UGT1基因簇的三维染色质结构与转录调控相关机制研究

The variable and constant genomic organization of UGT1 cluster, which encodes the UGT1 family of drug-metabolism enzymes, is highly conserved in vertebrate, including zebrafish. This striking organization is reminiscent of those of the protocadherin, immunoglobulin, and T-cell receptor gene clusters. The encoded UGT1 proteins have been shown to have glucuronosyltransferase activity in vertebrate, including zebrafish. Its expression pattern is significantly influenced by numerous in-vivo and in-vitro small molecules, such as hormones, drugs, and carcinogens, and varies delicatedly in a tissue-specific and developmental manner. We previously found that the transcription of members of the UGT1 cluster is significantly regulated by nuclear receptor family, and interestingly, several members of the UGT1 gene cluster are even co-regulated. In addition, we have identified 6 regulatory elements, including two CTCF-binding regions, potentially involved in the UGT1 regulation. In this application, we will set out to explore how these regulatory elements are organized and coordinated, in such a highly condense and dynamic 3D context, for the transcripition regulation of the UGT1 cluster by a combination of CRISPR DNA-fragment editing and chromosome conformation capture methods. Their functions will be verified in cellular and mouse models. This proposed work, if funded, may provide important insights on how genetic and environmental factors affect individual phenotypic variability of the UGT1 drug-metabolizing enzyme family and lay a solid foundation for future personalized drug-dosage treatment of human diseases.

人类药物代谢酶UGT1基因簇的一级线性基因组结构包含可变区和恒定区，与原钙粘蛋白、免疫球蛋白（抗体）以及T-细胞受体基因簇类似。我们前期发现UGT1基因簇的基因组结构在脊椎动物包括斑马鱼中保守，其编码的UGT1蛋白也具有UDP葡萄糖醛酸转移酶活性，经基因编辑、染色质构象捕获实验和转录组学分析，我们发现有6个调控区域对UGT1基因簇的细胞特异性表达和受核受体介导的转录调节发挥重要作用。它们包括两个CTCF结合区域和4个潜在的增强子区域，分散在UGT1基因簇的内部和上游远端区域。本申请将系统地研究这6个调控域在高度致密而动态的染色质三维结构中如何协同作用控制UGT1各成员的精确时空分布、参与核受体家族蛋白介导的转录调控，并解析哪些染色质架构蛋白参与维持这种三维调控网络。该工作将有助于揭示遗传和环境因素影响UGT1药物代谢酶家族个体表型差异的机制，为将来临床合理用药及人类疾病的预防提供科学依据。

人类药物代谢酶UGT1基因簇的线性基因组结构包含可变区和恒定区，与原钙粘蛋白、免疫球蛋白（抗体）以及T-细胞受体基因簇类似。该项目前期发现UGT1基因簇的基因组结构在脊椎动物包括斑马鱼中保守，其编码的UGT1蛋白具有UDP葡萄糖醛酸转移酶活性。经基因编辑、染色质构象捕获实验和转录组学分析，该团队发现有6个调控区域对UGT1基因簇的细胞特异性表达和受核受体介导的转录调节发挥重要作用。它们包括两个CTCF结合区域和4个潜在的增强子区域，分散在UGT1基因簇的内部和上游远端区域。该项目系统地研究这些调控域在高度致密而动态的染色质三维结构中如何协同作用控制UGT1各成员的精确表达、参与核受体家族蛋白介导的转录调控，并解析染色质架构蛋白如何参与维持这种三维调控网络。染色质架构蛋白CTCF方向性结合DNA在染色体三维结构建立中发挥关键作用。该项目首先分析和比较了人和小鼠UGT1基因簇的CTCF结合位点（CBS）的方向性分布，发现人和小鼠的UGT1基因簇中CBS分布存在很大差异，人UGT1基因簇内高度保守的CTCF结合位点在小鼠Ugt1基因簇相应位置未发现有CTCF富集。其次，解析了UGT1基因簇附近的染色质拓扑结构域（TAD）的结构，各基因的染色质远程互作蓝图以及增强子标志p300、H3K4me1和H3K27ac的分布，确定了潜在调控元件对UGT1的转录调控和它们之间的协同作用。然后，以人肝癌细胞系(HepG2)、肺癌细胞系（A549）和子宫内膜癌细胞系（Hec1B）为模型，采用CRISPR-Cas9介导的DNA片段编辑技术对潜在调控元件进行原位删除，并通过RNA-seq分析技术发现这些调控元件能够显著调控UGT1相关亚型的转录水平。最后，采用定量高分辨率染色质构象捕获技术（QHR-4C）检测了转录调控元件参与调控UGT1基因簇转录的局部空间构象。CRISPR-Cas9编辑系统干扰这些元件会造成局部染色质空间构象发生改变，从而影响UGT1基因簇的转录。该工作有助于揭示遗传和环境因素影响UGT1药物代谢酶家族个体表型差异的机制，为将来临床合理用药及人类疾病的预防提供科学依据。