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001 YEAST ONE HYBRID CLONING OF FACTORS WHICH BIND A CRITICAL CACC/SP1 ELEMENT IN THE HUMAN CARDIAC TROPONIN I GENE
M. Raman*, M. H. Yacoub, P. J. R. Barton, N. J. Brand.Heart Science Centre, National Heart and Lung Institute, Imperial College, Harefield, Middlesex, UB9 6JH, UK
The human cardiac troponin I (hTnIc) gene shows both cardiac-specific and developmentally-regulated expression. Previous studies have identified an overlapping CACC/Sp1 element in a critical region within the hTnIc promoter and electrophoretic mobility shift analysis (EMSA) identified four proteins present in cardiac muscle extracts, which bind this element. Two of these proteins were Sp1 and Sp3. The other two proteins, however, appeared to be novel CACC-box binding factors as, unlike other CACC-box binding factors identified to date, their characteristics of DNA binding suggests that they are not zinc finger factors and that they appear to be cardiac-enriched. These novel proteins were named HCB1 and HCB2 for Heart CACC-box binding proteins 1 and 2. We hypothesise that HCB1 and HCB2 may therefore be important novel tissue-restricted regulators of cardiac gene expression. In an attempt to clone the cDNA sequences for HCB1 and HCB2, we have employed yeast one hybrid cloning. A yeast reporter strain with the “bait” CACC/Sp1 element cloned upstream of the HIS3 selectable marker was transformed with a “prey” neonatal rat cardiac myocyte cDNA library. Plasmid DNA was isolated and sequenced from the 71 positive library clones obtained. These include Sp1, Leukaemia/Lymphoma-Related Factor (LRF), Kruppel-Like Factor 4 (KLF4), and Nuclear Factor I-A (NFI-A) clones. NFI-A is an HCB1/2 candidate as its DNA binding domain shows no apparent homology to any of the well characterised zinc finger motifs. Moreover, evidence …