Biology and MB&B

Graduate Student Career Retreat 2008

Name: Upasna Sharma

   Lab: Scott Holmes [MB&B]

 Abstract

Cell-cycle requirement for the establishment

of silencing at the budding yeast telomeres

Upasna Sharma, MBB Department, Wesleyan University

In Sacchromyces cerevisiae gene silencing is mediated by the formation of heterochromatin like structure at the telomeres, the mating type loci and the ribosomal DNA (rDNA) repeats. A model has been proposed for the formation of heterochromatin in yeast, wherein the initiation of silencing takes place by the association of DNA binding factors Rap1, Abf1 and Orc to the cis-acting “silencer” sequences. These factors are thought to recruit the Sir complex, which alters the N-terminal tails of the histone H3 and H4, mediating the formation of heterochromatin. Independent studies have shown that the establishment of silencing in yeast requires progression through the cell-cycle. In the Holmes lab, using an inducible Sir3 system, it has been shown that the establishment of silencing at the telomere requires passage from G2/M to the subsequent G1 phase (Martins-Taylor, 2004). This suggests that there are some specific factors or events in mitosis that affect the assembly of heterochromatin.  Alternatively some factors or event outside of mitosis can inhibit the establishment of silencing. Thus far the cell-cycle dependent events that are responsible for the assembly and stability of heterochromatin have not been defined. Determining the cell-cycle dependence for the establishment of silencing will help us better understand the epigenetic regulation of the silent chromatin.

To understand the mechanisms involved in the establishment of silencing, I want to define more precisely the cell-cycle interval when silencing is established at the yeast telomeres. To achieve this goal, I introduced temperature sensitive alleles of CDC genes in strains having inducible SIR3 construct. These strains when shifted to non-permissive temperature, arrest the cells at specific points in the cell-cycle. Using these strains I assayed for the repression of a telomere linked URA3 gene in cells arrested at metaphase and telophase. I found that telophase arrested cells show URA3 repression while the metaphase arrested cells do not show repression. Thus, I conclude from these experiments that the establishment of silencing occurs during the metaphase-telophase transition of the cell-cycle. All published studies carried out to define the timing of silencing have shown that the passage through a particular interval of cell-cycle was required for the establishment of silencing. Here we define a specific point in the cell-cycle at which silencing can be established in the absence of cell-cycle progression. Why is the establishment of silencing restricted to mitosis? What factors or events limit the establishment of silencing to the M-phase? My future experiments aim to study the factors and the molecular events that can restrict the assembly of heterochromatin to a specific interval of the cell-cycle.