Combined global localization analysis and transcriptome data identify genes that are directly coregulated by Adr1 and Cat8

Abstract

In Saccharomyces cerevisiae, glucose depletion causes a profound alteration in metabolism, mediated in part by global transcriptional changes. Many of the transcription factors that regulate these changes act combinatorially. We have analyzed combinatorial regulation by Adr1 and Cat8, two transcription factors that act during glucose depletion, by combining genome-wide expression and genome-wide binding data. We identified 32 genes that are directly activated by Adr1, 28 genes that are directly activated by Cat8, and 14 genes that are directly regulated by both. Our analysis also uncovered promoters that Adr1 binds but does not regulate and promoters that are indirectly regulated by Cat8, stressing the advantage of combining global expression and global localization analysis to find directly regulated targets. At most of the coregulated promoters, the in vivo binding of one factor is independent of the other, but Adr1 is required for optimal Cat8 binding at two promoters with a poor match to the Cat8 binding consensus. In addition, Cat8 is required for Adr1 binding at promoters where Adr1 is not required for transcription. These data provide a comprehensive analysis of the direct, indirect, and combinatorial requirements for these two global transcription factors.

FIG. 1.

Cat8 binds in vivo to positively regulated promoters. (A) Promoter maps, generated as described in reference , showing CSREs and Adr1-binding sites for seven promoters. All promoter maps show 700 bp upstream of the ATG except for HAP4, which shows 800 to 100 bp upstream. Dotted lines indicate the region amplified by PCR in panel B. (B) PCR using primers to the indicated promoters was performed on Cat8-bound ChIP DNA from logarithmically growing wild-type cells transferred to 0.05% glucose for 6 h. See Fig. 5 for additional ChIP data on Cat8.

FIG. 2.

Adr1 and Cat8 cooccupy coregulated promoters. Sequential ChIP was performed on cells with myc-tagged Adr1 and hemagglutinin-tagged Cat8 and derepressed for 6 h. A/C, sample was precipitated for bound Adr1 and then bound Cat8; C/A, sample was precipitated for bound Cat8 and then bound Adr1. PCR using primers to the indicated promoters was performed on input (whole-cell extract) and ChIP samples. As positive controls, each factor was subjected to sequential precipitations with the same antibody (lanes C/C and A/A are Cat8 and Adr1 precipitations, respectively). As negative controls, each factor was subjected to a single precipitation followed by a mock sequential precipitation using protein A-Sepharose only (lanes C/- and A/-).

FIG. 3.

Additive and synergistic interactions between Adr1 and Cat8. β-Gal activity was measured in derepressed strains bearing the indicated reporters on plasmids. The ADH2 plasmid is CEN, and the ACS1 plasmid is 2μm. Data for ACS1 from reference are used with permission. White bars are the activity from Δadr1, gray bars are from Δcat8, striped bars are the expected activity if Cat8 and Adr1 activation were additive, and black bars are the actual activity measured in CAT8 ADR1 wild type.

FIG. 4.

Adr1 binding alone assists Cat8 activation of ADH2. In-gel assays for ADH activity are shown for strains that are Δadr1CAT8 (TYY497) and Δadr1 Δcat8 (TYY495) with one of the following plasmids: empty vector pRS314 (-) (42); pJS21, which expresses the first 172 amino acids of Adr1, containing the entire DNA-binding domain but lacking the major activation domain (BD) (43); or pJS20, which expresses wild-type full-length Adr1 (ADR1) (43). Activity levels in Miller units from an integrated β-Gal reporter (16) in the same strains are shown below. In-gel activity assays were done with extracts prepared after 6 h of derepression in 0.05% glucose; reporter assays were done with cells derepressed for 24 h. The upper ADHI band from constitutively expressed ADH 1 served as a loading control for the in-gel activity assay.

FIG. 5.

Cat8 binding is affected by Δadr1 at some promoters. (A) Promoter maps for Adr1-bound and Cat8-bound and regulated promoters showing CSREs and Adr1-binding sites. Lines under the promoters indicate the region amplified by PCR in panel B. (B) PCR using primers to the indicated promoters was performed on Cat8-bound ChIP DNA from wild-type or isogenic Δadr1 cells after 6 h in 0.05% glucose.

FIG. 6.

Adr1 binding is affected by Δcat8 at some promoters. (A) Promoter maps for three Adr1- and Cat8-coregulated promoters (ACS1, ADH2, and ATO3) and three promoters that are bound but not positively regulated by Adr1 (FBP1, MDH2, and MLS1). Lines under the promoters indicate the region amplified by PCR in panel B. (B) PCR using primers to the indicated promoters was performed on Adr1-bound ChIP DNA from wild-type or isogenic Δcat8 cells after 6 h in 0.05% glucose.