Positive and negative cis-acting regulatory elements mediate expression of the mouse vascular smooth muscle...

Segments of the 5'-flanking region of the mouse vascular smooth muscle alpha-actin gene were assayed for promoter activity in transfected mouse BC3H1 myogenic cells and AKR-2B embryonic fibroblasts. The region between -150 and -191 that functions as a positive transcriptional element in myogenic and fibroblastic cells contains a mammalian-specific inverted CC(A/T)6GG-type consensus sequence. Expression was restricted to fully differentiated myogenic cells when an additional sequence spanning -191 to -224 was included in reporter gene constructs. This 33-base pair (bp) negative regulatory element is 70% conserved between the mouse and human genes and contains a 10-bp motif at its 3' end that only partially resembles a CC(A/T)6GG element. Retention of a GGGA motif at the 3' boundary of the 33-bp region is sufficient to maintain full transcriptional repression in fibroblasts and is partly responsible for repression in undifferentiated myoblasts. Complete muscle tissue-restrictive expression requires an additional 8 bp from the CC(A/T)6GG-like element immediately 5' to the GGGA motif, since replacement of this region with an unrelated 10-bp sequence completely eliminated restrictive transcriptional behavior in undifferentiated myoblasts. The distal portion of the 5'-flanking region between -224 and -1074 contains six E-box motifs (CANNTG) and mediates high level transcription only in postconfluent BC3H1 myoblasts. Analysis of reporter gene constructs including either the proximal E-box at -240 or all six E-boxes indicate that the five distal E-boxes are not required for high level transcription. A 724-bp segment of the 5'-flanking region consisting of the proximal E-box flanked upstream by a mammalian-specific 352-bp region was sufficient for maximal transcriptional activation in postconfluent BC3H1 myoblasts. Deletion of the 352-bp region restricts the early transcriptional response to high cell density in temporal studies of promoter activity during BC3H1 myogenic cell differentiation.