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Abstract
The genes of the Polycomb-Group (PcG) regulate homeotic gene expression patterns throughout the course of Drosophila development. The products of PcG genes maintain homeotic gene repression. Early homeotic gene expression patterns are established by transiently expressed segmentation genes, but PcG genes are required for the faithful maintenance of these patterns. When PcG genes are mutated, homeotic genes are expressed ectopically in the embryo. Little is known about the molecular mechanisms by which PcG proteins repress gene expression. However, PcG proteins are hypothesized to work at the level of chromatin structure, since many contain motifs known to modify or bind to chromatin.
I describe here the development of an in vitro system to study transcriptional repression by PcG proteins. The PcG complex PRC1, and a recombinant complex of PRC1's core PcG proteins (PRC1 core complex, or PCC) are able to repress transcription of both chromatin and naked DNA templates. Repression is due to an interaction between PcG proteins and the template, and is enhanced by the presence of nucleosomes, indicating that PcG proteins can collaborate with chromatin to repress transcription. Not all processes are blocked by PcG proteins, though. The activator Gal4-VP16, can bind to a PcG repressed template, and it can be cut by nucleases. Thus PcG proteins might establish a selectively inhibitory chromatin structure.
I also describe an analysis of the PcG protein Posterior Sex Combs (PSC), which is central to PCC and PRC1 activity in vivo and in vitro. Three separate lines of evidence were used to identify regions of the PSC protein responsible for its activities. We sequenced genetically identified alleles of Psc, tested the mutant proteins corresponding to these alleles in biochemical inhibition assays, and tested a similar series of engineered deletions of Psc in an in vivo assay for homeotic gene repression. We find that all three approaches identify a particular region in PSC's non-conserved C-terminus as a region essential for function. This correlation suggests strongly that in vitro inhibition activities of PSC reflect a key aspect of PSC function in vivo.