Abstract B81: SUMO and ubiquitin post-transcriptional modifications regulate the stability of the IRF2BP2 transcriptional cofactor

IRF2BP2 (Interferon Regulatory Factor 2 Binding Protein 2) protein is a gene expression regulator, acting as transcriptional cofactor. IRF2BP2 acts in different cellular process including DNA damage repair, apoptosis, and immune response. Even though a few targets are known, there are several putative IRF2BP2-regulated genes. Novel evidence suggests that IRF2BP2 plays central functions in gene expression regulation. Recently, our group characterized the IRF2BP2 as an NFAT1 transcriptional repressor. To further understand the IRF2BP2 role in cellular functions, this work aims to clarify mechanisms involved in IRF2BP2 regulation. Initially, we performed a yeast two-hybrid screening to identify IRF2BP2-interacting proteins using IRF2BP2 as a bait. Among others, the UBE2I SUMO-conjugating enzyme and SUMO1 were identified as possible IRF2BP2 partners. Confocal microscopy demonstrated that these proteins colocalized in the nucleus. Furthermore, analysis by His-pull down and FRET confirmed the interaction between IRF2BP2 and UBE2I. Moreover, yeast two-hybrid assays showed that the interaction occurs independently of N-terminus C4-zinc finger of IRF2BP2. Subsequently, we observed that IRF2BP2 protein is SUMO1-modified in vivo. Our results indicated that three SUMO-1 molecules are added at three lysine residues. Finally, we demonstrated that the SUMO-modification favors disperse nuclear IRF2BP2 distribution and does not affect its repressor activity on NFAT1 transcription factor. Nevertheless, different assays showed that IRF2BP2 has a long half-life and that SUMO-1 conjugation increases IRF2BP2 protein stability. On the other hand, we showed that IRF2BP2 protein is also poliubiquitinated. Interestingly, the modifications observed were mainly Lys6- and Lys11-linked ubiquitin chains. Moreover, our data suggest that IRF2BP2 is not degraded by the proteasome and that Lys11-linked chain may constitute a signal for lysosome degradation. In conclusion, SUMO-modification upregulates IRF2BP2 protein stability. However, the consequences of Lys6 and Lys11 atypical ubiquitin chains linkage remain to be elucidated. Citation Format: Renata Ramalho-Oliveira, Matheus Rajao, Vilenia Souza, Joao P. B. Viola. SUMO and ubiquitin post-transcriptional modifications regulate the stability of the IRF2BP2 transcriptional cofactor [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; Sao Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B81.