Abstract
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.
Highlights
Sumoylation is a conserved eukaryotic post-translational modification (PTM) that primarily affects nuclear proteins
We demonstrate that, in budding yeast, some components of general transcription factors (GTFs) are post-translationally modified by the SUMO peptide when they are assembled at promoters
We determined that the large subunit of TFIIF, Tgf1, is the major target of sumoylation among GTFs and that increasing Tfg1 sumoylation reduces the interaction of TFIIF with RNA polymerase II (RNAPII)
Summary
Sumoylation is a conserved eukaryotic post-translational modification (PTM) that primarily affects nuclear proteins. In genome-wide ChIP (ChIP-seq) analyses performed with Sko in budding yeast, human MITF, and the human androgen and glucocorticoid receptors, sumoylation-impairing mutations led to a dramatic increase in the number of genomic binding sites at loci not normally bound by these SSTFs [16,17,18,19,20,21] These findings point to general and conserved roles for sumoylation of SSTFs in promoting dissociation from nonspecific sites genome-wide, while limiting their association with authentic binding sites [3,22]
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