Characterization of Human Protein C Gene Promoter: Insights from Natural Human Mutants

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Abstract
Human protein C is a liver-produced plasma anticoagulant. Four heterozygous point mutations located in the promoter region have been identified in families with type I protein C deficiency and recurrent venous thrombosis. However, detailed analysis of regulatory elements and their interacting factors remains to be undertaken. This report presents results of biochemical and functional characterizations of several cis-elements located in the 5′-upstream regulatory region and the trans-acting factors that interact with them. A cloned DNA fragment from nucleotides (nt) −418 to +45 could confer tissue specificity, whereas nt −88 to +45 was sufficient for basal promoter activity of protein C gene. Five cis-elements corresponding to HNF-1, HNF-3, and NF-I/CTF binding sites have been identified. Four heterozygous mutations have been shown to disrupt HNF-3 [mutants of A(−32)G and T(−27)A] and HNF-1 [T(−14)C and C(−10)T] binding. Mutation in the NF-I-binding site also significantly impairs the promoter activity. Viewed as a whole, these results indicate that HNF-1, HNF-3, and NF-I/CTF play critical roles in transcriptional regulation of the protein C gene.