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. ? ?
