Role Of Human Primate-specific Zinc Finger Protein Znf480 In Myogenesis

Primate-specific KRAB zinc finger proteins account for at least 30% of KRAB zinc finger proteins in the whole human genome. While little related report regards to functional role of primate-specific zinc finger proteins has been published. Therefore, we choose a primate-specific gene ZNF480 as a model to investigate the potential role of primate-specific zinc finger proteins.ZNF480 is highly expressed in muscle and heart tissues, indicating that it may be involved in development of muscle and heart tissues. Half-quantitative RT-PCR and western blot suggest that mRNA and protein levels of ZNF480 are significantly upregulated during stable transfected C2C12 myogenesis, which suggests that ZNF480 may be involved in myogenesis.To investigate whether ZNF480 can regulate myogenesis in morphologic, we examined the information of myotube via morphologic observation and immunofluorescence analysis by muscle-specific antibody MHC. Compared with control group, overexpression of ZNF480 can promote the formation of myotubes and multi-nucleus cells, and the numbers of myotubes and multi-nucleus have been increased following the process of myogenesis.Myf-5, myogenin and MyoD are bHLH transcriptional factors involved in myogenesis. E47 and E12 are bHLH transcriptional factors without tissue-specific. During myogenesis, Myf-5 and MyoD are key genes whose expression will determine whether mesodermal progenitor turn to myoblast, while myogenin plays pivotal role in terminal differentiation of myoblast. Therefore, we examined the possibility whether ZNF480 can regulate these genes. Half-quantitative RT-PCR and western blot show that ZNF480 can upregulate mRNA level of muscle-specific bHLH transcriptional factor and muscle-specific gene MCK, and the protein levels of MHC and myogenin, which in turn documents that ZNF480 promotes myogenesis through regulating bHLH and MCK. Moreover, Western blot suggests that the protein level of p21 is upregulated obviously during cell myogenesis of overexpression of ZNF480. It indicates that ZNF480 cause cell cycle exiting and cell differentiation by upregulating the level of p21 protein.We performed luciferase activity assays which show that over-expression of ZNF480 in NIH3T3 cell can not enhance the activities of E12 or MyoD on MCK promoter, but ZNF480 enhances the activites of MCK promoters (MCK4800 and 4RTK) when cotransfected with E12/MyoD. It suggests that ZNF480 promotes the ability of E12/MyoD to transactivate MCK promoter activity.To probe whether ZNF480 can bind to the promoters of MCK and myogenin and regulate their transcription, we performed EMSA assay. The results show that ZNF480 can not bind to MCK promote region alone but it does through forming protein complex with E12/MyoD. At the same time, EMSA assay showed that ZNF480 can directly bind to the promoter region of myogenin. These results document that ZNF480 regulate transcriptional activity of MCK and myogenin by forming protein complex or binding directly to regulate myogenesis.To sum up, primate-specific human zinc finger protein ZNF480 may enhance myogenesis and play an important role in myogenesis through regulation of myogenic regulation factors Myf-5, MyoD and myogenin and muscle specific gene MCK...