EVIDENCE FOR SERUM RESPONSE FACTOR-MEDIATED REGULATORY NETWORKS GOVERNING SM22-ALPHA TRANSCRIPTION IN SMOOTH, SKELETAL, AND CARDIAC-MUSCLE-CELLS

SM22 alpha is an adult smooth muscle-specific protein that is expresse d in the smooth, cardiac, and skeletal muscle lineages during early em bryogenesis before becoming restricted specifically to all vascular an d visceral smooth muscle cells (SMC) in late fetal development and adu lthood. We have used the SM22 alpha gene as a marker to define the reg ulatory mechanisms that control muscle-specific gene expression in SMC s. Previously, we reported that the 445-base-pair promoter of SM22 alp ha was sufficient to direct transcription of a lacZ reporter gene in e arly cardiac and skeletal muscle cell lineages and in a subset of arte rial SMCs, but not in venous nor visceral SMCs in transgenic mice. Her e we describe two evolutionarily conserved CArG (CC(A/T)(6)GG) boxes i n the SM22 alpha promoter, both of which are essential for full promot er activity in cultured SMCs. In contrast, only the promoter-proximal CArG box is essential for specific expression in developing smooth, sk eletal, and cardiac muscle lineages in transgenic mice. Both CArG boxe s bind serum response factor (SRF), but SRF binding is not sufficient for SM22 alpha promoter activity, since overexpression of SRF in the e mbryonal teratocarcinoma cell line F9, which normally expresses low le vels of SRF, fails to activate the promoter. However, a chimeric prote in in which SRF was fused to the transcription activation domain of th e viral coactivator VP16 is able to activate the SM22 alpha promoter i n F9 cells. These results demonstrate the SM22 alpha promoter-proximal CArG box is a target for the regulatory programs that confer smooth, skeletal, and cardiac muscle specificity to the SM22 alpha promoter an d they suggest that SRF activates SM22 alpha transcription in conjunct ion with additional regulatory factors that are cell type-restricted. (C) 1997 Academic Press.