Promoter Specificity of Transcription Factors

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DESCRIPTION (provided by applicant): Specific gene expression is controlled by transcription factors binding to elements present in promoters and enhancers. In many instances multiple transcription factors can recognize the same DNA sequence, but each factor can activate different genes. Thus the DNA-binding characteristics of a transcription factor are not sufficient to determine promoter specificity. The yeast transcription factors Swi5 and Ace2 have nearly identical zinc finger DNA-binding domains, and both factors show similar pattern of cell cycle regulation, being present in the nucleus for a limited period in M and G1. Although Swi5 and Ace2 recognize the same DNA sequences in vitro, they are required for the activation of different cell cycle regulated genes in vivo. Swi5 activates transcription of HO, but not CTS1, and Ace2 activates CTS1, but not HO. Generally, Swi5 activates genes that promote cell cycle progression while Ace2 activates genes required for cell separation. Chromatin immunoprecipitation (ChIP) experiments show that there are two distinct mechanisms operative to prevent these factors from activating genes. In one case a factor binds to a promoter, but does not activate, and in the other case a factor does not bind in vivo to a promoter site that it recognizes in vitro. Swi5 binds to CTS1 but does not activate transcription. Our data suggests that the Fkh1 and Fkh2 proteins bound at the CTS1 promoter prevent Swi5 from activating transcription. Thus, the Fkh proteins act as a selective repressor, blocking Swi5 but not Ace2 from promoting CTS1 expression. Experiments are proposed to understand how this selective repression machinery works. Data suggest that Ace2 recruits a kinase to the promoter, and that this kinase overcomes this repression. Ace2 can bind to the HO promoter in vitro, but ChIPs show that it does not bind to the HO promoter in vivo. Experiments are proposed to identify both cis acting sites and trans acting regulators that prevent Ace2 from binding at HO, and to characterize this regulation. Ace2 binds well to the SIC1 promoter in vitro, but poorly in vivo. The data suggest that distinct portions of Ace2 form a structural unit that binds an inhibitor. Structural studies are proposed to characterize the nature of this region of the Ace2 protein, and a genetic screen is proposed to identify and characterize the inhibitor. A kinase mutation blocks Ace2 from binding DNA in vivo, although Ace2 is still in the present in the nucleus. This DNA-binding defect can be suppressed by mutations in the N-terminal region of Ace2. The data suggest that the N-terminal region acts as an autoinhibitor, blocking DNA-binding by the C-terminal DNA-binding domain, with phosphorylation in the N-terminal region of Ace2 relieving this inhibition. Biochemical and genetic experiments are proposed to characterize this autoinhibition. Project Narrative: Regulation of gene expression is essential for the proper differentiation and maintenance of distinct cell types in eukaryotes, and abnormalities in gene expression can cause specific diseases including cancer. The yeast Saccharomyces cerevisiae with its powerful genetic tools has proven to be the model eukaryotic organism for studies of gene regulation, and since the transcription regulatory machinery is conserved between yeast and vertebrates the insights gained from studies in yeast are generally universal. Here we study two transcription factors with identical DNA-binding domains that recognize the same DNA sequence in vivo, to determine what mechanisms restrict their ability to activate expression of specific genes in vivo.