遗传 ›› 2019, Vol. 41 ›› Issue (6): 509-523.doi: 10.16288/j.yczz.19-072

• 研究报告 • 上一篇    下一篇

染色质架构蛋白CTCF调控UGT1基因簇的表达

郑晓飞,黄海燕(),吴强()   

  1. 上海交通大学系统生物医学研究院比较生物医学研究中心,系统生物医学教育部重点实验室,上海 200240
  • 收稿日期:2019-03-15 修回日期:2019-04-08 出版日期:2019-06-20 发布日期:2019-05-27
  • 作者简介:郑晓飞,硕士研究生,专业方向：遗传学。E-mail: 1159171993@qq.com
  • 基金资助:
    国家自然科学基金项目资助(81872944);国家自然科学基金项目资助(31470820);国家自然科学基金项目资助(81302861)

Chromatin architectural protein CTCF regulates gene expression of the UGT1 cluster

Xiaofei Zheng,Haiyan Huang(),Qiang Wu()   

  1. Center for Comparative Biomedicine, Key laboratory of Systems Biomedicine (Ministry of Education), Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China
  • Received:2019-03-15 Revised:2019-04-08 Published:2019-06-20 Online:2019-05-27
  • Supported by:
    Supported by the National Natural Science Foundation of China(81872944);Supported by the National Natural Science Foundation of China(31470820);Supported by the National Natural Science Foundation of China(81302861)

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摘要：

尿苷二磷酸葡萄糖醛酸转移酶(UDP-glucuronosyltransferase, UGT)是一类重要的Ⅱ相药物代谢酶,通过葡萄糖醛酸接合反应代谢大量内外源小分子化合物,对机体维持内部动态平衡具有重要意义。UGT基因突变或表达异常会造成高胆红素血症等多种疾病、影响药物疗效或减弱代谢药物能力,因此探索UGT表达调控机制将会为人类疾病的预防和个体化医疗以及精准医学提供科学依据。脊椎动物UGT分为UGT1和UGT2两个亚家族,UGT1基因簇结构与原钙粘蛋白(protocadherin, Pcdh)、免疫球蛋白或B细胞受体(immunoglobulin or B-cell receptor)、T细胞受体(T-cell receptor)基因簇类似,但与UGT2结构不同,分为可变区和恒定区,可变区包含成串排列的外显子,任意一个外显子都可以被可变剪接到下游同一套恒定区外显子上,形成9种UGT1信使RNA并翻译成不同UGT1葡醛酸转移酶亚型。本实验室前期工作发现,染色质架构蛋白CTCF结合DNA的方向性在染色质三维结构构建中发挥重要作用。基于此,为了进一步解析UGT1复杂基因簇的三维转录调控机制,本研究分析和比较了人和小鼠UGT1基因簇的CTCF结合位点(CTCF binding site, CBS)的方向性分布,发现人和小鼠的UGT1基因簇中CBS分布差异很大。以人肺癌细胞系A549为模型,通过RNAi敲低细胞中CTCF和SMC3 (cohesin亚基),证明了CTCF和cohesin蛋白参与调控人UGT1基因簇的转录表达。进一步采用CRISPR介导的DNA片段编辑技术对hCBS1进行了原位反转(in situ inversion)和删除,并通过RNA-seq分析技术发现hCBS1删除能够显著降低UGT1A6、UGT1A7和UGT1A9的表达水平,然而hCBS1反转仅仅显著降低UGT1A7的表达水平。上述研究表明hCBS1参与UGT1A6、UGT1A7和UGT1A9的转录调节,是人UGT1基因簇的潜在转录调控元件。本研究为未来进一步探索UGT1基因簇的三维基因转录调控机制提供了实验基础。

关键词: UGT1, CTCF, CRISPR/Cas9, DNA片段编辑, 转录调控

Abstract:

UDP-glucuronosyltransferases (UGTs) are an important family of phase Ⅱ drug-metabolizing enzymes that catalyze the glucuronidation of numerous endogenous or exogenous small compounds. The aberrant expression of UGT isoforms causes many diseases, such as hyperbilirubinemia and affect drug efficacy or toxicity. Understanding mechanisms of UGT gene regulation will provide scientific foundations for disease prevention and personalized or precision medicine. Vertebrate UGT family genes can be divided into UGT1 and UGT2 subfamilies. Similar to the protocadherin, immunoglobulin, and T-cell receptor gene clusters and different from the UGT2 gene cluster, the UGT1 gene cluster is organized into variable and constant regions. The UGT1 variable region contains a tandem array of variable exons, each of which can be alternatively spliced to a single set of 4 downstream constant exons, generating at least nine UGT1 mRNAs that could be translated into different UGT1 glucuronyltransferase isoforms. Our previous work reveals that the relative orientations and locations of CTCF binding sites play a key role in the three-dimensional organization of the mammalian genomes in cell nuclei. Thus in order to study the transcriptional mechanisms of UGT1 gene cluster, the distributions and orientations of CTCF binding sites (CBSs) are analyzed and compared between human and mouse UGT1 gene clusters. We find that the CBSs in the UGT1 gene cluster are not conserved between human and mouse species. We show that CTCF and cohesin regulate the transcription of the UGT1 gene cluster by knocking down the CTCF or the cohesin subunit SMC3 in the human A549 cell line. By using CRISPR DNA-fragment editing, we deleted and inverted hCBS1. By RNA-seq experiments, we find that hCBS1 deletion results in a significant decrease of levels of the UGT1A6, UGT1A7, and UGT1A9 gene expression and that hCBS1 inversion results in a significant decrease of levels of the UGT1A7 gene expression. Our data suggest that the CTCF binding site hCBS1 plays an important regulatory role in the regulation of UGT1 gene expression, providing an experimental basis for further mechanistic studies of the 3D genome regulation of the UGT1 gene cluster.

Key words: UGT1, CTCF, CRISPR-Cas9, DNA fragment editing, transcriptional regulation