Promyelocytic leukemia (PML) protein forms the basis of PML nuclear bodies (PML NBs), which control many important processes. We have screened an shRNA library targeting ubiquitin pathway proteins for effects on PML NBs, and identified RNF8 and RNF168 DNAdamage response proteins as negative regulators of PML NBs. Additional studies confirmed that depletion of either RNF8 or RNF168 increased the levels of PML NBs and proteins, whereas overexpression induced loss of PML NBs. RNF168 partially localized to PML NBs through its UMI/MIU1 ubiquitin-interacting region and associated with NBs formed by any PML isoform. The association of RNF168 with PML NBs resulted in increased ubiquitylation and SUMO2 modification of PML. In addition, RNF168 was found to associate with proteins modified by SUMO2 and/or SUMO3 in a manner dependent on its ubiquitin-binding sequences, suggesting that hybrid SUMO-ubiquitin chains can be bound. In vitro assays confirmed that RNF168, preferentially, binds hybrid SUMO2-K63 ubiquitin chains compared with K63-ubiquitin chains or individual SUMO2. Our study identified previously unrecognized roles for RNF8 and RNF168 in the regulation of PML, and a so far unknown preference of RNF168 for hybrid SUMOubiquitin chains.

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