Abstract Epigenetic processes control the binary on-off states of specific gene sets, thereby creating heritable transcription patterns that drive development, and maintain cellular identity. The prominent epigenetic regulatory marks on eukaryotic chromatin are histone modifications and DNA cytosine methylation (5meCpG). These marks are placed by protein complexes, including members of the histone modifying and DNA methyltransferase (DNMT) enzyme families. It is hypothesized that errors in placement, removal, or reading of epigenetic marks can cause human disease through inappropriate silencing of specific genes. As the epigenetic marks that mediate gene silencing are reversible, there is interest in devising therapeutic strategies to reactivate epigenetically silent genes. Inhibitors of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) enzyme families can reverse epigenetic silencing and produce anti-tumor effects, possibly through reactivation of silent tumor suppressor genes (so-called epigenetic therapy). As these inhibitors show limited specificity within enzyme families, their precise mechanisms of action are not well understood. To identify cellular factors involved in maintenance of epigenetic silencing, we constructed a population of human cells harboring epigenetically silent GFP reporter genes. Using this cell population we have implemented a GFP reporter-based siRNA knockdown screen to identify novel factors and networks that maintain epigenetic silencing in human cells. We have now completed a genome-wide, high throughput siRNA-based screen (21,122 siRNA targets). The screen has produced 128 gene hits that have satisfied several validating criteria. The output of this screen was of high quality, as several of the factors were identified in an earlier independent screen. Among the newly identified hits, there was a significant enrichment for factors that mediate SUMOylation, as well as 25 factors corresponding to novel genes, including 9 with no predicted functions. This screen has the potential to identify novel cellular pathways and reveal new targets for epigenetic therapy of cancer and other diseases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3008. doi:10.1158/1538-7445.AM2011-3008

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