Identification of SENP6 target proteins by endogenous SUMO2/3 IP-MS

Cellular signaling by the ubiquitin-related SUMO pathway relies on coordinated conjugation and deconjugation events. SUMO-specific deconjugating enzymes counterbalance SUMOylation, but comprehensive insight into their substrate specificity and regulation is missing. By characterizing SENP6 we define an N-terminal multi-SIM domain as critical determinant in targeting SENP6 activity to SUMO chains. Using an integrated proteomic profiling approach we reveal a network of SENP6 functions at the crossroad of chromatin organization and DNA damage response (DDR). SENP6 acts as SUMO eraser on key organizers of telomeric and centromeric chromatin domains and determines the SUMOylation status and chromatin association of the cohesin complex. Importantly, SENP6 is part of the hPSO4/PRP19 complex that drives ATR-Chk1 activation during DDR. Intriguingly, SENP6 deficiency impairs chromatin-association of the ATR cofactor ATRIP thereby compromising the activation of Chk1 signaling in response to replicative stress and sensitizing cells to replicative stress-induced DNA damage. Collectively, we propose a general role of SENP6 in orchestrating chromatin dynamics and genome stability networks by balancing chromatin-residency of protein complexes.