DNA-Bound Bas1 Recruits Pho2 To Activate ADE Genes in Saccharomyces cerevisiae

Abstract

Expression of the genes in the ADE regulon of Saccharomyces cerevisiae is repressed by the presence of purine bases in the extracellular medium and derepressed when cells are grown in the absence of purines. Derepression requires the transcriptional activators Bas1 and Pho2, as well as the biosynthetic intermediates 5-phosphoribosyl-4-succinocarboxamide-5-aminoimidazole (SAICAR) and 5-phosphoribosyl-4-carboxamide-5-aminoimidazole (AICAR). In this study, we investigated if nuclear localization and binding to promoter DNA by the activators are regulated by purines. Using indirect immunofluorescence, we found that Bas1 is localized to the nucleus under both repressing and derepressing conditions. Importantly, we detected Bas1 bound to promoter DNA under both conditions using chromatin immunoprecipitation assays at several ADE promoters (ADE1, ADE2, ADE4, and ADE5,7) and HIS4. We analyzed the binding of Bas1 to wild-type and mutant sequences of the ADE5,7 promoters in vivo, and found that Bas1 binds independently to each of its two binding sites. Pho2 was not required for the association of Bas1 with chromosomal DNA, but it was required for an increase in Bas1-immunoprecipitated DNA. The presence of Pho2 at promoters was dependent on Bas1 and occurred only under derepressing conditions when the ADE genes are transcribed at elevated levels. We propose a model for regulation of the ADE genes in which DNA-bound Bas1 is inactive due to masking of its activation domain and Pho2 binds poorly to promoters when cells have sufficient purine nucleotides. Upon limitation for purines, the SAICAR/AICAR regulatory signal is transmitted to the nucleus to increase Bas1 and Pho2 interaction, recruiting Pho2 to promoters and freeing the activation domains for transactivation.

Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae

“…Further analyses using Bas1 and Pho2 fused to the transcription activation domain of VP16 suggested that Bas1 binds to the ADE gene promoters and recruits Pho2 (Pinson et al 2000). This was further demonstrated by chromatin immunoprecipitation (ChIP) analysis (Som et al 2005).…”

Eaf1 Links the NuA4 Histone Acetyltransferase Complex to Htz1 Incorporation and Regulation of Purine Biosynthesis

“…Previous studies have shown that Bas1 is constantly present at ADE promoters in an inactivated state. Upon induction, Bas1 undergoes a conformation change and recruits Pho2 (41). Interestingly, previous works also show that Pho2 directly interacts with NuA4 in vitro and in vivo, a step considered crucial to preset the promoter of the PHO5 gene for induction (26).…”

“…In an adenine-sufficient environment, the transcription of ADE genes is basal/repressed. The deprivation of available adenine will trigger the expression of a series of ADE genes in order to adapt to the environment (39)(40)(41). The fact that YPD media are poor in adenine allowed us to detect the implication of NuA4 in the regulation of ADE gene expression in the microarray experiment.…”

Cooperative Regulation of ADE3 Transcription by Gcn4p and Bas1p in Saccharomyces cerevisiae

“…Bas1p is required for the activation of adenylate biosynthetic genes under conditions of adenine depletion and in an SAGA-and SWI/SNF-dependent manner (18,33). In addition, Bas1p activates not only adenylate biosynthesis but also the one-carbon metabolic regulon under glycine-replete conditions (39).…”

“…Purine biosynthesis, one of the major metabolic pathways influenced by one-carbon metabolism, is executed by many ADE genes (4). It has been known that the expression of the one-carbon regulon and ADE genes were regulated mostly in a cooperative manner at the transcriptional level (18,24,33). Gcn4p and Bas1p are well-known transcription factors mediating this regulation (26,39).…”