1,10-phenanthroline inhibits sumoylation and reveals that yeast SUMO modifications are highly transient.

The steady-state levels of protein sumoylation depend on relative rates of conjugation and desumoylation. Whether SUMO modifications are generally long-lasting or short-lived is unknown. Here we show that treating budding yeast cultures with 1,10-phenanthroline abolishes most SUMO conjugations within one minute, without impacting ubiquitination, an analogous post-translational modification. 1,10-phenanthroline inhibits the formation of the E1~SUMO thioester intermediate, demonstrating that it targets the first step in the sumoylation pathway. SUMO conjugations are retained after treatment with 1,10-phenanthroline in yeast that express a defective form of the desumoylase Ulp1, indicating that Ulp1 is responsible for eliminating existing SUMO modifications almost instantly when de novo sumoylation is inhibited. This reveals that SUMO modifications are normally extremely transient because of continuous desumoylation by Ulp1. Supporting our findings, we demonstrate that sumoylation of two specific targets, Sko1 and Tfg1, virtually disappears within one minute of impairing de novo sumoylation. Altogether, we have identified an extremely rapid and potent inhibitor of sumoylation, and our work reveals that SUMO modifications are remarkably short-lived.

Sumoylation is Largely Dispensable for Normal Growth but Facilitates Heat Tolerance in Yeast.

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.

Dynamic sumoylation of promoter-bound general transcription factors facilitates transcription by RNA polymerase II.

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.