Key result
Two enhancer sequences upstream of the mouse alpha-cardiac actin gene increased transcription 100-fold, though myogenic factors alone were insufficient to activate transcription.
Effect estimate: 100-fold increase
The study identifies two muscle-specific enhancer sequences upstream of the mouse alpha-cardiac actin gene, demonstrating that while an E box is essential, myogenic factors alone are insufficient for full activation.
No immediate clinical impact; leaves open cofactor requirements for cardiac actin enhancer function in vivo.
A DNase I-hypersensitive site analysis of the 5'-flanking region of the mouse alpha-cardiac actin gene with muscle cell lines derived from C3H mice shows the presence of two such sites, at about -5 and -7 kb. When tested for activity in cultured cells with homologous and heterologous promoters, both sequences act as muscle-specific enhancers. Transcription from the proximal promoter of the alpha-cardiac actin gene is increased 100-fold with either enhancer. The activity of the distal enhancer in C2/7 myotubes is confined to an 800-bp fragment, which contains multiple E boxes. In transfection assays, this sequence does not give detectable transactivation by any of the myogenic factors even though one of the E boxes is functionally important. Bandshift assays showed that MyoD and myogenin can bind to this E box. However, additional sequences are also required for activity. We conclude that in the case of this muscle enhancer, myogenic factors alone are not sufficient to activate transcription either directly via an E box or indirectly through activation of genes encoding other muscle factors. In BALB/c mice, in which cardiac actin mRNA levels are 8- to 10-fold lower, the alpha-cardiac actin locus is perturbed by a 9.5-kb insertion (I. Garner, A. J. Minty, S. Alonso, P. J. Barton, and M. E. Buckingham, EMBO J. 5:2559-2567, 1986). This is located at -6.5 kb, between the two enhancers. The insertion therefore distances the distal enhancer from the promoter and from the proximal enhancer of the bona fide cardiac actin gene, probably thus perturbing transcriptional activity.
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Biben et al. (1994) studied this question. Enhancer sequences upstream of the cardiac actin gene was evaluated on Transcription from the proximal promoter (100-fold increase). Two enhancer sequences upstream of the mouse alpha-cardiac actin gene increased transcription 100-fold, though myogenic factors alone were insufficient to activate transcription.
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