PcG Function of YY1 in Transcription and Development

YY1 在转录和发育中的 PcG 功能

• 批准号: 7031075
• 负责人: Michael Lee Atchison
• 金额: $ 29.13万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7031075
• 关键词: DNA; DNA binding protein; Drosophilidae; RNA interference; arthropod genetics; cell cycle; cell differentiation; cell growth regulation; chromatin immunoprecipitation; developmental genetics; gene induction /repression; gene mutation; genetic regulation; genetic transcription; histones; intermolecular interaction; nucleic acid sequence; proliferating cell nuclear antigen; protein localization; protein sequence; protein structure function; site directed mutagenesis; transcription factor; ubiquitin

DESCRIPTION (provided by applicant): YY1 is a developmentally important transcription factor that regulates expression of numerous genes. The Drosophila Polycomb group (PcG) protein, Pleiohomeotic (PHO), bears sequence homology with YY1. We found that YY1 can functionally replace PHO as a PcG protein in vivo. YY1 expression in Drosophila results in PcG-dependent transcriptional repression and rescue of pho mutant flies. DNA binding by YY1 causes recruitment of PcG proteins and modification of histones by deacetylation and methylation. We found that YY1 sequences 201-226, when linked to the GAL4 DNA binding domain are necessary and sufficient for transcriptional repression. These YY1 residues physically interact with CtBP, PCNA and SUMO-1. We will address the following questions concerning YY1 PcG function: 1) What is the mechanism of PcG transcriptional repression by YY1? By ChIP assay we will determine the YY1 sequences needed for PcG recruitment to DNA and histone modification. In vivo rescue experiments will determine the sequences needed for organismal development. Genetic and ChIP approaches will determine the importance of PCNA, CtBP and SUMO-1 in YY1 medicated PcG repression. We will also explore the temporal requirements of YY1 DNA binding, PcG recruitment, and histone modification. 2) What is the mechanism of CtBP function in YY1 DNA binding and PcG repression? CtBP mutation results in reduced YY1 DNA binding and PcG recruitment in vivo. We will determine the effect of CtBP mutation on YY1 stability, DNA binding ability, intracellular location, and possible sequestration into a complex. EMSA and GST pull-down studies will explore the importance of CtBP sumoylation for interaction with YY1. We will use ChIP assays to characterize PcG binding to numerous PREs that bind to YY1. 3) How does YY1 function in mammalian PcG repression systems? A number of candidate mammalian PRE sequences have been identified that bind YY1 and other PcG proteins. We will use ChIP studies to determine whether CtBP, PCNA, or sumoylated proteins bind to mammalian PREs. We will use overexpression and RNAi knock-down of YY1, CtBP, SUMO-1 and PCNA to explore the effects on PcG recruitment, histone modification, and gene expression. PcG proteins are also implicated in muscle differentiation. Therefore, we will test the roles of YY1, CtBP, SUMO-1, and PCNA in differentation of myblasts into myotubes.

描述（由申请人提供）：YY1是一种对发育重要的转录因子，调节多种基因的表达。果蝇多梳族 (PcG) 蛋白多同形异型 (PHO) 与 YY1 具有序列同源性。我们发现YY1可以在体内功能性地替代PHO作为PcG蛋白。 YY1 在果蝇中的表达导致 PcG 依赖性转录抑制并拯救 pho 突变果蝇。 YY1 与 DNA 的结合导致 PcG 蛋白的募集以及组蛋白通过脱乙酰化和甲基化的修饰。我们发现，当连接到 GAL4 DNA 结合域时，YY1 序列 201-226 对于转录抑制来说是必要且充分的。这些 YY1 残基与 CtBP、PCNA 和 SUMO-1 发生物理相互作用。我们将解决以下有关YY1 PcG功能的问题：1）YY1抑制PcG转录的机制是什么？通过 ChIP 测定，我们将确定 PcG 募集至 DNA 和组蛋白修饰所需的 YY1 序列。体内救援实验将确定有机体发育所需的序列。遗传和 ChIP 方法将确定 PCNA、CtBP 和 SUMO-1 在 YY1 药物 PcG 抑制中的重要性。我们还将探讨 YY1 DNA 结合、PcG 募集和组蛋白修饰的时间要求。 2) CtBP在YY1 DNA结合和PcG抑制中的作用机制是什么？ CtBP 突变导致体内 YY1 DNA 结合和 PcG 募集减少。我们将确定 CtBP 突变对 YY1 稳定性、DNA 结合能力、细胞内定位以及可能隔离成复合物的影响。 EMSA 和 GST Pull-down 研究将探讨 CtBP sumoylation 对于与 YY1 相互作用的重要性。我们将使用 ChIP 检测来表征 PcG 与众多与 YY1 结合的 PRE 的结合。 3) YY1 在哺乳动物 PcG 抑制系统中如何发挥作用？已鉴定出许多可结合 YY1 和其他 PcG 蛋白的候选哺乳动物 PRE 序列。我们将使用 ChIP 研究来确定 CtBP、PCNA 或苏酰化蛋白是否与哺乳动物 PRE 结合。我们将利用 YY1、CtBP、SUMO-1 和 PCNA 的过表达和 RNAi 敲低来探索对 PcG 募集、组蛋白修饰和基因表达的影响。 PcG 蛋白也与肌肉分化有关。因此，我们将测试YY1、CtBP、SUMO-1和PCNA在成肌细胞分化为肌管中的作用。