调控因子在 CYP7A1（胆固醇 7α-羟化酶）启动子和增强子区域的相互作用调节 CYP7A1 的表达。
Interactions Between Regulatory Variants in CYP7A1 (Cholesterol 7α-Hydroxylase) Promoter and Enhancer Regions Regulate CYP7A1 Expression.
机构信息
Department of Cancer Biology and Genetics, Center for Pharmacogenomics, College of Medicine, Ohio State University, Columbus (D.W., K.H., W.S.).
Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville (D.W.).
出版信息
Circ Genom Precis Med. 2018 Oct;11(10):e002082. doi: 10.1161/CIRCGEN.118.002082.
BACKGROUND
CYP7A1 (cholesterol 7α-hydroxylase) catalyzes the rate-limiting step in bile acid biosynthesis from cholesterol-a main pathway for cholesterol removal from the body. CYP7A1 single-nucleotide polymorphisms (SNPs) are associated with total cholesterol and LDL (low-density lipoprotein) levels, risk of cardiovascular diseases, and other phenotypes; however, results are inconsistent, and causative variants remain uncertain, except for a frequent promoter SNP (rs3808607).
METHODS
We used chromatin conformation capture (4C assay), chromatin immunoprecipitation qPCR assay in hepatocytes, and CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing in hepatocellular carcinoma cell line cells to identify regulatory regions for CYP7A1. We then screened for SNPs located in regulatory regions, testing effects on reporter gene assays and on hepatic CYP7A1 expression by measuring allelic mRNA expression imbalance.
RESULTS
4C assays showed several regions interacting with CYP7A1 promoter. CRISPR-mediated genome editing in hepatocellular carcinoma cell line cells revealed a novel CYP7A1 enhancer and a repressor region, located >10 kb downstream of the CYP7A1 promoter. SNP screening with an allelic mRNA expression imbalance in human livers and reporter gene assays identified a frequent functional SNP (rs9297994) located in the downstream CYP7A1 enhancer region. SNP rs9297994 is in high linkage disequilibrium with promoter SNP rs3808607 but has opposite effects on CYP7A1 mRNA expression. Their combined effects using a 2-SNP model robustly associate with hepatic CYP7A1 mRNA expression, ranging >2 orders of magnitude. Moreover, only the 2-SNP model, but not each SNP alone, is significantly associated with LDL levels, risk of coronary artery disease, statin response, and diabetes mellitus in several clinical cohorts, including CATHGEN (Catheterization Genetics) and Framingham.
CONCLUSIONS
Two interacting regulatory SNPs modulate CYP7A1 expression and are associated with risk of coronary artery disease and diabetes mellitus.
背景
CYP7A1（胆固醇 7α-羟化酶）催化胆固醇生物合成胆汁酸的限速步骤-这是体内胆固醇清除的主要途径。CYP7A1 单核苷酸多态性（SNP）与总胆固醇和 LDL（低密度脂蛋白）水平、心血管疾病风险和其他表型有关；然而，结果不一致，且致病变体仍不确定，除了常见的启动子 SNP（rs3808607）。
方法
我们使用染色质构象捕获（4C 测定法）、肝细胞染色质免疫沉淀 qPCR 测定法和 CRISPR（成簇规律间隔短回文重复）介导的肝癌细胞系细胞中的基因组编辑来鉴定 CYP7A1 的调节区域。然后，我们筛选位于调节区域的 SNP，通过测量等位基因 mRNA 表达失衡，测试对报告基因测定和肝 CYP7A1 表达的影响。
结果
4C 测定法显示与 CYP7A1 启动子相互作用的几个区域。肝癌细胞系细胞中的 CRISPR 介导的基因组编辑揭示了一个新的 CYP7A1 增强子和一个位于 CYP7A1 启动子下游 >10kb 的抑制子区域。在人类肝脏和报告基因测定中进行的等位基因 mRNA 表达失衡 SNP 筛选确定了位于下游 CYP7A1 增强子区域的一个常见功能 SNP（rs9297994）。SNP rs9297994 与启动子 SNP rs3808607 高度连锁，但对 CYP7A1 mRNA 表达的影响相反。使用 2-SNP 模型的联合效应与肝 CYP7A1 mRNA 表达高度相关，范围 >2 个数量级。此外，只有 2-SNP 模型，而不是每个 SNP 单独，与 LDL 水平、冠状动脉疾病风险、他汀类药物反应和糖尿病在几个临床队列中显著相关，包括 CATHGEN（导管遗传学）和弗雷明汉。
结论
两个相互作用的调节 SNP 调节 CYP7A1 的表达，并与冠状动脉疾病和糖尿病的风险相关。
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