Myc Function in ES Cells Figure 4. Quantitation of H3K4me3 and H3K27me3 levels in response to c-Myc. (A) Response of H3K4me3 to Lentiviral mediated overexpression or knock-down c-Myc in mES cells as determined by western blot. (B) Response of H3K27me3 to Lentiviral mediated overexpression or knock-down c-Myc in mES cells as determined by western blot. (C) Effect of H3K4me3 and H3K27me3 level in response to c-Myc loss in early developmental embryos. Ratios based on normalization to c-tubulin internal control wwere calculated using ImageJ software . doi:10.1371/journal.pone.0007839.g004 proteins as well as other Myc target genes and genes bound by Polycomb but not Myc, selecting genes that are predominantly linked to PcG binding, differentiation, pluripotency, and chroma- tin modification (Table S4). We first carried out anti-Myc chromatin immunoprecipitation on this custom array permitting us to verify our initial array data and examine the effects of modulating Myc levels. We compared vector-alone treated mES with mES cells overexpressing c-myc (mES qc-myc) and mES cells in which c-myc has been knocked down (mES Q c-myc). Figure 5 shows c-Myc binding to a representative group of promoters. We plotted log2 enrichment profiles to permit us to more easily compare wildtype ES cells with the mES Q c-myc and mES qc- myc cells. Here negative enrichment ratios indicate that the signal from the anti-Myc ChIP was less than the input DNA against which it was normalized. Ebf1, Ebf2, Olig3, Gata6, and Sox 2 display significant endogenous c-Myc binding at several sites within their promoters in mES cells (Figure 5 blue bars, Figure S10) consistent with our promoter array data (Table S1). Hes1, Onecut1, and Jak2 were not identified as positive for Myc binding on the original promoter array, and show relatively low levels of endogenous c-Myc binding in this high density tiling array (Figure S10). Overexpression of c-Myc generally results in increased enrichment (red bars) at endogenous binding sites, although in some promoters we observe overexpressed c-Myc binding at a small number of sites where no or very low endogenous binding signal is detected (e.g. Ebf2, Hes1, Olig3, Gata6) consistent with previously observed occupation of low affinity binding sites at higher Myc levels [27]. By contrast, shRNA-mediated knockdown of c-Myc invariably leads to reduced Myc binding (green bars). These findings further validate our Myc binding assignments and indicate that the extent of binding is dependent on c-Myc levels. When we plotted histone H3 methylation profiles we found that nearly all promoters on our custom array (,95%) displayed changes in the intensity and position of H3-K27me3 and/or H3- K4me3 peaks in response to altered Myc levels (profiles for 427 promoters are provided in Figures S5, S6, S7, S8, and S9). However the extent and type of change appear to be largely promoter-specific. Fig. 6 shows H3-K27me3 (red) and H3-K4me3 (green) in wildtype mES, mESqc-myc, and mESQc-myc cells over a 3KB to 5KB region proximal to the TSS of a subset of genes that are illustrative of the types of changes observed more generally (see Figures S5, S6, S7, S8, and S9). Sox2 is a c-Myc bound and regulated gene (Figure 1, 2, 6) that is not known to be a PcG target and, as expected, we fail to observe peaks of H3-K27me3 in the PLoS ONE | www.plosone.org 6 November 2009 | Volume 4 | Issue 11 | e7839