BRAF35 Sumoylation is essential for the activity of the LSD1/COREST/HDAC complex

Abstract

SUMO modification of transcription factors and cofactors has generally been correlated with transcriptional repression. It has been described that SUMO contributes to gene-specific binding and activity of the LSD1/CoREST/HDAC repressor complex. In this complex, it has been described that LSDl, CoREST and HDACl can be sumoylated. We have found that BRAF35, another component of the LSDl/CoREST/HDAC complex, is SUMOylated in vivo. A mutant version of BRAF35 carrying points mutations in the SUMO attachment sequences impedes SUMOylation of BRAF35 in vivo, without affecting BRAF35 protein levels, and overexpression of this mutant in HeLa cells led to transcriptional activation of some neuronal-specific genes, including SCN1A, SCN2A2 and SCN3A, that are known CoREST/LSDl target genes. This SUMOylation-deficient BRAF35 mutant protein is still able to interact with the other subunits of the complex in co-immunoprecipitations assays, concluding that SUMOylation of BRAF35 is not necessary for the assembly of the complex. However, when we checked the effect of these mutations in neuronal differentiation, we found a defect in the repression of the differentiation process. Transfection of BRAF35 suppressed neuronal differentiation promoted hy NeuroD2 in P19 cells, while transfection of the BRAF35 mutant has no effect on the process of neurogenesis induced by NeuroD2, indicating that SUMOylation of BRAF35 is essential for its repressive activity. These results were corroborated using an in vivo model of neurogenesis, the differentiation of the neural tube of the chick embryo. When Neurogenin and BRAF35 were simultaneously expressed, cells can not be differentiated into neurons. By contrast, co-transfection of the SUMOylation-deficient BRAF35 mutant has no effect on the process of neurogenesis induced by Neurogenin. Together, our results demonstrate that SUMOylation of BRAF35 is essential for tbe repression of neuronal genes in non-neuronal tissues by the LSDl/CoREST/HDAC complex, supporting a role for SUMOylalion in transcriptional silencing of some neuronal-specific genes in non-neuronal cells.