Kurachi S, Furukawa M, Salier J P, Wu C T, Wilson E J, French F S, Kurachi K
Department of Human Genetics, University of Michigan Medical Center, Ann Arbor 48109-0618.
Biochemistry. 1994 Feb 15;33(6):1580-91. doi: 10.1021/bi00172a039.
Hemophilia B-Leyden is characterized by the gradual amelioration of bleeding after the onset of puberty. All Leyden phenotype mutations found to date lie within the Leyden-specific region, which spans roughly nt-40 to +20 in the 5' end of the human factor IX gene. With HepG2 cell nuclear extracts, the Leyden-specific region and its immediate neighboring region of the normal factor IX gene showed five DNase I footprints: 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 affinities of short oligonucleotides containing sequences of FP-I, FP-II, or FP-III were substantially reduced in the presence of Leyden phenotype mutations in these areas, correlating well with the negative effects 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 and FP-II but generated a new footprint, FP-III' (nt -34 to -23), partially overlapping with FP-III, indicating mutation-dependent competitive protein binding at these sites. Although the FP-III' area contains an androgen responsive element-like sequence, the nuclear protein that binds at FP-III' is not androgen receptor. The protein was not recognized by anti-androgen receptor antibody and, furthermore, was present not only in liver but also in both androgen receptor-positive and androgen 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 greatly reduced in the presence of exogenous androgen receptor, suggesting a possible interaction of this protein with androgen receptor. The affinities of both this protein and a protein which binds to FP-III (presumably HNF-4) to -32sub with a mutation at nt -26 were grossly lowered. These findings suggest that the amelioration of hemophilia B-Leyden with a mutation at nt -20 after puberty involves binding of a specific non-androgen receptor 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 receptor upon a surge of testosterone.(ABSTRACT TRUNCATED AT 400 WORDS)
莱登型乙型血友病的特征是青春期开始后出血症状逐渐改善。迄今为止发现的所有莱登表型突变都位于莱登特异性区域内，该区域大致跨越人类凝血因子IX基因5'端的nt -40至+20。利用HepG2细胞核提取物，正常凝血因子IX基因的莱登特异性区域及其紧邻区域显示出五个DNase I足迹：FP-I（nt +4至+19）、FP-II（nt -16至-3）、FP-III（nt -27至-19）、FP-IV（nt -67至-49）和FP-V（nt -99至-77）。在这些区域存在莱登表型突变的情况下，含有FP-I、FP-II或FP-III序列的短寡核苷酸的蛋白质结合亲和力大幅降低，这与这些突变对凝血因子IX基因表达的负面影响密切相关。nt -20（T突变为A）处的莱登表型突变导致FP-III和FP-II足迹均消失，但产生了一个新的足迹FP-III'（nt -34至-23），与FP-III部分重叠，表明这些位点存在突变依赖性竞争性蛋白质结合。尽管FP-III'区域包含一个雄激素反应元件样序列，但在FP-III'处结合的核蛋白不是雄激素受体。该蛋白不能被抗雄激素受体抗体识别，此外，在电泳迁移率变动分析中，它不仅存在于肝脏中，还存在于雄激素受体阳性和阴性细胞中。用睾酮处理HepG2细胞后，这种蛋白的核浓度显著增加。在存在外源性雄激素受体的情况下，它与含有FP-III'序列的寡核苷酸（-32sub）的结合亲和力大大降低，表明这种蛋白可能与雄激素受体相互作用。该蛋白和与FP-III结合的蛋白（可能是肝细胞核因子4）与nt -26处有突变的-32sub的亲和力均大幅降低。这些发现表明，青春期后nt -20处有突变的莱登型乙型血友病症状改善涉及一种特异性非雄激素受体核蛋白在FP-III'处的结合，并且在睾酮激增时，它能够通过与雄激素受体增强的相互作用，最佳地替代正常基因中与FP-III结合的蛋白的功能。（摘要截短于400字）
