Nuclear Receptor DNA Binding in Human Physiology and Disease

描述（由申请人提供）：核受体（NRs）是一种配体依赖性转录因子，可调节涉及人体生理和疾病几乎所有方面的多种基因的表达。它们在很大程度上是通过首先结合基因调控区域的特定DNA反应元件（RE）来实现的。虽然在过去的25年里，人们对NR-DNA结合有了基本的了解，但最近的研究表明，我们对NR-DNA相互作用及其影响因素还有很多需要了解的。此外，尽管人们对nr在生理和疾病中的作用进行了大量研究，并且它们本身也是许多成功药物的靶点，但我们仍然没有完全了解它们在疾病易感性中的作用，也没有完全了解它们对药物治疗的个体反应。个体之间的差异至少部分是由他们的基因组成决定的，单核苷酸多态性（snp）被认为在很大程度上解释了这种差异。虽然基因编码部分的许多snp与蛋白质功能的改变有关，但最近的全基因组研究表明，某些snp与附近基因（eSNPs）表达水平的变化有关。然而，缺乏的是eSNPs系统的功能表征。我们提出，很大一部分snp通过改变rna对其DNA应答元件的亲和力来影响基因表达。在本提案中，我们将在三个特定目标中通过整合一系列生化，分子，基因组和生物信息学方法来研究NR DNA结合特异性以及它如何受到snp的影响：在目标1中，我们将使用基于蛋白质结合微阵列（PBMs）的综合方法详尽地确定一组精选NR的DNA结合特异性。PBMs是一种新型的高通量（10- 100,000的反应）体外DNA结合试验。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，我们将把PBMs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的nr的亲和力改变snp （aaSNPs）。SNP PBMs的结果将与公开可用的数据库（GWAS、dbSNP、GTEx等）交叉参考，以确定影响疾病和药物代谢相关基因表达水平的aaSNPs。在Aim 3中，PBMs将用于研究多种因素对NR DNA结合的影响，包括不同的配体、NR伴侣、共调节分子。所有结果将在一个专门用于该项目的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查找和靶基因预测工具。它们将推进将NRs与疾病和药物代谢联系起来的快速跟踪研究的长期目标，从而有助于个性化医疗，并确保针对NRs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.