Cell-specific transcription of the smooth muscle gamma-actin gene requires both positive- and negative-acting cis...

We have characterized the function of putative regulatory sequences upon the smooth muscle transcription of the SMGA gene, using promoter deletion analyses. We demonstrate that the SMGA promoter contains four domains: a basal promoter (-1 to -100), a smooth muscle specifier sequence (-100 to -400), a negative regulator (-400 to -1000), and a smooth muscle-specific modulator (-1000 to -2000). The basal or core promoter supports equivalent transcription in both smooth and skeletal muscle cells. Addition of sequences containing a CArG motif juxtaposed to an E-box element stimulates smooth muscle transcription by five- to sixfold compared to skeletal muscle. This smooth muscle-specific segment is maintained for about 200 bp, after which is a segment of DNA that appears to inhibit the transcriptional capacity of the SMGA promoter in smooth muscle cells. Within the boundary between the smooth muscle specifier and negative regulatory sequences (-400 to -500) are three E-box elements. The smooth muscle modulator domain contains two CArG elements and multiple E-boxes. When added to the SMGA promoter it causes an additional three- to fivefold increase in smooth muscle-specific transcription over that stimulated by the smooth muscle specifier domain. Thus, our studies show that the appropriate cell-specific transcription of the SMGA gene involves complex interactions directed by multiple cis-acting elements. Moreover, our characterization of a cell culture system employing embryonic gizzard smooth muscle cells lays the foundation for further molecular analyses of factors that regulate or control SMGA and other smooth muscle genes during differentiation.