The heat shock response is among the most highly conserved genetic systems known; it is essential for all life, prokaryotic and eukaryotic. It represents the principal means by which cells endure physiologic stress, be it thermal, chemical, or anoxic, and thus likely plays a key role in cell surviving during fever, ethanol toxicity, and ischemia. Almost without exception, the response is coordinately regulated at the level of transcription. We wish to understand the molecular basis of this transcriptional regulation: what activates heat shock genes under non-inducing (basal) conditions, what causes their induction in response to environmental stress, and what limits their response during periods of continuous stress. To address these questions, we propose to use the HSP90 gene family of Saccharomyces cerevisiae as a model system. This family consists of two members which differ strikingly in their regulation but which encode a functionally indistinguishable gene product. HSP82 is expressed at a low basal level which is enhanced 10-to-20-fold by heat shock; HSC82 is expressed at a 10-fold higher constitutive level than is HSP82, but is induced only 2-fold further by stress. The specific questions we propose to address are the following. First, which cis- regulatory elements activate HSP82? We wish to perform a systematic 5'-deletion analysis of the gene's promoter region and, employing oligonucleotide-directed mutagenesis and gene transplacement techniques, introduce site-specific mutations into two sequence elements--the TATA box and the promoter-proximal heat shock element, HSE1--our prior work has shown are intimately engaged in protein/DNA interactions in vivo. Functional consequences will be assessed by Northern blot analysis; structural consequences by chromatin footprinting using chemical and enzymatic probes at both nucleosome- and nucleotide-resolution. Second, which cis-regulatory elements are responsible for the 10-fold higher basal level of expression of HSC82 vs. HSP82? To address this question, we propose to perform a complementary mutagenesis and footprinting analysis of the HSC82 promoter. Third, which trans-acting proteins activate these heat shock genes, and which are responsible for limiting the response during periods of chronic stress? Using antibodies to yeast heat shock factor (HSF), yeast TATA-binding factor, TFIID, the largest subunit of yeast RNA polymerase II, and yeast hsp70 (ssa1p), we propose to immunoprecipitate covalently crosslinked protein/DNA complexes purified from heat shocked and control cells, and identify by blot- hybridization those DNA sequences in intimate contact with each protein in vivo.
