Role of polyhomeotic and the cohesin protein Pds5 in silencing and S phase

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For normal development to occur, genes must be kept off (silent) where they are supposed to be off. Silencing plays a vital role in development because it prevents genes from being turned on too early, or from being kept on. Both forms of misregulation can lead to birth defects, or to cancer. A group of about 20 proteins, called the Polycomb group proteins work together to make a the DNA of a target gene inaccessible to the proteins that turn genes on. We are studying one of these proteins, called Polyhomeotic. In previously work previously funded by NSERC, a graduate student discovered that Polyhomeotic associates with a protein (Pds5) whose known role is in holding newly replicated DNA together before cell division. Another student has shown using genetic analysis that Pds5 has an unexpected role in silencing. One of the aims of this proposal is to determine the molecular role for Pds5 in silencing. In a separate study, my students have shown using genetic analysis that Polyhomeotic is required for normal cell division, and works together with Pds5. Very surprisingly, the stage of cell division where DNA is replicated is speeded up in Polyhomeotic mutants. Another aim of this proposal is to understand why this occurs. The most likely reasons are that mutations in Polyhomeotic make it easier for the protein machinery to replicate DNA, or that mutations make more starting points for DNA replication available to the replication machinery. Pds5 is recruited by the protein machinery that makes DNA. We want to test the hypothesis that Pds5 recruits Polyhomeotic to newly replicated DNA. To do this, we have to develop a system for purifying this newly synthesized DNA in a way that does not disrupt the interaction of proteins with DNA. This is the third aim of the proposal. This research will identify the molecular basis for new role(s) for Pds5, and has the potential to provide important insight about the molecular mechanisms of silencing.