title: Molecular cloning, characterization, and promoter analysis of the mouse Crp2/SmLim gene. Preferential expression of its promoter in the vascular smooth muscle cells of transgenic mice.
abstract: 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.
authors.affiliation: Cardiovascular Biology Laboratory, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
authors.forename: S F
authors.initials: SF
authors.lastname: Yet
authors.forename: S C
authors.initials: SC
authors.lastname: Folta
authors.forename: M K
authors.initials: MK
authors.lastname: Jain
authors.forename: C M
authors.initials: CM
authors.lastname: Hsieh
authors.forename: K
authors.initials: K
authors.lastname: Maemura
authors.forename: M D
authors.initials: MD
authors.lastname: Layne
authors.forename: D
authors.initials: D
authors.lastname: Zhang
authors.forename: P B
authors.initials: PB
authors.lastname: Marria
authors.forename: M
authors.initials: M
authors.lastname: Yoshizumi
authors.forename: M T
authors.initials: MT
authors.lastname: Chin
authors.forename: M A
authors.initials: MA
authors.lastname: Perrella
authors.forename: M E
authors.initials: ME
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000235:genetics*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D060588:LIM Domain Proteins; D007763:Lac Operon; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D016329:Sp1 Transcription Factor / Q000378:metabolism
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; C484376:Csrp2 protein, mouse; C484375:Csrp2 protein, rat; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D060588:LIM Domain Proteins; D009124:Muscle Proteins; D009687:Nuclear Proteins; D016329:Sp1 Transcription Factor