The proposed project's goals are to understand the function of the newly identified SAS gene family in chromatin-mediated transcriptional regulation and genomic silencing. A sequence signature shared by Sas proteins and enzymes known to have acetyltransferase activity suggests that the Sas proteins function by acetylating key chromatin components. SAS genes were discovered in the yeast Saccharomyces, but sequence homologs including human MOZ and Tip60, have significant implications for human health. MOZ is the common 5' partner in recurrent 8p11 translocations that lead to the M4/M5 subtype of acute myeloid leukemia. Tip60 is a human gene associated with HIV-Tat that facilitates increased levels of Tat-dependent transcription. Understanding the most conserved elements of SAS function may thus lead to increased understanding of leukemia and HIV related disease including AIDS and AIDS-related malignancies. Analysis of yeast SAS function revealed that SAS2 or SAS3 mutants are defective in transcriptional silencing. The third yeast gene, ESA1, is essential for viability. Because ESA1 is most closely related to the human homologs, much of the proposal focuses on its analysis. Conditional alleles, including mutations in the putative acetyltransferase domain, will identify critical regions of ESA 1. Analysis of conditional alleles will facilitate the proposed genetic and cell biological dissection of ESA 1. It will be determined if ESA 1 is required at a single or multiple points in the cell cycle and if loss of ESA1 function leads to silencing defects, thereby potentially identifying genetic loci whose repression is essential for normal mitotic growth. Biochemical characterization will test directly the hypothesis that SAS genes function through chromatin acetylation. Identification of relevant substrates and regulators of activity will be sought through biochemical and genetic approaches. Tests to determine limits of functional conservation between yeast and human SAS genes will be performed by determining if human genes can suppress yeast mutant phenotypes. Experiments will be performed to test the hypothesis that mis-localization of SAS activity may lead to disease by altering locally defined patterns of transcriptional regulation. Results from these diverse experimental approaches should establish mechanisms of SAS gene function and suggest how disruption of this function leads to alterations in genomic silencing and activation.
