Control of Cardiac Troponin T Gene Expression

心肌肌钙蛋白 T 基因表达的控制

• 批准号: 6853554
• 负责人: Jim Jung-Ching Lin
• 金额: $ 25.81万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6853554
• 关键词: DNA binding protein; cardiac myocytes; cardiogenesis; cell differentiation; cytoskeletal proteins; developmental genetics; gel mobility shift assay; gene expression; genetic promoter element; genetically modified animals; laboratory mouse; laboratory rabbit; laboratory rat; molecular cloning; muscle proteins; nucleic acid repetitive sequence; protein binding; protein structure function; recombinant proteins; tissue /cell culture; troponin

DESCRIPTION (provided by applicant): The overall goal of this project is to understand how cardiac muscle cells control their cardiac-specific gene expression. An understanding of cardiac-specific gene expression is fundamental to an eventual understanding of the molecular mechanisms that govern cardiac muscle differentiation and pathology. We previously show that the transgene expression driven by rat cardiac troponin T (cTnT) proximal promoter is faithfully recapitulated the endogenous cTnT expression throughout the embryonic development and the adult. This promoter contains two highly homologous modules, D2 and F. Each of which has a TCTG(G/C) direct repeat and an A/T-rich sequence, recognized by a cardiac-specific 42 kDa proteins and a ubiquitous high mobility group 2 (HMG2) protein, respectively. Additionally, F contains a MEF2-like motif, which has an A/T-rich core. Mutational analyses suggest that D2 acts as an enhancer but cannot totally substitute for the F function. Overexpression of HMG2 has differential effects on the promoter in cardiomyocytes versus fibroblasts, suggesting that HMG2 together with tissue-specific factors could direct a stimulatory or inhibitory effect on the cTnT gene expression in heart or non-heart tissue, respectively. This hypothesis is consistent with that a 5-bp change in the F module destroyed the direct repeat and A/T-rich site leads to a decrease in cardiac transgene expression and simultaneously an increase in ectopic transgene expression. Thus, the specific aims are: (1) To test whether F module alone is sufficient to confer the cardiac-specific expression in transgenic mice; (2) To investigate the mechanism by which HMG2 influences the cTnT promoter activity in cardiac and non-cardiac tissues. We will first use ligase-mediated circularization assay and circular permutation GMSA to test whether HMG2 can bend short DNA fragment containing D2 or F module. The interaction between recombinant HMG2 and the direct repeat binding proteins (DRBPs) purified from cardiac and non-cardiac extracts will be further evaluated in terms of DNA binding affinity in GMSA and promoter activity in transactivation experiments; and (3) To clone and characterize DRBPs that bind to novel TCTG(G/C) direct repeat. We anticipate that cardiac DRBPs should have molecular mass of 42 kDa, while non-cardiac DRBPs should have different size. Developmental expression patterns of these DRBPs will be determined by in situ hybridization and immunohistochemical studies.

描述（由申请人提供）：该项目的总体目标是了解心肌细胞如何控制其心脏特异性基因表达。 对心脏特异性基因表达的理解是对控制心肌分化和病理的分子机制的最终理解。 我们先前表明，由大鼠心脏肌钙蛋白T（CTNT）驱动的转基因表达忠实地概括了整个胚胎发育和成人的内源性CTNT表达。 该启动子包含两个高度同源的模块D2和F。每个模块都有TCTG（g/c）直接重复和一个富含A/T的序列，分别由心脏特异性的42 kDa蛋白和无处不在的高迁移率组2（HMG2）蛋白识别。 此外，F包含一个类似于MEF2的主题，该图案具有富含A/T的核心。 突变分析表明，D2充当增强子，但不能完全代替F函数。 HMG2的过表达对心肌细胞中的启动子与成纤维细胞具有差异作用，这表明HMG2与组织特异性因子一起可以分别指导对心脏或非心脏组织中CTNT基因表达的刺激或抑制作用。 该假设与F模块的5 bp变化破坏了直接重复，并且富含A/T的位点导致心脏转基因表达的降低，并且同时增加了异位转基因表达。 因此，具体目的是：（1）测试单独的F模块是否足以赋予转基因小鼠的心脏特异性表达； （2）研究HMG2影响心脏和非心脏组织中CTNT启动子活性的机制。 我们将首先使用连接酶介导的圆形化测定法和圆形置换GMSA来测试HMG2是否可以弯曲含有D2或F模块的短DNA片段。 重组HMG2与从心脏和非心脏提取物中纯化的直接重复重复结合蛋白（DRBP）之间的相互作用将根据GMSA中的DNA结合亲和力和反式激活实验中的启动子活性进一步评估； （3）克隆并表征与新型TCTG（g/c）直接重复结合的DRBP。 我们预计心脏DRBP的分子质量应为42 kDa，而非心脏DRBP应具有不同的大小。 这些DRBP的发育表达模式将由原位杂交和免疫组织化学研究确定。