SELECTION OF NEW HSF1 AND HSF2 DNA-BINDING SITES REVEALS DIFFERENCES IN TRIMER COOPERATIVITY

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 demonst rated that the two mouse HSFs (mHSF1 and mHSF2) interacted differently with the HSP70 heat shock element (HSE). To further address the issue s of cooperativity and the interaction of multiple HSFs with the HSE, we selected new mHSF1 and mHSF2 DNA-binding sites through protein bind ing and PCR amplification. The selected sequences, isolated from a ran dom 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 se lected in each binding site was four to five for mHSF1 and tno to thre e for mHSF2, suggesting differences in the potential for cooperative i nteractions between adjacent trimers. Our comparison of mHSF1 and mHSF 2 binding to selected sequences further substantiated these difference s in cooperativity as mHSF1, unlike mHSF2, was able to bind to extende d HSE sequences, confirming previous observations on the HSP70 HSE. Ce rtain selected sequences that exhibited preferential binding of mHSF1 or mHSF2 were mutagenized, and these studies demonstrated that the, af finity of an HSE for a particular HSF and the extent of HSF interactio n could be altered by single base substitutions. The domain of mHSF1 u tilized for cooperative interactions was transferable, as chimeric mHS F1/mHSF2 proteins demonstrated that sequences within or adjacent to th e mHSF1 DNA-binding domain were responsible. We have demonstrated that HSEs can have a greater affinity for a specific HSF and that in mice, mHSF1 utilizes a higher degree of cooperativity in DNA binding. This suggests two ways in which cells have developed to regulate the activi ty of closely related transcription factors: developing the ability to fully occupy the target binding site and alteration of the target sit e to favor interaction with a specific factor.