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。 年龄相关性黄斑变性 (AMD) 是一种视网膜神经退行性疾病，是视力丧失的主要原因 全世界的老年人中。尽管抗 VEGF 治疗可有效治疗新生血管 （“湿”）形式的疾病，对于更常见的萎缩（“干”）形式，尚无经过证实和批准的治疗方法 的疾病。更好地了解 AMD 的遗传学和疾病机制可能会有所帮助 制定更有效的治疗策略。全基因组关联研究 (GWAS) 已确定 大量与 AMD 风险增加相关的单核苷酸多态性 (SNP)。虽然这些 GWAS 研究引起了人们对补体系统在 AMD 中的作用的兴趣增加，其分子机制 哪些 AMD 风险等位基因会导致该疾病的风险增加，人们知之甚少。了解 AMD 风险 SNP 是 特别具有挑战性，因为它们大多数发生在基因组的非编码区域。作为解决这个问题的一种方法 问题，表达数量性状位点 (eQTL) 研究可以识别可能调节下游基因的 SNP 表达。然而，eQTL 不提供有关 SNP 结合蛋白的信息。确定相交的 GWAS 通过染色体免疫沉淀测序 (ChIP-seq) 获得具有转录因子 (TF) 结合位点的 SNP 是另一种方法 识别功能性 SNP 及其相互作用的 TF 的有用方法，但这种方法需要先验知识 相关的 TF。在此应用中，据我们所知，使用了一种以前未应用于 AMD 的方法 研究中，我们建议对非蛋白质实施疾病相关 SNP 的全蛋白质组分析 (PWAS) 研究 编码区 SNP 可识别 AMD 中等位基因特异性蛋白质-DNA 相互作用和调节活性的改变。这 这种方法的基本原理是我们的假设，即功能性 AMD 相关 DNA SNP 可能通过以下方式执行其功能： 与特定蛋白质的等位基因特异性相互作用。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，包含携带 SNP 的 DNA 探针，其中超过 1,700 个人类 可以同时检测每个探针的转录因子 (TF)/DNA 结合蛋白。鉴定出等位基因特异性 蛋白质-DNA 相互作用将通过一系列生物信息学分析进行优先排序，并使用人类视网膜进行验证 由人类干细胞分化而来的色素上皮细胞（RPE）和感光细胞（PR）。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。目标 2 将进行生化表征并 优先考虑目标 1 中确定的 TF。目标 3 将在功能上表征已确定的 AMD-SNP 等位基因特异性蛋白 AMD 相关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 AMD 相关应激源的反应。 总而言之，我们希望这些研究能够提供新的治疗相关的机制见解 AMD 的发展和进展的基础。