Characterization of the murine coagulation factor X promoter

Factor X (FX) is a vitamin K-dependent serine protease zymogen that functio ns in both the extrinsic and intrinsic pathways of blood coagulation. In th is study, the 5'-flanking region of the murine FX gene was analyzed to dete rmine those elements that govern its transcriptional activity and regulatio n. Consistent with other TATA-less promoters, murine FX contains two start sites of transcription, at bp-5 and -21 relative to the ATG translational i nitiation codon. The mRNA of FX was found in a number of tissues, including the liver, stomach, intestine, kidney, ovary, testes, spleen, skeletal mus cle, and lung. Using DNase I footprinting, three areas of protection have b een identified in the proximal 287 bp of the promoter, spanning bp -28 to - 218. Further examination of this region revealed transcription factor bindi ng 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 o f which were found by transient transfection analyses in HepG2 cells to inf luence the activity of the promoter. Ablation of the NF-Y site was most dra matic, reducing activity to 10% of that of the wild-type construct. Deletio n of the HNF-4 site led to an activity of 25% of wild-type, and a GATA-4 mu tation reduced activity to 63% of wild-type values. This investigation reve aled the identity of the factors bound at the proximal promoter of the FX g ene, and the relative importance of each. This is the first report of a mem ber of the GATA family of transcription factors being important in the regu lation of a coagulation-based gene.