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Activator Gcn4p and Cyc8p/Tup1p Are Interdependent for Promoter Occupancy at ARG1 In Vivo

Soon-ja Kim, Mark J. Swanson, Hongfang Qiu, Chhabi K. Govind, and Alan G. Hinnebusch^*

Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, Bethesda, Maryland 20892

Received 6 July 2005/ Returned for modification 8 August 2005/ Accepted 21 September 2005

The Cyc8p/Tup1p complex mediates repression of diverse genes in Saccharomyces cerevisiae and is recruited by DNA binding proteins specific for the different sets of repressed genes. By screening the yeast deletion library, we identified Cyc8p as a coactivator for Gcn4p, a transcriptional activator of amino acid biosynthetic genes. Deletion of CYC8 confers sensitivity to an inhibitor of isoleucine/valine biosynthesis and impairs activation of Gcn4p-dependent reporters and authentic amino acid biosynthetic target genes. Deletion of TUP1 produces similar but less severe activation defects in vivo. Although expression of Gcn4p is unaffected by deletion of CYC8, chromatin immunoprecipitation assays reveal a strong defect in binding of Gcn4p at the target genes ARG1 and ARG4 in cyc8 cells and to a lesser extent in tup1 cells. The defects in Gcn4p binding and transcriptional activation in cyc8 cells cannot be overcome by Gcn4p overexpression but are partially suppressed in tup1 cells. The impairment of Gcn4p binding in cyc8 and tup1 cells is severe enough to reduce recruitment of SAGA, Srb mediator, TATA binding protein, and RNA polymerase II to the ARG1 and ARG4 promoters, accounting for impaired transcriptional activation of these genes in both mutants. Cyc8p and Tup1p are recruited to the ARG1 and ARG4 promoters, consistent with a direct role for this complex in stimulating Gcn4p occupancy of the upstream activation sequence (UAS). Interestingly, Gcn4p also stimulates binding of Cyc8p/Tup1p at the 3' ends of these genes, raising the possibility that Cyc8p/Tup1p influences transcription elongation. Our findings reveal a novel coactivator function for Cyc8p/Tup1p at the level of activator binding and suggest that Gcn4p may enhance its own binding to the UAS by recruiting Cyc8p/Tup1p.

Supplemental material for this article may be found at http://mcb.asm.org/.

Present address: School of Biological Sciences, Louisiana Tech University, Ruston, LA 71272.

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