|M.Sc Student||Kotler Anat|
|Subject||Regulation of Meiosis Specific Gene Expression by the|
HDAC-Rpd3 in Budding Yeast
|Department||Department of Biology||Supervisor||Professor Emeritus Yona Kassir|
|Full Thesis text - in Hebrew|
The budding yeast Saccharomyces cerevisiae is a unicellular eukaryote which can live and reproduce in the presence of nutrients both as a haploid and a diploid. Upon nitrogen depletion in the presence of a non-fermentable carbon source haploid cells arrest at G1, while diploid cells enter meiosis. The meiotic pathway is governed by a transcriptional cascade which depends on Ime1. Histone modification is a pivotal epigenetic mechanism that regulates the transcription of genes. Rpd3, the main Histone Deacetylase in budding yeast is found in two subcomplexes, Rpd3S and Rpd3L. Three proteins are shared between the two complexes: Ume1, Sin3 and Rpd3, while the rest are complex specific subunits. Rpd3 functions as a positive regulator for the transcription of IME1, the network of middle meiosis-specific genes (MMG), and their activator, the middle early gene NDT80. Howeer, it serves as a negative regulator for the transcription of the early meiosis-specific genes (EMG).
The main goal of my research was to examine if the positive and negative roles of Rpd3 are mediated through the different complexes in which it operates. I constructed isogenic diploid strains deleted for either RCO1 or SDS3, specific components of the Rpd3S and Rpd3L, respectively, and determined the effect of these mutations on the transcription of representative meiosis specific genes. RT-q PCR analysis revealed that at early meiotic times Rpd3S serves as a positive regulator for the transcription of IME1 and EMG, while Rpd3L serves as the positive regulator for the transcription of NDT80 and the MMG. The decline in the transcription of IME1 and EMG at late meiotic times which is dependent on Rpd3 was found to be independent of either Rco1 or Sds3. We suggest that each Rpd3 complex, by itself suffices for down regulating the transcription of these genes. On the other hand, the decline in the transcription of NDT80 at late meiotic times was dependent solely on Rpd3L.
Ectopic expression of NDT80 in rpd3Δ cells led to partial suppression of MMG transcription as well as entry into nuclear division, suggesting that the positive effect of Rpd3 on the transcription of these genes was mediated through its effect on NDT80. Finally I showed that Rpd3 is required for the transcription of NDT80 either independent of the recombination checkpoint, or in an additional parallel signal as bypass of the recombination checkpoint in a rad17Δ rpd3Δ double mutant did not allow the expression of NDT80.