Molecular pathogenesis of Gilbert's syndrome:: decreased TATA-binding protein binding affinity of UGT1A1 gene promoter
Molecular pathogenesis of Gilbert's syndrome:: decreased TATA-binding protein binding affinity of UGT1A1 gene promoter
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DOI：
10.1097/fpc.0b013e328012d0da
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发表时间：
2007-04-01
影响因子：
2.6
通讯作者：
Chao, You-Chen
中科院分区：
文献类型：
--
作者：
Hsieh, Tsai-Yuan;Shiu, Tzu-Yue;Chao, You-Chen
目的 吉尔伯特综合征是一种先天性、非溶血性、非结合性高胆红素血症。吉尔伯特综合征最常见的基因型是 UDP-葡萄糖醛酸基转移酶 1A1 (UGT1A1) 基因启动子中的纯合多态性 A(TA)(7)TAA，在 TATA 盒样序列中插入胸腺嘧啶，导致 UGT1A1 活性降低。然而，导致 UGT1A1 活性降低的机制尚未阐明。为了阐明吉尔伯特综合征患者 UGT1A1 活性缺陷的机制。方法使用野生型 A(TA)(6)TAA 或突变型 A(TA)(7)TAA 启动子和荧光素酶报告基因在两种不同的肝癌细胞系中进行启动子活性测定。使用野生型或突变型 TATA 盒寡核苷酸探针和核提取物或 TATA 结合蛋白，通过竞争性电泳迁移率变动测定来评估对核蛋白复合物或 TATA 结合蛋白的结合亲和力。还通过定量电泳迁移率变动分析研究了TATA结合蛋白与野生型或突变型寡核苷酸探针之间复合物的形成。结果UGT1A1基因的启动子活性的TATA盒样序列中存在TA插入。竞争性电泳迁移率变动测定显示突变型 TATA 盒样序列 A(TA)7TAA 的核蛋白复合物结合亲和力和 TATA 结合蛋白结合亲和力降低。同时比较突变体A(TA)(5)TAA和A(TA)(8)TAA，定量电泳迁移率变动分析表明，随着TATA-box-like序列中TA重复次数的增加，TATA结合蛋白结合亲和力逐渐降低。结论UGT1A1启动子TATA-box-like序列中TA的插入影响了其与TATA结合蛋白的结合亲和力，导致其活性降低。这解释了吉尔伯特综合征的发病机制。
Objectives Gilbert's syndrome is a congenital, nonhemolytic, unconjugated hyperbilirubinemia. The most common genotype of Gilbert's syndrome is the homozygous polymorphism, A(TA)(7)TAA, in the promoter of the gene for UDP-glucuronosyltransferase 1A1 (UGT1A1), with a thymine adenine insertion in the TATA-box-like sequence, which results in a decrease in UGT1A1 activity. The mechanism responsible for this decrease in UGT1A1 activity, however, has not been elucidated. To clarify the mechanism underlying this deficiency in UGT1A1 activity in patients with Gilbert's syndrome.Methods The promoter activity assay using the wild-type A(TA)(6)TAA or the mutant A(TA)(7)TAA promoter and a luciferase reporter was performed in two different hepatoma cell lines. The binding affinity for a nuclear protein complex or for TATA-binding protein was evaluated by a competitive electophoretic mobility shift assay using wild-type or mutant TATA-box-like oligonucleotide probes and nuclear extract or TATA-binding protein. The formation of complexes between TATA-bincling protein and wild-type or mutant oligonucleotide probes was also studied by a quantitive electophoretic mobility shift assay.Results A TA insertion in the TATA-box-like sequence of the promoter activity of UGT1A1 gene. A competitive electrophoretic mobility shift assay showed a decrease in nuclear proteincomplex binding affinity and TATA-binding protein binding affinity of the mutant TATA-box-like sequence A(TA)7TAA. When the mutants A(TA)(5)TAA and A(TA)(8)TAA were also compared, quantitative electrophoretic mobility shift assay demonstrated that the TATA-binding protein binding affinity progressively decreased as the number of TA repeats in the TATA-box-like sequence increased.Conclusion TA insertion in the TATA-box-like sequence of the UGT1A1 promoter affected its binding affinity for TATA-binding protein, causing a decrease in its activity. This explains the pathogenesis of Gilbert's syndrome.