PcG Function of YY1 in Transcription and Development

描述（由申请人提供）：YY1是一种重要的发育转录因子，可调节多种基因的表达。果蝇Polycomb group （PcG）蛋白，Pleiohomeotic (PHO)，序列与YY1同源。我们发现YY1在体内可以代替PHO作为PcG蛋白。YY1在果蝇中的表达导致pcg依赖性的转录抑制和pho突变果蝇的拯救。YY1结合DNA导致PcG蛋白的募集和组蛋白的去乙酰化和甲基化修饰。我们发现YY1序列201-226，当与GAL4 DNA结合域连接时，对转录抑制是必要的和充分的。这些YY1残基与CtBP、PCNA和SUMO-1发生物理相互作用。我们将探讨以下关于YY1 PcG功能的问题：1)YY1抑制PcG转录的机制是什么？通过ChIP检测，我们将确定PcG募集到DNA和组蛋白修饰所需的YY1序列。体内拯救实验将确定生物体发育所需的序列。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被隔离到复合体中的影响。EMSA和GST下拉研究将探索CtBP酰化对YY1相互作用的重要性。我们将使用ChIP分析来表征PcG与许多与YY1结合的PREs的结合。3) YY1在哺乳动物PcG抑制系统中如何起作用？许多候选的哺乳动物PRE序列已经被确定，可以结合YY1和其他PcG蛋白。我们将使用ChIP研究来确定CtBP、PCNA或聚合蛋白是否与哺乳动物的PREs结合。我们将使用YY1、CtBP、SUMO-1和PCNA的过表达和RNAi敲除来探索对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在成肌细胞向肌管分化中的作用。

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.