The 5′-flanking region of the mouse vascular smooth muscle α-actin gene contains evolutionarily conserved sequence motifs within a functional promoter

The 5′-flanking, 5′-untranslated, and amino-terminal protein coding regions of the single-copy 13-kilobase mouse vascular smooth muscle (VSM) α-actin gene have been cloned and sequenced. Respectively, there is 73 and 89% homology from the start of transcription (+1) to a point 206 base pairs upstream when comparing mouse to chicken and mouse to human VSM α-actin 5′-flanking region sequences. Two proximal 16-base pair motifs containing putative cis- acting regulatory elements having the configuration CC(A/T)₆GG were found to be 100% conserved and present in the same position upstream from the transcription start site in all three species. A third more distal CC(A/T)₆GG-like motif was 100% conserved between only the mouse and human genes whereas a fourth motif was unique to the mouse gene. The two upstream motifs may be important in controlling VSM α-actin gene transcription in mammals. Cell transfection assays using hGH reporter gene fusion plasmids showed that all four CC(A/T)₆GG elements were required for tissue-specific, core promoter activity and were able to direct hGH expression in both mouse BC3H1 myogenic cells and early-passage rabbit aortic smooth muscle cells. The core promoter was not active in mouse fibroblasts suggesting that the region between -372 and -143 may mediate tissue-restrictive expression of the VSM α-actin gene. A putative "cell density responsive element" may be located between -1074 and -372 since fusion plasmids containing this portion of the VSM α-actin 5′-flanking region were significantly more active in promoting hGH expression in inducible, density-activated BC3H1 myoblasts compared to aortic smooth muscle cells which are largely constitutive for VSM α-actin expression.