Phenobarbital mediates an epigenetic switch at the constitutive androstane receptor (CAR) target gene Cyp2b10 in the liver of B6C3F1 mice [Expression array]

Evidence suggests that epigenetic perturbations are involved in the adverse effects associated with some drugs and toxicants, including certain classes of non-genotoxic carcinogens. Such epigenetic changes (altered DNA methylation and covalent histone modifications) may take place at the earliest stages of carcinogenesis and their identification holds great promise for biomedical research. Here, we evaluate the sensitivity and specificity of genome-wide epigenomic and transcriptomic profiling in phenobarbital (PB)-treated B6C3F1 mice, a well-characterized rodent model of non-genotoxic liver carcinogenesis. Methylated DNA Immunoprecipitation (MeDIP)-coupled microarray profiling of 17,967 promoter regions and 4,566 intergenic CpG islands was combined with genome-wide mRNA expression profiling to identify liver tissue-specific PB-mediated DNA methylation and transcriptional alterations. Only a limited number of significant anti-correlations were observed between PB-induced transcriptional and promoter-based DNA methylation perturbations. However, the constitutive androstane receptor (CAR) target gene Cyp2b10 was found to be concomitantly hypomethylated and transcriptionally activated in a liver tissue-specific manner following PB treatment. Furthermore, analysis of active and repressive histone modifications using chromatin immunoprecipitation revealed a strong PB-mediated epigenetic switch at the Cyp2b10 promoter. Our data reveal that PB-induced transcriptional perturbations are not generally associated with broad changes in the DNA methylation status at proximal promoters and suggest that the drug-inducible CAR pathway regulates an epigenetic switch from repressive to active chromatin at the target gene Cyp2b10. This study demonstrates the utility of integrated epigenomic and transcriptomic profiling for elucidating early mechanisms and biomarkers of non-genotoxic carcinogenesis. 29–32 days old male B6C3F1/Crl (C57BL/6 ♂ x C3H/He ♀) mice were obtained from Charles River Laboratories (Germany). Animals were allowed to acclimatise for 5 days prior to being randomly divided into two treatment groups (n = 10) and phenobarbital (Sigma 04710, 0.05% (w/v) in drinking water) was administered to one group through ad libitum access to drinking water for 28 days. Mice were checked daily for activity and behavior and sacrificed on the last day of dosing (day 28). Blood was withdrawn for PK analysis and target (liver) and non-target (kidney) tissues removed, split into several sections, frozen in liquid nitrogen and stored at 80°C for subsequent analyses. Total RNA from liver and kidney was purified and processed for Affymetrix gene expression profiling while genomic DNA was prepared for promoter array based methylome analysis using the Methylated DNA immunoprecipitation (MeDIP) procedure. Remaining tissue material was used for chromatin immunoprecipitation (ChIP) to analyze histone modifications at individual promoters. Plasma samples were also collected to evaluate phenobarbital exposure in individual animals by LC-MS.已有研究表明，表观遗传扰动（epigenetic perturbations）参与了部分药物与毒物（包括几类非遗传毒性致癌物）相关的不良效应。这类表观遗传改变（包括DNA甲基化异常与共价组蛋白修饰）可发生在癌变的最早阶段，对其的鉴定有望为生物医学研究带来重大突破。本研究针对经苯巴比妥（phenobarbital, PB）处理的B6C3F1小鼠——一种经过充分表征的非遗传毒性肝癌发生啮齿类模型——评估全基因组表观基因组与转录组分析的灵敏度与特异性。本研究将针对17967个启动子区域与4566个基因间CpG岛的甲基化DNA免疫沉淀（Methylated DNA Immunoprecipitation, MeDIP）耦联芯片分析，与全基因组mRNA表达谱分析相结合，以鉴定肝脏组织特异性的PB介导的DNA甲基化与转录改变。仅在少量PB诱导的转录扰动与启动子区域DNA甲基化扰动之间观察到显著负相关。但经PB处理后，组成型雄烷受体（constitutive androstane receptor, CAR）的靶基因Cyp2b10呈现肝脏组织特异性的伴随低甲基化与转录激活状态。此外，通过染色质免疫沉淀（chromatin immunoprecipitation, ChIP）分析活性与抑制性组蛋白修饰后发现，PB在Cyp2b10启动子区域介导了显著的表观遗传开关事件。本研究数据表明，PB诱导的转录扰动通常并不与近端启动子区域DNA甲基化的广泛改变相关，同时提示药物诱导的CAR通路可调控靶基因Cyp2b10处从抑制性染色质到活化染色质的表观遗传转换。本研究证实了整合表观基因组与转录组分析在阐明非遗传毒性致癌物致癌的早期机制与生物标志物方面的应用价值。 本研究使用的29~32日龄雄性B6C3F1/Crl（C57BL/6 ♂ × C3H/He ♀）小鼠购自德国Charles River实验室。小鼠在随机分为两组（每组n=10）前先适应环境5天；其中一组通过自由饮用含0.05%（w/v）苯巴比妥（Sigma 04710）的饮水给药28天。每日观察小鼠的活动与行为状态，并于给药结束当日（第28天）实施安乐死。采集血液用于药代动力学（PK）分析，摘取靶组织（肝脏）与非靶组织（肾脏），将其分割为多份后置于液氮中速冻，并于-80℃保存以备后续分析。纯化肝脏与肾脏的总RNA，用于Affymetrix基因表达谱分析；同时提取基因组DNA，通过甲基化DNA免疫沉淀（MeDIP）流程制备用于启动子芯片甲基化组分析的样本。剩余的组织样本则用于染色质免疫沉淀（ChIP）分析单个启动子区域的组蛋白修饰情况。此外还收集血浆样本，通过液相色谱-质谱联用（LC-MS）技术检测每只动物的苯巴比妥暴露水平。