Inducible Yeast Two-Hybrid with Quantitative Measures

The yeast two-hybrid (Y2H) assay has long been used to identify new protein-protein interaction pairs and to compare relative interaction strengths. Traditional Y2H formats may be limited, however, by use of constitutive strong promoters if expressed proteins have toxic effects or post-transcriptional expression differences in yeast among a comparison group. As a step toward more quantitative Y2H assays, we modified a common vector to use an inducible CUP1 promoter, which showed quantitative induction of several "bait" proteins with increasing copper concentration. Using mouse Nxf1 (homologous to yeast Mex67p) as a model bait, copper titration achieved levels that bracket levels obtained with the constitutive ADH1 promoter. Using a liquid growth assay for an auxotrophic reporter in multiwell plates allowed log-phase growth rate to be used as a measure of interaction strength. These data demonstrate the potential for quantitative comparisons of protein-protein interactions using the Y2H system.

Co-dependent recruitment of Ino80p and Snf2p is required for yeast CUP1 activation

In yeast, Ace1p-dependent induction of CUP1 is responsible for protecting cells from copper toxicity. Although the mechanism of yeast CUP1 induction has been studied intensively, it is still uncertain which chromatin remodelers are involved in CUP1 transcriptional activation. Here, we show that yeast cells are inviable in the presence of copper when either chromatin remodeler, Ino80p or Snf2p, is not present. This inviability is due to the lack of CUP1 expression in ino80Δ and snf2Δ cells. Subsequently, we observe that both Ino80p and Snf2p are present at the promoter and they are responsible for recruiting chromatin remodeling activity to the CUP1 promoter under induced conditions. These results suggest that they directly participate in CUP1 transcriptional activation. Furthermore, the codependent recruitment of both INO80 and SWI/SNF depends on the presence of the transcriptional activator, Ace1p. We also demonstrate that both remodelers are required to recruit RNA polymerase II and targeted histone acetylation, indicating that remodelers are recruited to the CUP1 promoter before RNA polymerase II and histone acetylases. These observations provide evidence for the mechanism of CUP1 induction. As such, we propose a model that describes novel insight into the order of events in CUP1 activation.

Targeted Histone Acetylation at the Yeast CUP1 Promoter Requires the Transcriptional Activator, the TATA Boxes, and the Putative Histone Acetylase Encoded by SPT10

ABSTRACT The relationship between chromatin remodeling and histone acetylation at the yeast CUP1 gene was addressed. CUP1 encodes a metallothionein required for cell growth at high copper concentrations. Induction of CUP1 with copper resulted in targeted acetylation of both H3 and H4 at the CUP1 promoter. Nucleosomes containing upstream activating sequences and sequences farther upstream were the targets for H3 acetylation. Targeted acetylation of H3 and H4 required the transcriptional activator (Ace1p) and the TATA boxes, suggesting that targeted acetylation occurs when TATA-binding protein binds to the TATA box or at a later stage in initiation. We have shown previously that induction results in nucleosome repositioning over the entire CUP1 gene, which requires Ace1p but not the TATA boxes. Therefore, the movement of nucleosomes occurring on CUP1 induction is independent of targeted acetylation. Targeted acetylation of both H3 and H4 also required the product of the SPT10 gene, which encodes a putative histone acetylase implicated in regulation at core promoters. Disruption of SPT10 was lethal at high copper concentrations and correlated with slower induction and reduced maximum levels of CUP1 mRNA. These observations constitute evidence for a novel mechanism of chromatin activation at CUP1, with a major role for the TATA box.

Template topology and transcription: chromatin templates relaxed by localized linearization are transcriptionally active in yeast.

To address the role of transient torsional stress in transcription, we have utilized the regulated expression of HO endonuclease in yeast to create double-strand breaks in DNA templates in vivo at preselected sites. Linearization of circular minichromosomes, either 2 kb upstream or immediately downstream of a lacZ reporter gene controlled by the yeast metallothionein gene (CUP1) promoter, did not alter the copper induction profile of lacZ RNA transcripts compared to that of nonlinearized controls. Constructs site-specifically integrated into yeast chromosome II gave similar results. In vivo cross-linking with psoralen as a probe for negative DNA supercoiling demonstrated that template linearization efficiently dissipated DNA supercoiling induced by transcription. Therefore, the efficient transcription of linearized, relaxed templates found here demonstrates that transient torsional tension is not required for transcription of chromatin templates in yeast.