RESUMEN
RNF4, a SUMO-targeted ubiquitin ligase (STUbL), localizes to the nucleus and functions in the DNA damage response during interphase of the cell cycle. RNF4 also exists in cells undergoing mitosis, where its regulation and function remain poorly understood. Here we showed that administration of etoposide, an anticancer DNA topoisomerase II poison, to mitotic human cervical cancer HeLa cells induced SUMO-2/3-dependent localization of RNF4 to chromosomes. The FK2 antibody signals, indicative of poly/multi-ubiquitin assembly, were detected on etoposide-exposed mitotic chromosomes, whereas the signals were negligible in cells depleted for RNF4 by RNA interference. This suggests that RNF4 functions as a STUbL in the etoposide-induced damage response during mitosis. Indeed, RNF4-depletion sensitized mitotic HeLa cells to etoposide and increased cells with micronuclei. These results indicate the importance of the RNF4-mediated STUbL pathway during mitosis for the maintenance of chromosome integrity and further implicate RNF4 as a target for topo II poison-based therapy for cancer patients.
Asunto(s)
Cromosomas Humanos/metabolismo , Etopósido/farmacología , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Reparación del ADN , ADN-Topoisomerasas de Tipo II , Células HEK293 , Células HeLa , Humanos , Ratones , Micronúcleos con Defecto Cromosómico/inducido químicamente , Mitosis , Proteínas Nucleares/efectos de los fármacos , Proteínas Nucleares/inmunología , Proteína SUMO-1/inmunología , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/inmunología , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/inmunologíaRESUMEN
Thymine DNA glycosylase (TDG) is a base excision repair enzyme that interacts with the small ubiquitin-related modifier (SUMO)-targeted ubiquitin E3 ligase RNF4 and functions in the active DNA demethylation pathway. Here we showed that both SUMOylated and non-modified forms of endogenous TDG fluctuated during the cell cycle and in response to drugs that perturbed cell cycle progression, including hydroxyurea and nocodazole. Additionally, we detected a SUMOylation-independent association between TDG and RNF4 in vitro as well as in vivo, and observed that both forms of TDG were efficiently degraded in RNF4-depleted cells when arrested at S phase. Our findings provide insights into the in vivo dynamics of TDG SUMOylation and further clarify the TDG-RNF4 interaction.