Characterization of cis-regulating elements and trans-activating factors of the rat cardiac troponin T gene.

To study the transcriptional regulation of the rat cardiac troponin T (cTnT) gene, chimeric genes composed of the upstream region (-757 to +193 base pairs (bp) relative to the transcription initiation site) of the cTnT gene and the bacterial chloramphenicol acetyltransferase (CAT) gene were constructed and transfected into primary cultures of neonatal cardiomyocytes and cardiac fibroblasts. Deletion analysis showed that a 41-bp fragment (-249 to -209 bp) containing the MEF-2-like motif is an essential element for minimal cardiac-specific expression of the rat cTnT gene. The proximal promoter (-208 to -1 bp) contains two consensus CArG boxes, one M-CAT motif, one AP2 site, and one TATA box. The construct (cTNT-208) composed of the CAT reporter gene driven by this proximal promoter did not show cardiac muscle-specific expression. Ligation of consensus MEF-2-like sequence into the upstream of this chimera only partially increased its ability to express in cardiomyocytes. These results suggest that the spacing among MEF-2-like motif and proximal promoter and/or the flanking sequences of the MEF-2-like motif are important in determining cardiac muscle-specific expression. By footprint analysis with a DNA fragment (-303 to +6 bp), we identified three novel regions (called A, B, and C) protected by protein extract from rat hearts, in addition to the known motifs such as MEF-2, M-CAT, and CArG. Gel retardation with the probe (-235 to -141 bp), containing the MEF-2-like motif, one of the CArG boxes, and the C region, or the 41-bp probe (-249 to -209 bp), containing the MEF-2-like motif, revealed different DNA-protein complexes formed by heart, skeletal muscle, and liver extracts. By using DNA affinity purification, DNA-binding proteins with apparent molecular masses of 22-26 kDa were identified from rat heart extract but not from skeletal and liver extracts, suggesting the involvement of cardiac-specific proteins in regulating the cTnT gene expression.