Transcriptional control of the factor IX gene: analysis of five cis- acting elements and the deleterious effects of naturally occurring hemophilia B Leyden mutations

Abstract

Hemophilia B Leyden is a rare form of inherited factor IX deficiency in which patients experience spontaneous postpubertal recovery of factor IX levels. The mutations resulting in this disorder are localized in a 40-nucleotide region encompassing the major transcriptional start site for factor IX. Here we report the further characterization of five cis- acting elements in the factor IX promoter and the effects on protein binding and transcriptional activation of five Leyden mutations (at nucleotides +13, -5, -6, -20, and -26) that occur within the proximal three elements (sites 1 through 3). Bandshift studies using nuclear extracts from four different rat tissues have shown that at least some of the proteins binding to each of the five sites are ubiquitous in nature. The pattern of DNA binding at site 1 suggests that this element plays an important role in mediating the liver-specific expression of factor IX. Additional studies with liver nuclear extracts obtained at several different points in development have shown an increase in DNA binding at sites 1, 4, and 5 between 1 day and 1 week. Using DNase I footprint analysis and competition bandshift studies, we have shown that the binding of nuclear proteins to each of the mutant sites is disrupted to a variable extent. There appears to be some, although reduced, protein binding to all of the mutant oligonucleotides apart from the -26 mutant. In vitro transcription assays have shown that each of the mutations reduces the global proximal promoter activity by approximately 40%. Two double mutant promoters did not show any additional downregulation in the in vitro transcription assay. In experiments designed to assess the relative transcriptional activity mediated from each of the five sites independently, we have tested artificial homopolymer promoters of each site in the in vitro transcription assay. These studies show that sites 4 and 5 are the strongest activators and that transactivation from site 5 is further enhanced by the albumin D site-binding protein. In summary, these investigations show deleterious effects of each of the Leyden mutations tested on the binding of trans-acting factors and also show disruption of transcriptional activation in a functional in vitro transcription assay. Our results also show that cis-acting elements 4 and 5 are the principal activators of this locus.

Androgen effects on factor IX expression: in‐vitro and in‐vivo studies in mice

“…In combination with the observations of Kurachi et al (1994) described above, these findings suggest that AR requires an association with other protein(s) (deficient in untransfected HepG2 cells such as C/EBPa) to mediate effective transactivation of the factor IX promoter. Additional previous evidence from our laboratory, relating to the role of C/EBPa, D-site Binding Protein (DBP) and GA-Binding Protein (GABP) interactions at the sequence between nts ¹190 and ¹220 of the factor IX promoter (Boccia et al, 1996;Picketts et al, 1994Picketts et al, , 1993 further emphasizes the complexity of the factors potentially mediating peri-pubertal transcriptional regulation at this locus.…”

The Diagnosis and Management of Congenital Hemophilia

“…Additionally, there is one unusual form of hemophilia B (hemophilia B Leyden) that results from a point mutation in the FIX promoter region. 15 In this disorder, FIX:C levels are markedly reduced in early childhood, leading to a diagnosis of hemophilia, but following puberty, the synthesis of testosterone leads to increased FIX promoter activity and increased FIX production. Ultimately, many such individuals may have normal FIX:C levels as adults.…”

Molecular testing for disorders of hemostasis

“…Aside from inhibitor risk assessment, genotype data can also occasionally be used to generate novel information concerning the expected coagulation phenotype. The most interesting and positive example of this potential is the identification of hemophilia B Leiden mutations in the promoter region of the F9 gene [16]. These cases of, initially moderately severe, hemophilia B undergo spontaneous phenotypic correction following puberty such that by age 30 -40 years, the FIX levels are usually normal.…”