epigenetic marks associated with gene repression. This includes, in comparison to non-pluripotent cells, decreased DNA methylation and histone H3 lysine 27 trimethylation (H3-K27me3) as well as augmentation of positive marks such as histone H4 acetylation and H3-K4me3 [19,20] (for review see [3]). Nonetheless, the association of Polycomb complexes with a subset of Sox2-Oct4- Nanog bound genes that are important for cell fate transitions [16–18] would suggest that a highly regulated form of repression must be important in self-renewing ES cells. Indeed several studies have confirmed the presence of H3-K27me3 at the promoters of Polycomb bound loci, many of which overlap with Sox2-Oct4- Nanog binding sites. Interestingly, a subgroup of these promoters contain islands of H3-K4me3 within a larger domain of H3- K27me3, constituting what has been termed a ’’bivalent‘‘ chromatin structure [20]. A number of these bivalent genes lose Polycomb binding and H3-K27 methylation upon differentiation, leading to the proposal that bivalency marks genes that are poised for activation upon ES cell differentiation [16,20–23]. Such bivalent domains are not restricted to ES cells and their formation and resolution is likely to be a widespread and dynamic process [21–23]. While loss of function of Polycomb subunits and abolishment of H3-K27me3 results in upregulation of differenti- ation-related bivalent genes in ES cells, these cells continue to self- renew, indicating that Polycomb-mediated repression of these genes is not the sole determinant of the transcriptional regulation that maintains the undifferentiated state [24,25]. These experi- ments prompt questions concerning the extent to which histone modifications represent a cause or a consequence of pluripotency. A related issue concerns the precise mechanisms through which widespread epigenetic changes occur. Recently the histone H3-K9 demethylases Jmjd1a and Jmjd2c have been shown to be targets of Oct4 and are implicated in the activities of the Sox2-Oct4-Nanog network and the maintenance of pluripotency-related gene expression [26]. However it is still unclear how these and other histone modifying complexes are recruited to specific gene targets in ES cells. Another transcriptional regulatory protein that has been connected with ES cell function is Myc. The myc oncogene family encodes the c-, N- and L-Myc, bHLHZ proteins that hetero- dimerize with Max and bind DNA at E-box sequences. Myc family proteins have been associated with cell growth and proliferation through widespread binding to DNA (on the order of 10–20% of genomic loci are bound) and modulation of the expression of hundreds to thousands of genes [27–31]. These include genes encoding ribosomal RNA and ribosomal proteins as well as many factors involved in translation and metabolism [32–36]. myc gene expression is itself regulated by many different mitogens, cytokines, and growth factors, suggesting that Myc serves as an integrator of signal transduction. As such, Myc proteins are likely to link extracellular signals to an acute cellular growth response [37–39]. It is therefore unsurprising that Myc is expressed in different types of stem cells including ES cells [40–43]. Yet there are several lines of evidence indicating that Myc may function as a regulator of pluripotency. For example, although Myc expression has long been associated with the inhibition of terminal differentiation, recent studies have indicated that it can also drive diverse progenitor cell types down differentiation pathways [44–46]. Moreover, c-myc loss of function in hematopoietic stem cells leads to their retention in the stem cell niche and inhibition of differentiation [47] while deletion of both N- and c-myc results in their ablation [48] . Importantly, in ES cells c-Myc appears to be critical in maintaining self-renewal and inhibiting differentiation [41,49]. Moreover, myc family genes have been shown to cooperate with Sox2, Oct3/4 and Klf4 in the Myc Function in ES Cells conversion of murine embryo fibroblasts (MEFs) into induced pluripotent stem cells (iPS) [50–52] (for review see [9]). In the generation of iPS cells myc can be replaced by other factors but at the cost of greatly diminished efficiency [53,54]. Fibroblasts reprogrammed by Myc, Oct4, Sox2 and Klf4 display widespread epigenetic changes characteristic of the chromatin in ES cells [55]. In this regard it is interesting that myc loss of function has been associated with a reversible global change in histone acetylation and methylation [56]. Furthermore induction of c-Myc in a B cell line results in increased histone H3 and H4 acetylation at a defined group of Myc target genes [57]. Reasoning that Myc may have gene targets and functions that are unique to ES cells we have carried out a genome location (ChIP-chip) analysis of c-Myc binding in mES cells and examined the consequences of altering Myc levels on histone methylation. Our data indicate that Myc is involved in the transcriptional regulation of several members of the ES cell pluripotency network and in widespread histone methylation mediated by the Polycomb complex. Results Identification of Genes Bound and Regulated by c-Myc in ES Cells We mapped genomic binding sites for endogenous c-Myc in murine embryonic stem (ES) cells by employing DNA derived from anti-Myc chromatin-immunoprecipitation to probe DNA microarrays (ChIP-chip) comprised of 21,632 mouse gene promoters. For these experiments we employed sorted SSEA-1 positive R1 and AK7 ES cells and a pre-absorbed antibody with reactivity restricted to c-Myc protein (see Materials and Methods). We imposed a cut-off of four-fold enrichment following chromatin immunoprecipitation with anti-Myc compared to input, and also applied the widely used ACME/R analysis software to identify significant peaks across gene promoters by assigning a probability value (p-value) to each probe in the array (we used p- value,0.0001 as our cut-off) [58,59] (see Materials and Methods and Figure. S1, Table S1). Using these approaches we identified 3189 promoter binding sites representing 12.8% of promoters analyzed. Examples of enrichment profiles (ChIP-enriched versus total genomic DNA) observed for Myc binding within a ,5 kb region of the transcriptional start sites of the genes encoding the histone chaperone HirA, the histone demethylase SMCX, and the Sox2 transcription factor are shown in Figure 1A (see Table S1 for list of c-Myc-bound genes). The percentage of c-Myc bound promoters identified is comparable with earlier promoter and genomic binding experiments in Drosophila and mammalian cells [27–29,31]. We further validated association of c-Myc with a subset of the genomic loci identified in our ChIP-chip experiments by employing qChIP-PCR in four independent experiments with c-Myc antibody, carried out using two different ES cells lines (AK7 and R1) (Figure 1B, Figure S2). We randomly selected 44 genes for validation. Nineteen of these genes (Dlx-1, Evx2, Nkx2.2, Nkx2.9, Gata1, Pax5, Sox21, Sox7, Pax9, Pax6, Onecut1, Hes1, Oct3/4, Gata4, Cdx2, Mash1, Irx5, Math1, Cdk4) were not bound by Myc in the ChIP-chip assay and did not meet our criteria (.2 fold enrichment) for Myc binding in the qChIP-PCR. Two genes (Irx2 and Msx1) were negative in our ChIP-chip assay but positive in qChIP-PCR, and one gene (NeuroD2) was positive in the ChIP- chip assay but fell below the cut-off in the qChIP-PCR. The remaining genes were positive in both assays (Figure 1B). From these data we calculate an average false discovery rate of 6.8% AK7 ES cells and 11.3% in R1 ES cells. To determine whether c-Myc regulates the levels of expression of genes identified in our analysis we examined the transcript levels PLoS ONE | www.plosone.org 2 November 2009 | Volume 4 | Issue 11 | e7839