Regulation of Nuclear Receptor Expression and Function

最近的进展。我的研究计划的总体目标是增加我们对化学生物学和细胞和分子因子的复杂网络的理解，这些因子控制着核受体超家族成员的表达和功能，如雌甾烷X受体（PXR），组成型雄烷受体（CAR）和维生素D受体（VDR）。这些受体调节基因的表达，这些基因参与许多化学物质的运输，生物活化和解毒，包括药物，环境化学物质和我们体内存在的化学物质，如类固醇激素和维生素。在过去的五年里，我的研究重点是研究PXR，CAR和VDR的化学调控，阐明PXR表达的转录后机制，并发现PXR和CAR的新生物学作用。这些研究的结果为我的NSERC发现资助更新申请提供了科学基础。目标.目的是通过研究参与控制PXR和VDR表达和功能的特定microRNA的作用来研究PXR和VDR的共调节，鉴定和描述共激活因子对PXR和CAR调节的贡献，并研究其他受体对PXR和CAR的调节。我的研究计划的长期目标是提高我们的核受体的系统生物学的基础知识。方法论人肝细胞原代培养物、人肠细胞原代培养物和LS 180人腺癌细胞将作为实验模型。实验方法将包括使用微小RNA的模拟物和抑制剂、药理学拮抗和反向激动、基因编辑、基于细胞的荧光素酶报告基因测定、时间分辨荧光共振能量转移竞争性配体结合测定、荧光显微镜、共免疫沉淀、下拉测定、哺乳动物双杂交测定、染色质免疫沉淀测定、实时聚合酶链式反应、和免疫印迹分析。HQP我的研究计划将为2名硕士生，2名博士生和5名本科暑期学生提供学习机会。这项研究将构成每个研究生论文的基础，并为本科生提供研究经验。冲击产生的基础科学知识将有助于增加我们对影响PXR和CAR表达和功能的各种因素的理解。 这些信息将预测和解释我们对内源性物质、药物和其他化学物质的反应的个体差异。我提出的研究计划将为受体生物学领域的下一代科学家提供培训。具备生物化学、细胞生物学和药理学方面的技能和经验，我的HQP将为加拿大生命科学/生物技术/制药部门的研究和开发工作贡献他们的专业知识。

Recent Progress. The overall goal of my research program is to increase our understanding of the chemical biology and the intricate network of cellular and molecular factors that govern the expression and function of members of the superfamily of nuclear receptors, such as pregnane X receptor (PXR), constitutive androstane receptor (CAR), and vitamin D receptor (VDR). These receptors regulate the expression of genes involved in the transport, bioactivation, and detoxification of many chemicals, including drugs, environmental chemicals, and those present in our body, such as steroid hormones and vitamins. My studies in the past five years have focused on studying chemical regulation of PXR, CAR, and VDR, elucidating post-transcriptional mechanism of PXR expression, and discovering novel biological actions of PXR and CAR. Results from these studies provide a scientific foundation for my NSERC Discovery Grant renewal application. Objectives. The objectives will be to study co-regulation of PXR and VDR by investigating the role of specific microRNAs involved in controlling the expression and function of PXR and VDR, identify and delineate the contribution of coactivators to the regulation of PXR and CAR, and investigate the regulation of PXR and CAR by other receptors. The longer-term objective of my research program is to enhance our fundamental knowledge of the systems biology of nuclear receptors. Methodology. Primary cultures of human hepatocytes, primary cultures of human intestinal cells, and LS180 human adenocarcinoma cells will be the experimental models. Experimental approaches will include the use of a mimic and an inhibitor of a microRNA, pharmacological antagonism and inverse agonism, gene editing, cell-based luciferase reporter gene assays, time-resolved fluorescence resonance energy transfer competitive ligand binding assay, fluorescence microscopy, co-immunoprecipitation, pull down assays, mammalian two-hybrid assay, chromatin immunoprecipitation assay, real-time polymerase chain reaction, and immunoblot analyses. HQP. My proposed research program will provide learning opportunities for 2 MSc students, 2 PhD students, and 5 undergraduate summer students. The research will form the basis of each graduate student's thesis and provide research experience to undergraduate students. Impact. The fundamental scientific knowledge generated will help increase our understanding of the various factors that influence PXR and CAR expression and function. The information will predict and explain inter-individual differences in how we respond to endogenous substances, drugs, and other chemicals. My proposed research program will provide training to the next generation of scientists in the field of receptor biology. Equipped with skills and experiences in biochemistry, cell biology, and pharmacology, my HQP will contribute their expertise to the research and development efforts in the life sciences/biotechnology/pharmaceutical sectors in Canada.