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. ? ?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL072910-03
Application #
6853554
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Program Officer
Evans, Frank
Project Start
2003-04-01
Project End
2007-03-31
Budget Start
2005-04-01
Budget End
2006-03-31
Support Year
3
Fiscal Year
2005
Total Cost
$258,125
Indirect Cost
Name
University of Iowa
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Harlan, Shannon M; Reiter, Rebecca S; Sigmund, Curt D et al. (2008) 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. Anat Rec (Hoboken) 291:1574-86
Lin, Jim Jung-Ching; Grosskurth, Shaun E; Harlan, Shannon M et al. (2007) Characterization of cis-regulatory elements and transcription factor binding: gel mobility shift assay. Methods Mol Biol 366:183-201