CELL-SPECIFIC REGULATION OF THE ZFHEP PROMOTER IN NEUROGENIC AND LYMPHOBLAST CELLS.Manavella, Pablo A., Hapney, Kristine L. *, Darling, Douglas S. * and Cabanillas, Ana M. Facultad de CCQQ, UN Cordoba, Argentina.* University of Louisville, USA. amcaba@bioclin.fcq.unc.edu.ar
Zfhep transcription factor is involved in myogenesis, neurogenesis and T-cell differentiation. Zfhep protein is down regulated during neurodifferentiation in the embryonic brain, and in P19 embryocarcinoma cells. We have isolated the human Zfhep promoter, and aim to define the major regulatory elements in different cell types. Constructs contained 1000, 400, 212, or 12 bp of the promoter in a luciferase reporter vector. The reporter (0.7 mg) and 0.3mg of CMVb-gal (to normalize transfection efficiency) were transfected into P19, CHO-K1 (ovary) or Jurkat (lymphocytic) cells. Data are expressed as activation relative to Z1p.12Luc (control) which contains only 12 bp of promoter. The Z1p.1000Luc reporter was active in each cell line assayed. The highest activity was obtained in Jurkat cells where the activity of Z1p.1000Luc and Z1p.400Luc was 31 times higher than Z1p.12Luc (P< 0.001). In Jurkat cells, Z1p.212 was activated 3-fold above Z1p.400Luc (P < 0.01), suggesting the presence of a negative cis-acting element in the promoter between -400 and -212. This element is not observed in CHO or P19 cells. Transfection of differentiating P19 cells showed decreased promoter activity compared to control P19 cells, indicating the presence of a neurogenic response element within the first 212 bp. Conclusion: We have localized two different regulatory regions of the Zfhep promoter utilized in either lymphocytes (-400 to -212) or in neuroblasts (-212 to cap site).
