PcG Function of YY1 in Transcription and Development

YY 1是一种发育重要的转录因子，调节许多基因的表达。的 果蝇Polycomb group（PcG）蛋白，Pleiohomeotic（PHO），与YY 1具有序列同源性。我们 发现YY 1可以在体内功能上取代PHO作为PcG蛋白。YY 1在果蝇中的表达结果 在PcG依赖的转录抑制和拯救pho突变苍蝇。YY 1引起的DNA结合 PcG蛋白的募集以及通过去乙酰化和甲基化修饰组蛋白。我们发现 YY 1序列201-226，当与GAL 4 DNA结合结构域连接时，是必需的和足够的， 转录抑制这些YY 1残基与CtBP、PCNA和SUMO-1物理相互作用。我们将 本文主要探讨了YY 1 PcG功能的相关问题：1）PcG的作用机制 转录抑制YY 1？通过ChIP检测，我们将确定PcG所需的YY 1序列 招募DNA和组蛋白修饰。体内拯救实验将确定 需要有机体的发展。遗传和ChIP方法将决定PCNA的重要性， CtBP和SUMO-1在YY 1介导的PcG抑制中的作用。我们还将探讨 YY 1 DNA结合、PcG募集和组蛋白修饰。2)CtBP的作用机制是什么？ YY 1 DNA结合和PcG抑制？CtBP突变导致YY 1 DNA结合和PcG减少 体内招募。我们将确定CtBP突变对YY 1稳定性、DNA结合能力、 细胞内定位，并可能螯合到复合物中。EMSA和GST下拉研究将 探索CtBP类小泛素化对于与YY 1相互作用的重要性。我们将使用ChIP检测， 表征PcG与许多结合YY 1的PRE的结合。3)YY 1在哺乳动物中的作用 PcG抑制系统？已经鉴定了许多候选哺乳动物PRE序列， 结合YY 1和其他PcG蛋白。我们将使用ChIP研究来确定CtBP、PCNA或 sumoylated蛋白质与哺乳动物PRE结合。我们将使用YY 1的过表达和RNA干扰敲低， CtBP、SUMO-1和PCNA，以探讨对PcG募集、组蛋白修饰和基因表达的影响。 表情PcG蛋白也参与肌肉分化。因此，我们将测试 YY 1、CtBP、SUMO-1和PCNA在成肌细胞向肌管分化中的作用。

YY1 is a developmental^ 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.