Hepatocyte nuclear factor 1 and the regulation of the human factor IX gene : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Biology at Massey University

Factor IX is a serine protease involved in the mammalian blood clotting cascade. An absence of functional factor IX protease in the bloodstream results in Haemophilia B. Mutations in the regulatory region of the factor IX gene can produce a rare form of the disease called Haemophilia B Leyden. Single nucleotide substitutions at positions -5 and -6 of the human factor IX promoter, which result in Haemophilia B Leyden, disrupt the binding of an unidentified transcription factor which interacts in the region -13 to +3. A group of transcription factors which may interact with the factor IX promoter in this region is called the hepatocyte nuclear factor 1 (HNF1) family. The aim of this research was to investigate the potential role of the HNF1 proteins in the regulation of factor IX promoter gene expression. This study was bipartite, involving research into the ability of the HNF1 transcription factors to bind the factor IX promoter in vitro, and to regulate the initiation of its transcription. The HNF1 cDNAs were firstly subcloned into an expression vector suitable for use in mammalian tissue culture. Gel mobility shift assays were employed to examine the binding of the HNF1 proteins to the wildtype factor IX promoter. The ability of these proteins to bind the factor IX promoter region carrying the -5 or -6 mutations was also investigated. Luciferase reporter gene assays using a human hepatoma cell line were used to study the regulatory effects of the HNF1 transcription factors on transcription from the normal and mutant factor IX promoters. A variant form of the HNF1 transcription factor was shown to bind to the -14 to +6 region of the normal sequence of the factor IX promoter as well as that containing some of the -5 and -6 mutations. A protein from rat liver nuclear extracts which displayed an HNFI-like binding activity was delected using gel mobility shift assays with the factor IX promoter region. All forms of the HNF1 transcription factors could regulate the transcription of a reporter gene driven by the wildtype factor IX promoter. Two forms of the HNF1 transcription factor down-regulated the wildtype factor IX promoter-reporter gene construct by at least 50%, while the same construct was up-regulated by a third form of the transcription factor. Unfortunately time constraints resulted in the premature conclusion of planned experimentation. The results generated during this research have been unable to confirm a role for the HNF1 family in the regulation of the factor IX promoter, but have provided a basis for further research