SUMOPiv

SUMO-conjugation has recently emerged as an essential post-translational modification, controlling protein/protein interactions, subcellular localization, stability, both in the basal state and under stress. Three SUMO paralogs have been identified and there is evidence that SUMO1 and SUMO2/3 are differently regulated in particular by cellular stress. We have recently shown that PML, a protein that nucleates PML nuclear bodies (NBs), controls the sumoylation of the numerous partner proteins that it recruits, implying that PML is a key regulator of this process. PML NBs are implicated in several biological processes, like cell death, DNA repair or stemness. Since formation of PML NBs is exquisitely sensitive to stress, PML could be implicated in stress-responsive modulation of sumoylation. To compare the in vivo sumoylation by the different SUMO paralogs and its regulation by oxidative stress and PML NBs, we will first collaborate with Niels Brose’s laboratory to obtain knock-in mouse where a (His)x6 and HA tags are fused in frame to SUMO1. This laboratory is also generating (His)x6-HA-SUMO2, and are pleased to collaborate to this project by sharing these animals with the coordinator. Proteins conjugated by (His)x6-HA-SUMO1 or 2 will be purified and identified by mass spectrometry. Thus, these animals will allow a precise comparison of the basal sumoylation pattern in a tissue-specific and stress-responsive manner. We will then cross those mice on a pml-/- background to establish any role of PML in the modulation of SUMO1 or SUMO2-specific conjugation in vivo, both in the basal state and in response to oxidative stress that promotes PML NB-biogenesis. We will then examine how conjugation of SUMO1 or SUMO2 regulates the subcellular localization of a set of PML partner proteins including DAXX, HIPK2, SP100 and the newly identified proteins with PML-dependent sumoylation. PML is itself heavily sumoylated. Since PML undergoes multiple post-translational modifications proposed to modulate its own sumoylation and possibly that of partner proteins, we will directly identify by mass spectrometry the phosphorylation, acetylation and ubiquitination status of SUMO1 or SUMO2-conjugated and unconjugated PML in vivo. The target amino-acids will be mutated and these mutant PML genes will be reintroduced in primary cells to analyze PML own sumoylation and consequence on partners sumoylation as well. Collectively, these studies should provide important insights into the fine-tuning of sumoylation in vivo. They will provide important tools for the scientific community and may have some spinoff for drug discovery, as our studies have previously established that the curative activity of arsenic is enforced through its direct targeting of PML and induction of sumoylation.