Defining Genome Stability Mechanisms and their Regulation by SUMO and Ubiquitin - The overarching goal of our proposal is to define how the posttranslational modifiers (PTMs) SUMO and ubiquitin orchestrate diverse cellular pathways that prevent genome instability, fight viral infections, and maintain cellular health. Both PTMs are covalently attached to lysine residues by a cascade consisting of E1/E2 enzymes, and E3 ligases, which provide target protein specificity. The effects of SUMO and ubiquitin on target protein function are multifaceted, and include changes in protein binding partners, cellular localization, phase separation e.g. PML nuclear bodies (PML NBs), and proteasomal degradation. This proposal centers on two factors, a SUMO- targeted ubiquitin ligase (STUbL) and the structural maintenance of chromosomes (SMC) complex SMC5/6, which integrate signaling through SUMO and ubiquitin to support key health-related processes. STUbL is an E3 ubiquitin ligase that selectively recognizes and ubiquitinates SUMOylated proteins to promote their proteasomal degradation and/or extraction from chromatin or other protein complexes (e.g. PML NBs) by the p97 ATPase. In this way, STUbL regulates multiple aspects of genome stability, including the fate of dysfunctional telomeres. Moreover, together with p97 and the proteasome, STUbL mediates the therapeutic effect of arsenic trioxide (ATO) in acute promyelocytic leukemia (APL). ATO induces the SUMOylation and STUbL-dependent degradation of the oncogenic PML-RARA fusion protein, which results in the differentiation or apoptosis of APL blasts. SMC5/6 is functionally related to cohesin and condensin but uniquely, can modify targets with SUMO and ubiquitin. Also unique among SMC complexes, SMC5/6 not only plays key roles in genome stability, supporting the DNA damage response and replication, but surprisingly, also represses the transcription of extrachromosomal circular DNA (eCDNAs) including multiple pathogenic viruses, and plasmids. Despite these important advances, many crucial aspects of both STUbL and SMC5/6 function remain mechanistically undefined. To provide a framework for functional insights, we used proximity labeling to reliably map the proteomic environments of STUbL and SMC5/6 in key health-related settings, including at dysfunctional ALT cancer cell telomeres, and viral replication centers. One SMC5/6 cofactor we identified binds SUMO and directs the complex to both SUMO-rich phase-separated ALT telomere-associated PML NBs, and sites of viral replication, thereby unifying these seemingly disparate processes. Hence, we would define functions for SMC5/6 and its new SUMO-binding cofactor in each of these processes to reveal common mechanisms. Moreover, a novel genetic screening method identified key modulators of ALT, including STUbL and SUMO, which when integrated with our proteomic hits, will provide new insights into telomere and chromosome maintenance. Overall, our analysis of STUbL and SMC5/6 using proteomic, genetic, cell biological, biochemical, and structural methods would synergize to define key health-related mechanisms at the nexus of the SUMO and ubiquitin pathways, ultimately providing targets and guidance for therapeutic interventions.
