Identification of cis elements in the cardiac troponin T gene conferring specific expression in cardiac muscle of transgenic mice

To investigate the underlying mechanism regulating cardiac gene expression, transgenic mice carrying the rat cardiac troponin T proximal promoter (-49 7 bp from the transcriptional start site) fused to a LacZ or chloramphenico l acetyltransferase (CAT) reporter gene were analyzed, The LacZ expression pattern throughout development was very similar to that of the endogenous c ardiac troponin T gene. Within this promoter, a high degree of sequence hom ology was found at 2 sites, modules D (-335 to -289 bp) and F (-249 to -209 bp). Both regions contain at least a TCTG(G/C) direct repeat and an A/T-ri ch site, whereas only the F module has a muscle enhancer factor 2 (MEF2)-li ke motif. No significant decrease in CAT transgene expression was observed when only the MEF2 core sequence was mutated. However, when the MEF2 core s equence and its flanking TCTGG site were mutated (Mut5), CAT transgene expr ession was significantly decreased in the heart, and ectopic expression of the transgene was also observed. When mutations were introduced into this p romoter to destroy all upstream TCTG(G/C) direct repeats in the D module (M utD), CAT expression remained cardiac specific, but the expression level wa s dramatically decreased. Relaxation of cardiac-specific transgene expressi on became even more severe in transgenic mice carrying double mutations (Mu t[D + 5]). In addition, CAT activity in the heart was nearly abolished. The se results suggest that D and F modules have an additive function in determ ining the level of expression in the heart and only the F module confers ca rdiac-specific expression.