Purification and characterization of polyhomeotic associated proteins from Drosophila Kc1 cells

The Polycomb group (PcG) genes encode repressors of homeotic and other genes, that are required to maintain silencing of target loci. Tethered P cG proteins repress reporter genes in cell lines and in Drosophila embryos, showing that P cG proteins are repressors. PcG genes are required for the maintenance but not the initiation of homeotic gene repression, because in P cG mutants, initiation of homeotic gene expression is normal, and then breaks down after a lag of several hours. Coimmunoprecipitation and cofractionation of P cG proteins, colocalization of P cG proteins on polytene chromosomes, and synergistic mutant phenotypes in double heterozygous mutants of PcG genes suggest that P cG proteins act through multimeric protein complexes. However, the mechanisms of PcG-mediated homeotic gene silencing are not known. One approach to this problem is to identify proteins that associate with PcG proteins in vivo in an effort to generate testable hypotheses about PcG-mediated silencing. Using epitope tagging followed by immunopurification, I purified Polyhomeotic (PH) proximal and its associated proteins from Drosophila K c l cell nuclear extracts. I subsequently identified the PH-associated proteins using mass spectrometry sequencing and western blotting analysis. I showed that molecular chaperones are associated with P H and that a mutation in chaperone Hsc70.4 enhances the extra sex combs phenotype of ph and Pc. These results suggest that chaperones may participate in the formation of PH-containing complexes, or may be required for silencing. I demonstrated that the histone deacetylase Rpd3 and histone binding protein p55 are associated with PH, and that the Rpd3 mutation enhances the extra sex combs phenotype of ph and Pc. Surprisingly, histone deacetylase activity was not detected in immunopurified PH. I showed using western blotting analysis that the TATA-binding protein (TBP) and its associating proteins TAFn 4 2 and TAFn85 are also associated with PH. PH and PC were coimmunoprecipitated by anti- TBP antibody. In addition, Tbp mutants enhance the extra sex combs phenotype of ph but not Pc. Together, these findings suggest that P cG proteins use different means to silence gene expression including modifying histones and targeting the basal transcriptional machinery.Science, Faculty ofZoology, Department ofGraduat