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An analysis of polycomb group protein interactions

University of British Columbia

The Polycomb Group (PcG) of proteins are global regulators of transcription. PcG mutants display posterior homeotic transformations, the result of ectopic expression of homeotic selector genes of the Bithorax and Antennapedia Complex, demonstrating that the PcG is required for the repression of target genes outside of their normal spatial boundaries of expression. Coimmunoprecipitation, cofractionation, and colocation on larval salivary gland chromosomes suggest that PcG proteins act through large multimeric complexes formed at their target sites. This thesis is a characterization of the protein interactions that underlie multimeric complex formation. Using the yeast two-hybrid system and an in vitro co-affinity precipitation assay, I demonstrate direct interactions between Polycomb (Pc) and Posterior Sex Combs (Psc), and between Psc and polyhomeotic (ph). I also show that Psc, ph, and Asx have self-interacting domains, and perform a detailed analysis of the selfinteracting domain of ph. For the most part, these interacting domains are highly conserved between the Drosophila proteins and their mammalian counterparts. Because Asx shows no direct interact interactions with Pc, Psc, or ph, I screen Asx for interacting proteins within a two-hybrid library and within a two-hybrid panel of other chromatin proteins. Several interactors are identified, including the Drosophila homologue of cyclin G, and z40, a previously unknown protein which interacts strongly with Pc. In addition, an interaction is demonstrated between the respective carboxyl termini of Asx and trithorax (trx), a protein required for activation of homeotic selector genes. I show that Psc can repress transcription in Saccharomyces cerevisiae, and show that this repression does not require interactions with a variety of yeast proteins required for repression of various loci in the S. cerevisiae genome. These data enlarge our understanding of the structure of PcG complexes, and suggest that PcG proteins interact with one another promiscuously, enabling them, in theory, to form a large number of different complexes each tailored to a particular chromosomal neighbourhood.

Thesis/Dissertation

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2009-06-22

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http://hdl.handle.net/2429/9524

Zoology

University of British Columbia

UBCV

DSpace