A SERUM RESPONSE FACTOR-DEPENDENT TRANSCRIPTIONAL REGULATORY PROGRAM IDENTIFIES DISTINCT SMOOTH-MUSCLE CELL SUBLINEAGES

The SM22 alpha promoter has been used as a model system to define the molecular mechanisms that regulate smooth muscle cell (SMC) specific g ene expression during mammalian development. The SM22 alpha gene is ex pressed exclusively in vascular and visceral SMCs during postnatal dev elopment and is transiently expressed in the heart and somites during embryogenesis. Analysis of the SM22 alpha promoter in transgenic mice revealed that 280 bp of 5' flanking sequence is sufficient to restrict expression of the lacZ reporter gene to arterial SMCs and the myotoma l component of the somites. DNase I footprint and electrophoretic mobi lity shift analyses revealed that the SM22 alpha promoter contains six nuclear protein binding sites (designated smooth muscle elements [SME s]-1 to -6, respectively), two of which bind serum response factor (SR F) (SME-1 and SME-4). Mutational analyses demonstrated that a two-nucl eotide substitution that selectively eliminates SRF binding to SME-3 d ecreases SM22 alpha promoter activity in arterial SMCs by approximatel y 90%. Moreover, mutations that abolish binding of SRF to SME-I and SM E-4 or mutations that eliminate each SME-3 binding activity totally ab olished SM22 alpha promoter activity in the arterial SMCs and somites of transgenic mice. Finally, we have shown that a multimerized copy of SME-4 (bp -190 to -110) when linked to the minimal SM22 alpha promote r (bp -90 to +41) is necessary and sufficient to direct high-level tra nscription in an SMC lineage-restricted fashion. Taken together, these data demonstrate that distinct transcriptional regulatory programs co ntrol SM22 alpha gene expression in arterial versus visceral SMCs, Mor eover, these data are consistent with a model in which combinatorial i nteractions between SRF and other transcription factors that bind to S ME-4 (and that bind directly to SRF) activate transcription of the SM2 2 alpha gene in arterial SMCs.