Interaction of a transcriptional repressor with the RNA polymerase II holoenzyme plays a crucial role in repression

Abstract

The yeast transcriptional repressor Tup1, tethered to DNA, represses to strikingly different degrees transcription elicited by members of two classes of activators. Repression in both cases is virtually eliminated by mutation of either member of the cyclin-kinase pair Srb10/11. In contrast, telomeric chromatin affects both classes of activators equally, and in neither case is that repression affected by mutation of Srb10/11. In vitro, Tup1 interacts with RNA polymerase II holoenzyme bearing Srb10 as well as with the separated Srb10. These and other findings indicate that at least one aspect of Tup1's action involves interaction with the RNA polymerase II holoenzyme.

Figure 1

Tup1 represses transcription mediated by nonclassical activators particularly efficiently. (A) The reporter used in the repression assays contains five Gal4 DNA-binding sites upstream of two lexA sites which are, in turn, positioned 50 bp upstream from the natural GAL1 TATA box. (B and C) Cells harboring the reporter gene were cotransformed with plasmids expressing Gal4-Tup1 and a variety of LexA-fusion activators. In each case, LexA-fusion activators consisted of full-length protein with lexA replacing the respective DNA-binding domain where applicable. Gal4-Tup1 consisted of Gal4 (residues 1–100) fused to full-length Tup1. Cells were assayed for GAL1-lacZ expression by β-galactosidase activity (B). The numbers represent the average of assays on three independent colonies performed in duplicate. The standard errors were typically 10–15%. The fold repression, shown in C, was calculated as the ratio of values obtained with and without the repressor function of Tup1.

Figure 2

Classical and nonclassical activators equally overcome telomeric repression, and that repression does not require Srb10. Yeast strains harboring a URA3-based reporter gene located at a telomeric (A) or internal (B) chromosomal locus were assayed for URA3 expression in the context of coexpressed classical (Gal4) and nonclassical (Gal11) activator molecules. Serial dilution spot assays on cell viability were performed by using plates with (+FOA) or without (−FOA) FOA. Decreased viability on +FOA plates indicates URA3 gene expression, whereas growth indicates URA3 repression by telomere position effect. (A) Promoter bound Gal4 and Gal11 can overcome telomeric repression equally well in both wild-type and ΔSrb10 or ΔSrb11 cells. The URA3 reporter contains binding sites for Gal4. Activators were tethered to the promoter via fusions to the DBD of Gal4 (residues 1–100). (B) When placed at an internal chromosomal locus, the URA3 reporter gene is expressed at sufficient levels even in the absence of an activator to render cells sensitive to FOA.

Figure 3

Tup1 interacts with the holoenzyme, at least in part, through its interactions with Srb10. (A) Tup1 immunoprecipitates with the holoenzyme found in wild-type extracts but much less so with holoenzyme found in extracts from a strain deleted for Srb10. Purified Tup1 was incubated with equivalent amounts of nuclear extracts of either the wild-type strain or an isogenic strain lacking Srb10 (Input). Each extract contained equivalent levels of Rpb1 (largest subunit of the RNA polymerase II), Srb5 (an integral component of the holoenzyme), and Tbp (the TATA binding protein which does not stably associate with the RNA pol II holoenzyme). Holoenzyme and proteins bound to it were precipitated with affinity-purified antibodies against-Srb5 (Pellet). As expected, Tbp did not immunoprecipitate well, whereas both Srb5 and Rpb1 did immunoprecipitate efficiently. (B) Tup1 interacts with Srb10/11 complex. Purified Tup1 was incubated with purified Srb10/11 complex (Input) and then immunoprecipitated by using affinity-purified anti-Srb10 antibodies (Pellet). Srb10–3 has a point mutation in the kinase domain that eliminates the catalytic function of the enzyme without altering its ability to interact with its cyclin partner or the holoenzyme. (C) Tup1 interacts with Srb10. Purified, flag epitope-tagged Tup1 was incubated with four components of the repression subcomplex of the holoenzyme. Anti-flag monoclonal antibody was used to immunoprecipitate the tagged Tup1 incubated individually with insect-cell extracts containing overexpressed Srb8, 9, 10, and 11. The input (I), the supernatant (S), the wash (W), and the precipitated (P) levels of each protein in the reaction are shown. Ovalbumin was used as a control for nonspecific aggregation. In lane P of the Srb11 subpanel, the faster migrating band is not a degradation product of Srb11 but a chain of the flag monoclonal antibody that is detected by the secondary antibody used in the immunoblot.

Figure 4

Tup1 repression occludes the holoenzyme and Tbp from the promoter. (A) Tup1 tightly regulates mating-specific gene expression. To determine the relative expression levels of a subset of mating type-specific genes we performed quantitative reverse transcription–PCR analysis on RNA isolated from the isogenic cells of opposite mating types. A constant amount of chromosomal DNA was used in the analysis of each gene to control for PCR efficiency of each primer pair. Actin gene expression was monitored to control for total RNA recovery. (B) Tup1 recruited to α2 repressed genes occludes the holoenzyme and Tbp from a-specific gene promoters. Chromosomal immunoprecipitation assays were performed on yeast cells of opposite mating type. Antibodies specific for PolII (anti-ctd), Gal11 (anti-Gal11) and Tbp (anti-Tbp) were used to immunoprecipitate in vivo formaldehyde cross-linked chromatin. Promoter DNA coupled to complexes containing PolII, Gal11, and Tbp were analyzed by quantitative PCR studies. Primer pairs encompassed the core promoter region of each gene. The histograms indicate DNA immunoprecipitated relative to the Actin promoter region for each gene. Plotted values correspond to the mean values obtained with three independent experiments. SEM = 15–25%.