Cardiac troponin T gene expression in muscle

We have been analyzing the regulatory regions of the cardiac troponin T gene promoter as a mean toward understanding the mechanisms that govern the transcription of genes which are cross-expressed in cardiac and skeletal muscles during development. By analyzing the activities of mutant cardiac troponin T gene promoter by transient transfection of primary embryonic muscle cells, we showed that both common and distinct elements are required for activity of the cardiac troponin T promoter in these embryonic muscle cells. In skeletal muscle the minimal promoter sufficient to direct activity of the cardiac troponin T promoter is only 99 nucleotides upstream from the transcription initiation site. Within the distal half of this promoter are two tandem copies of a conserved hexanucleotide sequence (5'-CATTCCT-3') we termed the 'M-CAT motif'. Since mutation of either one of the M-CAT motifs abolishes promoter activity, we concluded that both M-CAT motifs are essential for activity of the promoter. The above minimal promoter is insufficient to confer promoter activity in embryonic cardiocytes. In these cells an additional 48-nucleotide region approximately 100 nucleotides upstream of the minimal promoter is needed for efficient promoter activity. We have named this region the 'cardiac element'. This element contains a conserved sequence motif found in other muscle gene promoter. The cardiac element can also act irrespective of orientation and is relatively independent of position, characteristics that are like transcriptional enhancers. This element alone, however, is insufficient to direct cardiac promoter activity. Activity of the 48-nucleotide cardiac element is dependent on either direct or indirect interaction with the downstream M-CAT motifs because mutation of either M-CAT motif also abolishes promoter activity in cardiac cells. The third regulatory region (nucleotide position -550 to -268) is not essential for promoter activity but can enhance activity of the cTNT promoter or a heterologous promoter in both cardiac and skeletal muscle cells by three to five folds. Within this region are sequences which show similarity to motifs found in other muscle gene enhancers. In vitro DNA-protein binding studies showed that the above three regulatory regions interact with nuclear factors. A direct correlation exists between promoter activity and sequence specific binding of a nuclear factor we termed the 'M-CAT binding factor' to the M-CAT motifs. Similar interaction of nuclear regulatory molecules with sequences within the cardiac element and the upstream enhancer region is likely to be the mechanisms which control the action of these regulatory regions.(ABSTRACT TRUNCATED AT 400 WORDS)