Myc Function in ES Cells Figure 2. Transcriptional response of c-Myc target genes to c-Myc overexpression or knockdown. (A) Response of a subset of c-Myc target genes to Lentiviral mediated overexpression c-Myc in mES cells as determined by Real-Time PCR. Each bar represents the average (DDCt) from triplicate sets of experiments on a log2 scale after normalization to internal controls and vector alone. The fold differences detected range from ,2– 20 fold. Note an approximately eight-fold increase in c-myc RNA levels (bar at far left). Color-coded genes are read from left to right (inset). (B) Response of a subset of c-Myc target genes to RNAi mediated knock-down of c-Myc in mES cells as determined by Real-Time PCR. Each bar represents the average (DDCt) from a triplicate set of experiments on a log2 scale after normalization to internal controls and vector alone. c-myc RNA levels decreased 4 fold (bar at far left). Color-coded genes are read from left to right (inset). * indicates previously identified bivalent gene. (C) Sox2 immunostaining in mES cells. mES cells were infected with empty lentiviral vector alone (WT; left panel); lentiviral vector expressing shRNA against c- myc (middle panel); lentiviral vector expressing c-myc (right panel). Scale bar indicates 50 mm See Supplementary Figure S4 for immunoblots of Sox2 protein. doi:10.1371/journal.pone.0007839.g002 direct qChIP-PCR (Figure 1B). Moreover, sox2 expression increases ,8-fold in response to c-myc overexpression and decreases 2-fold when endogenous c-myc expression is reduced by shRNA (Figure 2A, B). As expected, we also observe changes in Sox2 protein level following modulation of c-Myc expression (Figure 2C, Figure S4). Because Sox2 and its binding partner Oct3/4 are key components of a transcription factor network that can induce pluripotency in human and murine fibroblasts [51–53] it is possible that Myc participates in this network, at least in part, through its ability to stimulate Sox2 expression. c-Myc Associates with a Subset of Polycomb-Regulated and Bivalent Genes in ES Cells In Drosophila, dmyc and polycomb (PcG) interact genetically [62] and genes in the trithorax group, that oppose PcG function, have been demonstrated to interact with dmyc genetically and physically [63,64]. To test the hypothesis that c-Myc impinges on PcG function in mES cells, we compared c-Myc and PcG binding more extensively. In ES cells, PcG complex binding has been previously identified through genome-wide location analysis and found to associate with, and transcriptionally repress developmental regulatory genes [16,18]. We compared our mES c-Myc binding data and the mES PcG ChIP-chip data from the study of Boyer et al, [16] (Table S2) and found that of our 2282 c- Myc-bound promoters represented on the Boyer et al. array 9.5% are H3-K27 methylated and associated with the PcG protein Suz12 (p = 3.4E203, Chi-square calculation). This represents 18% of Suz12 + H3-K27me3 genes and 17% of all genes displaying H3K27 trimethylation (p = 2.9E203) (Figure 3B, Table S2). The overlapping Suz12/H3-K27me3/c-Myc genes predominantly PLoS ONE | www.plosone.org 4 November 2009 | Volume 4 | Issue 11 | e7839