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是调节许多基因表达的发展上重要的转录因子。果蝇多蛋白酶基（PCG）蛋白（pHO）具有与YY1的序列同源性。我们发现YY1可以在体内功能替代PHO作为PCG蛋白。果蝇中的YY1表达会导致PCG依赖性转录抑制和拯救Pho突变体蝇。通过YY1结合DNA会导致PCG蛋白募集，并通过脱乙酰化和甲基化对组蛋白进行修饰。我们发现，YY1序列201-226与GAL4 DNA结合结构域链接时，需要进行转录抑制。这些YY1残基与CTBP，PCNA和SUMO-1物理相互作用。我们将解决有关YY1 PCG功能的以下问题：1）YY1的PCG转录抑制的机制是什么？通过CHIP测定，我们将确定PCG募集到DNA和组蛋白修饰所需的YY1序列。体内救援实验将确定有机发育所需的序列。遗传和芯片方法将确定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）YY1 DNA结合和PCG抑制中CTBP功能的机制是什么？ CTBP突变导致YY1 DNA结合和体内PCG募集降低。我们将确定CTBP突变对YY1稳定性，DNA结合能力，细胞内位置以及可能的隔离为复合物的影响。 EMSA和GST下拉研究将探讨CTBP Sumoylation与YY1相互作用的重要性。我们将使用CHIP测定法表征PCG与与YY1结合的许多PRE的结合。 3）YY1在哺乳动物PCG抑制系统中如何发挥作用？已经确定了许多结合YY1和其他PCG蛋白的候选哺乳动物前序列。我们将使用CHIP研究来确定CTBP，PCNA或Sumoylated蛋白是否与哺乳动物PRES结合。我们将使用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在分化为肌管中的作用。