Discovery of Epigenetic Marks in Human Cells by High Throughput siRNA Screening

描述（由申请人提供）： 表观遗传沉默介导发育和细胞分化期间特定基因的遗传性转录关闭。这些过程是通过染色质上的表观遗传标记的酶促放置或去除来执行的。已知的标记包括DNA甲基化和各种翻译后组蛋白修饰。这些标记被抑制复合物读取，并且也可能直接影响转录机器对染色质的可接近性。据推测，表观遗传标记的放置、去除或阅读中的错误可通过特定基因的不适当沉默引起人类疾病。由于介导基因沉默的表观遗传标记是可逆的，因此有兴趣设计治疗策略来重新激活表观遗传沉默基因。因此，确定介导表观遗传沉默的人类因素和途径的完整补充至关重要。一个全基因组的，基于siRNA的基因敲低筛选将被用来发现在人类细胞中维持表观遗传沉默的新因子。该功能筛选基于特定表观遗传沉默因子的siRNA敲低将导致沉默基因的重新激活的原理。设计了HeLa细胞报告系统，其中表观遗传学沉默的绿色荧光蛋白（GFP）基因的再活化被用作高通量读出。使用富集靶向表观遗传调节因子的预选siRNA组验证该测定。通过GFP再激活的高通量读出，将使用全基因组siRNA文库功能性地询问每个人基因，以揭示表观遗传沉默中的直接或间接作用。该提议的具体目的是：1）使用该siRNA筛选来鉴定维持表观遗传沉默的新型表观遗传调节因子和标记。将使用生物信息学方法分析新的命中以预测功能，并通过染色质免疫沉淀来监测沉默基因座的定位。2)开发新的报告细胞，为发现新的沉默因子拓展平台。这些筛选有可能识别标记染色质表观遗传沉默的新细胞途径，并揭示癌症和其他疾病表观遗传治疗的新靶点。公共卫生相关性：众所周知，DNA突变会导致基因突变，导致癌症和其他人类疾病。现在认识到，这种失活也可以通过基因沉默机制发生，由此基因保持完整但不起作用。这一过程被称为“表观遗传沉默”，是可逆的，拟议研究的目标是确定导致表观遗传沉默的新细胞过程，以便设计新的治疗方法。

DESCRIPTION (provided by applicant): Epigenetic silencing mediates the heritable transcriptional shutoff of specific genes during development and cellular differentiation. Such processes are executed by enzymatic placement, or removal, of epigenetic marks on chromatin. The known marks include DNA methylation and a variety of posttranslational histone modifications. These marks are read by repressive complexes, and may also directly influence the accessibility of chromatin by the transcriptional machinery. 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. It is therefore critical to identify the entire complement of human factors and pathways that mediate epigenetic silencing. A genome-wide, gene-by-gene siRNA-based knockdown screen will be used to discover new factors that maintain epigenetic silencing in human cells. This functional screen is based on the principle that siRNA knockdown of specific epigenetic silencing factors will lead to reactivation of silent genes. A HeLa cell reporter system was devised whereby reactivation of an epigenetically silent green fluorescent protein (GFP) gene is used as a high throughput readout. This assay was validated using a pre-selected siRNA set that is enriched for targeting epigenetic regulators. By means of high throughput readout of GFP reactivation, each human gene will be functionally interrogated using a genome-wide siRNA library to reveal direct or indirect roles in epigenetic silencing. The Specific Aims of this proposal are: 1) To identify novel epigenetic regulators and marks that maintain epigenetic silencing using this siRNA screen. Novel hits will be analyzed using bioinformatics methods to predict function, and by chromatin immunoprecipitation to monitor localization at the silent locus. 2) To develop new reporter cells that will expand the platform for discovery of novel silencing factors. These screens have the potential to identify novel cellular pathways that mark chromatin for epigenetic silencing and reveal new targets for epigenetic therapy of cancer and other diseases. PUBLIC HEALTH RELEVANCE: It is well understood that DNA mutations can inactivate genes, leading to cancer and other human diseases. It is now being appreciated that such inactivation can also occur by a gene silencing mechanism, whereby the gene remains intact but nevertheless does not function. This process, termed "epigenetic silencing," is reversible, and the goal of the proposed research is to identify novel cellular processes that cause epigenetic silencing such that new therapies can be devised.