The transcriptional coactivators SAGA, SWI/SNF, and mediator make distinct contributions to activation of glucose-repressed genes

Abstract

The paradigm of activation via ordered recruitment has evolved into a complicated picture as the influence of coactivators and chromatin structures on gene regulation becomes understood. We present here a comprehensive study of many elements of activation of ADH2 and FBP1, two glucose-regulated genes. We identify SWI/SNF as the major chromatin-remodeling complex at these genes, whereas SAGA (Spt-Ada-Gcn5-acetyltransferase complex) is required for stable recruitment of other coactivators. Mediator plays a crucial role in expression of both genes but does not affect chromatin remodeling. We found that Adr1 bound unaided by coactivators to ADH2, but Cat8 binding depended on coactivators at FBP1. Taken together, our results suggest that commonly regulated genes share many aspects of activation, but that gene-specific regulators or elements of promoter architecture may account for small differences in the mechanism of activation. Finally, we found that activator overexpression can compensate for the loss of SWI/SNF but not for the loss of SAGA.

FIGURE 1.

Adr1 and Cat8 show differential dependences on coactivators for stable binding. A, ChIP for Adr1 at ADH2 in coactivator mutants (RBY3, RBY110, RBY111, RBY117, and RBY119). Values, based on QPCR, were first normalized to a negative control locus (TEL) and to an input sample, and then expressed as the -fold over the WT repressed (R) value. B, ChIP for Cat8 at FBP1 in coactivator mutants (CTYTY18, RBY126, RBY127, and RBY123). Values as in A. C, mRNA analysis of CAT8 in WT (W303a) and coactivator mutants (LLTY72, LLTY73, KKTY3, and RBY93). Values, based on QPCR, were first normalized to ACT1 and then expressed as a percent of the maximum WT Derepressed (D) value. D, ChIP for Cat8 with different cross-linkers was performed as described under “Experimental Procedures” with either dimethyl adipimate (DMA) plus formaldehyde (FMA), just FMA, or EGS plus FMA at FBP1 in med17 ts (RBY123) and snf5Δ (RBY127). ChIP/Input values (obtained as in A) were expressed as the percent of ChIP/Input value of the WT strain (RBY135). E, mRNA analysis of FBP1 in WT (W303a), cat8Δ (RBY19), gcn5Δ (EAY12), gcn5Δcat8Δ (EAY16), snf2Δ (KKTY3), and snf2Δcat8Δ (RBY160). Values, based on QPCR, were first normalized to ACT1, and then expressed as the -fold over repressed (R).

FIGURE 2.

SAGA, SWI/SNF, and Mediator are required for full expression of ADH2 and FBP1. mRNA analysis throughout 6 h of derepression of (A) ADH2 and (B) FBP1 from gcn5Δ (LLTY72), ada1Δ (LLTY73), snf2Δ (KKTY3), and med17 ts (RBY93). Values, based on QPCR, were normalized to ACT1 and then expressed as the percent of the wild-type (W303a) level after 4-h derepression. Error bars represent the standard deviation of two biological replicates.

FIGURE 3.

Coactivators binding is interdependent at ADH2 and FBP1. ChIP for coactivators in gcn5Δ (RBY110, RBY115, RBY116, and RBY155) (A); ada1Δ (RBY106, RBY108, RBY111, RBY113, and RBY154) (B); snf2Δ (RBY109, RBY112, RBY117) (C); and med17 ts (RBY119, RBY120, RBY121) at ADH2 (light bars) and FBP1 (black bars) (D). Data for coactivator occupancy in a WT strain was obtained using RBY3, RBY5, RBY8, RBY9, and RBY11. Values are expressed as the -fold over the WT repressed (R) value, as in Fig. 1A (see “Experimental Procedures” for details). WT data is shown for 4-h derepression (no error bar) or an average of 4- and 6-h derepression (with error bars representing the standard deviation) and data for all mutant strains is based on the average for 4- and 6-h derepression. Error bars of the mutant values indicate the standard deviation of at least three independent biological replicates.

FIGURE 4.

Coactivators differentially contribute to chromatin remodeling at the ADH2 promoter. A, supercoiling analysis on strains containing a plasmid with the ADH2 promoter was performed as described under “Experimental Procedures.” Samples from WT (W303a) stain containing the WT plasmid, pLLTY1 or from WT strain containing a plasmid in which the ADH2 TATAA box was mutated to GAGAA, pLLTY3, were repressed (R) or 4-h derepressed (DR). The locations of nicked DNA and Band 1 are indicated. Quantitation of supercoiling assays in WT (W303a) (B), gcn5Δ (LLTY72) (C), ada1Δ (LLTY73) (D), snf2Δ (KKTY3) (E), and med15Δ (LLTY120) (F), all carrying pLLTY1. Samples were from cells either repressed (dashed), or derepressed for 2.5 h (gray) or 5 h (black) and subjected to supercoiling analysis. The topoisomer distributions were quantified using phosphorimaging. The data are presented as relative intensity for each of eight bands. Error bars represent the standard deviation of three independent experiments. A shift to the right represents a decrease in overall nucleosome density.

FIGURE 5.

Chromatin remodeling at the FBP1 promoter requires SWI/SNF, but not SAGA. A, schematic of the nucleosome positions in the FBP1 promoter in a WT strain under repressed (R) and derepressed (DR) conditions (12). The star indicates the location of the FBP1 TATAA box. NuSA was used to determine the nucleosome positioning at the FBP1 promoter in wild type (W303a) (B), ada1Δ (LLTY73) (C), and snf2Δ (KKTY3) (D). Cells were either repressed (gray lines) or derepressed for 4 h (black lines). Each point shows the relative protection from micrococcal nuclease digestion for each amplicon.

FIGURE 6.

Recruitment of coactivators is nearly simultaneous. Occupancy of transcription factors and coactivators in a wild-type strain (RBY143) at different times of derepression at ADH2 (A) and FBP1 (B). Values, based on QPCR, were normalized to the highest value for each protein over the time course.