EXPRESSION OF THE ZFHEP GENE DURING NEUROGENESIS AND MYOGENESIS
Roqueiro, G, Manavella, P., Aoki, P, Gea, S, Darling, D. and Cabanillas, A.M. CIBICI-CONICET. Dpto. Bioquímica Clínica, Fac CCQQ, UNC, Córdoba. Gonzalo_roqueiro@hotmail.com
Zfhep (Zinc Finger Homeodomain Enhancer-binding Protein) is a transcription factor involved in differentiation processes such as myogenesis, neurogenesis and lymphopoiesis. The promoter has been recently isolated and so far, there is no direct assessment of its regulation during cell differentiation. Our goal was to evaluate the activity of Zfhep promoter during myocyte and neuron development. The embryonal carcinoma (P19) and a mouse myoblast (C2C12) cell lines were differentiated for inducing neurogenesis (by pretreatment with retinoic acid) or myogenesis (by incubation with 2% horse serum), respectively. Western blot and immunocytochemistry methods were used to validate the model. Human Zfhep promoter was cloned into pGL3-basic and several mutant deletions were made. The reporter chimeras and CMVb-gal (to normalize transfection efficiency) were transfected into C2C12 and P19 cells by calcium phosphate and FuGENE6, respectively; then they were induced to differentiate. Luciferase and b-galactosidase activities were assessed in cell lysates by standard methods. The promoter was active in all of the constructs tested in both, undifferentiated C2C12 and P19 cells. The activity of the Zfhep promoter, decreased significantly after differentiation of P19 to neurons. The activity was also lowered by 30 fold in differentiated C2C12 cells. These results first show that the Zfhep gene is dynamically regulated during myogenesis and neurogenesis. The repression of the gene could be related to a lack of function of Zfhep during the differentiated cell state.
