EXPRESSION OF THE ZFHEP GENE DURING LYMPHOPOIESIS
Cabanillas, A.M., Doan, L.*, Grimes, H-L.*, Darling, D.S.* CIBICI-CONICET, FAC. CCQQ, UN CBA. (*) BIOCHEM & MOLEC BIOLOGY DPMNT, UNIV OF LOUISVILLE, USA.
T lymphocytes go through differentiation process in the thymus from common lymphoid precursor cells to double negative (DN1-4), double positive (DP) and finally to single positive (SP) CD8+ or CD4+ T cells. Zfhep is a transcription factor, which mutation causes severe T cell deficiency. There is no direct assessment of Zfhep expression during thymocyte (THY) development. Our aim was to define Zfhep expression pattern during THY differentiation and the regulatory elements in the Zfhep promoter. Zfhep expression of sorted DN, DP, CD4+ and CD8+ SP THY was assessed by western blot (WB) and intranuclear flow cytometry (IFC) with surface staining for CD4/CD8 and CD44/CD25 (to sort DN population). 5?deletions of Zfhep promoter were transfected into Jurkat cells by Fugene6. Zfhep is expressed in total THY; WB and IFC showed that sorted DP THY clearly express more Zfhep than SP cells. Zfhep increased between the DN1 and DN2 cells; DN3-DN4 populations also had a high Zfhep expression. Maximal gene activity was seen in the first 212 bases of Zfhep promoter (93 fold activation relative to control). We localized a G-rich segment (-9 to -53) strictly required for activity.
Zfhep shows a regulated expression during lymphopoiesis. Zfhep high expression in DN2-4 and DP thymocytes is compatible with a role of Zfhep in the control of early steps of thymocyte differentiation. Transfection studies have localized two regulatory regions of the Zfhep promoter in T lymphocytes.
