DIFFERENTIAL CELL EXPRESSION OF ALTERNATIVE ISOFORMS OF ZFHEP PROMOTER. Torgnelli, M.S., Darling, D.S. and Cabanillas, A.M.
Dpto. Bioquímica Clínica. Facultad Ciencias Químicas. Universidad Nacional de Córdoba. amcaba@bioclin.fcq.unc.edu.ar
Zfhep (Zinc Finger Homeodomain Enhancer Protein) is a member of the Zn++ fingers/homeodomain family of transcription factors which is highly expressed in rat central nervous system and heart as two isoforms, Zfhep-1 and Zfhep-2. The Zfhep-2 isoform lacks the first exon of the gene. Zfhep is involved in lymphopoiesis, neurogenic and myogenic differentiation. Zfhep gene regulation has not been studied yet. Our first goal was to characterize Zfhep promoter. We examined whether Zfhep isoforms expression is due to the usage of alternative promoters. RT-PCR from human total RNA showed that Zfhep-1 and Zfhep-2 transcripts are contained in distinct exons and that 5?-flanking sequence of Zfhep-2 is downstream of the first exon of Zfhep-1 suggesting that both isoforms would be regulated for two different promoters. We then isolated both promoters, Z-1p and Z-2p by PCR of human genomic DNA which were subcloned into a luciferase expression vector. The constructs were incorporated to the cells by calcium phosphate transfection to several cell lines: P19, C2C12, COS-7, CHO-K1, and JEG-3. Z-1pLuc activity was 15-20 (COS-7), 12 (JEG-3), 50-70 (C2C12 and CHO-K1), and 10-20 (P19) times higher than the respective controls. Z-2pLuc activity was 20-22 fold over control in C2C12 and CHO-K1 and absent in COS-7 and JEG-3. This result shows that both Zfhep-1 and Zfhep-2 promoters are active and that their expression is dependent on cell types.
