EMSA And Mutation Analysis Of Several Cis Elements In The Promoter Region Of Sheep Musclin Gene

As a kind of bioactive factor, musclin plays an important role in regulating muscle growth and metabolism, and it was found in mice skeletal muscle through signal sequence trap by Nishizawa in 2004. For the research on the transcriptional regulatory mechanism of musclin gene, especially on the function of various important cis elements in the sheep musclin gene promoter, there is no detailed research report so far. In the previous study, our group found some components which might be important through bioinformatics analysis in sheep musclin gene promoter. Therefore, this test is intended to explore the function of these cis-acting elements by EMSA technology and mutation analysis, which can lay the foundation for revealing the transcriptional regulatory mechanism of sheep musclin gene.Based on the musclin gene promoter of sheep cloned by our research group in the previous study, in this test we replaced the cis elements with restriction enzyme cutting site, and designed the primers separately for PCR amplification. Then using EGFP as report gene, we constructed a series of expression vectors MUSCLINproXM-EGFP for the musclin promoter containing mutant elements which had the potential relationship with muscle such as E-box, GA-box, MEF2, MEF3, FoxO, HMTB, SMAD and VTBP. The vectors were transfected into mice C2C12 cells, and through fluorescence microscopy it was found that the element mutations had different influence on the transcriptional regulation activity of the musclin promoter. The results of RTFQ-PCR of EGFP showed that the mutation of other E-boxes down regulated the transcription activity of the musclin promoter except the mutation of E2 or E8 up regulated the transcription activity of the musclin promoter, and for other elements the mutation of MEF2, MEF3, FoxO, HMTB, SMAD or VTBP up regulated the transcription activity of the musclin promoter while the mutation of GA-box had the opposite effect.In order to explore the function of the elements further, the elements of E-boxes, FoxO, VTBP, SMAD, HMTB and MEF3 were selected to test by EMSA. The results showed that there were really some transcription factors which can combine with E1, E2, E5, E8, FoxO, HMTB and SMAD in the nucleoprotein of sheep muscle tissue. All of the above indicated that those elements played an important role in the transcriptional regulation of the sheep musclin gene.