Role of chromatin structure in regulating gene silencing

染色质结构在调节基因沉默中的作用

• 批准号: 6883158
• 负责人: MARY E BRYK
• 金额: $ 26.19万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6883158
• 关键词: SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; cell cycle; chromatin; enzyme complex; fungal genetics; gene induction /repression; genetic regulation; histones; methylation; nonhistone nucleoprotein; nucleic acid structure; nucleosomes; polymerase chain reaction; posttranslational modifications; protein protein interaction; ribosomal DNA; site directed mutagenesis

DESCRIPTION (provided by applicant): The long-term objective of this project is to learn more about how chromatin structure regulates transcriptional gene silencing in eukaryotes. The basic mechanisms used to generate inactive or silent chromatin are largely conserved in eukaryotic organisms ranging from yeast to humans. Our studies focus on understanding the roles of chromatin-associated factors, such as histones, histone-modifying enzymes, and silencing proteins, in regulating the silencing of RNA polymerase II-transcribed genes located in the ribosomal DNA locus (rDNA) of the budding yeast Saccharomyces cerevisiae. The experiments in this proposal will use a combination of genetic, molecular, and biochemical approaches to address how histones and silencing factors affect gene silencing and chromatin structure in the rDNA. First, studies to characterize the histone H3 methyltransferase complex, COMPASS, will be performed. Gene silencing is one of the only cases in S. cerevisiae, where H3 methylation has a clear effect on gene expression. The proposed experiments will investigate the role of COMPASS proteins in rDNA silencing, determine how rDNA chromatin is altered by methylation of histone H3, and address the possibility that methylated histone H3 recruits silencing factors to the rDNA. Mutagenesis studies focusing on the catalytic subunit of COMPASS coupled with in vivo and in vitro assays for COMPASS function will advance the molecular characterization of this evolutionarily conserved catalytic domain. Second, the histone composition of rDNA chromatin will be analyzed using molecular and genetic approaches. These studies will not only shed light on how histones and silencing factors function in gene silencing in S. cerevisiae, they will provide information about how related proteins function in humans. Furthermore, gene silencing is critical for normal growth and development. When the processes that regulate gene silencing are disrupted in mammalian cells, inappropriate gene expression and excessive recombination can lead to diseases, including cancer and leukemia. Our studies investigating the mechanisms that cells use to control gene silencing will provide insight into how changes in regulatory pathways can lead to malignancy and will contribute to the identification of targets for future therapeutics.

描述（由申请人提供）：该项目的长期目标是更多地了解染色质结构如何调节真核生物中的转录基因沉默。用于产生无活性或沉默染色质的基本机制在从酵母到人类的真核生物中很大程度上是保守的。我们的研究重点是了解染色质相关因子（例如组蛋白、组蛋白修饰酶和沉默蛋白）在调节芽殖酵母核糖体 DNA 基因座 (rDNA) 中 RNA 聚合酶 II 转录基因沉默中的作用酿酒酵母。该提案中的实验将结合遗传、分子和生化方法来解决组蛋白和沉默因子如何影响 rDNA 中的基因沉默和染色质结构。首先，将进行组蛋白 H3 甲基转移酶复合物 COMPASS 特征研究。基因沉默是酿酒酵母中仅有的情况之一，其中 H3 甲基化对基因表达有明显影响。拟议的实验将研究 COMPASS 蛋白在 rDNA 沉默中的作用，确定组蛋白 H3 甲基化如何改变 rDNA 染色质，并解决甲基化组蛋白 H3 将沉默因子招募到 rDNA 的可能性。针对 COMPASS 催化亚基的诱变研究，结合体内和体外 COMPASS 功能测定，将推进这一进化保守催化结构域的分子表征。其次，将使用分子和遗传学方法分析 rDNA 染色质的组蛋白组成。这些研究不仅将揭示组蛋白和沉默因子如何在酿酒酵母基因沉默中发挥作用，还将提供有关相关蛋白质如何在人类中发挥作用的信息。此外，基因沉默对于正常生长和发育至关重要。当哺乳动物细胞中调节基因沉默的过程被破坏时，不适当的基因表达和过度重组可能导致疾病，包括癌症和白血病。我们研究细胞用于控制基因沉默的机制，将深入了解调节途径的变化如何导致恶性肿瘤，并将有助于确定未来治疗的靶点。