Control of Cardiac Troponin T Gene Expression

心肌肌钙蛋白 T 基因表达的控制

• 批准号: 7033053
• 负责人: Jim Jung-Ching Lin
• 金额: $ 25.21万
• 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 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7033053
• 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(由申请者提供)：本项目的总体目标是了解心肌细胞如何控制其心脏特异基因的表达。对心脏特异基因表达的了解是最终了解支配心肌分化和病理的分子机制的基础。我们先前的研究表明，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概括了整个胚胎发育和成体过程中内源性cTnT的表达。该启动子含有两个高度同源的模块D2和F，每个模块都有一个TCTG(G/C)直接重复序列和一个富含A/T的序列，分别被心脏特异的42 kDa蛋白和普遍存在的高迁移率族2(HMG2)蛋白识别。此外，F含有一个类似MEF2的基序，它有一个富含A/T的核心。突变分析表明，D2是一种增强子，但不能完全替代F功能。HMG2的过表达对心肌细胞和成纤维细胞中cTnT基因启动子的表达有不同的影响，提示HMG2与组织特异性因子一起可能分别对心脏和非心脏组织中cTnT基因的表达产生刺激或抑制作用。这一假设与F模块上5个碱基的改变破坏了直接重复序列和A/T富含部位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，我们的具体目标是：(1)测试F模块是否足以在转基因小鼠中实现心脏特异性表达；(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环化实验和循环排列GMSA来检测HMG2是否能够弯曲含有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(DRBPs)与重组HMG2之间的相互作用将进一步从GMSA中的DNA结合亲和力和反式激活实验中的启动子活性来评估；以及(3)克隆和鉴定与新的TCTG(G/C)直接重复序列结合的DRBPs。我们预测心脏DRBPs的分子质量应该为42 kDa，而非心脏DRBPs的大小应该不同。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。

项目成果

Requirement of TCTG(G/C) Direct Repeats and Overlapping GATA Site for Maintaining the Cardiac-Specific Expression of Cardiac troponin T in Developing and Adult Mice.

• DOI: 10.1002/ar.20772
• 发表时间: 2008-12
• 期刊: ANATOMICAL RECORD-ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY
• 影响因子: 2
• 作者: Harlan, Shannon M.;Reiter, Rebecca S.;Sigmund, Curt D.;Lin, Jenny Li-Chun;Lin, Jim Jung-Ching
• 通讯作者: Lin, Jim Jung-Ching

Characterization of cis-regulatory elements and transcription factor binding: gel mobility shift assay.

顺式调控元件和转录因子结合的表征：凝胶迁移率变化测定。

• DOI: 10.1007/978-1-59745-030-0_10
• 发表时间: 2007
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Lin,JimJung-Ching;Grosskurth,ShaunE;Harlan,ShannonM;Gustafson-Wagner,ElisabethA;Wang,Qin
• 通讯作者: Wang,Qin