1. A new mutation in the HNF4 binding region of the factor VII promoter in a patient with severe factor VII deficiency
by Josephine A. Carew, Eleanor S. Pollak, Stanislaw Lopaciuk, and Kenneth A. Bauer
Blood
Volume 96(13):
December 15, 2000
©2000 by American Society of Hematology

2. Binding of fVII oligonucleotides
Binding of fVII oligonucleotides.(A) The −55 C to T mutation diminished binding of HNF4 to fVII promoter sequence.
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.
Josephine A. Carew et al. Blood 2000;96:
©2000 by American Society of Hematology

3. Cotransfection of an HNF4 expression vector alters expression of reporter gene from vectors with WT and MT55 fVII promoter sequence.A total of 3 μg of WT or MT55 reporter vectors were transiently transfected into HepG2 cells, with or without 6 μg of the pCD...
Cotransfection of an HNF4 expression vector alters expression of reporter gene from vectors with WT and MT55 fVII promoter sequence.A total of 3 μg of WT or MT55 reporter vectors were transiently transfected into HepG2 cells, with or without 6 μg of the pCDNAI-HNF4 expression vector or pUC-19 plasmid DNA, as noted. The growth hormone values were corrected for expression from the promoterless pOGH vector under parallel conditions, and for transfection efficiency, then normalized to expression from the WT vector in the absence of coexpressed HNF4. The results shown (3.97% ± 2.87% [1 SD] for MT55 plasmid versus 100% ± 13.1% [1 SD] for WT plasmid, in the absence of pCDNAI-HNF4; 187% ± 99% [1 SD] for MT55 plasmid versus 610% ± 185% [1 SD] for WT plasmid, in the presence of pCDNAI-HNF4) were the averages from 3 experiments. The total number of replicates in each group is shown.
Josephine A. Carew et al. Blood 2000;96:
©2000 by American Society of Hematology