Myc Function in ES Cells Figure 6. c-Myc levels influence the pattern of histone H3 lysine methylation in mES cells. (A) a group of genes bound by c-Myc (B) a group of non-Myc target genes. c-Myc levels were manipulated by (1) Lentiviral-delivered overexpression (qc-Myc) (2) knock-down (Q c-Myc), or (3) empty lentiviral vector control in ES cells. ChIP DNA isolated with anti-H3K4me3 or anti-H3-K27me3 was applied to the custom-designed arrays (see text). Enrichment ratios (log2 scale) for ChIP-enriched versus total input genomic DNA were processed by ACME and the given p values (2log10; y axis) identifying significant sites were plotted (see Methods). Red peaks present H3-K27me3 and green peaks present H3K4me3. Tick marks on the y- axis are spaced 400 bp apart. Asterisks mark positions of overlapping (bivalent) H3-K4me3 and H3-K27me3 peaks. The arrows at bottom indicate transcriptional start sites. doi:10.1371/journal.pone.0007839.g006 Drosophila cells indicated widespread binding by Myc [27–31]. Genomic loci associated with Myc binding were found to encompass a wide range of biological functions including metab- olism, protein biosynthesis, and cell signaling – functions generally consistent with the ability of Myc proteins to stimulate cell growth. Our ChIP-chip analysis of Myc bound loci in mES cells has identified a similarly broad range of functional categories related to growth and metabolism among 3189 binding sites. However, among the mES cell c-Myc targets identified here, we also detected genes involved in developmental processes (e.g. Sox2, Ebf1, Atoh1, En1, Hand1, Bmi1, Suz12) many of which had not been previously connected to Myc. We also detected genes not previously identified as Myc targets that are involved in chromatin assembly and modification including HirA, HirIP5, SMCX, SMRCA2, Anp32b, and Anp32e. Examination of gene targets using qChIP-PCR and/ or RT-PCR confirmed that many are bound and regulated by Myc. That Myc can elicit cell-type specific gene regulatory programs had been earlier suggested by a series of expression profiling experiments in pancreatic b-cells that uncovered a striking, and most likely direct, regulation of pancreatic cell-specific differentiation related genes by c-Myc [71]. Our data indicate that in mES cells Myc participates in binding and regulation of genes important in controlling aspects of chromatin structure and pluripotency. That Myc affects ES cell biology is consistent with an earlier study employing overexpression of mutant forms of Myc and demon- strated that c-Myc promoted mES cell self-renewal through inhibition of differentiation [41] as well as recent data demonstrat- ing that Myc regulates ES cell differentiation through the induction of a specific subset of microRNAs [60]. While this work was in progress three reports were published describing Myc binding sites in ES cells [72–74]. Two of these studies focused on binding by multiple core transcription factors [72,73] and the third examined Stat3 and Myc [74]. In these reports Myc binding was largely examined in the context of binding and gene regulation by these other factors. In this paper we have used cells sorted for the ES cell marker SSEA-1 to achieve a highly purified and phenotypically uniform population of ES cells. In addition we not only identified genes bound by endogenous c-Myc in ES cells but have examined the effects of Myc overexpression and knockdown on promoter occupancy and the landscape of H3-K4 and H3-K27 methylation. In general while our results agree with these reports in finding widespread binding of Myc to many genes critically involved in ES cell function, we have also found evidence implicating Myc in histone methylation and in bivalency in a subset of PcG target genes. Participation of Myc in ES Cell Transcription Circuitry It is noteworthy that sox2, encoding a critical transcription factor regulating pluripotency, is regulated and bound by Myc. Moreover histone H3 methylation at the sox2 promoter is altered in response to changes in c-myc abundance. This result is consistent with a previous study demonstrating elevated Sox2, among other pluripotency factors, in an ES cell-like expression module discovered in diverse human epithelial cancers [76]. Recently several pluripotency genes have been shown to be regulated by N- Myc overexpression in neuroblastoma [75]. This raises the possibility that Myc may participate in the Sox2 pluripotency network by directly up regulating Sox 2. We also detected overlap between Myc targets and genes marked by H3-K27me3 and Suz12 binding. Other studies have reported only a low correlation between c-Myc targets and H3-K27me3 [65,73]. Our data are consistent with this work in that we find only a fraction of Myc PLoS ONE | www.plosone.org 8 November 2009 | Volume 4 | Issue 11 | e7839