Figure 5. c-Myc binding sites on a subset of gene promoters in mES cells. c-Myc levels were manipulated by Lentiviral-delivered overexpression or knock-down of c-Myc in mES cells followed by anti- Myc ChIP-chip analysis on custom- designed arrays (see text). Unprocessed enrichment ratios (log2 scale) present ChIP-enriched versus total input genomic DNA (crosslinked, sonicated and processed identically to the ChIP sample) (y axis) for all probes within a genomic region 2 kb upstream and 1 kb downstream of the TSS. The TSS and direction of transcription are denoted by arrows at the bottom of the figure. Blue bars: endogenous c-Myc binding in WT mES cells, Red bars: Myc overexpressing mES cells, Green bars: Myc knock-down mES cells. Negative values occur when there is no enrichment in ChIP DNA relative to input. doi:10.1371/journal.pone.0007839.g005 vicinity of the sox2 promoter in mES or in mES qc-myc cells. However, H3-K4me3 is apparent in the sox 2 promoter region in mES and shows, in mESqc-myc cells, an increased signal near the sox 2 TSS compared to wildtype mES cells (Figure 6A). The presence of an augmented H3-K4me3 peak is consistent with our finding that Sox2 is induced by c-myc overexpression (Figure 2A) (see Discussion). In contrast, c-myc knockdown abolishes H3- Myc Function in ES Cells K4me3 and leads to repression of sox2 (Figure 2B). Interestingly, a small peak of H3-K27me3 is present at the sox2 promoter in mESQc-myc cells, suggesting that loss of c-Myc binding may lead to PcG activity, at least within this promoter region (Figure 6A). In ,13% of the Myc targeted promoters on our custom array the overexpression of c-myc increased the levels of H3K4me3 and decreased H3K27me3 levels. Genes with enriched H3K4me3 in mESqc-myc cells, such as Ebf1, Ebf2, and Sox2 (Figure 6A) encode transcription factors involved in early development. Their expression levels increase in mESqc-myc and decrease in mESQc-myc cells relative to wildtype mES cells (Figure 2). In addition, Ebf1, Ebf2 and Olig 3 were previously identified as bivalent genes [20] and indeed we observe that their H3-K4me3 and H3-K27me3 peaks overlap in wildtype mES cells (Figure 6 blue asterisks). For these promoters, augmentation of c-Myc levels results in an apparent resolution of the bivalency. Knockdown of c-Myc had little effect on H3-K4me3 distribution compared to wildtype mES cells although H3-K27me3 peaks are enhanced and clearly display changes in position. Loss of bivalency with c-myc overexpression was observed in ,32% of genes on the custom array (Table S3). A different pattern emerged from our analysis of another subset of promoters. Here, overexpression of c-Myc frequently results in enrichment of promoter proximal H3-K27me3. These include genes involved in differentiation such as MyoD1, mesenchyme homeobox 2 (Meox2), and Smarca2 (also known as Brahma, a component of the SWI/SNF related chromatin remodeling complex) or genes highly expressed in differentiated cells such as very low density lipoprotein receptor (VLDLR), glutamate receptor (Grm7), dopamine receptor D4 (Drd4), and the protocadherin cluster (see Table S3). Within the GATA6 promoter, Myc overexpression induced an H3-K4me3 peak as well as an overlapping H3-K27me3 peak, thereby creating a new bivalent region (Figure 6). GATA6 expression is repressed under these conditions (Figure 2A,B). Knockdown of c-Myc results in decreased H3-K4me3 in the presence of widespread H3-K27me3. Therefore induction of GATA6 expression upon c-Myc knock- down (Figure 2A,B) is presumably dependent on other changes in chromatin modification or remodeling independent of H3 methylation (Figure 2). Analysis of a group of genes that did not score as Myc targets showed no or only small changes in methylation patterns (Figure 6B). However we find that some non-Myc targets such as Jak2, Hes1, and Onecut1 reveal changes in H3 methylation (Figure 6B). Jak2 shows an increase and spreading of H3-K27me3 as well as H3-K4me3 over the promoter region. mESQc-myc cells display a nearly complete loss of the H3-K27me3 and a diminished intensity of H3-K4me3 in the Jak2 promoter region. Onecut1 and Hes1 also display changes in both H3-K27me3 and/ or H3-K4me3 in response to changes in Myc levels. These effects are likely due to secondary or long-range effects of Myc on chromatin (see Discussion). Overall our data suggest that altering Myc levels induces varied and widespread changes in methylation patterns. Discussion The recent findings that Myc family proteins can collaborate with other transcription factors (Oct4, Sox2, Klf4) to reprogram mouse and human fibroblasts [50,51,68–70] as well as earlier work showing the importance of c-Myc in maintenance of mES cell self- renewal [41] prompted us to examine the nature of c-Myc target genes in mES cells using ChIP-chip analysis. Previous studies employing ChIP-chip and Chip-PET in human cells and DamID in PLoS ONE | www.plosone.org 7 November 2009 | Volume 4 | Issue 11 | e7839