Zilberman A, Dave V, Miano J, Olson E N, Periasamy M
Division of Cardiology and Cardiovascular Research Center, University of Cincinnati, Ohio 45267, USA.
Circ Res. 1998 Mar 23;82(5):566-75. doi: 10.1161/01.res.82.5.566.
In recent years, significant progress has been made toward understanding skeletal muscle development. However, the mechanisms that regulate smooth muscle development and differentiation are presently unknown. To better understand smooth muscle-specific gene expression, we have focused our studies on the smooth muscle myosin heavy chain (SMHC) gene, a highly specific marker of differentiated smooth muscle cells. The goal of the present study was to isolate and characterize the mouse SMHC gene promoter, since the mouse promoter would be particularly suited for in vivo promoter analyses in transgenic mice and would serve as a tool for targeting genes of interest into smooth muscle cells. We report 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 transcription factors. Transient transfection analysis of promoter deletion constructs in primary cultures of smooth muscle cells showed that the region between -1208 and -1050 bp is critical for maximal SMHC promoter activity. A comparison of SMHC promoter sequences from mouse, rat, and rabbit revealed the presence of a highly conserved region located between -967 and -1208 bp. This region includes three CArG/CArG*-like elements, two SP-1 binding sites, a NF-1-like element, an Nkx2-5 binding site, and an Elk-1 binding site. Gel mobility shift assay and DNase I footprinting analyses show that all three CArG/CArG*-like elements can form DNA-protein complexes with nuclear extract from vascular smooth muscle cells. Protein binding to the CArG* 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 part of the protein complex. In addition, we show that cotransfection with the SRF dominant-negative mutant expression vector abolishes SMHC promoter activity, suggesting that SRF protein plays a critical role in SMHC gene regulation.
近年来，在理解骨骼肌发育方面已取得显著进展。然而，目前尚不清楚调节平滑肌发育和分化的机制。为了更好地理解平滑肌特异性基因表达，我们将研究重点放在平滑肌肌球蛋白重链（SMHC）基因上，它是分化的平滑肌细胞的高度特异性标志物。本研究的目的是分离并鉴定小鼠SMHC基因启动子，因为小鼠启动子特别适合用于转基因小鼠的体内启动子分析，并可作为将感兴趣的基因靶向平滑肌细胞的工具。我们在此报告小鼠SMHC启动子及其5'侧翼区域的分离和鉴定。对启动子2.6 kb部分的DNA序列分析确定了几个已知转录因子的潜在结合位点。平滑肌细胞原代培养物中启动子缺失构建体的瞬时转染分析表明，-1208至-1050 bp之间的区域对于最大程度的SMHC启动子活性至关重要。对小鼠、大鼠和兔子的SMHC启动子序列进行比较，发现-967至-1208 bp之间存在一个高度保守的区域。该区域包括三个CArG/CArG样元件、两个SP-1结合位点、一个NF-1样元件、一个Nkx2-5结合位点和一个Elk-1结合位点。凝胶迁移率变动分析和DNase I足迹分析表明，所有三个CArG/CArG样元件都能与血管平滑肌细胞核提取物形成DNA-蛋白质复合物。与血清反应元件或心脏α-肌动蛋白基因的真实CArG元件均可竞争蛋白质与CArG*元件的结合。使用血清反应因子（SRF）抗体，我们证明SRF是蛋白质复合物的一部分。此外，我们表明与SRF显性负突变体表达载体共转染可消除SMHC启动子活性，这表明SRF蛋白在SMHC基因调控中起关键作用。
