Gene Regulation and Epigenetic Remodeling in Murine Embryonic Stem Cells by c-Myc Chin-Hsing Lin1, ChenWei Lin2, Hisashi Tanaka3, Matthew L. Fero2, Robert N. Eisenman 1 * 1 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 2 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 3 Department of Molecular Genetics, Cleveland Clinic Foundation, Cleveland, Ohio, United States of America Abstract Background: The Myc oncoprotein, a transcriptional regulator involved in the etiology of many different tumor types, has been demonstrated to play an important role in the functions of embryonic stem (ES) cells. Nonetheless, it is still unclear as to whether Myc has unique target and functions in ES cells. Methodology/Principal Findings: To elucidate the role of c-Myc in murine ES cells, we mapped its genomic binding sites by chromatin-immunoprecipitation combined with DNA microarrays (ChIP-chip). In addition to previously identified targets we identified genes involved in pluripotency, early development, and chromatin modification/structure that are bound and regulated by c-Myc in murine ES cells. Myc also binds and regulates loci previously identified as Polycomb (PcG) targets, including genes that contain bivalent chromatin domains. To determine whether c-Myc influences the epigenetic state of Myc-bound genes, we assessed the patterns of trimethylation of histone H3-K4 and H3-K27 in mES cells containing normal, increased, and reduced levels of c-Myc. Our analysis reveals widespread and surprisingly diverse changes in repressive and activating histone methylation marks both proximal and distal to Myc binding sites. Furthermore, analysis of bulk chromatin from phenotypically normal c-myc E7 embryos demonstrates a 70–80% decrease in H3-K4me3, with little change in H3- K27me3, compared to wild-type embryos indicating that Myc is required to maintain normal levels of histone methylation. Conclusions/Significance: We show that Myc induces widespread and diverse changes in histone methylation in ES cells. We postulate that these changes are indirect effects of Myc mediated by its regulation of target genes involved in chromatin remodeling. We further show that a subset of PcG-bound genes with bivalent histone methylation patterns are bound and regulated in response to altered c-Myc levels. Our data indicate that in mES cells c-Myc binds, regulates, and influences the histone modification patterns of genes involved in chromatin remodeling, pluripotency, and differentiation. Citation: Lin C-H, Lin C, Tanaka H, Fero ML, Eisenman RN (2009) Gene Regulation and Epigenetic Remodeling in Murine Embryonic Stem Cells by c-Myc. PLoS ONE 4(11): e7839. doi:10.1371/journal.pone.0007839 Editor: Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America Received September 16, 2009; Accepted October 17, 2009; Published November 13, 2009 Copyright: 2009 Lin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Supported by NIH/NCI research grant RO1CA20525. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: eisenman@fhcrc.org Introduction ES cells must be capable of self-renewal while simultaneously retaining the capacity to commit to a wide range of differentiation lineages. The notion that the determination and maintenance of embryonic stem (ES) cell pluripotency and self-renewal is related to an epigenetic state characterized by an open chromatin conformation has received considerable support over the last several years [1–6]. Open chromatin is thought to contribute to pluripotency by permitting relatively broad accessibility to transcriptional regulation and is itself likely to be the result of diverse activities including nucleosome assembly, positioning, and remodeling, incorporation of histone variants, binding of chroma- tin modifying factors, epigenetic modifications, sub-nuclear compartmentalization, and other dynamic processes that maintain active chromatin (for reviews see [2,7,8]). Much recent work on ES cell pluripotency has focused on two aspects of transcriptional regulation: the actions of the Sox2-Oct4- Nanog transcription factor network and the nature of epigenetic changes associated with pluripotency [9]. The Sox2-Oct4-Nanog transcription factors have been known for about a decade to be required for early embryonic development and for ES cell self- renewal [10–13]. Genome-wide binding analyses have indicated that in both human and murine ES cells the Sox2, Oct4, and Nanog factors occupy hundreds of gene promoters [14,15]. Importantly, these gene targets include many developmental regulators, a subset of which, encoding transcription factors and chromatin modifying activities, are associated with RNA poly- merase II and are expressed in ES cells. A second subset of Sox2- Oct4-Nanog bound genes are involved in lineage-specific differentiation – these genes are associated with Polycomb complex components (including Suz12, Eed, EZH2) and are repressed in ES cells [16–18]. Therefore the Sox2-Oct4-Nanog factors are arguably functioning as selectors of genes whose activation or repression in ES cells are critical for pluripotency and self-renewal. It is likely that one reflection of the open chromatin conformation proposed for ES cells is the relative paucity of PLoS ONE | www.plosone.org 1 November 2009 | Volume 4 | Issue 11 | e7839