Cis-acting regulatory elements regulating CYP3A4 transcription in human liver.
Cis-acting regulatory elements regulating CYP3A4 transcription in human liver.
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DOI：
10.1097/fpc.0000000000000402
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发表时间：
2020-07
影响因子：
2.6
通讯作者：
Wang D
中科院分区：
文献类型：
--
作者：
Collins JM;Wang D
CYP3A4酶是肝脏中最丰富的药物代谢酶，代谢约50%的常用药物。CYP3A4在表达和酶活性方面表现出很大的人与人之间的差异，原因不明。本研究旨在鉴定控制CYP3A4转录的顺式调控元件，在原代培养的人肝细胞和肝细胞系中，采用染色质构象捕获（4C和3C测定）、染色质免疫沉淀后的qPCR （ChIP-qPCR）、crispr介导的基因组区域缺失和报告基因测定。4C分析鉴定了四个区域（R1-R4）与CYP3A4启动子相互作用，其中一个与先前鉴定的上游增强子CLEM4/XREM （R2）重叠，而其他三个是新的。ChIP-qPCR、报告基因检测和crispr介导的缺失实验表明R2和R4都具有调控作用。有趣的是，R4的缺失增加了CYP3A4的表达，同时降低了CYP3A43的表达，这可能是由于CYP3A集群内竞争性的域-域相互作用，而R4的缺失增加了CYP3A4启动子与R2之间的相互作用。我们还在R4中发现了一个SNP rs62471956，在报告基因试验中，变异等位基因a的转录活性增加。在136个肝脏样本中，rs62471956 A等位基因与CYP3A43的高表达和CYP3A4的低表达相关，进一步支持R4对CYP3A4和CYP3A43的相反作用。rs62471956与CYP3A4*22完全失活，可能导致CYP3A4*22表达降低。这些结果验证了先前确定的CYP3A4增强子（CLEM4和XREM），并通过CYP3A簇内竞争性域-域相互作用证明了CYP3A4转录控制的其他调控机制。
The CYP3A4 enzyme is the most abundant drug-metabolizing enzyme in the liver, metabolizing ~50% of commonly used medications. CYP3A4 displays large inter-person variability in expression and enzyme activity with unknown causes. This study aims to identify cis-acting regulatory elements controlling the transcription of CYP3A4, using chromatin conformation capture (4C and 3C assays), chromatin immunoprecipitation followed by qPCR (ChIP-qPCR), CRISPR-mediated deletions of genomic regions and reporter gene assays in primary culture human hepatocytes and hepatic cell lines. 4C assays identified four regions (R1–R4) interacting with the CYP3A4 promoter, one of which overlaps with the previously identified upstream enhancers CLEM4/XREM (R2) while the other three are novel. ChIP-qPCR, reporter gene assays and CRISPR-mediated deletion experiments indicate regulatory roles for both R2 and R4. Interestingly, deletion of R4 increased CYP3A4 while decreasing CYP3A43 expression, possibly due to competitive domain-domain interactions within the CYP3A cluster, supported by deletion of R4 increasing interaction between the CYP3A4 promoter and R2. We also identified a SNP rs62471956 within R4, with the variant allele A having increased transcriptional activity in a reporter gene assay. The rs62471956 A allele is associated with higher CYP3A43 expression and lower CYP3A4 expression in a cohort of 136 liver samples, further supporting the opposing effects of R4 on CYP3A4 and CYP3A43. rs62471956 is in complete LD with CYP3A4*22, potentially contributing to reduced expression of CYP3A4*22. These results validate previously identified enhancers (CLEM4 and XREM) of CYP3A4 and demonstrate additional regulatory mechanisms underlying CYP3A4 transcriptional control via competitive domain-domain interactions within the CYP3A cluster.