Autonomous activity of the alternate aldolase A muscle promoter is maintained by a sequestering mechanism

Abstract

The mouse aldolase A gene contains two closely-spaced alternate promoter/first exons. The more distal of the two, the M promoter, is muscle-specific while the 3' promoter, the H promoter, is expressed constitutively. Various segments from these promoter regions were linked to a reporter gene and used to transfect the myogenic cell line C2C12 and the hepatoma cell line BWTG3. A muscle-specific enhancer, MEN1, responsible for 80% of promoter M activity and containing 4 consensus MyoD binding sites was localized between -2578 to -2723 of the M promoter. Another muscle-specific enhancer and a restrictive element, MEN2/MSE, were found in the interval -1100 to -350. The MSE restrictive element was found to prohibit inappropriate up-regulation of the M promoter by selectively sequestering it from H promoter elements in both myoblasts and myotubes. Among the H promoter elements was found an enhancer, HEN, situated between -533 and -200 which did not function in myotubes. These studies also show that H promoter elements can act synergistically with a non-specific element, MAE, located between -350 and -130 of the M cap site greatly stimulating M promoter transcription in all cell types when the MSE restrictive element was absent. Through the analysis of interactions between these elements and the aldolase A and HSV-TK promoters we showed that neither the enhancers nor the promoter proximal sequences by themselves contain adequate information to reproduce the native pattern of aldolase A promoter modulation. Rather, the sequestering of the M promoter by the MSE restrictive element and the relative positioning and context of promoters M and H appear critical to the regulated expression of aldolase A.