Transcription factors (TFs) are among the most frequently detected targets of sumoylation, and effects of the modification have been studied for about 200 individual TFs to date. TF sumoylation is most often associated with reduced target gene expression, which can be mediated by enhanced interactions with corepressors or by interference with protein modifications that promote transcription. However, recent studies show that sumoylation also regulates gene expression by controlling the levels of TFs that are associated with chromatin. SUMO can mediate this by modulating TF DNA-binding activity, promoting clearance of TFs from chromatin, or indirectly, by influencing TF abundance or localization.

Transcription-related proteins are frequently identified as targets of sumoylation, including multiple subunits of the RNA polymerase II (RNAPII) general transcription factors (GTFs). However, it is not known how sumoylation affects GTFs or whether they are sumoylated when they assemble at promoters to facilitate RNAPII recruitment and transcription initiation. To explore how sumoylation can regulate transcription genome-wide, we performed SUMO ChIP-seq in yeast and found, in agreement with others, that most chromatin-associated sumoylated proteins are detected at genes encoding tRNAs and ribosomal proteins (RPGs). However, we also detected 147 robust SUMO peaks at promoters of non-ribosomal protein-coding genes (non-RPGs), indicating that sumoylation also regulates this gene class. Importantly, SUMO peaks at non-RPGs align specifically with binding sites of GTFs, but not other promoter-associated proteins, indicating that it is GTFs specifically that are sumoylated there. Predominantly, non-RPGs with SUMO peaks are among the most highly transcribed, have high levels of TFIIF, and show reduced RNAPII levels when cellular sumoylation is impaired, linking sumoylation with elevated transcription. However, detection of promoter-associated SUMO by ChIP might be limited to sites with high levels of substrate GTFs, and promoter-associated sumoylation at non-RPGs may actually be far more widespread than we detected. Among GTFs, we found that TFIIF is a major target of sumoylation, specifically at lysines 60/61 of its Tfg1 subunit, and elevating Tfg1 sumoylation resulted in decreased interaction of TFIIF with RNAPII. Interestingly, both reducing promoter-associated sumoylation, in a sumoylation-deficient Tfg1-K60/61R mutant strain, and elevating promoter-associated SUMO levels, by constitutively tethering SUMO to Tfg1, resulted in reduced RNAPII occupancy at non-RPGs. This implies that dynamic GTF sumoylation at non-RPG promoters, not simply the presence or absence of SUMO, is important for maintaining elevated transcription. Together, our findings reveal a novel mechanism of regulating the basal transcription machinery through sumoylation of promoter-bound GTFs.

The small ubiquitin-like modifier (SUMO) is implicated in various cellular activities, including transcriptional regulation. We previously showed that the yeast activator Gcn4 becomes sumoylated during activation, facilitating its eventual promoter eviction and transcriptional shut off. Here we show that the corepressor Tup1 is sumoylated, at two specific lysines, under various stress conditions. Mutation of these sites has no effect on Tup1 recruitment or RNAP II promoter occupancy immediately following induction. However, Tup1 levels subsequently decrease, while RNAP II and transcription increase in Tup1 mutant cells. Consistent with this, a Tup1 mutant displaying increased sumoylation led to reduced transcription. We also show that coordinated sumoylation of Gcn4 and Tup1 enhances Gcn4 promoter eviction, and that multiple Tup1-interacting proteins become sumoylated after stress. Together, our studies provide evidence that coordinated sumoylation of Gcn4, Tup1, and likely other factors, dampens activated transcription by stabilizing Tup1 binding and stimulating Gcn4 and RNAP II removal.

The Saccharomyces cerevisiae transcription factor Gcn4 is expressed during amino acid starvation, and its abundance is controlled by ubiquitin-mediated proteolysis. Cdk8, a kinase component of the RNA polymerase II Mediator complex, phosphorylates Gcn4, which triggers its ubiquitination/proteolysis, and is thought to link Gcn4 degradation with transcription of target genes. In addition to phosphorylation and ubiquitination, we previously showed that Gcn4 becomes sumoylated in a DNA-binding dependent manner, while a nonsumoylatable form of Gcn4 showed increased chromatin occupancy, but only if Cdk8 was present. To further investigate how the association of Gcn4 with chromatin is regulated, here we examine determinants for Gcn4 sumoylation, and how its post-translational modifications are coordinated. Remarkably, artificially targeting Gcn4 that lacks its DNA binding domain to a heterologous DNA site restores sumoylation at its natural modification sites, indicating that DNA binding is sufficient for the modification to occur in vivo. Indeed, we find that neither transcription of target genes nor phosphorylation are required for Gcn4 sumoylation, but blocking its sumoylation alters its phosphorylation and ubiquitination patterns, placing Gcn4 sumoylation upstream of these Cdk8-mediated modifications. Strongly supporting a role for sumoylation in limiting its association with chromatin, a hyper-sumoylated form of Gcn4 shows dramatically reduced DNA occupancy and expression of target genes. Importantly, we find that Cdk8 is at least partly responsible for clearing hyper-sumoylated Gcn4 from DNA, further implicating sumoylation as a stimulus for Cdk8-mediated phosphorylation and degradation. These results support a novel function for SUMO in marking the DNA-bound form of a transcription factor, which triggers downstream processes that limit its association with chromatin, thus preventing uncontrolled expression of target genes. KEYWORDS Gcn4; sumoylation; Cdk8; transcription; gene activation T HE expression of numerous genes is controlled by genespecific transcription factors (TFs), which bind to target DNA sequences and activate transcription. TFs contain DNA binding domains that recognize DNA elements referred to as upstream activator sequences (UAS) in budding yeast, located proximal to their cognate promoters, or enhancers in higher eukaryotes, which can be situated several 100 kb away from the genes that they regulate (Hahn and Young 2011;Shlyueva et al. 2014;Vernimmen and Bickmore 2015). Once bound, TFs trigger the ordered assembly of the general transcription factors (GTFs) on target gene promoters, and the recruitment of RNA polymerase II (RNAP II), to form the transcriptional preinitiation complex (PIC). To promote PIC formation, many DNA-bound TFs make physical contact between their activation domains and specific GTF components, either directly, through coactivators, or through the Mediator complex (Thomas and Chiang 2006;Hahn and Young 2011). DNA binding is critical for TF function, as unbound ac...

Numerous proteins are sumoylated in normally growing yeast and SUMO conjugation levels rise upon exposure to several stress conditions. We observe high levels of sumoylation also during early exponential growth and when nutrient-rich medium is used. However, we find that reduced sumoylation (∼75% less than normal) is remarkably well-tolerated, with no apparent growth defects under nonstress conditions or under osmotic, oxidative, or ethanol stresses. In contrast, strains with reduced activity of Ubc9, the sole SUMO conjugase, are temperature-sensitive, implicating sumoylation in the heat stress response, specifically. Aligned with this, a mild heat shock triggers increased sumoylation which requires functional levels of Ubc9, but likely also depends on decreased desumoylation, since heat shock reduces protein levels of Ulp1, the major SUMO protease. Furthermore, we find that a ubc9 mutant strain with only ∼5% of normal sumoylation levels shows a modest growth defect, has abnormal genomic distribution of RNA polymerase II (RNAPII), and displays a greatly expanded redistribution of RNAPII after heat shock. Together, our data implies that SUMO conjugations are largely dispensable under normal conditions, but a threshold level of Ubc9 activity is needed to maintain transcriptional control and to modulate the redistribution of RNAPII and promote survival when temperatures rise.