targets that overlap with PcG target genes- thus Myc in general does not associate with H3-K27 methylated gene regions. However the 218 genes that do display Myc, Suz12 and H3- K27me3 overlap include many transcription factors involved in developmental regulation of differentiation functions (predomi- nantly ectoderm and neuronal differentiation). The genes encoding these transcription factors are repressed by PcG complexes (including the Suz12 subunit) in ES cells [16–18]. Importantly, of the 17 common Myc-PcG target genes tested we found that 14 (Fgf18, EBF1, EBF2, Zic4, En2, Bmi1, Lhx2, Fgf5, Ngn2, Ngn3, Olig 2, Olig3, Atoh1, and HoxB3) are activated upon Myc overexpression and repressed subsequent to Myc knockdown in mES cells (Figure 2A, B). Because many of these promoters marked by H3-K27me3 are considered bivalent [21–23] we surmise that Myc is involved in the maintenance of the bivalent state. Myc Influences the H3 Lysine Methylation Landscape While there is a compelling connection between Myc binding and the induction of histone acetylation [57,65,77] it is less clear how Myc influences histone methylation. In a human B cell line Myc binding is strongly associated with promoters marked by H3- K4me3 prior to the induction of Myc [65]. In Drosophila dMyc binds to and inhibits the activity of the H3-K4me3 demethylase Lid, [63] suggesting that at least in this system Myc may act to maintain H3-K4 methylation levels by blocking the action of a demethylase [63]. When we analyzed c-Myc regulation of H3-K4 and H3-K27 methylation in bulk chromatin derived from ES cells we found that H3-K3me3 levels positively correlate with c-Myc levels while H3-K27me3 levels were unaffected by c-Myc abundance (Figure 4A, B). Surprisingly, phenotypically normal E7 embryos from c-myc mice (approximately 3 days prior to the advent of embryonic lethality) have strikingly reduced H3- K4me3, while H3-K27me3 was essentially unchanged (Figure 4C). This result extends our earlier work demonstrating global decreases in H3-K9 methylation and H3/H4 acetylation in bulk chromatin in the nervous system and in cell lines upon Myc loss of function [56]. To examine the effects of c-Myc levels on individual genes we analyzed H3-K4 and H3-K27 trimethylation within the subset of Myc associated genes linked to PcG binding, differentiation, pluripotency, and chromatin modification. Our results show broad and complex changes in the trimethylation patterns of both H3- K27 and H3-K4 in nearly all the promoters on the array in response to altered Myc levels. These changes display several noteworthy features. First, H3 methylation changes in response to altered Myc levels are generally, but not exclusively, observed in promoter regions directly bound by Myc. However, while Myc binding sites are frequently proximal to altered H3 methylation sites, peaks of Myc binding can also be found several kb away. Second, the patterns of H3 methylation changes observed appear to be promoter specific. A recent study in human B cells also reported that induction of Myc resulted in histone methylation changes at individual promoters at both Myc and non-Myc target genes [57]. Here we find that methylation changes can take the form of shifts in position within the promoter and/or quantitative changes that presumably reflect changes in the fraction of mES cells containing the altered methylation mark. Third, H3-K27 and K3-K4 methylation bivalency is both lost and gained depending on the specific promoter and the levels of Myc. It is important to note that while the changes observed in H3-K4 and H3-K27 trimethylation patterns can be construed as consistent with activation or repression in response to changes in c-Myc levels, we have also observed cases where altered methylation patterns do Myc Function in ES Cells not correspond to changes in gene expression. This is consistent with a previous study demonstrating that during differentiation active PcG recruitment to some genes can result in increased H3K27me3 but not to inhibition of transcription [25]. H3-K4 and H3-K27 methylation patterns represent only a part of the complex interplay of modifications and factors involved in chromatin structure and gene regulation [73,78] underscoring the impor- tance of more detailed analysis of other histone marks and transcription factor interactions at individual promoters. Mechanisms of Epigenetic Regulation Mediated by Myc How does Myc elicit changes in the histone H3 methylation patterns of promoters in mES cells? A great deal of previous work has indicated that the Myc protein recruits multiple activities involved in chromatin modification – these include histone acetyltransferases (GCN5, Tip60, p300), potential chromatin remodelers (p400, Ini1, Tip48/49), ubiquitin ligases (Fbw7, Skp2), the H3-specific kinase Pim1, the HDAC1 histone deacetylase, and the JARID1A/LID histone H3-K4 demethylase (for reviews see [38,61,79]). Furthermore, studies in Drosophila indicate that dMyc interacts with members of the Trithorax group, including components of histone methyltransferases and Swi/Snf remodeling complexes [63,64]. It is likely that the precise mix of co-regulators recruited by Myc to DNA is determined in a context- specific manner and, given the short half-life of Myc proteins, is subject to rapid exchange. The consequences of these multiple interactions are predicted to have profound effects on chromatin proximal to Myc binding sites and may in principle lead to erasure or establishment of H3 methylation patterns. Such modification patterns may be spread along chromatin, as has been suggested for H3-K9 and H3-K27 methylation and heterochromatin formation [80]. These effects may be reinforced by the nature of the target genes induced by Myc which include chromatin modification enzymes (GCN5, SMCX) and remodelers (SMARC). Moreover, recent work has shown that nucleosome replacement mediated by factors controlling assembly and disassembly may underlie epigenetic changes that govern accessibility of transcription factor to promoters (for review see [8]). In this regard it is of interest that the gene encoding the histone assembly factor HirA is bound and regulated by Myc. HirA levels have been previously shown to regulate unbound histones and ES cell differentiation [1]. Therefore Myc may stimulate a more generalized nucleosome instability leading to widespread changes in epigenetic marks. Such generalized effects of Myc -through spreading of chromatin modifications, through induction of Myc target genes which themselves modify chromatin, and through nucleosome instability – may account for the fact that we detect changes in histone methylation patterns in regions distant from Myc binding sites and in promoters in which Myc is not bound. We surmise that the ability of Myc to broadly affect the epigenetic marks and expression of genes important in pluripotency and differentiation is related for Myc’s role as a reprogramming factor critical in the generation of iPS cells. Materials and Methods Cell Culture The ES cell lines, R1 and AK7, the most commonly used cell lines for generating transgenic mice, were obtained from Andras Nagy and Philippe Soriano respectively. Both mES cell lines were cultured in DMEM supplied with 10% FBS, L-glutamine, non- essential amino acid, sodium pyruvate, LIF (leukemia inhibitory factor), and b-mercaptoethanol. ES cells were thawed on plates with a mitomycin C-treated mouse embryonic fibroblast feeder PLoS ONE | www.plosone.org 9 November 2009 | Volume 4 | Issue 11 | e7839