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.
Aside from identical twins, no two individuals are completely identical genetically. Most differences between individuals are due to single nucleotide changes or polymorphisms (SNPs) in the genome. SNPs are being increasingly recognized as playing a major role in phenotypic variations (e.g., eye and hair color, basal body weight, muscle tone, responsiveness to alcohol consumption, etc.) as well as susceptibility to diseases such as cancer, diabetes, heart disease and mental disorders. There is also tremendous variability in individuals'response to drug treatments that makes some drugs life-saving for certain people but cause serious side effects for others. Many SNPs introduce structural or functional changes in the proteins encoded by genes;other SNPs, the vast majority, are in the regulatory regions of genes that determine the level of expression of genes. The ultimate goal of this proposal is to examine the effect of SNPs on a special class of proteins called nuclear receptors that bind DNA and regulate the expression of many important genes in response to hormones, vitamins and drugs. SNPs in the DNA sequences that the nuclear receptors bind will be identified using a very powerful new technology called protein binding microarrays. SNPs in the nuclear receptors themselves will also be examined for their effect on nuclear receptor DNA binding. The functional characterization of both types of SNPs will help predict disease susceptibility and response to drug treatments. This characterization will help lay the foundation for personalized medicine which will ultimately lead to more effective and hence less costly health care costs.
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