RESUMEN
The Na(+)-Ca(2+) exchanger (NCX) regulates intracellular calcium homeostasis. We report on an upstream region of the rat NCX1 multipartite promoter that is active in cardiac myocytes. Although inactive in most non-cardiac cell lines, its activity can be rescued by cotransfection with GATA-4 and -6, but not GATA-5 transcription factors. In transgenic mice and similar to endogenous NCX1 mRNA expression, the upstream promoter region directs uniform beta-galactosidase expression in cardiac myocytes from approximately 7.75dpc. In adult mouse hearts, promoter activity is, however, significantly reduced and heterogeneous, except in the conduction system (sinoatrial and atrioventricular node, atrioventricular bundles). The upstream NCX1 promoter region thus directs appropriate spatial and temporal control of cardiac expression throughout development.
Asunto(s)
Miocardio/metabolismo , Regiones Promotoras Genéticas , Intercambiador de Sodio-Calcio/genética , Animales , Sitios de Unión , Western Blotting , Células COS , Núcleo Celular/metabolismo , Células Cultivadas , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Exones , Factor de Transcripción GATA4 , Factor de Transcripción GATA5 , Factor de Transcripción GATA6 , Genes Reporteros , Operón Lac , Ratones , Ratones Transgénicos , Plásmidos/metabolismo , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Intercambiador de Sodio-Calcio/química , Factores de Tiempo , Distribución Tisular , Factores de Transcripción/metabolismo , Transfección , beta-Galactosidasa/metabolismoRESUMEN
OBJECTIVES: The expression of the human cardiac troponin I (hTnIc) gene is developmentally regulated and tissue-specific. In analysing the putative binding elements within the proximal promoter, a CACC-box sequence overlapping a consensus Sp1 element has been identified. The aim of this study was to characterise the factors binding to this element and to determine their importance in the transcriptional activity of the promoter. METHODS: A combination of supershift and competition electrophoretic mobility shift assays (EMSA) were used to identify the binding of factors to the overlapping CACC-box/Sp1 consensus element. The functional importance of this element was tested by transient transfection into primary neonatal rat cardiac myocytes in culture. RESULTS: At least four factors were able to interact with this region including the zinc finger proteins Sp1, Sp3 and two potentially novel factors. Whereas both Sp1 and Sp3 bound to the consensus Sp1 element, and to a lesser extent the CACC-box, two of the complexes required the intact CACC-box for binding. Site-directed mutagenesis of this region showed that the CACC-box is essential for hTnIc promoter-reporter activity. Further characterisation using EMSA indicated that the factors binding the hTnIc CACC-box are unlikely to be zinc finger proteins as they are insensitive to the addition of divalent cation chelating agents. They were also unable to bind to other known CACC-box elements. These factors are present in both human and rat cardiac muscle but absent from a number of cell lines including several derived from skeletal muscle. CONCLUSION: The human cardiac troponin I gene promoter requires an upstream CACC-box element for full activity. This element binds at least two complexes which represent novel, tissue-restricted DNA-binding activity present in the heart which we have named HCB1 and HCB2 for heart CACC-box binding factors.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Troponina I/genética , Animales , Técnicas de Cultivo de Célula , Línea Celular , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Plásmidos , Ratas , Ratas Sprague-Dawley , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3 , Factores de Transcripción/genética , Transfección , Troponina I/metabolismo , Dedos de Zinc/genéticaRESUMEN
The human cardiac troponin I (TnIc) gene exhibits both cardiac-specific and developmentally regulated expression. The structure and expression of this gene as well as the identification of putative regulatory elements have been described previously. This study shows that a minimal promoter containing 98 bp of sequence is sufficient to drive transcription in neonatal rat cardiac myocytes. This region contains several putative cis -regulatory elements including an Initiator element surrounding the start site of transcription, an A/T-rich (TATA/MEF-2) element, two GATA elements and a cytosine-rich region containing overlapping CACC box and Sp1 elements. Using electrophoretic mobility shift assays (EMSAs) this study demonstrates the binding of MEF-2, Oct-1, and recombinant TBP to the A/T-rich element and of GATA-4 to both GATA elements. The CACC/Sp element binds the zinc finger transcription factors Sp1 and Sp3 in addition to an unidentified complex present in neonatal rat cardiac myocytes. Mutation of each of these sites has a deleterious effect on promoter activity as assayed by transient transfection into cardiac myocytes. The data suggest that transcriptional activity of the human TnIc gene can be driven by a compact promoter region and that within this region GATA, MEF-2 Sp1 and CACC box-binding factors are required for optimal activity. Furthermore, a comparison with data obtained for identical elements in the promoters of rodent TnIc genes identifies differences between species which may be of consequence for species-specific promoter function.