Identification of an enhancer in the first intron involved in the regulation of mouse vascular smooth muscle α-actin gene

We have cloned and determined nucleotide sequences of the first intron region of the mouse vascular smooth muscle (VSM) α-actin gene since Min et al. (1990) suggested that transcriptional regulatory elements might be located in the first intron. The first intron contains 2697 nucleotides, and consensus donor and acceptor boundary sequences (5′GT-AG3′) for RNA splicing. Comparing the nucleotide sequences of the first intron with the equivalent region of human and chicken VSM α-actin gene, we found that there were relatively highly conserved regions having from 65% to 85% sequence homology. From transient human growth hormone (hGH) reporter gene transfection assays in BC3H1 myogenic cells, we showed that deletion of the 550 bp BfrI fragment containing the homologous region from the first intron showed a 2.5 fold reduction in the level of hGH expression when compared to the plasmid pSB12b-8-hGH. This plasmid contained the 5′-flanking region plus the first exon, the first intron, and part of the second exon ligated to the hGH gene. In addition, the insertion of the 550 bp BfrI fragment upstream of the thymidine kinase (TK) promoter showed transcriptional activity 3 times higher than that of the control plasmid pTKGH. This conserved region contains CArG box, AP-1, Sp1 binding sites, human immunoglobulin kappa enhancer, and polyoma virus enhancer. Taken together, we show that not only the promoter region but also the first intron may be important for the regulation of mouse; VSM α-actin gene expression.