title: Two mutations in the promoter region of the human protein C gene both cause type I protein C deficiency by disruption of two HNF-3 binding sites.
abstract: Protein C is a vitamin K-dependent zymogen of a serine protease that inhibits blood coagulation by the proteolytic inactivation of factors Va and VIIIa. Individuals affected with protein C deficiency are at risk for thrombosis. Genetic analyses of affected individuals, to determine the cause of the protein C deficiency, revealed a large variety of mutations in the protein C gene, including several in the promoter region of this gene. Comparison of the region around two of these mutations, A-32-->G and T-27-->A, with transcription factor consensus sequences suggested the presence of two overlapping and inversely oriented HNF-3 binding sites. Direct evidence for the presence of the two HNF-3 binding sites in the protein C promoter was obtained using electrophoretic mobility shift assays and UV cross-linking experiments. These experiments revealed that HNF-3 can bind specifically to both putative HNF-3 sites in the wild-type protein C promoter. Due to the T-27-->A mutation, one binding site is completely lost, while the other site still binds HNF-3, but with strongly reduced affinity. As a consequence of the A-32-->G mutation, the protein C promoter loses all its HNF-3 binding capacity. Transient transfection experiments demonstrated that the binding of HNF-3 to the protein C promoter is of physiological significance. This followed from experiments in which the introduction of the A-32-->G or T-27-->A mutation resulted in a 4-5-fold reduced promoter activity in HepG2 cells. Furthermore, transactivation of the wild-type protein C promoter construct with HNF-3 showed a 4-5-fold increased promoter activity in HepG2 cells. In HeLa cells, significant wild-type promoter activity was only observed after transactivation with HNF-3. When a promoter construct containing the T-->A mutation at position -27 was used, the transactivation potential of HNF-3 was 2-fold reduced in HepG2 cells, whereas in HeLa cells no transactivation was observed. With the promoter construct containing the A-32-->G mutation, no transactivation by HNF-3 was found either in HepG2 or in HeLa cells.
authors.affiliation: Department of Hematology, University Hospital, Leiden, The Netherlands.
authors.forename: C A
authors.initials: CA
authors.lastname: Spek
authors.forename: J S
authors.initials: JS
authors.lastname: Greengard
authors.forename: J H
authors.initials: JH
authors.lastname: Griffin
authors.forename: R M
authors.initials: RM
authors.lastname: Bertina
authors.forename: P H
authors.initials: PH
authors.lastname: Reitsma
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006367:HeLa Cells; D015186:Hominidae / Q000235:genetics*; D006801:Humans; D008113:Liver Neoplasms; D008114:Liver Neoplasms, Experimental; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011486:Protein C / Q000235:genetics*; D020151:Protein C Deficiency; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; D011486:Protein C; D011994:Recombinant Proteins; D014157:Transcription Factors; D004247:DNA