Identification of a novel polymorphic enhancer of the human CYP3A4 gene
Identification of a novel polymorphic enhancer of the human CYP3A4 gene
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DOI：
10.1124/mol.65.2.326
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发表时间：
2004-02-01
影响因子：
3.6
通讯作者：
Kamataki, T
中科院分区：
文献类型：
--
作者：
Matsumura, K;Saito, T;Kamataki, T
CYP 3A 4是成人肝脏中最丰富的细胞色素P450形式，其活性在个体间存在广泛差异。这种变异性被认为主要是由转录和遗传因素引起的，但对潜在的机制知之甚少。本研究的目的是阐明CYP 3A 4基因转录的调控机制，并寻找CYP 3A 4基因5 ′侧翼区的遗传多态性。用一系列CYP 3A 4启动子-荧光素酶报告质粒瞬时转染人肝癌HepG 2细胞和正常人肝细胞，发现从-11.4到-10.5个酶的区域，命名为CYP 3A 4的组成性肝增强子模块（CLEM 4），对CYP 3A 4基因的组成性激活是重要的。使用从HepG 2细胞制备的核提取物的凝胶位移测定显示，HNF-1 α、HNF-4 α、USF 1和AP-1与CLEM 4相互作用。此外，将突变引入其结合位点表明，基本上所有位点都是最大增强子活性所需的。通过对法国人基因组DNA中CLEM 4的遗传多态性进行筛查，我们发现了一种新的变异体，TGT插入在-11，129和-11，128之间（-11，129_-11，128insTGT），其等位基因频率为3.1%。~ 11，129_~ 11，128 insTGT导致USF 1结合的破坏和增强子活性的36%降低。这些结果表明，CLEM 4是肝脏中CYP 3A 4基因的组成性增强子，并且-11，129_-11，128insTGT可能至少部分地导致CYP 3A 4表达的个体间差异。
CYP3A4, the most abundant form of cytochrome P450 in the human adult liver, shows wide interindividual variation in its activity. This variability is thought to be caused largely by transcriptional and genetic factors, yet the underlying mechanisms are poorly understood. The purpose of this study was to clarify the mechanisms controlling the CYP3A4 gene transcription and to search for genetic polymorphisms in the 5'-flanking region of the CYP3A4 gene. Transient transfection of human hepatoma HepG2 cells and of normal human hepatocytes with a series of CYP3A4 promoter-luciferase reporter plasmids revealed that a region from - 11.4 to - 10.5 kilobases, designated the constitutive liver enhancer module of CYP3A4 (CLEM4), was important for the constitutive activation of the CYP3A4 gene. Gel shift assay using nuclear extracts prepared from HepG2 cells showed that HNF-1alpha, HNF-4alpha, USF1, and AP-1 interacted with CLEM4. Furthermore, the introduction of mutations into their binding sites demonstrated that essentially all sites were required for the maximal enhancer activity. Screening for genetic polymorphisms within CLEM4 in genomic DNA from French persons, we identified the novel variant, TGT insertion between - 11,129 and - 11,128 ( - 11,129_ - 11,128insTGT), whose allele frequency was 3.1%. The - 11,129_ - 11,128insTGT resulted in the disruption of USF1 binding and a 36% reduction of the enhancer activity. These results suggest that CLEM4 is a constitutive enhancer of the CYP3A4 gene in the liver and that - 11,129_ - 11,128insTGT may at least partly contribute to the interindividual variability of CYP3A4 expression.