CHARACTERIZATION OF CIS-REGULATING ELEMENTS AND TRANSACTIVATING 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 neona tal cardiomyocytes and cardiac fibro blasts. Deletion analysis showed that a 41-bp fragment (-249 to -209 bp) containing the MEF-2-like moti f is an essential element for minimal cardiac-specific expression of t he rat cTnT gene. The proximal promoter (-208 to -1 bp) contains two c onsensus 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 t his proximal promoter did not show cardiac muscle-specific expression. Ligation of consensus MEF-2-like sequence into the upstream of this c himera only partially increased its ability to express in cardiomyocyt es. These results suggest that the spacing among MEF-2-like motif and proximal promoter and/or the flanking sequences of the MEF-2-like moti f 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), containin g the MEF-2-like motif, one of the CArG boxes, and the C region, or th e 41-bp probe (-249 to -209 bp), containing the MEF-2-like motif, reve aled different DNA-protein complexes formed by heart, skeletal muscle, and liver extracts, By using DNA affinity purification, DNA-binding p roteins with ap parent molecular masses of 22-26 kDa were identified f rom rat heart extract but not from skeletal and liver extracts, sugges ting the involvement of cardiac-specific proteins in regulating the cT nT gene expression.