2 was observed to interact with DNA methyltransferases (DNMTs) and was required for DNA methylation of EZH2-target promoters. Alternatively, several lines of evidence suggest the coordination between DNAme and H3K27me3 may be antagonistic. A proteomic analysis has shown the PRC2 components EED and SUZ12 are excluded from methylated DNA [13], and in neural stem cells Dnmt3a deficiency leads to increased H3K27me3 [14]. Also, our lab has previously shown that at the imprinted locus Rasgrf1 DNAme and H3K27me3 are mutually exclusiveDNAme and H3K27me3 in Mouse Embryonic Stem Cells[15]. Finally, additional studies suggest that an important relationship between DNAme and H3K27me3 is disrupted in cancer cells. Polycomb group Homatropine methobromide targets are more likely to have cancerspecific promoter DNA hypermethylation than non-targets [16?18]. However, embryonic carcinoma cells lack DNA hypermethylation at PRC targets [19], and knockdown of EZH2 in cancer cells may lead to hypomethylation [20]. Thus the evidence of interaction is conflicting, but it is clear that the relationship between these marks is important in both normal and cancerous cells. Here, we attempt to address the relationship between DNAme and H3K27me3 by undertaking a genome-wide analysis to examine the effect loss of one mark has upon the placement of the other. We use mouse embryonic stem cells with defective PRC2 activity to examine the effect on the placement of DNAme, and use cells with defective DNA methyltransferase activity to i.Ctional domains. Considerable evidence suggests that posttranslational modifications to DNA and histones define a `chromatin state’ that dictates a distinct cellular state and thus a particular transcriptional program (Reviewed in [1?]). Genome-wide maps of chromatin state have been made for numerous modifications in a variety of cell types. The resulting maps show that modifications often exist in specific combinations corresponding to unique functional genomic features. For example, trimethylation of histone H3 at lysine 4 (H3K4me3) and lysine 27 (H3K27me3) exists at the promoters of a subset of genes in ES cells [4,5]. Such `bivalent’ genes tend to be associated with developmental functions and are repressed in ES cells, but poised for activation upon differentiation. A more recent study examined nine histone modifications in nine human cell types and found 15 chromatin states with distinct profiles of chromatin marks and functional enrichments [6]. Epigenetic modifications may also be antagonistic. In Arabidopsis thaliana the histone Hvariant H2A.Z and DNA methylation (DNAme) are mutually antagonistic [7]. DNA methylation is associated with repression while H2A.Z promotes transcriptional competence. Mutation of the PIE1 subunit of the Swr1 complex that deposits H2A.Z leads to genome-wide hypermethylation, while mutation of the MET1 DNA methyltransferase engenders opposite changes in DNA methylation and H2A.Z deposition. In addition to the examples described, coordinate regulation of epigenetic modifications has been demonstrated in a number of studies, consistent with the hypothesis of a histone code [8?1]. DNA methylation and H3K27me3 are both involved in the establishment and maintenance of epigenetic gene silencing. There are data showing coordinate regulation between the marks. Some evidence points toward a cooperative relationship. For example, the polycomb group protein EZH2 has been shown to positively regulate DNA methylation [12]. In these studies, EZH2 was observed to interact with DNA methyltransferases (DNMTs) and was required for DNA methylation of EZH2-target promoters. Alternatively, several lines of evidence suggest the coordination between DNAme and H3K27me3 may be antagonistic. A proteomic analysis has shown the PRC2 components EED and SUZ12 are excluded from methylated DNA [13], and in neural stem cells Dnmt3a deficiency leads to increased H3K27me3 [14]. Also, our lab has previously shown that at the imprinted locus Rasgrf1 DNAme and H3K27me3 are mutually exclusiveDNAme and H3K27me3 in Mouse Embryonic Stem Cells[15]. Finally, additional studies suggest that an important relationship between DNAme and H3K27me3 is disrupted in cancer cells. Polycomb group targets are more likely to have cancerspecific promoter DNA hypermethylation than non-targets [16?18]. However, embryonic carcinoma cells lack DNA hypermethylation at PRC targets [19], and knockdown of EZH2 in cancer cells may lead to hypomethylation [20]. Thus the evidence of interaction is conflicting, but it is clear that the relationship between these marks is important in both normal and cancerous cells. Here, we attempt to address the relationship between DNAme and H3K27me3 by undertaking a genome-wide analysis to examine the effect loss of one mark has upon the placement of the other. We use mouse embryonic stem cells with defective PRC2 activity to examine the effect on the placement of DNAme, and use cells with defective DNA methyltransferase activity to i.
