Essential transcription factor-DNA interactions for the transcriptional autoregulation of the Candida glabrata AMT1 gene.

Abstract

The interaction of DNA binding transcription factors with their cognate target DNA elements is a decisive factor in the modulation of target gene expression. This study investigates the role of Amt1p-DNA interactions in vitro and in vivo in fostering copper (Cu) ion resistance to the yeast Candida glabrata. One of the primary mechanisms for preventing the cytotoxic accumulation of Cu within the eukaryotic cell is the rapid biosynthesis of metallothioneins, small metal binding proteins. In C. glabrata, transcriptional activation of the metallothionein gene family is performed by the Amt1p Cu-metalloregulatory transcription factor. Importantly, the essential primary step in this cascade of Cu-dependent gene activation is the rapid and robust transcriptional autoactivation of the AMT1 gene. Purified recombinant Amt1p was used for in vitro protein-DNA interaction studies with an AMT1 promoter DNA fragment containing the single metal response element (MRE). The results of these studies demonstrate that monomeric Amt1p interacts with the MRE with 0.2-0.3 nM affinity, utilizing adjacent major and minor groove interactions and critical phosphate backbone contacts. The Amt1p DNA binding domain was found to have a potential glycine-arginine-proline DNA minor groove interacting domain, which when mutated severely compromises DNA binding in vitro and completely abolished Cu resistance to C. glabrata in vivo. Previous experiments in our laboratory established a role for the upstream A16 element in the AMT1 promoter in providing essential nucleosomal access to Amt1p in vivo. Studies are presented that address the length and positional requirements for the A16 element on the AMT1 promoter in C. glabrata, and the A16 element was shown to facilitate nucleosomal access to the activated mammalian glucocorticoid receptor in S. cerevisiae. Importantly, chromatin alterations were found to be necessary for establishing the A16 element structure, as is evident by the requirement for the yeast SWI/SNF nucleosome remodeling complex and the Gcn5p histone acetyltransferase for Amt1p access in vivo. These studies provide insight into the mode of DNA binding for a unique class of Cu-dependent DNA binding domains, and evidence for the potential universality of homopolymeric (dA:dT) elements in providing nucleosomal access to transcription factors in all eukaryotic organisms.