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.

The cardiac-specific -497 bp promoter of rat cardiac troponin T (cTnT) contains two similar modules, D and F, each of which possesses TCTG(G/C) direct repeats and A/T-rich sites. To identify cis-elements critical for cardiac specificity, a -249 bp promoter containing only module F and its site-directed mutations were used to generate transgenic mice. Transgene expression of the -249 bp promoter remained cardiac-specific, despite low and nonuniform expression. The nonuniform expression pattern of the transgene coincided with differential expression of HMGB1, which appeared to be the predominant form of HMGB family proteins in the heart. The HMGB1 binds to the A/T-rich/MEF2-like sites of the cTnT promoter, as determined by chromatin immunoprecipitation assays. Mice carrying the -249 bp promoter with point mutations disrupting the direct repeats expressed transgene at lower levels in the heart and ectopically in the brain. Ectopic expression of transgene was also observed in developing limbs and head. These results suggest an important role for the direct repeat in determining the cardiac specificity. Furthermore, mice carrying a mutant promoter simultaneously disrupting the direct repeats and overlapping GATA site failed to express the transgene in any tissues tested. Therefore, the direct repeat and overlapping GATA site are critical for the expression level and cardiac specificity. The F module controls one level of cardiac specificity. For a uniform and high level of cardiac-specific expression, the upstream element (-497 to -250 bp) is further required, possibly through the D enhancer module and the combination of Nkx2.5 and GATA sites.