Cooperative Binding of Heat Shock Factor to the Yeast HSP82 Promoter In Vivo and In Vitro

The ability to respond rapidly to a large constellation of environmental stresses is crucial to the survival of all organisms, from bacteria to humans (32). Almost without exception, this response is regulated at the transcriptional level. In eukaryotes, environmental stress is sensed, directly or indirectly, by a sequence-specific transcriptional activator termed heat shock factor (HSF) (31, 54). In insects and vertebrates, HSF exists in a non-DNA binding, monomeric form predominantly localized within the cytoplasm (37, 53). Upon heat shock (or other stress), HSF rapidly trimerizes via arrays of amphipathic α-helical residues in the N-terminal domain, translocates into the nucleus, and binds to its cognate promoter elements within chromatin (50). In all metazoan heat shock genes which have been examined, the promoter regions are maintained in a transcriptionally poised, nucleosome-free state by other sequence-specific regulators, which create an environment conducive for rapid, inducible HSF binding (24). Thus, a specific architecture needs to be established within the upstream regulatory regions of stress-responsive genes; HSF is in fact incapable of binding to the Drosophila hsp70 promoter in the absence of GAGA, TATA, or initiator elements (38). In contrast, heat shock promoters of the budding yeast Saccharomyces cerevisiae appear to be maintained in a transcriptionally poised state by HSF itself. At HSC82, a 4-bp substitution within the high-affinity heat shock element (HSE) abolishes promoter-associated DNase I hypersensitivity and restriction enzyme accessibility, despite the continuous presence of a second activator, GRF2/REB1, bound adjacent to the mutated HSE (6). Similarly, deletion or substitution of the high-affinity HSE at HSP82 (creating hsp82 alleles termed ΔHSE1 and ΔHSE1·, respectively) reduces transcription ≥100-fold and results in a dramatic alteration of promoter chromatin structure: the nuclease-hypersensitive region is replaced by two stably positioned nucleosomes, one centered over the mutated HSE and the other centered over the TATA initiation site (16). However, a double-point mutation within HSE1 (termed P2), despite seriously weakening sequence-specific interactions within the HSP82 enhancer (reference 28 and this study), has no effect on promoter-associated DNase I hypersensitivity (21, 28) or on the pattern of micrococcal nuclease (MNase) cleavage (8, 12). Thus, the phenotype of the ΔHSE1 and ΔHSE1· alleles suggests an HSF-dependent mechanism for establishment of the nucleosome-free state, whereas the phenotype of hsp82-P2 argues for an HSF-independent mechanism. To clarify the role of HSF in regulating HSP82, we have used a combined mutagenesis and footprinting strategy. This approach has confirmed a central role for HSF in potentiating HSP82 promoter function and has led to the discovery that HSF binds cooperatively to the HSP82 upstream region, both in vivo and in vitro. These cooperative interactions, which are maintained even in the presence of a double-point mutation within HSE1, likely underlie the phenotypic difference between P2 and ΔHSE1.