The transcriptional regulation of HO has some unique features, and our studies of yeast HO regulation have uncovered several novel transcriptional regulators. Saccharomyces cerevisiae divides asymmetrically, producing mother and daughter cells, and HO expression only occurs in mothers in a limited period of the cell cycle. Mother cells retain the """"""""memory to transcribe HO, as HO is still expressed after an extend G1 period during which essentially all of an essential transcriptional activator, Swi5p, has been degraded. The Swi5p protein plays a critical role in the mother cell specificity of HO expression. However, Swi5p apparently enters the nucleus and is degraded before HO is activated, suggesting that Swi5p leaves an activating """"""""imprint"""""""" on the promoter. Swi5p binds to two HO promoter sites separated by 500 bp, and our data suggest that Swi5p may facilitate a loop between these two sites. Experiments are propOsed to further investigate these long range interactions. The cell cycle kinetics of transcription factor activation and expression of HO are complex, and experiments are proposed to investigate when Swi5p is bound to the promoter. Changes in the structure of HO chromatin during the cell cycle could explain many of the observed regulatory phenomena, and chromatin structure during the cell cycle will be examined directly. SIN3 is a negative transcriptional regulator of HO and other yeast genes. Sin3p does not bind to DNA, and our data suggest that Sin3p is targeted to promoters by binding to DNA-binding proteins. We propose to characterize the mechanism by which Sin3p achieves transcriptional repression in several ways. Mad, a mammalian DNA-binding protein, interacts with Yeast Sin3p and represses transcription in yeast in a SIN3-dependent manner, and we will further characterize the Mad/Sin3 interaction. We have identified several yeast DNA-binding proteins that interact with Sin3p, and will characterize their role in transcriptional repression. SIN4 negatively regulates some genes, including HO, and SIN4 is required for full expression of others. Many of the phenotypes in sin4 mutants suggest that chromatin is involved. Sin4p genetically and physically interacts with the Rgr1p protein, and both of these proteins are present in the """"""""holoenzyme"""""""" or """"""""mediator"""""""" complex required for activator dependent transcription. Sin4p appears to be in a subcomplex of the holoenzyme with Gal11p and p50; Rgr1p is either part of the subcomplex, or acts to bind the subcomplex. Genetic and biochemical experiments are proposed to investigate the structure of this subcomplex and its role in transcriptional regulation. Our work with SIN4 led to the identification of ACT3. Mutations in ACT3 cause defects in transcriptional regulation and affect the structure of chromatin. ACT3 encodes an actin-related protein that is predicted to bind ATP, and we have shown that Act3p is present in the nucleus. We show that act3 mutations cause transcriptional patterns to be inherited in a metastable or epigenetic fashion, using a specific reporter gene whose activity can be assayed by colony color. We will investigate this novel transcriptional regulator using biochemical and genetic techniques.
Showing the most recent 10 out of 28 publications