SUMOylation Of DEC1 And Its Relative Functional Research

Differentiated embryo-chondrocyte expressed gene 1(DEC1) belongs to the family of the basic helix-loop-helix (bHLH) protein that is very important in regulating the gene expression through as a transcription factor. Moreover, DEC1 is also involved in many signal pathways, such as nuclear receptor, hypoxia-inducible factor (HIF) and the p53, and regulates the processes of the cell growth, proliferation and differentiation. On the other hand, DEC1 has been implicated in the development of breast cancer.It was found that the expression of DEC1 showed robust circadian rhythms and it as a new clock protein regulated the related clock gene expression. In the mammalian clock system, DEC1 represses the CLOCK/BMAL1-mediated the transcriptional activity by binding to the E—box in the clock gene promoter. However, it is not clear that the precise regulating mechinasm of DECl in molecular clock systems.SUMOylation as an important posttranslational modificaton affects many proteins function, including the protein localizations, interactions and the transcriptional activity. Therefore, our research is focused on the SUMOyltaion of DEC1 and its relative function in localization and gene expression.Firstly, we showed that DEC1 can be modified by SUMO1,2 and 3 in COS-7 cells which co-transfected the DEC1 and SUMO expressing plasmids. Interestingly, DEC1 was preferentially modified by SUMO1 compared to SUM02 and 3. Furthermore, the endogenous DEC1 was also modified by SUMO1 in MCF-7 cells.Secondly, it is found that DEC1 has two potential SUMOylation sites in its C terminal domain after analysis of the amio acids sequences. And then K159 and K279 were identified as two major SUMOyaltion sites in DEC1 through constructing the site-specific mutagenesis expressing plasmids, K159R, K279R and 2K/2R (substitution of both K159 and K279 with arginine) for Western blotting in COS-7 cells. Moreover, K279R mutant was more easily to influence the SUMOylation levels of DEC1 compared to K159R mutant.Lastly, we found that the SUMOylation sites mutant could lead DEC1 transfer to cytoplasm and decrease the levels of DEC1 protein in the nucleo through the immunofluorescence and the nucleo-cytoplasmic separation assays in MCF-7 cells. It is worth notice that the total mutant DEC1 protein levels were also decreased in nucleo and cytoplasmic compared to the wild-type DEC1,which indicated that SUMOyaltion may be also important for the stability of DEC1. On the other hand, we also discovered that SUMOylation of DEC1 repressed the CLOCK/BMAL1-mediated transcriptional activity through the HDAC1 recruitment in MCF-7 cells utilizing the luciferase reporter gene and IP expriments.Our research confirmed that DEC1 was modified by SUMO and then SUMOyaltion promoted its nucleo localization. Furthermore, it is found that SUMOyaltion promoted DEC1 to repress the CLOCK-mediated transcriptional activity through recruiting the HDAC1 in breast cancer cells, which providing an important theoretic foundation for further to understanding the development of breast cancer and the biological clock regulating machnism.