The related yeast transcriptional activators SWI5 and ACE2 have related zinc finger/DNA-binding domains, and bind to the same sequence. Yet SWI5 activates HO (endonuclease for mating type switching) and ACE2 activates CTS1 (chitinase). Both activators work in G1 of the cell cycle. What is the basis of their specificity? In the prior period, Dr. Stillman has made several findings. First, binding sites for ACE2 in the CTS1 promoter were identified and binding of ACE2 and SWI5 to CTS1 and HO sites compared in vitro. This led to the conclusion that SWI5 and ACE2 bind to the same sequences with the same affinities. Interestingly, a third gene SIC1 (cdk inhibitor) was identified which is activated by both SWI5 and ACE2. Second, SWI5 binds to the HO UAS in concert with another activator, PHO2, in vitro. In vivo, PHO2 is not required for transcription of wild type HO, but is important when the SWI- binding sites in HO have been weakened by mutation. PHO2 works in concert with different yeast activators for genes such as PHO5 and HIS4. It plays no role in expression of CTS1 and does not cooperate with ACE2. Third, by making SWI5-ACE2 chimeras, large regions in these proteins were identified which are distinct from the DNA-binding domains and dictate target gene specificity. Fourth, a negative element in the CTS1 promoter was roughly defined which when removed allows SWI5 to activate. It is suggested that one activity of ACE2 must be to counteract this element. This element also will inhibit the heterologous CYC1 activators in promoter fusions. Possible trans-acting repressors for this element were identified by mutations in genes (NCE) that allow SWI5 to activate CTS1. Fifth. regions in both PHO2 and SWI5 required for cooperation at HO were identified. The PHO2 mutations were deletions and the SWI5 mutations included single amino acid changes, which clustered in one region. Sixth, mutations in the two distant SWI5 binding sites of HO were used to expose a PHO2 requirement, leading to a picture that a SWI5-PHO2 complex mediates the long range interactions between these two sites. In the next period, the PI proposes to 1. further characterize the PHO2-SWI5 interaction. 2. Characterize the negative regulation in the CTS1 promoter. 3. Characterize the role of negative regulation in HO promoter specificity. 4. Characterize how ACE2 confers promoter specific activation. 5. Characterize the regulation of other genes regulated by SWI5 and ACE2.
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