Genomic cloning and promoter analysis of aortic preferentially expressed gene-1. Identification of a vascular smooth muscle-specific promoter mediated by an E box motif.
Aortic preferentially expressed gene-1 (APEG-1) was originally identified as a 1.4-kilobase (kb) transcript preferentially expressed in differentiated vascular smooth muscle cells (VSMC). Its expression is markedly down-regulated in de-differentiated VSMC, suggesting a role for APEG-1 in VSMC differentiation. We have now determined that APEG-1 is a single-copy gene in the human, rat, and mouse genomes and have mapped human APEG-1 to chromosome 2q34. To study the molecular mechanisms regulating its expression, we characterized the genomic organization and promoter of mouse APEG-1. APEG-1 spans 4.5 kb in the mouse genome and is composed of five exons. Using reporter gene transfection analysis, we found that a 2. 7-kb APEG-1 5'-flanking sequence directed a high level of promoter activity only in VSMC. Its activity was minimal in five other cell types. A repressor region located within an upstream 685-base pair sequence suppressed the activity of this 2.7-kb promoter. Further deletion and mutation analyses identified an E box motif as a positive regulatory element, which was bound by nuclear protein prepared from VSMC. In conjunction with its flanking sequence, this E box motif confers VSMC-specific enhancer activity to a heterologous SV40 promoter. To our knowledge, this is the first demonstration of an E box motif that mediates gene expression restricted to VSMC.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015415:Biomarkers; D002478:Cells, Cultured; D002889:Chromosomes, Human, Pair 2; D003001:Cloning, Molecular; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009219:Myosin-Light-Chain Kinase; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases; D051381:Rats; D017384:Sequence Deletion