Binding of fVII oligonucleotides.

(A) The −55 C to T mutation diminished binding of HNF4 to fVII promoter sequence. A radiolabeled WT oligonucleotide encompassing the HNF4 binding site (−77 to −47 base pairs prior to the translation start site) in the human fVII promoter region showed binding to HNF4 protein prepared by in vitro transcription/translation (lane 3), and corresponding oligonucleotides having the −55 mutation (MT55, lane 5) or the −61 mutation (MT61, lane 7) showed weaker and undetectable binding, respectively. All 3 oligonucleotides bound nonspecifically to components of the mock transcription/translation reaction mixture (lanes 2, 4, 6), which migrated to various different positions on the gel. The binding reaction with a control oligonucleotide (the HNF4 binding site from the ApoCIIIB promoter) is shown in lane 1. The amounts of WT, MT55, and MT61 oligonucleotides used per lane were identical, but less control oligonucleotide was used to compensate for its comparatively strong binding to HNF4. (B) Competition assays show specificity of binding to HNF4. The radiolabeled WT oligonucleotide bound to HNF4 prepared by in vitro transcription/translation (lane 2), and binding was subject to competition by inclusion of increasing concentrations of unlabeled WT oligonucleotide (5 ×, 10 ×, 50 ×, and 100 × relative to the concentration of the labeled oligonucleotide, lanes 3-6 successively). There was also competition, though less effective, by unlabeled MT55 oligonucleotide (10 ×, 50 ×, 100 ×, and 200 ×, lanes 7-10 successively) but not at all by unlabeled MT61 oligonucleotide (100 × and 200 ×, lanes 11 and 12). Binding of the control ApoCIIIB-labeled oligonucleotide to the HNF4 protein is shown in lane 13, and binding between the WT fVII probe and components of the mock transcription/translation reaction mixture is shown in lane 1.