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.
Structural and functional analyses suggest that Zfhep isoforms are regulated by alternate usage of two promoters; the 5´flanking sequence of Z-2 is downstream of the first exon of Z-1.
Zfhep gene regulation has not been thoroughly studied. We demonstrated that cotransfection of Zfhep-1 repressed its own promoter in JEG-3 and COS-7 cells. Our present goal was to study the autoregulation of Zfhep gene by Zfhep isoforms. Transfection assays were performed by the Ca-P precipitation in C2C12 (myoblast) and CHO-K1 cells, which express both, Z-1 and Z-2 promoter. CMVb and 1 mg Z-1pLuc or Z-2pLuc and 1 mg expression plasmids of Zfhep-1 or Zfhep-2 were transfected. Luciferase and b-galactosidase activities were assayed 48 hrs after transfection.
Over-expression of Zfhep-1 down regulated Z1p activity in both CHO-K1 (49.0 %+/- 7.4, mean +/- SEM, n=5) and C2C12 (74.1 % +/- 5.8, n=3) cells but showed no effect on the Z2p. Besides, down regulation of the Z1p was dependent on the amount of Zfhep-1. Over-expression of Zfhep-2 had no effect on Z-2p or Z-1p in CHO-K1. However, Zfhep-2 cotransfection significantly activated both Z-1p (P< 0.001) and Z-2p (P< 0.01) promoters in myoblasts.
Our data indicate that the Zfhep gene is differentially autoregulated by isoforms of Zfhep. The isoforms seem to use different pathways since the Z -1 promoter is repressed by Zfhep-1, and activated by Zfhep-2. This is the first report on a differential activity of isoform Zfhep-2 respect to the full length isoform Zfhep-1.
