| Summary: | 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 of === Zoology, Department of === Graduate
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