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。 了解心脏特异性基因表达对于最终了解控制心肌分化和病理学的分子机制至关重要。 我们之前表明，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年过程中的内源性 cTnT 表达。 该启动子包含两个高度同源的模块，D2 和 F。每个模块都有一个 TCTG(G/C) 同向重复序列和一个富含 A/T 的序列，可被心脏特异性 42 kDa 蛋白和普遍存在的高迁移率基团 2 识别。 (HMG2) 蛋白，分别。 此外，F 包含类似 MEF2 的基序，其核心具有富含 A/T 的核心。 突变分析表明，D2 作为增强子，但不能完全替代 F 功能。 HMG2 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影响，这表明 HMG2 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。 该假设与 F 模块中的 5 bp 变化破坏了同向重复序列和富含 A/T 的位点导致心脏转基因表达减少并同时异位转基因表达增加的假设相一致。 因此，具体目标是：（1）测试单独的F模块是否足以赋予转基因小鼠心脏特异性表达； (2)探讨HMG2影响心脏和非心脏组织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同向重复结合蛋白（DRBP）之间的相互作用将在GMSA中的DNA结合亲和力和反式激活实验中的启动子活性方面得到进一步评估； (3) 克隆并表征与新型 TCTG(G/C) 同向重复序列结合的 DRBP。 我们预计心脏 DRBP 的分子量应为 42 kDa，而非心脏 DRBP 的大小应不同。 这些 DRBP 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