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

Abstract

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.

Ethanol stress stimulates sumoylation of transcription factor Cst6 which restricts expression of its target genes

“…Extending our previous work on Gcn4 and Sko1, we have now shown that another bZIP transcription factor, Cst6, is also regulated by sumoylation in budding yeast (Rosonina et al 2012;Sri Theivakadadcham et al 2019). Gcn4 expression (and consequently, its sumoylation) is dependent on amino acid starvation, but like Sko1, a fraction of Cst6 molecules is sumoylated during normal growth conditions, suggesting that the modification regulates properties of the transcription factor not related to stress.…”

“…For some transcription factors, sumoylation was shown to occur specifically after they bind to target promoters and the modification facilitates their subsequent removal from DNA, thereby enabling gene deactivation or restricting expression levels (e.g. Gcn4, Ikaros, and Sko1; (Rosonina et al 2012;Apostolov et al 2016;Sri Theivakadadcham et al 2019)). As mentioned above, however, in other cases, sumoylation promotes the interaction of transcription factors with DNA (e.g.…”

“…Supporting that Cst6 is sumoylated, bands were detected on the SUMO immunoblot for the Cst6-6HA IP ( Figure 1A). As a positive control, Cst6-6HA sumoylation levels were compared alongside another bZIP transcription factor, Sko1, which is known to be polysumoylated during non-stress conditions (Sri Theivakadadcham et al 2019). In addition to Sko1-6HA or Cst6-6HA-specific bands, a band of variable intensity was detected at ~250 kDa in the SUMO immunoblots of HA IPs (arrow in Figure 1A and throughout).…”

“…Sumoylation of bZIP transcription factors can function in both promoter clearance (e.g. c-Fos, Gcn4 and Sko1; (Rosonina et al 2012;Tempe et al 2013;Sri Theivakadadcham et al 2019)) and enhancing DNA binding (e.g. CREB1; (Chen et al 2014)).…”

“…Denatured IP samples were prepared using a method previously described by Sri Theivakadadcham et al, (2019). Briefly, 50 mL of yeast cultures were grown in rich medium, harvested by centrifugation, and washed with 20% Trichloroacetic acid (TCA).…”