Wednesday, May 18, 2022
5/18/2022
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.
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