Targeting Dynamics of CAR and PXR in the Mouse and Human Genomes

摘要：在哺乳动物生物中，异种感应受体CAR（组成型雄甾烷受体；NR1I3）和PXR（孕烷X受体；NR1I2）作为化学暴露的毒理学和生理反应的介质发挥着重要作用。作为核受体，CAR和PXR都是一个大型基因网络的转录调节因子，编码一个功能反应网络，包括代谢和运输外源物质，调节脂质和能量稳态，以及调节细胞增殖。小鼠受体生物学模型已被广泛应用于表征这些特征。尽管它们很重要，但老鼠的受体并不等同于人类。在其他方面，各自的受体在其配体特异性上存在根本差异，这是由定义其配体结合袋的氨基酸接触残基的显着差异编码的。这些受体在功能上也有所不同，例如，在非基因毒性受体激活剂（如直接配体TCPOBOP）或间接激活剂（如苯巴比妥（PB））的促进下，CAR在小鼠肝细胞癌的发展中被认为是必要的。然而，在人群中进行的广泛流行病学研究已经确定，长期接触铅不会增加癌症风险。引人注目的是，CAR和PXR在结合DNA目标的能力上有重叠和不同的偏好。令人惊讶的是，使用染色质免疫沉淀和生物信息学分析的结果揭示了物种之间转录因子结合位点的广泛差异。每种受体的物种选择性剪接变异进一步定义了物种反应的内在差异。为了对哺乳动物物种的受体功能进行基于生物学和科学上可辩护的推断，以准确预测潜在的人类毒性，描述这些差异反应背后的分子机制至关重要。本研究计划的中心假设是，小鼠和人类的CAR和PXR所贡献的独特生物学作用，是通过它们各自的动态和差异能力与它们的基因组目标相互作用而在最基本的水平上被编程的。此外，我们假设在人类中，CAR的CAR2和CAR3剪接变体贡献了一层额外的生物多样性，部分是通过与它们自己独特的DNA相互作用的差异相互作用来编程的。先进的策略需要使用独特的生物学模型和应用强大和无偏倚的染色质免疫沉淀方法，再加上下一代测序和生物信息学分析。总的来说，这些研究将揭示由这些受体连接的全球相互作用，确定共享和独特的受体结合位点，最终驱动这些关键的异种受体在哺乳动物基因网络中的生物学和毒理学功能。

DESCRIPTION (provided by applicant): Abstract In mammalian organisms, the xenobiotic-sensing receptors, CAR (constitutive androstane receptor; NR1I3) and PXR (pregnane X receptor; NR1I2), contribute critically as mediators of toxicological and physiological responses to chemical exposure. As nuclear receptors, both CAR and PXR function as transcriptional regulators for a large network of genes encoding a functional web of responses that include the metabolism and transport of xenobiotics, regulation of lipid and energy homeostasis, and modulation of cell proliferation. Mouse models of receptor biology have been deployed widely to characterize these features. Their importance notwithstanding, the mouse receptors are not equivalent to human. Among other aspects, the respective receptors differ fundamentally in their ligand specificity, encoded by marked variation in amino acid contact residues that define their ligand binding pockets. The receptors differ functionally as well, for example with CAR identified as necessary in the development of hepatocellular carcinoma in mice following promotion by non-genotoxic receptor activators, such as the direct ligand, TCPOBOP, or indirect activators such as phenobarbital (PB). However, extensive epidemiological studies in human populations have ascertained no excess risk of cancers following chronic PB exposures. Strikingly, CAR and PXR share overlapping as well as distinct preferences for their abilities to bind DNA targets. Surprisingly, results using chromatin immunoprecipitation and bioinformatics analyses have revealed extensive divergence among transcription factor binding sites between species. Species-selective splice variation in each receptor further defines inherent differences in species response. To allow biologically-based and scientifically defensible extrapolations of receptor function across mammalian species that accurately predict potential human toxicities, it is critical to delineate the molecular mechanisms underlying these differential responses. The central hypothesis of this research program is that unique biological roles contributed by mouse and human CAR, and PXR, are programmed at their most basic level by their respective dynamic and differential abilities to interact with their genomic targets. Further, we hypothesize that in humans, the CAR2 and CAR3 splice variants of CAR contribute an added layer of biological diversity, programmed in part through differential interaction with their own distinct DNA interactions. The strategies advanced entail the use of unique biological models and application of powerful and unbiased chromatin immunoprecipitation approaches, coupled with next-generating sequencing and bioinformatics analyses. Overall, these studies will reveal the global interactome bridged by these receptors, identifying both shared and distinct sites of receptor binding that ultimately drive the biological and toxicological functions contributed by these critical xenoreceptors across mammalian gene networks.