Connecting AMD SNPs to Functions Using Allele-specific Interactions

使用等位基因特异性相互作用将 AMD SNP 连接到功能

• 批准号: 10538627
• 负责人: Jiang Qian
• 金额: $ 48.11万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10538627
• 关键词: ATAC-seq; Affect; Affinity; Age related macular degeneration; Alleles; American; Atrophic; Base Pairing; Binding; Binding Proteins; Binding Sites; Biochemical; Bioinformatics; Biological; Biological Assay; Blindness; CRISPR interference; Cell Differentiation process; Cell physiology; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Code; DNA; DNA Binding; DNA Probes; DNA-Binding Proteins; DNA-Protein Interaction; Data; Development; Disease; Dryness; ES Cell Line; Effectiveness; Elderly; Electrophoretic Mobility Shift Assay; Engineering; Functional disorder; Gene Expression; Genetic Predisposition to Disease; Genetic Risk; Genetic Transcription; Genetic study; Genome; Goals; Heterozygote; Human; Immunoprecipitation; In Vitro; Individual; Interferometry; Investigation; Knowledge; Lead; Length; Luciferases; Maps; Molecular; Neurodegenerative Disorders; Phenotype; Photoreceptors; Population; Predisposition; Protein Analysis; Protein Array; Protein Microchips; Proteins; Proteome; Public Health; Quantitative Trait Loci; RNA-Binding Proteins; Reporter; Research; Resources; Retina; Risk; Role; Series; Single Nucleotide Polymorphism; Site; Specificity; Stress; Structure of retinal pigment epithelium; Surveys; Technology; Testing; Transcriptional Regulation; Transposase; Untranslated RNA; Validation; XCL1 gene; bevacizumab; cell behavior; cell type; chromatin immunoprecipitation; complement system; disorder risk; effective therapy; genetic risk factor; genetic variant; genome editing; genome wide association study; genomic locus; high risk; high throughput screening; human embryonic stem cell; human stem cells; insight; interest; loss of function; neovascular; novel; novel therapeutics; response; risk variant; screening; stressor; success; transcription factor; treatment strategy

PROJECT SUMMARY. Age-related Macular Degeneration (AMD) is a retinal neurodegenerative disease that is a major cause of vision loss among the elderly worldwide. Although anti-VEGF treatments can be effective in the treatment of the neovascular (“wet”) form of the disease, there are no proven and approved treatments for the more common atrophic (“dry”) form of the disease. Greater understanding of the genetics and disease mechanisms underlying AMD has the potential to aid in the development of more effective treatment strategies. Genomewide association studies (GWAS) have identified a large number of single nucleotide polymorphisms (SNPs) that are associated with increased risk of AMD. Although these GWAS studies have led to increased interest in the role of the complement system in AMD, the molecular mechanisms by which AMD risk alleles lead to increased risk for the disease are poorly understood. Understanding AMD risk SNPs is particularly challenging because most of them occur in non-coding regions of the genome. As one approach to this problem, expression quantitative trait loci (eQTLs) studies can identify SNPs that are likely to modulate downstream gene expression. However, eQTLs do not provide information on SNP-binding proteins. Determining intersecting GWAS SNPs with transcription factor (TF) binding sites by chromosomal immunoprecipitation sequencing (ChIP-seq) is another useful approach to identify functional SNPs and their interacting TFs, but this approach requires a priori knowledge of the relevant TFs. In this application, using an approach that has not, to our knowledge, been previously applied to AMD research, we propose to implement a Proteome-Wide Analysis of disease-associated SNPs (PWAS) study of non-protein coding region SNPs to identify allele-specific protein-DNA interactions and alteration of regulatory activity in AMD. The rationale for this approach is our hypothesis that functional AMD-related DNA SNPs likely execute their function via allele-specific interactions with specific proteins. We will survey the entire human TF and RNA-binding protein repertoires with SNP-carrying DNA probes using a protein array-based approach in which greater than 1,700 human transcription factors (TFs)/DNA binding proteins can be simultaneously surveyed for each probe. Identified allele-specific protein-DNA interactions will be prioritized using a series of bioinformatics analyses and validated using human retinal pigment epithelial (RPE) and photoreceptor (PR) cells differentiated from human stem cells. In Aim 1 we will identify TFs that show differential binding to allele-specific AMD-associated SNPs. Aim 2 will biochemically characterize and prioritize the TFs identified in Aim 1. Aim 3 will functionally characterize the identified AMD-SNP allele-specific protein interactions in AMD-relevant cell types, and explore how they affect cell behavior and response to AMD-related stressors. Taken together, we hope that these studies will provide new therapeutically relevant insights in the mechanisms underlying the development and progression of AMD.

项目摘要。 与年龄相关的黄斑变性（AMD）是一种视网膜神经退行性疾病，是视力丧失的主要原因 在全球的古老。尽管抗VEGF治疗可以有效治疗新生血管 （“湿”）该疾病的形式，对于更常见的萎缩（“干”）形式没有经过验证和批准的治疗方法 疾病。对AMD潜在的遗传学和疾病机制有更多的了解有可能帮助 在制定更有效的治疗策略中。全基因组关联研究（GWAS）已确定 与AMD风险增加有关的大量单核苷酸多态性（SNP）。虽然这些 GWAS研究导致人们对完成系统在AMD中的作用的兴趣增加，AMD是分子机制 AMD风险等位基因会导致疾病风险增加。了解AMD风险SNP是 特别是挑战，因为它们大多数发生在基因组的非编码区域中。作为一种方法 问题，表达定量性状位置（EQTL）研究可以鉴定可能调节下游基因的SNP 表达。但是，EQTL不提供有关SNP结合蛋白的信息。确定相交的GWA 通过染色体免疫沉淀测序（CHIP-SEQ）具有转录因子（TF）结合位点的SNP是另一个 识别功能性SNP及其相互作用的TFS的有用方法，但是这种方法需要先验了解 相关的TFS。在此应用中，据我们所知，使用一种未应用于AMD的方法 研究，我们建议对非蛋白质的疾病相关SNP（PWAS）研究实施全蛋白质组的分析 编码区域SNP以鉴定等位基因特异性蛋白-DNA相互作用以及AMD中调节活性的改变。这 这种方法的理由是我们的假设，即功能性AMD相关的DNA SNP可能会通过 等位基因特异性与特定蛋白质的相互作用。我们将调查整个人类TF和RNA结合蛋白 使用基于蛋白质阵列的方法，具有SNP携带DNA问题的曲目，其中大于1,700人 转录因子（TFS）/DNA结合蛋白可以简单地针对每个探针进行调查。确定的等位基因特定 蛋白-DNA相互作用将使用一系列生物信息学分析优先考虑，并使用人类视网膜进行验证 色素上皮（RPE）和感光细胞（PR）细胞与人类干细胞区分开。在目标1中，我们将确定 显示与等位基因特异性AMD相关SNP的差异结合的TF。 AIM 2将在生化特征和 优先考虑在AIM 1中识别的TF。AIM 3在功能上表征已识别的AMD-SNP等位基因特异性蛋白 与AMD相关的细胞类型中的相互作用，并探讨它们如何影响细胞行为和对与AMD相关的应激源的反应。 综上所述，我们希望这些研究能为机制提供新的治疗相关见解 AMD的发展和发展的基础。