Functional analysis of pig myostatin gene promoter with some adipogenesis- and myogenesis-related factors

Myostatin (MSTN) is primarily expressed in muscle and plays an important role in muscle and fat development in pigs. However, there is little information about the regulation of pig MSTN. In order to elucidate whether pig MSTN could be regulated by muscle- and fat- related factors, the porcine MSTN promoter was amplified and cloned into pGL3-basic vector, and transfected into cells to analyze the transcriptional activity of promoter with muscle- and fat-related factors through Dual-lucifer- ase reporter assays. 5 0 -deletion expression showed that there was a negative-regulatory region located between nucleotides -1519 and -1236 bp, and there were some positive-regulatory regions located between -1236 and -568 bp. The longest fragment (1.7 kb) was cotransfected with muscle-related transcription factor myogenic differ- entiation 1 (MyoD), resulting in promoter transcriptional activity upregulation. The fragment was treated by the adipogenic agents (DIM) including dexamethasone, insu- lin, and isobutyl-1-methylxanthine (IBMX). We found that MSTN promoter transcriptional activity can be regulated by IBMX, but not by DIM. CCAAT/enhancer binding protein (C/EBP) a and C/EBPb, two proteins which are induced by DIM during adipogenesis were cotransfected with the 1.7-kb fragment, respectively, resulting in promoter tran- scriptional activity downregulation. Treating the fragment with rosiglitazone which induce the expression of peroxi- some proliferator-activated receptor c (PPARc), resulting in promoter transcriptional activity upregulation. Cotrans- fection experiments confirmed this result. Taken together, we showed that porcine MSTN could be upregulated by IBMX, MyoD, and PPARc but downregulated by C/EBPa and C/EBPb.