Nuclear Receptor DNA Binding in Human Physiology and Disease

说明（由申请人提供）：核受体（NR）是配体依赖性转录因子，其调节涉及人类生理和疾病的几乎所有方面的多种基因的表达。它们在很大程度上通过首先结合基因调控区中的特异性DNA反应元件（RE）来实现。虽然过去的25年多已经导致了对NR DNA结合的基本理解，但最近的研究表明，我们对NR-DNA相互作用以及影响它的因素有更多的了解。此外，虽然NR在生理学和疾病中的作用已经得到了大量的研究，并且它们本身也是许多成功药物的靶点，我们仍然没有完全了解它们在疾病易感性或对药物治疗的个体反应中的作用。个体之间的变异性至少部分由其遗传组成决定，单核苷酸多态性（SNP）被认为是造成这种变异性的主要原因。虽然基因编码部分的许多SNP与蛋白质功能改变有关，但最近的全基因组研究表明，某些SNP与附近基因（eSNP）表达水平的变化有关。然而，缺乏的是系统的，功能性的eSNPs表征。我们提出，一个显着的比例SNP影响基因表达，通过改变其DNA响应元件的NR的亲和力。 在这项提案中，我们将研究NR DNA结合特异性，以及它是如何通过整合一系列的生物化学，分子，基因组和生物信息学的方法在三个特定的目标SNP的影响：在目标1，我们将详尽地确定一组选择的NR的DNA结合特异性使用基于蛋白质结合微阵列（PBM）的集成方法。PBM是一种新型的、高通量（10- 100，000次反应）的体外DNA结合测定。PBM结果将用于在基因组中搜索潜在的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中NR的亲和力改变SNP（aaSNP）。SNP PBM的结果将与公开可用的数据库（GWAS、dbSNP、GTEx等）交叉引用。以鉴定对疾病和药物代谢相关基因的表达水平具有影响的aaSNPs。 目的3：利用PBMs研究多种因素对NR DNA结合的影响，包括不同配体、NR配偶体、共调节分子等。 所有结果将在该项目专用网站以及其他公共数据库上公开，并将开发基于网络的基序发现和靶基因预测工具。他们将推进快速跟踪研究的长期目标，将NR与疾病和药物代谢联系起来，从而帮助个性化医疗，并确保靶向NR的药物可以更有效地使用。

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.