Cooperative binding of heat shock factor to the yeast HSP82 promoter in vivo and in vitro

Previous work has shown that heat shock factor (HSF) plays a central role i n remodeling the chromatin structure of the yeast HSP82 promoter via consti tutive interactions with its high-affinity binding site, heat shock element 1 (HSE1), The HSF-HSE1 interaction is also critical for stimulating both b asal (noninduced) and induced transcription. By contrast, the function of t he adjacent, inducibly occupied HSE2 and -3 is unknown. In this study, we e xamined the consequences of mutations in HSE1, HSE2, and HSE3 on HSF bindin g and transactivation, We provide evidence that in vivo, HSF binds to these three sites cooperatively. This cooperativity is seen both before and afte r heat shock, is required for full inducibility, and can be recapitulated i n vitro on both linear and supercoiled templates. Quantitative in vitro foo tprinting reveals that occupancy of HSE2 and -3 by Saccharomyces cerevisiae HSF (ScHSF) is enhanced similar to 100-fold through cooperative interactio ns with the HSF-HSE1 complex, HSE1 point mutants, whose basal transcription is virtually abolished, are functionally compensated by cooperative intera ctions with HSE2 and -3 following heat shock, resulting in robust inducibil ity. Using a competition binding assay, we show that the affinity of recomb inant HSF for the full-length HSP82 promoter is reduced nearly an order of magnitude by a single-point mutation within HSE1, paralleling the effect of these mutations on noninduced transcript levels, We propose that the remod eled chromatin phenotype previously shown for HSE1 point mutants (and lost in HSE1 deletion mutants) stems from the retention of productive, cooperati ve interactions between HSF and its target binding sites.