Myc Function in ES Cells Figure 1. c-Myc-bound promoters in mouse embryonic stem cells identified by ChIP-chip analysis and validated by qChIP-PCR. (A) Examples of three gene promoter regions identified using chromatin immunoprecipitation with anti-c-Myc antibody to probe DNA microarrays (see Methods and Materials for details). Shown are the fold enrichment ratios for anti-Myc ChIP-enriched versus total input genomic DNA (y axis) for all probes within the genomic regions indicated. Numbers represent the beginning and the end probe positions (x axis). The transcriptional start sites and direction of transcription are noted by arrows. (B) Validation by qChIP-PCR of putative c-Myc target genes in AK7 mES cells. Forty-four genes were selected at random for validation and included c-Myc target and non-c-Myc target genes as determined from the initial promoter array. Equal amounts of anti-c-Myc ChIP DNA and total input DNA were used for quantitative PCR employing SYBR Green detection with an ABI7900HT system. Bar heights represent the average fold enrichment ratios from 2 independent sets of anti-Myc ChIP-enriched versus total input genomic DNA. qChIP- PCR data derived for R1 mES cells is shown in Supplementary Figure S2). doi:10.1371/journal.pone.0007839.g001 of a 33 gene subset of the promoters identified as bound by c-Myc in our ChIP-chip experiments (Figure 2A, B). We manipulated c- Myc abundance in ES cells by employing Lentiviral vectors to drive overexpression of c-myc, or to introduce shRNAs designed to knockdown c-myc expression. We observed an 8-fold average increase in c-myc RNA levels following infection with c-myc expressing Lentiviruses and a 4-fold decrease in c-myc RNA after introduction of shRNA directed against c-myc (Figure 2A, B, far left). Myc protein levels were also shown to respond to introduction of the c-myc shRNA and overexpression vectors [60]. These changes in c-Myc protein level induced altered proliferation rates where increased c-Myc resulted in increased BrdU incorporation and decreased population doubling time, while c-Myc knockdown has the opposite effect (Figure S3). Nearly 90% (30/33) of the c-Myc target genes examined showed a two- fold or greater augmentation of expression following c-myc overexpression (Figure 2A). Twenty-four genes in this group displayed decreased levels of expression subsequent to c-myc knockdown (Figure 2B). Of the 33 genes examined, three genes (Gata6, Pcdh-a. Pcdh-c) appeared to be either marginally or substantially downregulated upon c-myc overexpression (Figure 2A). GATA 6 also displayed increased expression upon c-myc knockdown, indicating that it is repressed by c-Myc. This is consistent with considerable data showing that myc has a repressive, as well as an activating, role in transcription (for review see [61]). c-Myc Associates with ES Cell Genes Involved in Chromatin Structure and Pluripotency Ontology analysis of genes associated with endogenous c-Myc in ES cells indicates, as expected from previous studies, that they fall into multiple functional classes involving growth, metabolism, cell signaling pathways, cell cycle progression, and apoptosis. In mES cells we also detect c-Myc binding to genes involved in developmental processes (e.g. neurogenesis and ectoderm devel- opment) (see Figure 3A) as well as genes involved in chromatin structure, such as the histone chaperone HirA, the histone lysine demethylase SMCX, and the SWI/SNF related SMARC genes (see Figure 1, Table S1). Such genes have not been previously linked to c-Myc binding or regulation and likely represent mES cell-specific c-Myc targets. Of particular interest is our finding that the sox2 gene is bound by c-Myc proximal to the transcription start site (TSS) in ES cells (Figure 1A), an interaction confirmed by PLoS ONE | www.plosone.org 3 November 2009 | Volume 4 | Issue 11 | e7839