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

通过高通量 siRNA 筛选发现人类细胞中的表观遗传标记

• 批准号: 7692303
• 负责人: RICHARD ALAN KATZ
• 金额: $ 34.9万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7692303
• 关键词: Bioinformatics; Biological Assay; Biological Models; Cell Line; Cell physiology; Cells; Chromatin; Complement; Complex; DNA Methylation; Disease; Enzymes; Epigenetic Process; Excision; Gene Mutation; Gene Silencing; Genes; Goals; Green Fluorescent Proteins; Hela Cells; Histones; Human; Human Cell Line; Individual; Lead; Libraries; Location; Malignant Neoplasms; Mediating; Methods; Monitor; Pathway interactions; Phase; Procedures; Process; Proteins; Reader; Reading; Reporter; Reporter Genes; Research; Role; Screening procedure; Small Interfering RNA; System; Therapeutic; base; cancer therapy; cellular development; chromatin immunoprecipitation; genome-wide; histone modification; human disease; interest; novel; protein function; public health relevance

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.

描述（由申请人提供）： 表观遗传沉默介导发育和细胞分化过程中特定基因的可遗传转录关闭。这些过程是通过酶促放置或去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物读取，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、移除或读取错误可能会通过特定基因的不适当沉默而导致人类疾病。由于介导基因沉默的表观遗传标记是可逆的，因此人们对设计重新激活表观遗传沉默基因的治疗策略感兴趣。因此，确定介导表观遗传沉默的全部人类因素和途径至关重要。基于全基因组、逐个基因的 siRNA 敲除筛选将用于发现维持人类细胞表观遗传沉默的新因素。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重新激活的原理。设计了 HeLa 细胞报告系统，利用表观遗传沉默绿色荧光蛋白 (GFP) 基因的重新激活作为高通量读数。该测定使用预先选择的 siRNA 组进行了验证，该 siRNA 组针对表观遗传调节因子进行了富集。通过 GFP 重新激活的高通量读出，每个人类基因将使用全基因组 siRNA 文库进行功能性询问，以揭示表观遗传沉默中的直接或间接作用。该提案的具体目标是： 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。新的命中将使用生物信息学方法进行分析以预测功能，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，扩大发现新型沉默因子的平台。这些筛选有可能识别出标记染色质用于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 突变会使基因失活，从而导致癌症和其他人类疾病。现在人们认识到，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发挥作用。这个过程被称为“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，以便设计新的疗法。