Abstract
To identify the 5' sequences of the murine coagulation factor VII (fVII) gene that resulted in its efficient transcription, a variety of 5'-flanking sequences up to 7 kilobase pairs upstream of the translation ATG initiation codon were fused to the reporter gene, bacterial chloramphenicol acetyltransferase, and relative expression levels of this gene in mouse Hepa 1-6 cells were determined. It was found that the 5' region extending approximately 85 base pairs (bp) upstream of the transcriptional initiation site served as the minimal DNA region that provided full relative promoter activity for chloramphenicol acetyltransferase expression. This region of the gene also contains consensus sequences for liver-enriched transcription factors, C/EBP beta and HNF4, as well as for the ubiquitous protein factors, AP1, H4TF1, NF1, and Sp1. In vitro DNase I footprinting of the 200-bp proximal region of the promoter with a murine Hepa 1-6 cell nuclear extract revealed a clear footprint of a region corresponding to -80 to -28 bp of the murine fVII gene, suggesting that liver factors interact with this region of the DNA. Competitive gel shift and supershift assays with different synthetic oligonucleotide probes demonstrate that proteins contained in the nuclear extract, identified as C/EBP beta, H4TF1, and HNF4, bind to a region of the murine fVII DNA from 85 to 32 bp upstream of the transcription start site. Purified Sp1 also interacts with this region of the DNA at a site that substantially overlaps, but is not identical to, the H4TF1 binding locus. Binding of Sp1 to the mouse DNA was not observed with the nuclear extract as the source of the transcription factors, suggesting that Sp1 is likely displaced from its binding site by H4TF1 in the crude extract. In vivo dimethyl sulfate footprint analysis confirmed the existence of these sites and additionally revealed two other binding regions slightly upstream of the CCAAT/enhancer-binding protein (C/EBP) binding locus that are homologous to NF1 binding sequences. The data demonstrate that appropriate transcription factor binding sites exist in the proximal promoter region of the murine fVII gene that are consistent with its strong liver-based expression in a highly regulated manner.
Highlights
Factor VII1 is a vitamin K-dependent plasma protein that serves as the precursor for fVIIa, a serine protease that functions as a procoagulant in the extrinsic blood coagulation pathway
CAT expression plasmids containing as possible promoter elements a variety of 5Ј sequences immediately upstream of the transcription initiation site of the murine fVII gene (16) were constructed, and their comparative abilities to drive expression of the CAT gene were assessed
A synthetic mutant HNF4 consensus oligonucleotide was not competitive for this binding. These results suggest that a HNF4 transcription site is harbored in residues Ϫ33 to Ϫ50 of the murine fVII gene
Summary
Ported, few patients present total fVII deficiency states. very low levels of this zymogen (Ͻ2% of normal) are associated with severe coagulation disorders (18 –22). The clinical manifestations of less severe deficiencies are variable (18, 19, 23–25) Such observations suggest that only low levels of fVII activity are required to maintain hemostasis in unchallenged patients. The fVII-deficient mice suffer severe perinatal lethality mainly from intra-abdominal bleeding. Those surviving birth expire within the several weeks, primarily from intracranial bleeding (30). As well as the fVII-deficiency state produced in mice, indicates that fVII activity levels are important in hemostatic and cardiovascular pathology, we undertook an investigation aimed at defining the factors that regulate transcription of the fVII gene.
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