Selection of New HSF1 and HSF2 DNA-Binding Sites Reveals Differences in Trimer Cooperativity

Abstract

Multiple heat shock transcription factors (HSFs) have been discovered in several higher eukaryotes, raising questions about their respective functions in the cellular stress response. Previously, we had demonstrated that the two mouse HSFs (mHSFl and mHSF2) interacted differently with the HSP70 heat shock element (HSE). To further address the issues of cooperativity and the interaction of multiple HSFs with the HSE, we selected new mHSFl and mHSF2 DNA-binding sites through protein binding and PCR amplification. The selected sequences, isolated from a random population, were composed primarily of alternating inverted arrays of the pentameric consensus 5′-nGAAn-3′, and the nucleotides flanking the core GAA motif were nonrandom. The average number of pentamers selected in each binding site was four to five for mHSFl and two to three for mHSF2, suggesting differences in the potential for cooperative interactions between adjacent trimers. Our comparison of mHSFl and mHSF2 binding to selected sequences further substantiated these differences in cooperativity as mHSFl, unlike mHSF2, was able to bind to extended HSE sequences, confirming previous observations on the HSP70 HSE. Certain selected sequences that exhibited preferential binding of mHSFl or mHSF2 were mutagenized, and these studies demonstrated that the affinity of an HSE for a particular HSF and the extent of HSF interaction could be altered by single base substitutions. The domain of mHSFl utilized for cooperative interactions was transferable, as chimeric mHSFl/mHSF2 proteins demonstrated that sequences within or adjacent to the mHSFl DNA-binding domain were responsible. We have demonstrated that HSEs can have a greater affinity for a specific HSF and that in mice, mHSFl utilizes a higher degree of cooperativity in DNA binding. This suggests two ways in which cells have developed to regulate the activity of closely related transcription factors: developing the ability to fully occupy the target binding site and alteration of the target site to favor interaction with a specific factor.