Myc Function in ES Cells Figure 3. c-Myc, and Polycomb proteins share target genes. (A) Gene Ontology categories of c-Myc associated genes in murine ES cells. Functional categories derived for genes enriched at least 4-fold for c-Myc binding and possessing an ACME p,0.0001 in the ChIP-chip assay (see Materials and Methods and Supplementary Table S1). The numbers to the right represent the p-values for the statistical over-representation in each category. (B) Venn diagram depicting the overlap among murine ES cell genes bound by c-Myc (this paper), Suz12, and those displaying trimethylation of H3-K27 [16]. Nearly 96% of promoters bound by Suz12 also show the H3-K27me3 mark. Of genes associated with Myc (2282) 17% are associated with the H3-K27me3 mark and 9.5% associated with both Suz12 and H3-K27me3 (for gene list see Supplementary Table S3). (C) Gene ontology categories of gene displaying overlap among c-Myc, Suz12 binding and H3-K27me3 marks. Functional categories were derived for genes enriched at least 4-fold for c-Myc binding in the ChIP-chip assay (see Methods and Supplementary Table S1). Suz12 and H3-K27me3 bound genes were previously described [16]. Numbers to the right are p-values for the statistical over-representation among genes within a functional category. doi:10.1371/journal.pone.0007839.g003 encode transcription factors involved in development and differentiation (Figure 3C) such as Ebf2, MyoD1, Hand1, Isl1 and Atoh1, the homeobox gene family members Hox b,c,d, and genes involved in critical signaling pathways such as Wnt and FGF (for gene list see Table S2). Our ChIP-chip analysis also revealed that among the group of PcG bound genes associated with c-Myc are many genes previously characterized as possessing bivalent chromatin marks [20,21]. Furthermore when we examined a group of these known bivalent genes by qPCR we found that they are induced by c-myc overexpression and repressed following c-myc knockdown (e.g. Zic4, Ebf1 and Foxp2, Fgf5, Fgf18, Ebf2, Hand1, Olig2, Lhx2, HoxB3, Olig 3, Tcfap2b, En2, Atoh1, Ngn2, Ngn3, Gata6 marked by asterisks in Figure 2). These findings indicate that c-Myc binds to and can regulate PcG silenced genes in ES cells. c-Myc Influences the Level and Pattern of Histone H3 Lysine Methylation in ES Cells Myc has been linked to histone methylation through its inhibitory interaction with the LID histone H3-K4me3 demethy- lase in Drosophila [63] and by Myc’s apparent preference for binding to promoters that are methylated at H3-K4 [57,65]. Above we have described Myc binding to PcG target genes that contain an H3-K27me3 mark (see Figure 3B). To determine if changes in c-Myc abundance affects the levels of H3-K4me3 and H3-K27me3, we carried out lentiviral-mediated Myc expression and determined the presence of these two histone marks in bulk chromatin using Western blotting. Introduction of the c-myc shRNA and overexpression vectors results in the expected changes in Myc protein levels (Figure 4A). Our results show that an approximately 8-fold increase c-Myc augments H3K4me3 abun- dance by 50–60% (Figure 4A). By contrast, H3K4me3 levels are decreased ,30% subsequent to c-Myc knock-down (Figure 4A). When we examined H3-K27me3 under the same conditions we were unable to detect any significant change. To determine whether loss of c-Myc expression has a significant effect on H3K4me3 level during embryonic development we examined embryos from wildtype and c-myc mice. Targeted deletion of both c-myc alleles results in embryonic lethality at E10.5 [66] due to defective hematopoiesis [67]. When we analyzed c-myc embryos at E7, a time when no phenotypic change is apparent between wildtype and c-myc embryos, we found a dramatic (,70–80%) decrease in H3K4me3 levels, but no significant change in H3K27me3 compared to wildtype. Our results suggests that c-Myc expression is involved in maintaining H3K4me3 levels in ES cells and in early embryonic development (Figure 4C). To examine whether changes in Myc levels influence the H3 methylation landscape at individual Myc-bound genes in ES cells we extended our ChIP-chip analysis using antibodies against H3- K27me3 and H3-K4me3. We employed a custom-designed oligonucleotide tiling array of 815 promoter regions including promoters of genes that were bound by both Myc and Polycomb PLoS ONE | www.plosone.org 5 November 2009 | Volume 4 | Issue 11 | e7839