EVOLUTIONARILY CONSERVED PROMOTER REGION CONTAINING CARG-STAR-LIKE ELEMENTS IS CRUCIAL FOR SMOOTH-MUSCLE MYOSIN HEAVY-CHAIN GENE-EXPRESSION

In recent years, significant progress has been made toward understandi ng skeletal muscle development. However, the mechanisms that regulate smooth muscle development and differentiation are presently unknown. T o better understand smooth muscle-specific gene expression, we have fo cused our studies on the smooth muscle myosin heavy chain (SMHC) gene, a highly specific marker of differentiated smooth muscle cells. The g oal of the present study was to isolate and characterize the mouse SMH C gene promoter, since the mouse promoter would be particularly suited for in vivo promoter analyses in transgenic mic and would serve as a tool for targeting genes of interest into smooth muscle cells. We repo rt here the isolation and characterization of the mouse SMHC promoter and its 5' flanking region. DNA sequence analysis of a 2.6-kb portion of the promoter identified several potential binding sites for known t ranscription factors. Transient transfection analysis of promoter dele tion constructs in primary cultures of smooth muscle cells showed that the region between -1208 and -1050 bp is critical for maximal SMHC pr omoter activity. A comparison of SMHC promoter sequences from mouse, r at, and rabbit revealed the presence of a highly conserved region loca ted between -967 and -1208 bp. This region includes three CArG/CArG(st ar)-like elements, two SP-1 binding sites, a NF-1-like element, an Nkx 2-5 binding site, and an Elk-1 binding site. Gel mobility shift assay and DNase I footprinting analyses show that all three CArG/CArG(star)- like elements can be competed out by either serum response element or by an authentic CArG element from the cardiac alpha-actin gene. Using a serum response factor (SRF) antibody, we demonstrate that SRF is par t of the protein complex. In addition, we show that cotransfection wit he SRF dominant-negative mutant expression vector abolishes SMHC promo ter activity, suggesting that SRF protein plays a critical role in SMH C gene regulation.