Inhibition of DNA Binding by Differential Sumoylation of Heat Shock Factors

Abstract

Covalent modification of proteins by the small ubiquitin-related modifier SUMO regulates diverse biological functions. Sumoylation usually requires a consensus tetrapeptide, through which the binding of the SUMO-conjugating enzyme Ubc9 to the target protein is directed. However, additional specificity determinants are in many cases required. To gain insights into SUMO substrate selection, we have utilized the differential sumoylation of highly similar loop structures within the DNA-binding domains of heat shock transcription factor 1 (HSF1) and HSF2. Site-specific mutagenesis in combination with molecular modeling revealed that the sumoylation specificity is determined by several amino acids near the consensus site, which are likely to present the SUMO consensus motif to Ubc9. Importantly, we also demonstrate that sumoylation of the HSF2 loop impedes HSF2 DNA-binding activity, without affecting its oligomerization. Hence, SUMO modification of the HSF2 loop contributes to HSF-specific regulation of DNA binding and broadens the concept of sumoylation in the negative regulation of gene expression.

We thank Ronald Hay, Jorma J. Palvimo, and Jacob Seeler for generously providing plasmids. Jorma J. Palvimo is further acknowledged for valuable discussions and suggestions. We are thankful to Helena Saarento for excellent technical assistance. We acknowledge Johanna K. Ahlskog, Henri Blomster, Eva Henriksson, Aura Kaunisto, Minna Poukkula, and Anton Sandqvist for valuable suggestions and comments on the manuscript.

This work was supported by the Academy of Finland (L.S., K.D., and M.S.J.), the Sigrid Jusélius Foundation (L.S., and M.S.J.), the Finnish Cancer Organizations (L.S.), the Finnish Life Insurance Companies (L.S.), the Turku Graduate School of Biomedical Sciences (V.H.), the Paulo Foundation (V.H.), the Finnish Cultural Foundation (V.H.), and the U.S. National Institutes of Health (grant GM59911, D.J.T.).