Sumoylation of DNA-bound transcription factor Sko1 prevents its association with nontarget promoters

Sequence-specific transcription factors (TFs) represent one of the largest groups of proteins that is targeted for SUMO post-translational modification, in both yeast and humans. SUMO modification can have diverse effects, but recent studies showed that sumoylation reduces the interaction of multiple TFs with DNA in living cells. Whether this relates to a general role for sumoylation in TF binding site selection, however, has not been fully explored because few genome-wide studies aimed at studying such a role have been reported. To address this, we used genome-wide analysis to examine how sumoylation regulates Sko1, a yeast bZIP TF with hundreds of known binding sites. We find that Sko1 is sumoylated at Lys 567 and, although many of its targets are osmoresponse genes, the level of Sko1 sumoylation is not stress-regulated and the modification does not depend or impinge on its phosphorylation by the osmostress kinase Hog1. We show that Sko1 mutants that cannot bind DNA are not sumoylated, but attaching a heterologous DNA binding domain restores the modification, implicating DNA binding as a major determinant for Sko1 sumoylation. Genome-wide chromatin immunoprecipitation (ChIP-seq) analysis shows that a sumoylation-deficient Sko1 mutant displays increased occupancy levels at its numerous binding sites, which inhibits the recruitment of the Hog1 kinase to some induced osmostress genes. This strongly supports a general role for sumoylation in reducing the association of TFs with chromatin. Extending this result, remarkably, sumoylation-deficient Sko1 binds numerous additional promoters that are not normally regulated by Sko1 but contain sequences that resemble the Sko1 binding motif. Our study points to an important role for sumoylation in modulating the interaction of a DNA-bound TF with chromatin to increase the specificity of TF-DNA interactions. Author summary Transcription factors bind the genome at specific sites to control the expression of target genes. Although the DNA sequence of these sites is critical for determining where they bind, additional factors must act to ensure that only appropriate sites remain bound and regulated by transcription factors. Here we demonstrate that SUMO post-translational modification functions in transcription factor binding site selection. We show that a yeast transcription factor, Sko1, becomes sumoylated after it binds DNA, and that the modification reduces the interaction of Sko1 with DNA. We propose that this is important to ensure that Sko1 remains associated only with actual Sko1 genomic binding sites. Indeed, we find that a mutant form of Sko1 that cannot be sumoylated binds hundreds of sites on the genome that are not normally bound by Sko1. Most of these additional binding sites contain sequences that somewhat resemble the normal Sko1 binding site sequence, suggesting that sumoylation increases the stringency of Sko1 binding site selection. As numerous transcription factors are known to be targets of SUMO modification, our work suggests a general role for sumoylation in promoting specificity of binding to genomic sites in eukaryotic cells.