Promoter Specificity of Transcription Factors

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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 the same pattern of cell cycle regulation, being present in the nucleus for a limited period in G1. Although Swi5 and Ace2 recognize the same DNA sequences in vitro, they are required for the activation of two different G1-specific genes in vivo. Swi5 activates transcription of HO, but not CTS1, and Ace2 activates CTS1, but not HO. The Swi5 and Ace2 transcription factors function at the M/G1part of the cell cycle. A combination of S1 nuclease protection and microarray assays will be used to characterize all of the genes activated by these two factors, providing tools for mechanistic studies of gene regulation. Our analysis thus far suggests that 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 can bind to the CTS1 promoter, but it does activate transcription. We have identified cis-acting sequences within the CTS1 promoter and trans-acting factors that prevent activation by Swi5. Experiments are proposed to investigate how promoter bound factors can block promoter bound Swi5 from activating transcription, while allowing Ace2 to function in transcriptional activation. Although Swi5 and Ace2 enter the nucleus at approximately the same time, several genetic experiments suggest that Ace2 accumulation in the nucleus may be slightly delayed at anaphase relative to Swi5. Recent work shows that Ace2 has a novel nuclear export sequence. Based on cis- and trans- mutations that affect Ace2 activity, we believe we have identified set of cell cycle genes that function to inhibit nuclear export of Ace2 and thus allow it to activate transcription. Experiments are proposed to dissect this nuclear export. Ace2 can bind to the HO promoter in vitro, but chromatin immunoprecipitation shows that it does not bind to the HO promoter in vivo. If Swi5 enters the nucleus slightly before Ace2 during mitosis, then it is possible that after Swi5 binds the promoter acquires a block to Ace2 binding. We propose to characterize the promoter sites, regulatory proteins, and cell cycle machinery that produce this block to transcription factor binding.