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Cohesion of Silenced Chromatin by Sirtuins in Saccharomyces cerevisiae

by
Yu-Fan Chen
MS, National Yang Ming University, 2008

Thesis Advisor: Marc Gartenberg, PHD

Tuesday, April 28, 2015
9:00 AM


Abstract

Assembly of yeast heterochromatin requires Sir2, the founding member of the conserved family of
NAD+-dependent protein deacetylases known as the sirtuins. These proteins serve as metabolic
sensors involved in cellular lifespan. They have also been linked to cancer and numerous human
diseases. Previous work in the Gartenberg lab showed an unanticipated role for yeast Sir2: the
protein is required for cohesion of yeast heterochromatin. Tethered Sir2 fragments mediate cohesion
in the absence of other Sir proteins whereas synthetic heterochromatin domains that lack Sir2 do not
mediate cohesion at all. The domain of Sir2 responsible for cohesin recruitment maps to the Cterminus.
Using mutagenesis guided by Sir2 crystal structures, I identified a small surface patch within
this domain that is required for cohesin recruitment and cohesion. At least one additional domain of
Sir2 contributes to cohesin recruitment but the secondary domain is not sufficient for cohesion.
The Sir2 surface mutations that block cohesion do not disrupt heterochromatic repression in
most standard phenotypic assays. In more specialized assays, however, defects in silencing can be
detected. Elevated expression of Sir4, a Sir2 binding partner, suppresses the silencing defects without
restoring heterochromatic cohesion. These studies show that the silencing and cohesion functions of
Sir2 can be uncoupled, and that silencing can be achieved without cohesion of heterochromatin.
To investigate how Sir2 mediates cohesin recruitment, I used two approaches to find proteins
that interact with the C-terminus: yeast two-hybrid screening and copurification followed by mass
spectroscopy. Candidate factors from these approaches are discussed.


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