Molecular Cloning, Characterization, and Promoter Analysis of the Mouse Crp2/SmLim Gene

Several members of the LIM protein family have important roles in development and differentiation. We recently isolated a rat cDNA encoding a new member of this family, CRP2/SmLIM, that contains two LIM domains and is expressed preferentially in vascular smooth muscle cells (VSMC). To study the molecular mechanisms that regulate VSMC-specific transcription of the Crp2/SmLim gene, we cloned the cDNA and gene of mouse Crp2/SmLim. Mouse Crp2/SmLim is a single copy gene of six exons and five introns spanning approximately 20 kilobases of genomic DNA. By 5'-rapid amplification of cDNA ends and S1 nuclease protection assay, we determined that the transcription start site is an A residue 80 base pairs 5' of the translation initiation codon. A TATA-like sequence is located 27 base pairs 5' of the transcription start site, and there are potential cis-acting elements (GATA, Sp1, AP-2, E box, CCAC box, and GArC motif) in the 5'-flanking sequence. In transient transfection assays in rat aortic smooth muscle cells in primary culture, 5 kilobases of the Crp2/SmLim 5'-flanking sequence generated a high level of luciferase reporter gene activity. By deletion analysis and gel mobility shift assay, we found that the region between bases -74 and -39 of this 5 kilobase DNA fragment binds Sp1 and confers basal promoter activity in the Crp2/SmLim gene. In vitro, the 5-kilobase fragment was active in multiple cell types. In vivo, however, the 5-kilobase fragment directed high level expression of the lacZ reporter gene preferentially in the VSMC of transgenic mice, indicating the presence of VSMC-specific element(s) in this fragment.