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UBC Theses and Dissertations

For genes that encode one component of a multimeric protein complex, measuring only one phenotype often gives a biased view of function: SU(VAR)3-9 and chromatin architecture as an example Kalas, Pamela

Abstract

Eukaryotic genomes are organized into chromatin, a highly dynamic complex of DNA and proteins, which plays a critical role in the regulation of gene expression. This thesis focuses on the study of a non-histone chromatin protein, the SET domain-containing H3K9 methyltransferase (HMTase) SU(VAR)3-9, and its role in the packaging and regulation of a euchromatic locus, the histone genes cluster (HIS-C). SU(VAR)3-9 was discovered in Drosophila melanogaster, but it is highly conserved from yeast to mammals. It has two conserved domains, the chromo- and the SET domains, and both are required for its function in gene silencing. The SET domain is responsible for the catalytic activity of SU(VAR)3-9, while the exact function of the chromo domain is still unclear. To gain an insight on the role(s) of SU(VAR)3-9 in the regulation of gene silencing, we first characterized a collection of Su(var)3-9 EMS-induced mutants that had been isolated in a genetic screen for strong, dominant suppressors of position-effect variegation (PEV). These mutants were characterized at the molecular, enzymatic, and cellular level, and their effect on gene silencing was also examined. We found that all mutants have single amino acid substitutions in the conserved preSET/SET/postSET domain, and that they all display a dramatic or complete loss of HMTase activity, strongly suggesting that suppression of PEV is linked to SU(VAR)3-9’s ability to methylate H3K9. The HIS-C is a natural, euchromatic target of SU(VAR)3-9, and mutations in Su(var)3-9 can alter its chromatin structure (Ner et al., 2002). To investigate the exact role(s) of SU(VAR)3-9 in the regulation of this locus, we analyzed the effects of a series of Su(var)3-9 missense mutants on the chromatin architecture of the HIS-C and on the expression of the histone genes. We detected a drastic reduction in the levels of H3K9me2 and HP1 associated with the his genes in all Su(var)3-9 missense mutants, although the mutant SU(VAR)3-9s still associate with the HIS-C. In addition, these mutants have elevated amounts of histone H2A and histone H3 RNA, suggesting that the enzyme function of SU(VAR)3-9 is critical for the regulation of the histone genes.

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