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Am J Physiol Cell Physiol 276: C1420-C1431, 1999;
0363-6143/99 $5.00
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Vol. 276, Issue 6, C1420-C1431, June 1999

Differential activation of the SMalpha A promoter in smooth vs. skeletal muscle cells by bHLH factors

A. Daniel Johnson1 and Gary K. Owens2

1 Department of Biology, Wake Forest University, Winston-Salem, North Carolina 27109; and 2 Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908

E-box/basic helix-loop-helix (bHLH)-dependent regulation of promoters for skeletal muscle-specific genes is well established, but similar regulation of smooth muscle-selective promoters has not been reported. Using transient transfection assays of smooth muscle alpha -actin (SMalpha A) promoter-chloramphenicol acetyltransferase (CAT) reporter constructs in rat vascular smooth muscle cells (SMCs) and L6 skeletal myotubes, we identified two activator elements, smE1 and smE2, with sequences corresponding to E-box (5'-CAnnTG-3') motifs. In L6 myotubes, 4-bp mutations of smE1 or smE2 E-box motif alone completely abolished promoter activity. In contrast, mutation of smE1 and smE2 was required to reduce promoter activity in SMCs. Supershift analyses identified a myogenin-containing complex as the predominant smE1 and smE2 binding activity in skeletal muscle, and myogenin overexpression transactivated the promoter. Supershift analyses with SMC extracts demonstrated that the bHLH protein upstream stimulatory factor (USF) bound smE1, and USF overexpression transactivated the promoter in an smE1-dependent manner. In summary, our results provide novel evidence implicating E-box elements in directing expression of the SMalpha A promoter through distinct bHLH factor complexes in skeletal vs. smooth muscle.

alpha -actin promoter; basic helix-loop-helix protein; E-box; upstream stimulatory factor; myogenin


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