Abstract
BackgroundThe 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 FindingsTo 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 null 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/SignificanceWe 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.
Highlights
embryonic stem (ES) cells must be capable of self-renewal while simultaneously retaining the capacity to commit to a wide range of differentiation lineages
The recent findings that Myc family proteins can collaborate with other transcription factors (Oct4, Sox2, Klf4) to reprogram mouse and human fibroblasts [50,51,68,69,70] as well as earlier work showing the importance of c-Myc in maintenance of mES cell selfrenewal [41] prompted us to examine the nature of c-Myc target genes in mES cells using Chromatin Immunoprecipitation (ChIP)-chip analysis
Our ChIP-chip analysis of Myc bound loci in mES cells has identified a broad range of functional categories related to growth and metabolism among 3189 binding sites
Summary
ES cells must be capable of self-renewal while simultaneously retaining the capacity to commit to a wide range of differentiation lineages. Genome-wide binding analyses have indicated that in both human and murine ES cells the Sox, Oct, and Nanog factors occupy hundreds of gene promoters [14,15]. These gene targets include many developmental regulators, a subset of which, encoding transcription factors and chromatin modifying activities, are associated with RNA polymerase II and are expressed in ES cells. 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 still unclear as to whether Myc has unique target and functions in ES cells
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