Cloning Of Myomaker Gene 5' Regulatory Region In Sheep And Effects Of GC And (n)GRE On Its Transcriptional Regulatory Activity

Myomaker is a muscle-specific membrane protein which plays an important role in the regulation of myoblasts fusion and its transcriptional regulatory mechanism has not yet been elucidated till now. At present, the myomaker gene promoter sequence of sheep is only predicted but it has not been testified by studies. In pretest, glucocorticoids (GC) response elements such as GRE and nGRE were found in the predicted myomaker gene 5'regulatory region of sheep by bioinformatics analysis. Therefore, this study aimed to further investigate the effects of GC and (n)GRE on the transcriptional regulatory activity of sheep myomaker gene 5'regulatory region, which can lay a foundation for revealing the transcriptional regulatory mechanism of this gene and provide basis for the healthy and efficient farming of domestic animals and the prevention and cure of human muscle-related diseases.In this study, the 1585bp 5'rgulatory region of sheep myomaker gene was amplified and cloned for the first time. Many such potential regulatory elements as GRE and nGRE were predicted by bioinformatics software. Then using EGFP as report gene, the wild type and (n)GRE-mutant MyomakerPro-EGFP vectors were constructed and were transfected into C2C12 cells. The indexes were detected by fluorescence observation and measurement, quantitative PCR and EMSA. Results showed that:1) The mRNA expression of EGFP in differentiated cells was significantly higher than that in undifferentiated cells after transfected for 24h but it was decreased with the extension of differentiation time. Compared with the group transfected for 24h, the mRNA expression of EGFP in undifferentiated cells had a significant increase after transfected for 72h.2) Effects of dexamethasone (Dex, a synthetic glucocorticoid) on EGFP mRNA expression varied with the change of its doses and the cellular status. 10nM Dex significantly increased the mRNA expression of EGFP while 100 or 1000nM Dex significantly reduced it in undifferentiated cells after treated with Dex for 72h. Three doses of Dex obviously increased the mRNA expression of EGFP in differentiated cells after treated with Dex for 72h.3) Effects of Dex on EGFP mRNA expression were blocked by GC receptor antagonist (RU486) in undifferentiated cells. However, RU486 enhanced the effects of Dex in differentiated cells, which might be associated with the complexity of Dex function.4) The mRNA expression of EGFP was affected by the mutation of GRE and nGRE. EMSA results confirmed that there were specific transcription factors named GC receptor which could combine with (n)GRE elements in nucleoprotein of fetal sheep muscle. In addition, the relationship between the functional mechanism of GC with GC receptor and (n)GRE was proved once again by the EMSA results of cells in different treatment groups.In summary, the regulatory role of GC on the myomaker gene 5' regulatory region of sheep was related to the dose of GC and the status of cells and was mediated at least partially by the combination of GC receptor and (n)GRE elements.