TRANSCRIPTIONAL CONTROL OF THE FACTOR-IX GENE - ANALYSIS OF 5 CIS-ACTING ELEMENTS AND THE DELETERIOUS EFFECTS OF NATURALLY-OCCURRING HEMOPHILIA-B LEYDEN MUTATIONS

Hemophilia B Leyden is a rare form of inherited factor IX deficiency i n which patients experience spontaneous postpubertal recovery of facto r IX levels. The mutations resulting in this disorder are localized in a 40-nucleotide region encompassing the major transcriptional start s ite for factor IX. Here we report the further characterization of five cis-acting elements in the factor IX promoter and the effects on prot ein binding and transcriptional activation of five Leyden mutations (a t nucleotides +13. -5, -6, -20, and -26) that occur within the proxima l three elements (sites 1 through 3). Band-shift studies using nuclear extracts from four different rat tissues have shown that at least som e 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 elemen t plays an important role in mediating the liver-specific expression o f factor IX. Additional studies with liver nuclear extracts obtained a t several different points in development have shown an increase in DN A 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 t hat the binding of nuclear proteins to each of the mutant sites is dis rupted to a variable extent. There appears to be some, although reduce d, protein binding to all of the mutant oligonucleotides apart from th e -26 mutant. In vitro transcription assays have shown that each of th e mutations reduces the global proximal promoter activity by approxima te to 40%. Two double mutant promoters did not show any additional dow nregulation in the in vitro transcription assay. In experiments design ed 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 stu dies show that sites 4 and 5 are the strongest activators and that tra nsactivation from site 5 is further enhanced by the albumin D site-bin ding protein. In summary, these investigations show deleterious effect s of each of the Leyden mutations tested an the binding of trans-actin g factors and also show disruption of transcriptional activation in a functional in vitro transcription assay. Our results also show that ci s-acting elements 4 and 5 are the principal activators of this locus. (C) 1994 by The American Society of Hematology.