Transcriptional control of the factor IX gene: analysis of five cis- acting
elements and the deleterious effects of naturally occurring hemophilia B
Leyden mutations
DJ Picketts, CR Mueller and D Lillicrap
Department of Pathology, Queen's University, Kingston, Ontario, Canada.
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.
Volume 84,
Issue 9,
pp. 2992-3000,
11/01/1994
Copyright © 1994 by The American Society of Hematology
