Identification of novel, cardiac-restricted transcription factors binding to a CACC-box within the human cardiac troponin I promoter

Objectives: The expression of the human cardiac troponin I (hTnIc) gene is developmentally regulated and tissue-specific. In analysing the putative bi nding elements within the proximal promoter, a CACC-box sequence overlappin g 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 im portance in the transcriptional activity of the promoter. Methods: A combin ation 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 te sted by transient transfection into primary neonatal rat cardiac myocytes i n culture. Results: At least four factors were able to interact with this r egion 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 C ACC-box for binding. Site-directed mutagenesis of this region showed that t he CACC-box is essential for hTnIc promoter-reporter activity. Further char acterisation using EMSA indicated that the factors binding the hTnIc CACC-b ox are unlikely to be zinc finger proteins as they are insensitive to the a ddition of divalent cation chelating agents. They were also unable to bind to other known CACC-box elements. These factors are present in both human a nd rat cardiac muscle but absent from a number of cell lines including seve ral 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-restric ted DNA-binding activity present in the heart which we have named HCB1 and HCB2 for heart CACC-box binding factors. (C) 2001 Elsevier Science B.V. All rights reserved.