Characterization of the murine coagulation factor X promoter

Factor X (FX) is a vitamin K-dependent serine protease zymogen that functions in both the extrinsic and intrinsic pathways of blood coagulation. In this study, the 5'-flanking region of the murine FX gene was analyzed to determine those elements that govern its transcriptional activity and regulation. Consistent with other TATA-less promoters, murine FX contains two start sites of transcription, at bp -5 and -21 relative to the ATG translational initiation codon. The mRNA of FX was found in a number of tissues, including the liver, stomach, intestine, kidney, ovary, testes, spleen, skeletal muscle, and lung. Using DNase I footprinting, three areas of protection have been identified in the proximal 287 bp of the promoter, spanning bp -28 to -218. Further examination of this region revealed transcription factor binding sites for NF-Y, HNF-4, and a GATA factor. Electrophoretic mobility shift analysis (EMSA) confirmed the identities of NF-Y, HNF-4, and GATA-4, all of which were found by transient transfection analyses in HepG2 cells to influence the activity of the promoter. Ablation of the NF-Y site was most dramatic, reducing activity to 10% of that of the wild-type construct. Deletion of the HNF-4 site led to an activity of 25% of wild-type, and a GATA-4 mutation reduced activity to 63% of wild-type values. This investigation revealed the identity of the factors bound at the proximal promoter of the FX gene, and the relative importance of each. This is the first report of a member of the GATA family of transcription factors being important in the regulation of a coagulation-based gene.