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Molecular and Cellular Biology, March 1999, p. 1627-1639, Vol. 19, No. 3
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Alexander M. Erkine, Serena F. Magrogan, Edward A. Sekinger, and David S. Gross*

Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, Louisiana 71130

Received 20 August 1998/Returned for modification 12 October 1998/Accepted 11 November 1998

Previous work has shown that heat shock factor (HSF) plays a central role in remodeling the chromatin structure of the yeast HSP82 promoter via constitutive interactions with its high-affinity binding site, heat shock element 1 (HSE1). The HSF-HSE1 interaction is also critical for stimulating both basal (noninduced) and induced transcription. By contrast, the function of the adjacent, inducibly occupied HSE2 and -3 is unknown. In this study, we examined the consequences of mutations in HSE1, HSE2, and HSE3 on HSF binding and transactivation. We provide evidence that in vivo, HSF binds to these three sites cooperatively. This cooperativity is seen both before and after heat shock, is required for full inducibility, and can be recapitulated in vitro on both linear and supercoiled templates. Quantitative in vitro footprinting reveals that occupancy of HSE2 and -3 by Saccharomyces cerevisiae HSF (ScHSF) is enhanced ~100-fold through cooperative interactions with the HSF-HSE1 complex. HSE1 point mutants, whose basal transcription is virtually abolished, are functionally compensated by cooperative interactions with HSE2 and -3 following heat shock, resulting in robust inducibility. Using a competition binding assay, we show that the affinity of recombinant 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 remodeled chromatin phenotype previously shown for HSE1 point mutants (and lost in HSE1 deletion mutants) stems from the retention of productive, cooperative interactions between HSF and its target binding sites.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, LA 71130. Phone: (318) 675-5027. Fax: (318) 675-5180. E-mail: dgross{at}lsumc.edu.

Molecular and Cellular Biology, March 1999, p. 1627-1639, Vol. 19, No. 3
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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