Identification of cis elements in the cardiac troponin T gene conferring specific expression in cardiac muscle of transgenic mice
Identification of cis elements in the cardiac troponin T gene conferring specific expression in cardiac muscle of transgenic mice
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DOI：
10.1161/01.res.86.4.478
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发表时间：
2000-03-03
影响因子：
20.1
通讯作者：
Lin, JJC
中科院分区：
文献类型：
--
作者：
Wang, Q;Sigmund, CD;Lin, JJC
为了研究心肌基因表达调控的潜在机制，分析了携带大鼠心肌肌钙蛋白T近端启动子（距转录起始位点约497 bp）与LacZ或氯霉素乙酰转移酶（CAT）报告基因融合的转基因小鼠。在该启动子内，在模块D（-335至-289 bp）和F（-249至-209 bp）的2个位点处发现高度的序列同源性。这两个区域都含有至少一个TCTG（G/C）直接重复序列和一个富含A/T的位点，而只有F模块具有肌肉增强因子2（MEF 2）样基序。当仅MEF 2核心序列突变时，未观察到CAT转基因表达的显著降低。然而，当MEF 2核心序列及其侧翼TCTGG位点突变（Mut 5）时，心脏中CAT转基因表达显著降低，并且还观察到转基因的异位表达。当将突变引入该启动子以破坏D模块（MutD）中的所有上游TCTG（G/C）直接重复时，CAT表达保持心脏特异性，但表达水平显著降低。心脏特异性转基因表达的松弛在携带双突变的转基因小鼠中变得更加严重（Mut[D + 5]）。此外，心脏中的CAT活性几乎消失。这些结果表明，D和F模块在确定心脏中的表达水平方面具有相加功能，并且只有F模块赋予心脏特异性表达。
To investigate the underlying mechanism regulating cardiac gene expression, transgenic mice carrying the rat cardiac troponin T proximal promoter (-497 bp from the transcriptional start site) fused to a LacZ or chloramphenicol acetyltransferase (CAT) reporter gene were analyzed, The LacZ expression pattern throughout development was very similar to that of the endogenous cardiac troponin T gene. Within this promoter, a high degree of sequence homology was found at 2 sites, modules D (-335 to -289 bp) and F (-249 to -209 bp). Both regions contain at least a TCTG(G/C) direct repeat and an A/T-rich site, whereas only the F module has a muscle enhancer factor 2 (MEF2)-like motif. No significant decrease in CAT transgene expression was observed when only the MEF2 core sequence was mutated. However, when the MEF2 core sequence and its flanking TCTGG site were mutated (Mut5), CAT transgene expression was significantly decreased in the heart, and ectopic expression of the transgene was also observed. When mutations were introduced into this promoter to destroy all upstream TCTG(G/C) direct repeats in the D module (MutD), CAT expression remained cardiac specific, but the expression level was dramatically decreased. Relaxation of cardiac-specific transgene expression became even more severe in transgenic mice carrying double mutations (Mut[D + 5]). In addition, CAT activity in the heart was nearly abolished. These results suggest that D and F modules have an additive function in determining the level of expression in the heart and only the F module confers cardiac-specific expression.