Molecular Genetic Analysis of the Yeast Repressor Rfx1/Crt1 Reveals a Novel Two-Step Regulatory Mechanism

Abstract

In Saccharomyces cerevisiae, the repressor Crt1 and the global corepressor Ssn6-Tup1 repress the DNA damage-inducible ribonucleotide reductase (RNR) genes. Initiation of DNA damage signals causes the release of Crt1 and Ssn6-Tup1 from the promoter, coactivator recruitment, and derepression of transcription, indicating that Crt1 plays a crucial role in the switch between gene repression and activation. Here we have mapped the functional domains of Crt1 and identified two independent repression domains and a region required for gene activation. The N terminus of Crt1 is the major repression domain, it directly binds to the Ssn6-Tup1 complex, and its repression activities are dependent upon Ssn6-Tup1 and histone deacetylases (HDACs). In addition, we identified a C-terminal repression domain, which is independent of Ssn6-Tup1 and HDACs and functions at native genes in vivo. Furthermore, we show that TFIID and SWI/SNF bind to a region within the N terminus of Crt1, overlapping with but distinct from the Ssn6-Tup1 binding and repression domain, suggesting that Crt1 may have activator functions. Crt1 mutants were constructed to dissect its activator and repressor functions. All of the mutants were competent for repression of the DNA damage-inducible genes, but a majority were "derepression-defective" mutants. Further characterization of these mutants indicated that they are capable of receiving DNA damage signals and releasing the Ssn6-Tup1 complex from the promoter but are selectively impaired for TFIID and SWI/SNF recruitment. These results imply a two-step activation model of the DNA damage-inducible genes and that Crt1 functions as a signal-dependent dual-transcription activator and repressor that acts in a transient manner.

The Cyc8–Tup1 complex inhibits transcription primarily by masking the activation domain of the recruiting protein

“…Importantly, these events occur long before redeposition of nucleosomes to promoters, indicating that Tup1 is able to block transcription before any repressive chromatin structure is established; such repression in the absence of nucleosomes has been previously demonstrated (Bryant et al 2008). Previous studies (Redd et al 1996;Papamichos-Chronakis et al 2002Proft and Struhl 2002;Zhang and Reese 2005) and our analysis of Gcn4 binding in the ''mechanistic strain'' indicate that Tup1 does not inhibit activator binding. As recruitment of all three coactivators is mediated by direct and independent interactions with activation domains of DNA-bound activator proteins, the kinetic profile of molecular events at target promoters indicates that Tup1 directly blocks activator-mediated recruitment of these coactivators.…”

“…In principle, Cyc8-Tup1 could block activator-mediated recruitment of coactivator complexes or block binding of the activator to target promoters, although previous studies suggest that activator binding is not inhibited (Redd et al 1996;Papamichos-Chronakis et al 2002Proft and Struhl 2002;Zhang and Reese 2005). As an independent approach to address the effect of Cyc8-Tup1 on activator binding, we generated a ''mechanistic'' (A) Pol II association with the indicated coding regions in the Tup1-anchor-away or Tup1/TBP-anchor-away strains that were or were not treated with rapamycin for 1 h. (B) H3 occupancy with the indicated promoters in the Tup1-anchor-away or Tup1/ TBP-anchor-away strains that were or were not treated with rapamycin for 1 h. (C) H3 occupancy with the indicated promoters in the Tup1-anchor-away and Tup1/Snf2-anchor-away strains that were or were not treated with rapamycin for 1 h. Averages and standard errors of three individual experiments are shown.…”

“…Interestingly, in response to osmotic or carbon source stress, Cyc8-Tup1 does not dissociate from target promoters, and in fact it contributes to recruitment of the Swi/Snf and SAGA coactivator complexes (Papamichos-Chronakis et al 2002;Proft and Struhl 2002;Mennella et al 2003). In these and other cases (Fragiadakis et al 2004;Zhang and Reese 2005;Hickman and Winston 2007), the DNA-binding repressor also appears to function as an activator protein under appropriate stress conditions.…”

Ccr4 contributes to tolerance of replication stress through control ofCRT1mRNA poly(A) tail length

“…To address possible countervailing effects of elongated poly(A) tails and concomitantly decreased CRT1 mRNA in the ccr4⌬ strain, Crt1 protein levels were assessed with a polyclonal antibody to the N-terminus of Crt1 (Zhang and Reese, 2005). In contrast to the observed drop in CRT1 mRNA, Crt1 protein was not decreased during vegetative growth in a ccr4⌬ strain when compared with wild-type controls (Fig.…”

“…Extracts were resolved by 8% SDS-PAGE and transferred to nitrocellulose, followed by blocking with 5% skimmed milk in TBS-0.05% Tween (TBST). Crt1 protein levels were detected with a rabbit polyclonal antibody raised against the N-terminus of Crt1, provided by Joseph Reese (Zhang and Reese, 2005), and donkey anti-rabbit IgG-HRP secondary antibody. Control Pgk1 protein levels were detected with mouse monoclonal anti-yeast Pgk1 IgG (Molecular Probes) and sheep anti-mouse IgG-HRP.…”