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

描述(由申请人提供)： 表观遗传沉默介导了特定基因在发育和细胞分化过程中可遗传的转录关闭。这样的过程是通过酶法放置或移除染色质上的表观遗传标记来执行的。已知的标记包括DNA甲基化和各种翻译后组蛋白修饰。这些标记由抑制复合体读取，也可能直接影响转录机制对染色质的可及性。据推测，表观遗传标记的放置、移除或读取错误可通过不适当地沉默特定基因而导致人类疾病。由于介导基因沉默的表观遗传标记是可逆的，人们有兴趣设计治疗策略来重新激活表观遗传沉默基因。因此，确定调节表观遗传沉默的全部人类因素和途径是至关重要的。一种全基因组的、基于逐个基因的siRNA敲除筛选将被用来发现维持人类细胞表观遗传沉默的新因素。这一功能筛选是基于这样的原理，即特定表观遗传沉默因子的siRNA敲除将导致沉默基因的重新激活。设计了一种HeLa细胞报告系统，通过重新激活表观遗传沉默的绿色荧光蛋白(GFP)基因作为高通量读出。这项检测是使用预先选择的siRNA集进行验证的，该siRNA集被富含用于靶向表观遗传调节因子。通过高通量读出GFP重新激活，将使用全基因组siRNA文库对每个人类基因进行功能查询，以揭示在表观遗传沉默中直接或间接的作用。这项建议的具体目的是：1)利用这一siRNA筛选来识别维持表观遗传沉默的新的表观遗传调节因子和标记。新的HITS将使用生物信息学方法来预测功能，并通过染色质免疫沉淀来监测沉默位置的定位。2)开发新的报告细胞，为发现新的沉默因子提供平台。这些屏幕有可能识别新的细胞通路，标记染色质的表观遗传沉默，并揭示癌症和其他疾病的表观遗传治疗的新靶点。与公共卫生相关：众所周知，DNA突变会使基因失活，导致癌症和其他人类疾病。现在人们意识到，这种失活也可以通过基因沉默机制发生，从而使基因保持完整，但仍然不起作用。这一过程被称为“表观遗传沉默”，是可逆的，拟议研究的目标是识别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.