Evidence for serum response factor-mediated regulatory networks governing SM22α transcription in smooth, skeletal, and cardiac muscle cells

SM22α is an adult smooth muscle-specific protein that is expressed in the smooth, cardiac, and skeletal muscle lineages during early embryogenesis before becoming restricted specifically to all vascular and visceral smooth muscle cells (SMC) in late fetal development and adulthood. We have used the SM22α gene as a marker to define the regulatory mechanisms that control muscle-specific gene expression in SMCs. Previously, we reported that the 445-base-pair promoter of SM22α was sufficient to direct transcription of a lacZ reporter gene in early cardiac and skeletal muscle cell lineages and in a subset of arterial SMCs, but not in venous nor visceral SMCs in transgenic mice. Here we describe two evolutionarily conserved CArG (CC(A/T)₆GG) boxes in the SM22α promoter, both of which are essential for full promoter activity in cultured SMCs. In contrast, only the promoter-proximal CArG box is essential for specific expression in developing smooth, skeletal, and cardiac muscle lineages in transgenic mice. Both CArG boxes bind serum response factor (SRF), but SRF binding is not sufficient for SM22α promoter activity, since overexpression of SRF in the embryonal teratocarcinoma cell line F9, which normally expresses low levels of SRF, fails to activate the promoter. However, a chimeric protein in which SRF was fused to the transcription activation domain of the viral coactivator VP16 is able to activate the SM22α promoter in F9 cells. These results demonstrate the SM22α promoter-proximal CArG box is a target for the regulatory programs that confer smooth, skeletal, and cardiac muscle specificity to the SM22α promoter and they suggest that SRF activates SM22α transcription in conjunction with additional regulatory factors that are cell type-restricted.