AUTOREGULATION OF ZFHEP-1 IS MEDIATED BY
THE BINDING TO A SPECIFIC E2-BOX
Manavella, P.A*, Roqueiro, G*, Darling, D. ? &Cabanillas, AM*. *CIBICI-CONICET. Dpto. Bioquímica Clínica, Fac CCQQ, UNC,
Zfhep (Zinc Finger Homeodomain Enhancer-binding Protein) is a transcription factor expressed as two isoforms, Zfhep-1 and Zfhep-2. Zfhep-2 lacks the N-terminal DNA-binding domain of the larger Zfhep-1. Zfhep is involved in lymphopoiesis, neurogenesis and myogenesis. We previously demonstrated that
Zfhep-1 repressed its own promoter. Sequence analysis of the Zfhep-1 promoter (TRANSFAC database), revealed the presence of two potentials Zfhep binding sites. Our goal was to identify the sequences involved in Zfhep gene regulation by the Zfhep isoforms. Band shift assays were performed with Jurkat nuclear extracts and Zfhep-2 programmed rabbit reticulocyte lysates in the presence of [32P]-labeled DNA probe. Probes include the sequence of the putative binding site 1 (BS1), binding site 2 (BS2) or both binding sites (BS1+2). Both Zfhep isoforms bound either BS2 or BS1+2 probes, but none of the isoforms bound BS1 probe. Retardation complexes were competed by either anti-Zfhep antibodies or an excess of cold probe. The mutation of the E2-Box present in the BS2 region (CACCTG→CAtaTG) disrupted the binding of both Zfhep isoforms to the BS2. Competitive band shift assays shown that Zfhep-2 is able to compete and displace Zfhep-1 from its Binding site. Our data indicate that Zfhep-1 autoregulation is mediated by the binding of the protein to an E2-Box present between 216 and 228 of the Zfhep-1 promoter. We also demonstrate that Zfhep-2 is able to compete with Zfhep-1 for this binding site acting as a negative dominant isoform.
