Long-Range Interactions at theHOPromoter

Abstract

The SWI5 gene encodes a zinc finger DNA-binding protein required for the transcriptional activation of the yeast HO gene. There are two Swi5p binding sites in the HO promoter, site A at ؊1800 and site B at ؊1300. Swi5p binding at site B has been investigated in some detail, and we have shown that Swi5p binds site B in a mutually cooperative fashion with Pho2p, a homeodomain protein. In this report, we demonstrate that Swi5p and Pho2p bind cooperatively to both sites A and B but that there are differences in binding to these two promoter sites. It has been shown previously that point mutations in either Swi5p binding site only modestly reduce HO expression in a PHO2 strain. We show that these mutant promoters are completely inactive in a pho2 mutant. We have created stronger point mutations at the two Swi5p binding sites within the HO promoter, and we show that the two binding sites, separated by 500 bp, are both absolutely required for HO expression, independent of PHO2. These results create an apparent dilemma, as the strong mutations at the Swi5p binding sites show that both binding sites are required for HO expression, but the earlier binding site mutations allow Swi5p to activate HO, but only in the presence of Pho2p. To explain these results, a model is proposed in which physical interaction between Swi5p proteins bound to these two sites separated by 500 bp is required for activation of the HO promoter. Experimental evidence is presented that supports the model. In addition, through deletion analysis we have identified a region near the amino terminus of Swi5p that is required for PHO2-independent activation of HO, suggesting that this region mediates the long-range interactions between Swi5p molecules bound at the distant sites.The Saccharomyces cerevisiae HO gene encodes an endonuclease which is responsible for initiating mating type switching in yeast (for reviews, see references 8 and 20). When yeast cells divide, they do so asymmetrically, producing a large mother cell and a small daughter cell. Only the mother cell is able to switch its mating type, and the ability to switch mating type requires transcription of the HO gene. Daughter cells do not express HO and are therefore not capable of switching their mating type. The SWI5 gene was identified originally as a positive transcriptional regulator of HO transcription, and SWI5 has been implicated in the asymmetric expression of HO (19,21,27). Recent work has shown that the ASH1 gene product is required to repress HO transcription in daughter cells (2,25).SWI5 encodes a 709-amino-acid protein which contains three zinc finger DNA-binding domains near its carboxy terminus (26). Swi5p binds DNA in vitro via these zinc finger motifs to a site in the HO promoter located approximately 1,300 nucleotides (nt) upstream of the HO ATG start codon (16,26). Recently, a second Swi5p binding site within the HO promoter has been identified that is also required for proper transcriptional regulation of HO in vivo (27).The new Swi5p binding site is located approximately...

Cooperative Pho2-Pho4 Interactions at the PHO5 Promoter Are Critical for Binding of Pho4 to UASp1 and for Efficient Transactivation by Pho4 at UASp2

“…At the HO promoter, a Pho2 binding site is located next to a Swi5 binding site, and it was shown that the two proteins bind to their sites cooperatively in vitro (7). However, recent in vivo data indicate that the role of Pho2 in HO regulation is complex (18). In the case of the HIS4 promoter, a Pho2 (Bas2)-protected region largely overlaps the Bas1 footprint.…”

“…In the framework of this model, the deletion of amino acids 200 to 247 in our experiments, which results in the in-VOL. 18,1998 Pho2-Pho4 INTERACTIONS AT THE PHO5 PROMOTER 2637 on May 9, 2018 by guest http://mcb.asm.org/ ability of Pho4 to interact with Pho2, would destroy the internal interaction in the Pho4 protein, thus exposing the activation domain and in effect bypassing the Pho2 requirement. A second role for Pho2 can also explain our finding that the loss of promoter activity due to mutation of Pho2 cis elements can be almost fully compensated for by overexpression of Pho4 in a PHO2 strain, while the same is not true for the loss of promoter activity due to elimination of Pho2 itself.…”

“…Together with Swi5, it binds cooperatively to a regulatory element in the HO promoter (7) and plays a complex role in its regulation (18). Also named Bas2, Pho2 is involved in the regulation of HIS4 (3), TRP4 (6), and certain ADE genes (8).…”

Transcriptional Regulation inSaccharomyces cerevisiae: Transcription Factor Regulation and Function, Mechanisms of Initiation, and Roles of Activators and Coactivators

“…The complex dynamics of gene expression during the cell cycle and after subjecting cells to stress have been assayed by ChIP analysis of the regulatory TFs and coactivators (McBride et al 1997;Bhoite et al 2001;Simon et al 2001;Horak et al 2002;Tan et al 2008;Ni et al 2009). Similarly, during sporulation (Kahana et al 2010) and glucose starvation (Young et al 2003;Tachibana et al 2005;Ratnakumar and Young 2010) multiple TFs bind upstream of the promoter of coregulated genes.…”

Residues in the Swi5 Zinc Finger Protein That Mediate Cooperative DNA Binding with the Pho2 Homeodomain Protein

“…Swi5 recognizes two sites in the HO promoter, called site A and site B, located approximately 1,800 and 1,300 nucleotides, respectively, upstream from the transcription start site (24,37). Swi5 binds to both of these sites with relatively low affinity, and binding by Pho2 is extremely weak.…”