REGULATORY MECHANISM OF HUMAN FACTOR-IX GENE - PROTEIN-BINDING AT THELEYDEN-SPECIFIC REGION

Hemophilia B-Leyden is characterized by the gradual amelioration of bl eeding after the onset of puberty. All Leyden phenotype mutations foun d to date lie within the Leyden-specific region, which spans roughly n t -40 to +20 in the 5' end of the human factor IX gene. With HepG2 cel l nuclear extracts, the Leyden-specific region and its immediate neigh boring region of the normal factor IX gene showed five DNase I footpri nts: FP-I (nt +4 to +19), FP-II (nt -16 to -3), FP-III (nt -27 to -19) , FP-IV (nt -67 to -49), and FP-V (nt -99 to -77). Protein binding aff inities of short oligonucleotides containing sequences of FP-I, FP-II, or FP-III were substantially reduced in the presence of Leyden phenot ype mutations in these areas, correlating well with the negative effec ts of these mutations on factor IX gene expression. A Leyden phenotype mutation at nt -20 (T to A) caused a loss of both footprints FP-III a nd FP-II but generated a new footprint, FP-III' (nt -34 to -23), parti ally overlapping with FP-III, indicating mutation-dependent competitiv e protein binding at these sites. Although the FP-III' area contains a n androgen responsive element-like sequence, the nuclear protein that binds at FP-III' is not androgen receptor. The protein was not recogni zed by anti-androgen receptor antibody and, furthermore, was present n ot only in liver but also in both androgen receptor-positive and andro gen receptor-negative cells in electrophoretic mobility shift assays. The nuclear concentration of this protein increased significantly upon treatment of the HepG2 cells with testosterone. Its binding affinity to an oligonucleotide (-32sub) containing the FP-III' sequence was gre atly reduced in the presence of exogenous androgen receptor, suggestin g a possible interaction of this protein with androgen receptor. The a ffinities of both this protein and a protein which binds to FP-III (pr esumably HNF-4) to -32sub with a mutation at nt -26 were grossly lower ed. These findings suggest that the amelioration of hemophilia B-Leyde n with a mutation at nt -20 after puberty involves binding of a specif ic non-androgen recepter nuclear protein at FP-III' and it is able to substitute for the function of a protein bound at FP-III in the normal gene optimally through its elevated interaction with androgen recepto r upon a surge of testosterone. The major transcriptional initiation s ite of the factor IX gene in human liver was determined to be at nt -1 76, localizing the entire Leyden-specific region to the 5'-untranslate d region.