Abstract
Blood coagulation Factor X and its activated form Factor Xa play an essential role in the midphase of the clotting cascade. To delineate the mechanisms governing the liver-specific expression of Factor X, we have previously characterized the complete 2.8 kilobase pairs of the 5'-flanking region of Factor X and demonstrated that the first 209 base pairs is sufficient to confer maximal promoter activity in HepG2 cells, a hepatoma cell line that expresses Factor X. We have also shown that mutations at ACTTTG and CCAAT elements located at -56 to -51 and -120 to -116, respectively, significantly reduce the promoter activity. In this report, we demonstrate that Factor X mRNA is primarily but not exclusively expressed in the liver. Using DNase I footprinting analysis, we determine four protein binding sites within the 209-base pair fragment, designated site 1 (-73) to -44), site 2 (-128 to -94), site 3 (-165 to -132), and site 4(-195 to -169). Using gel mobility shift assays in combination with competition and supershift experiments, we demonstrate that hepatocyte nuclear factor 4 and Sp1 bind at site 1, the site which contains the ACTTTG element. Methylation interference assays reveal that HNF-4 and Sp1 contact adjacent sites with minor overlap. HNF-4 and Sp1 appear to bind site 1 in a mutually exclusive fashion. We also demonstrate that HNF-4 can transactivate the Factor X promoter in HeLa cells; mutation at the adjacent Sp1 site further increases the transactivation. Heteromeric transcription factor NF-Y was identified as the protein that binds the CCAAT box at site 2. We conclude that HNF-4 and NF-Y play crucial roles in modulating the activity of the proximal promoter of Factor X.
Highlights
The vitamin K-dependent proteins F.VII,1 F.IX, F.X and prothrombin are the precursors of the major enzymes of the coagulation cascade
In the case of the most proximal site, site 1, we show that two transcription factors, one ubiquitous (Sp1), and the other found in only a few tissues (HNF-4) bind at the site in a competitive fashion; we present evidence that HNF-4 binds at a similar site in the promoters of F.VII and F.IX
For site 2, which contains a CCAAT box, we have shown that the ubiquitous transcription factor NF-Y binds at this site; this is distinct from the transcription factor that binds at the CCAAT box in the F.IX promoter, which occupies a similar position with respect to the translation start site
Summary
The vitamin K-dependent proteins F.VII, F.IX, F.X and prothrombin are the precursors of the major enzymes of the coagulation cascade. In previous work [3], we had defined the start sites of transcription of the F.X gene and had carried out a functional characterization of the F.X promoter, which demonstrated that the proximal 209 bp of the promoter were adequate to confer maximal activity in HepG2 cells. Using site-directed mutagenesis and reporter gene assays, we defined two areas within the F.X promoter that bound proteins from HepG2 nuclear extracts and that were required for promoter activity. In the case of the most proximal site, site 1, we show that two transcription factors, one ubiquitous (Sp1), and the other found in only a few tissues (HNF-4) bind at the site in a competitive fashion; we present evidence that HNF-4 binds at a similar site in the promoters of F.VII and F.IX. For site 2, which contains a CCAAT box, we have shown that the ubiquitous transcription factor NF-Y binds at this site; this is distinct from the transcription factor that binds at the CCAAT box in the F.IX promoter, which occupies a similar position with respect to the translation start site
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