Control of Cardiac Troponin T Gene Expression

描述（由申请人提供）：本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。 了解心肌特异性基因表达是最终了解心肌分化和病理学分子机制的基础。 我们以前的研究表明，由大鼠心肌肌钙蛋白T（cTnT）近端启动子驱动的转基因表达忠实地再现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。 每一个都具有TCTG（G/C）直接重复序列和富含A/T的序列，分别被心脏特异性42 kDa蛋白和普遍存在的高迁移率族2（HMG 2）蛋白识别。 此外，F含有MEF 2样基序，其具有富含A/T的核心。 突变分析表明，D2作为增强子，但不能完全取代F功能。 HMG 2的过表达对心肌细胞和成纤维细胞中的启动子具有不同的作用，表明HMG 2与组织特异性因子一起可以分别对心脏或非心脏组织中的cTnT基因表达产生刺激或抑制作用。 这一假设与F模块中5-bp的变化破坏了直接重复序列和A/T富集位点导致心脏转基因表达减少，同时异位转基因表达增加是一致的。 因此，具体的目的是：（1）测试单独的F模块是否足以在转基因小鼠中赋予心脏特异性表达;（2）研究HMG 2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环化试验和环状排列GMSA来测试HMG 2是否可以弯曲含有D2或F模块的短DNA片段。 从心脏和非心脏提取物中纯化的直接重复序列结合蛋白（direct repeat binding proteins，DRBPs）与重组HMG 2的相互作用将通过GMSA中的DNA结合亲和力和反式激活实验中的启动子活性来进一步评估;（3）克隆和表征与新型TCTG（G/C）直接重复序列结合的DRBPs。 我们预计心脏DRBPs的分子量应为42 kDa，而非心脏DRBPs的分子量应不同。 这些DRBPs的发育表达模式将通过原位杂交和免疫组织化学研究来确定。

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.