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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.

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The aldolase isozyme family is composed of three members, A, B, and C, which are encoded by separate genes. The proteins are expressed in a tissue-restricted manner during development and in the adult. To elucidate the regulation of aldolase mRNA in the mouse liver, we analyzed its expression by a number of methods including Northern blot, RNA dot blot, and nuclear run-on assays. Our experiments demonstrate that the expression of aldolase A in the liver is primarily regulated by post-transcriptional control. In contrast, we found that changes in the level of aldolase B mRNA are due to changes in the rate of initiation of transcription. In addition, we examined the regulation of aldolase expression in the adult kidney. We found that although the kidney has eight times more aldolase B than the liver, the rate of initiation of transcription is similar in both tissues. Also, the rate of initiation of transcription of aldolase A is the same in the adult kidney and liver although there is 40 times more steady state aldolase A mRNA in the kidney than in the liver.