PMC

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.

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.

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.

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/-).

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. for additional ChIP data on Cat8.

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 (-) (); pJS21, which expresses the first 172 amino acids of Adr1, containing the entire DNA-binding domain but lacking the major activation domain (BD) (); or pJS20, which expresses wild-type full-length Adr1 (ADR1) (). Activity levels in Miller units from an integrated β-Gal reporter () 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.