The STUbL RNF4 regulates protein group SUMOylation by targeting the SUMO conjugation machinery.

SUMO-targeted ubiquitin ligases (STUbLs) mediate the ubiquitylation of SUMOylated proteins to modulate their functions. In search of direct targets for the STUbL RNF4, we have developed TULIP (targets for ubiquitin ligases identified by proteomics) to covalently trap targets for ubiquitin E3 ligases. TULIP methodology could be widely employed to delineate E3 substrate wiring. Here we report that the single SUMO E2 Ubc9 and the SUMO E3 ligases PIAS1, PIAS2, PIAS3, ZNF451, and NSMCE2 are direct RNF4 targets. We confirm PIAS1 as a key RNF4 substrate. Furthermore, we establish the ubiquitin E3 ligase BARD1, a tumor suppressor and partner of BRCA1, as an indirect RNF4 target, regulated by PIAS1. Interestingly, accumulation of BARD1 at local sites of DNA damage increases upon knockdown of RNF4. Combined, we provide an insight into the role of the STUbL RNF4 to balance the role of SUMO signaling by directly targeting Ubc9 and SUMO E3 ligases.