Slx5/Slx8‐dependent ubiquitin hotspots on chromatin contribute to stress tolerance

Chromatin is a highly regulated environment, and protein association with chromatin is often controlled by post‐translational modifications and the corresponding enzymatic machinery. Specifically, SUMO‐targeted ubiquitin ligases (STUbLs) have emerged as key players in nuclear quality control, genome maintenance, and transcription. However, how STUbLs select specific substrates among myriads of SUMOylated proteins on chromatin remains unclear. Here, we reveal a remarkable co‐localization of the budding yeast STUbL Slx5/Slx8 and ubiquitin at seven genomic loci that we term “ubiquitin hotspots”. Ubiquitylation at these sites depends on Slx5/Slx8 and protein turnover on the Cdc48 segregase. We identify the transcription factor‐like Ymr111c/Euc1 to associate with these sites and to be a critical determinant of ubiquitylation. Euc1 specifically targets Slx5/Slx8 to ubiquitin hotspots via bipartite binding of Slx5 that involves the Slx5 SUMO‐interacting motifs and an additional, novel substrate recognition domain. Interestingly, the Euc1‐ubiquitin hotspot pathway acts redundantly with chromatin modifiers of the H2A.Z and Rpd3L pathways in specific stress responses. Thus, our data suggest that STUbL‐dependent ubiquitin hotspots shape chromatin during stress adaptation. Synopsis SUMO‐targeted ubiquitin ligases (STUbLs), involved in nuclear quality control, genome maintenance and transcription, co‐localize with ubiquitin to seven distinct genomic loci on yeast chromatin, which are determined by the transcription factor‐like protein Euc1 and contribute to stress tolerance. Seven “ubiquitin hotspots” are sites of Cdc48‐dependent protein turnover within the yeast genome. The previously uncharacterized protein Ymr111c/Euc1 binds a ubiquitin‐hotspot DNA motif and recruits the STUbL Slx5/Slx8. SUMO‐dependent and ‐independent interactions between Euc1 and Slx5 cooperate for a novel bipartite Slx5/Slx8 recruitment mechanism. Euc1 and ubiquitin hotspots are required for cellular stress tolerance when gene expression control and chromatin maintenance by Rpd3‐HDACs or SWR1‐C are impaired. Graphical Abstract Yeast SUMO‐targeted ubiquitin ligases (STUbLs) and ubiquitin co‐localize to seven genomics loci marked by the transcription factor‐like protein Euc1.