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Silent Chromatin and Sister Chromtids Cohesion in Saccharomyces Cerevisiae
by
Ching-Shyi Wu
MS, 2001
National Yang Ming University
Taiwan
Thesis Advisor: Marc R. Gartenberg, PhD
Graduate Program in Cellular and Molecular Pharmacology
Robert Wood Johnson Medical School
4th Floor Conference Room
Tuesday, November 3, 2009
12:00 noon
Abstract
The cohesin complex ensures accurate segregation of genomes at mitosis and meiosis by holding sister chromatids together. The protein complex binds at specific spots along chromosomes including centromeres and heterochromatin regions. In budding yeast, cohesin is found at silenced chromosomal domains that functionally resemble heterochromatin, known as silent chromatin (HM mating-type loci, rDNA repeats and telomeres). The repressive structure at the HMR mating-type locus requires the Sir protein complex that nucleates at cis acting silencer elements. Sister chromatid cohesion at HMR and the connection between cohesin and silent chromatin have been examined. Using chromatin immunoprecipitation (ChIP) and GFP-tagged extrachromosomal HMR fragments, I find that cohesin binds to and holds silenced domain together in a Sir protein dependent manner. Importantly, cohesin binds to silent chromatin topologically in a way that is not consistent with double embrace model.
To further investigate whether individual Sir protein can mediate recruitment of cohesin at HMR loci, I tethered proteins of interest to a GFP-tagged extrachromosomal fragment that does not cohese. Those proteins that convey cohesion convert unpaired GFP foci to a single bright spot. The results show that Sir2 supports cohesion in this context independently of its histone deacetylase activity and the other Sir proteins.
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