STRUCTURE AND EXPRESSION OF THE HUMAN SM22-ALPHA GENE, ASSIGNMENT OF THE GENE TO CHROMOSOME-11, AND REPRESSION OF THE PROMOTER ACTIVITY BY CYTOSINE DNA METHYLATION

To investigate the molecular mechanisms that control expression of smo oth muscle cell (SMC) differentiation genes, we have isolated the huma n SM22 alpha gene, which is composed of five exons and four introns, s panning an approximately 6-kilobase (kb) genomic DNA at chromosome reg ion 11q23.2. Expression of the SM22 alpha messenger RNA was detected i n serum-stimulated cell cultures including SMC, undifferentiated skele tal muscle-lineage cells, and fibroblasts, and it was down-regulated i n SMC of balloon-injured atheromatous human vessels. A major transcrip tion start site of the SM22 alpha gene is located at 75 base-pairs (bp ) upstream of the ATG start codon. Analysis of the 2.6 kb 5'-upstream sequence demonstrated that two CArG/SRF-boxes and two GC-box/Sp1-bindi ng sites were present at bp -147 and -274, and at bp -233 and -1635, r espectively. The nucleotide sequences of the two CArG/SRF-boxes and th e proximal GC-box/Sp1-binding site are 100% conserved with those of th e murine SM22 alpha genes [Solway, J., Seltzer, J., Samaha, F.F., Kim, S., Alger, L.E., Niu, Q., Morriesey, E.E., Ip, H.S., and Parmacek, M. S. (1995) J. Biol. Chem. 270, 13460-13469; Kemp, P.R., Osbourn, J.K., Grainger, D.J., and Metcalf, C. (1995) Biochem. J. 310, 1037-1043]. Ce ll transfection assays using a luciferase reporter gene construct cont aining the 455-bp 5'-flanking region (positions -26 to -480) showed th at methylation of the CpG dinucleotides within this segment reduces it s transcriptional activity. The results imply a novel mechanism for tr anscriptional control of the SMC differentiation-specific gene promote r.