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Investigating the role of histone acetylation in yeast FACT complex function

University of British Columbia

The FACT (facilitates chromatin transactions) complex, a heterodimer comprised of Spt l6p and Pob3p, is involved in a number of processes involving DNA, including transcription initiation, elongation, and repression, DNA replication, and DNA repair. FACT is thought to facilitate these processes through its ability to bind histones and reversibly alter nucleosomal structure modulating DNA accessibility. Recent data has shown that the Sptl6p subunit of FACT interacts with Sas3p of the NuA3 HAT (histone acetyl transferase) complex. Interestingly, mutants of NuA3 share none of the many phenotypes observed in FACT mutants, suggesting that this putative interaction may be redundant to other unknown FACT interactions. In the first part of this study, we confirmed the interaction between FACT and NuA3, and proceeded to describe, for the first time, an interaction between FACT and the ADA HAT complex both in vivo and in vitro. The interaction of FACT with NuA3 and ADA was then mapped to the Nterminal 482 residues of the Sptl6Δp FACT subunit. However, attempts to implicate this interaction with known FACT functions were unsuccessful. Biochemical approaches similarly failed to demonstrate a role for FACT in NuA3 and ADA complex function. The second part of this study focused on the novel spt16Δ922 mutant, which expresses 182 residues more of the Sptl6Δp polypeptide than the sptl6ΔNTD allele, yet possesses a number of mutant phenotypes. Our results show that the spt16Δ922 mutation causes a loss in histone H3 acetylation, supporting the hypothesis that the protein encoded by spt16Δ922 is interfering with normal histone interactions. This loss in histone acetylation was shown to parallel the generation of an spt phenotype, suggesting a possible role for histone acetylation in preventing aberrant transcription. However, further work examining the role of histone acetylation with respect to FACT yielded conflicting results. We conclude that spt16Δ922 is interfering with histone modifications, although the molecular basis of this is as of yet unknown.

Text

2010-01-14

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http://hdl.handle.net/2429/18150

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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