Nuclear Receptor DNA Binding in Human Physiology and Disease

描述(申请人提供)：核受体(NRs)是一种配体依赖的转录因子，调节多种基因的表达，涉及人类生理和疾病的几乎所有方面。这在很大程度上是通过首先结合基因调控区域中的特定DNA反应元件(RE)来实现的。虽然在过去的25年里，人们对NR与DNA的结合有了基本的了解，但最近的研究表明，我们对NR-DNA的相互作用以及影响它的因素有更多的了解。此外，尽管神经递质在生理学和疾病中的作用已被大量研究，并且本身就是许多成功药物的靶标，但我们仍然不完全了解它们在疾病易感性或个体对药物治疗的反应中的作用。个体之间的变异性至少部分由他们的遗传构成决定，单核苷酸多态(SNPs)被认为是这种变异性的主要原因。虽然基因编码部分的许多SNPs与蛋白质功能改变有关，但最近的全基因组研究表明，某些SNPs与邻近基因(ESNPs)表达水平的变化有关。然而，缺乏对eSNPs的系统的、功能性的描述。我们认为，相当大比例的SNPs通过改变NRs对其DNA反应元件的亲和力来影响基因的表达。在这项建议中，我们将通过结合生化、分子、基因组和生物信息学的一系列方法来研究NRDNA结合的特异性以及SNPs是如何影响它的。在目标1中，我们将使用基于蛋白质结合微阵列(PBM)的综合方法来详尽地确定一组NRs的DNA结合特异性。PBMS是一种新型的、高通量(10-100,000‘S反应)的体外DNA结合分析方法。PBM结果将用于搜索基因组中潜在的NR靶基因，然后与全基因组定位和表达分析交叉参考。在目标2中，我们将把PBM扩展到100万个反应，以确定与疾病和药物代谢相关的基因调控区域中NRs的亲和力改变SNPs(AaSNPs)。SNP项目管理系统的结果将与可公开获得的数据库(Gwas、DBSNP、GTEx等)相互参照。为了找出影响疾病和药物代谢相关基因表达水平的aaSNPs。在目标3中，将使用PBMS来研究各种因素对NR DNA结合的影响，包括不同的配体、NR伙伴、共调节分子。所有结果将在专门用于该项目的网站以及其他公共数据库上公布，并将开发基于网络的工具，用于寻找基序和目标基因预测。他们将推进将NRS与疾病和药物新陈代谢联系起来的快速跟踪研究的长期目标，从而帮助个性化药物，并确保针对NRS的药物能够以更有效的方式使用。

DESCRIPTION (provided by applicant): Nuclear receptors (NRs) are ligand-dependent transcription factors that regulate the expression of a wide variety of genes involved in nearly al aspects of human physiology and disease. They do so in large part by first binding specific DNA response elements (RE) in regulatory regions of genes. While the past 25+ years has led to a basic understanding of NR DNA binding, recent studies indicate that we have much more to learn about the NR-DNA interaction, and the factors that influence it. Furthermore, while NRs have been investigated heavily for their role in physiology and disease and are themselves targets of many successful drugs, we still do not have a complete understanding of their role in disease susceptibility nor in individual responses to drug treatments. Variability between individuals is determined at least partially by their genetic make-up and single nucleotide polymorphisms (SNPs) are thought to account for much that variability. While many SNPs in the coding portion of genes have been associated with altered protein function, recent genome-wide studies show that certain SNPs are associated with changes in levels of expression of nearby genes (eSNPs). However, what is lacking is a systematic, functional characterization of eSNPs. We propose that a significant proportion SNPs affect gene expression by altering the affinity of NRs for their DNA response elements. In this proposal, we will examine NR DNA binding specificity and how it is influenced by SNPs by integrating a range of biochemical, molecular, genomic and bioinformatics approaches in three Specific Aims: In Aim 1, we will exhaustively determine the DNA binding specificity of a select group of NRs using an integrated approach based on protein binding microarrays (PBMs). PBMs are a novel, high throughput (10- 100,000's reactions) in vitro DNA binding assay. The PBM results will be used to search the genome for potential NR target genes and then cross referenced with genome-wide location and expression analysis. In Aim 2, we will expand PBMs to 1 million reactions in order to identify affinity altering SNPs (aaSNPs) for NRs in regulatory regions of genes associated with disease and drug metabolism. The results from the SNP PBMs will be cross referenced with publicly available databases (GWAS, dbSNP, GTEx, etc.) in order identify aaSNPs that have effects on expression levels of genes relevant to disease and drug metabolism. In Aim 3, PBMs will be used to investigate the effect of a variety of factors on NR DNA binding, including different ligands, NR partners, co-regulatory molecules. All results will be made publically available on a website dedicated to the project, as well as other public databases, and web-based tools for motif finding and target gene prediction will be developed. They will advance the long term goal of fast tracking research linking NRs to disease and drug metabolism, and thereby help personalize medicine and ensure that drugs that target NRs can be used in a more effective fashion.