Wang G, Yeh H I, Lin J J
Department of Biological Sciences, University of Iowa, Iowa City 52242-1324.
J Biol Chem. 1994 Dec 2;269(48):30595-603.
To study the transcriptional regulation of the rat cardiac troponin T (cTnT) gene, chimeric genes composed of the upstream region (-757 to +193 base pairs (bp) relative to the transcription initiation site) of the cTnT gene and the bacterial chloramphenicol acetyltransferase (CAT) gene were constructed and transfected into primary cultures of neonatal cardiomyocytes and cardiac fibroblasts. Deletion analysis showed that a 41-bp fragment (-249 to -209 bp) containing the MEF-2-like motif is an essential element for minimal cardiac-specific expression of the rat cTnT gene. The proximal promoter (-208 to -1 bp) contains two consensus CArG boxes, one M-CAT motif, one AP2 site, and one TATA box. The construct (cTNT-208) composed of the CAT reporter gene driven by this proximal promoter did not show cardiac muscle-specific expression. Ligation of consensus MEF-2-like sequence into the upstream of this chimera only partially increased its ability to express in cardiomyocytes. These results suggest that the spacing among MEF-2-like motif and proximal promoter and/or the flanking sequences of the MEF-2-like motif are important in determining cardiac muscle-specific expression. By footprint analysis with a DNA fragment (-303 to +6 bp), we identified three novel regions (called A, B, and C) protected by protein extract from rat hearts, in addition to the known motifs such as MEF-2, M-CAT, and CArG. Gel retardation with the probe (-235 to -141 bp), containing the MEF-2-like motif, one of the CArG boxes, and the C region, or the 41-bp probe (-249 to -209 bp), containing the MEF-2-like motif, revealed different DNA-protein complexes formed by heart, skeletal muscle, and liver extracts. By using DNA affinity purification, DNA-binding proteins with apparent molecular masses of 22-26 kDa were identified from rat heart extract but not from skeletal and liver extracts, suggesting the involvement of cardiac-specific proteins in regulating the cTnT gene expression.
为研究大鼠心肌肌钙蛋白T（cTnT）基因的转录调控，构建了由cTnT基因上游区域（相对于转录起始位点为-757至+193碱基对（bp））和细菌氯霉素乙酰转移酶（CAT）基因组成的嵌合基因，并将其转染至新生心肌细胞和心脏成纤维细胞的原代培养物中。缺失分析表明，一个包含MEF-2样基序的41-bp片段（-249至-209 bp）是大鼠cTnT基因最小心脏特异性表达的必需元件。近端启动子（-208至-1 bp）包含两个共有CArG框、一个M-CAT基序、一个AP2位点和一个TATA框。由该近端启动子驱动的CAT报告基因构建体（cTNT-208）未显示出心肌特异性表达。将共有MEF-2样序列连接到该嵌合体上游仅部分增强了其在心肌细胞中的表达能力。这些结果表明，MEF-2样基序与近端启动子之间的间距和/或MEF-2样基序的侧翼序列在决定心肌特异性表达中很重要。通过用DNA片段（-303至+6 bp）进行足迹分析，我们除了鉴定出已知基序如MEF-2、M-CAT和CArG外，还鉴定出大鼠心脏蛋白提取物保护的三个新区域（称为A、B和C）。用包含MEF-2样基序、一个CArG框和C区域的探针（-235至-141 bp）或包含MEF-2样基序的41-bp探针（-249至-209 bp）进行凝胶阻滞分析，揭示了心脏、骨骼肌和肝脏提取物形成的不同DNA-蛋白质复合物。通过使用DNA亲和纯化，从大鼠心脏提取物中鉴定出表观分子量为22-26 kDa的DNA结合蛋白，而从骨骼肌和肝脏提取物中未鉴定出，这表明心脏特异性蛋白参与调节cTnT基因表达。
