RESUMEN
Transcriptional activation of the yeast HO gene involves the sequential action of DNA-binding and chromatin-modifying factors. Here we examine the role of the SAGA complex and the Nhp6 architectural transcription factor in HO regulation. Our data suggest that these factors regulate binding of the TATA-binding protein (TBP) to the promoter. A gcn5 mutation, eliminating the histone acetyltransferase present in SAGA, reduces the transcription of HO, but expression is restored in a gcn5 spt3 double mutant. We conclude that the major role of Gcn5 in HO activation is to overcome repression by Spt3. Spt3 is also part of SAGA, and thus two proteins in the same regulatory complex can have opposing roles in transcriptional regulation. Chromatin immunoprecipitation experiments show that TBP binding to HO is very weak in wild-type cells but markedly increased in an spt3 mutant, indicating that Spt3 reduces HO expression by inhibiting TBP binding. In contrast, it has been shown previously that Spt3 stimulates TBP binding to the GAL1 promoter as well as GAL1 expression, and thus, Spt3 regulates these promoters differently. We also find genetic interactions between TBP and either Gcn5 or the high-mobility-group protein Nhp6, including multicopy suppression and synthetic lethality. These results suggest that, while Spt3 acts to inhibit TBP interaction with the HO promoter, Gcn5 and Nhp6 act to promote TBP binding. The result of these interactions is to limit TBP binding and HO expression to a short period within the cell cycle. Furthermore, the synthetic lethality resulting from combining a gcn5 mutation with specific TBP point mutations can be suppressed by the overexpression of transcription factor IIA (TFIIA), suggesting that histone acetylation by Gcn5 can stimulate transcription by promoting the formation of a TBP/TFIIA complex.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Proteínas Nucleares/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Proteínas de Unión al ADN/genética , Desoxirribonucleasas de Localización Especificada Tipo II/biosíntesis , Proteínas HMGN , Histona Acetiltransferasas , Sustancias Macromoleculares , Modelos Biológicos , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Quinasas/genética , Proteínas Quinasas/fisiología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteína de Unión a TATA-Box/genética , Factores de Transcripción/fisiología , Transcripción GenéticaRESUMEN
The yeast PHO2 gene encodes a homeodomain protein that exemplifies combinatorial control in transcriptional activation. Pho2 alone binds DNA in vitro with low affinity, but in vivo it activates transcription with at least three disparate DNA-binding proteins: the zinc finger protein Swi5, the helix-loop-helix factor Pho4, and Bas1, an myb-like activator. Pho2 + Swi5 activates HO, Pho2 + Pho4 activates PHO5, and Pho2 + Bas1 activates genes in the purine and histidine biosynthesis pathways. We have conducted a genetic screen and identified 23 single amino acid substitutions in Pho2 that differentially affect its ability to activate its specific target genes. Analysis of the mutations suggests that the central portion of Pho2 serves as protein-protein interactive surface, with a requirement for distinct amino acids for each partner protein.