| Myostatin gene is a member of transforming growth factor β (transforming growthfactor-beta, TGF-β) superfamily, an important negative regulator of the animal muscle growthand development process. Myostatin gene coding region conserved among different species,and its encoded protein precursor consists of three components: N terminal signal peptide,N-terminal propeptide and biologically active carboxyl terminus. Because of its low aminoacid homology of C-terminus to the other TGF-β family members (up to45%), it is alsoclassified as a member of new class of GDF-8(Growth-differentiation factor8) family.1. The research of porcine Myostatin promoter upstream regulatory region of potentialbinding sitesIn this study, first we cloned and amplified up to1969kb upstream of the porcineMyostatin gene promoter region. The different length fragments of promoter were connectedto the report vectors, then transfected into C2C12myoblasts and NIH3T3fibroblasts, whichrevealed that the promoter activity was specific found in myoblasts, indicating that theMyostatin gene promoter is tissue-specific promoter. Then, using a serial deletion strategy, wegot five new length fragments of the promoter, according to the luciferase assay, the resultsshowed that the upstream of the transcription start site there exist two positive transcriptionalregulatory regions among1bp to-137bp,-137bp to-466bp, and exist a negativetranscriptional regulatory region within the-852bp to-1969bp. Then, for obvious effect ofincreased transcriptional activity of-137bp to-466bp, we conducted more report vectors andfinally determined the possible positive regulation region of cis-acting elements located siteswithin the-137bp to-218bp. Finally, according to the existing research papers and sequencecharacteristics, we designed and synthesised four biotin-labeled double-stranded DNA probesto do the Electrophoretic Mobility Shift Assay (EMSA), and verified-206bp to-189bp regioncould be the specific sites of transcription factor protein binding to. Then by using themethods of site-specific cold probe competition,-206bp to-189bp region mutation,-206bpto-189bp region-specific deletion vectors of luciferase assay in the detection experiments,and ultimately we determined the region for the transcription factor protein the specificbinding site was in-206bp to-189bp. 2. Identification of transcription factor-binding proteinsFirst, according to biological software and reported research papers, we predicted thetranscription factor binding sites, suggesting that trans-acting factor might be the member ofMEF2protein family. Then, Eukaryotic expression vector pC-MEF2A, pC-MEF2C wereconstructed, by luciferase assay and the concentration gradient experiments, overexpressionMEF2C, but not MEF2A increased Myostatin promoter activity with MEF2motifs by two tosix folds, in both myoblasts and myotubes. The initially evidence proved that the MEF2Ctranscription factor protein should be the one combined to the binding sites. Then, by usingEMSA supershift methods, the identification was proved that MEF2C was the specifictranscription factor binding to the upstream regulatory region of Myostatin promoter.Ultimately, designed and synthesised the fragments of endogenous MEF2C interference, wecarried out real-time quantitative PCR reaction and Western blotting to show thatoverexpression of MEF2C, the mRNA levels of Myostatin gene were significantly increasedtwo to four folds and protein expression levels were meanwhile increased; while silence ofendogenous MEF2C, the mRNA levels and protein expression levels of the Myostatin genewere significantly decreased. These results proved that the transcription factor MEF2C couldmodulate and restrain myogenesis by Myostatin activation and Myostatin-dependent genedevelopment processing in porcine. Our research could provide a potential target site and aneffective transcription factor to regulate Myostatin expression and gave a new researchdirection to explore the functional performance of Myostatin. |
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