Genetic and epigenetic regulation of retinal development, aging and evolution

National Eye Institute

Investigators

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Abstract

Transcriptional regulation by NRL and associated transcription factors To determine the direct targets, we performed cut&run experiments and delineated the genomic occupancy of NRL and CRX in developing mouse retina. To explore how NRLs interaction with other proteins modulates its transcriptional activity, we are using multiple approaches. We have purified NRL-containing protein complexes from bovine retinal nuclear extracts and performed immunoprecipitation using anti-NRL antibody, followed by mass-spectrometry analyses. NRL interactors were also isolated by GST-pulldown followed by mass spectrometry and by yeast two-hybrid screening. We have identified over 50 candidate interactors of NRL. We are focusing on YBX1, CASQ1 and ATF4. Interaction of NRL with several RNA binding proteins, specifically those associated with R-loops, have expanded the role of this key transcription factor beyond rod cell fate determination and transcriptional initiation. We have shown that NRL works with DHX9 and DDX5 to resolve R-loops in the retinal rods and that it augments their expression to sustain high level of transcription of genes such as rhodopsin. NRLs transcriptional activity is modulated by post-translational modifications. Therefore, we are purifying distinct NRL isoforms and identifying specific amino acid residues that are targeted by various signaling pathways. We have generated a new mouse line Nrl_P2A-iCreT2A-Tdtomato for lineage tracing and are exploring the temporal and spatial features of Nrl promoter activation. This line will also allow us to examine in vivo NRL interactions during development and aging. Regulation of human retina development in stem cell-derived organoids We have conducted bulk and single-cell RNA-sequencing of human iPSC-derived retinal organoids at multiple developmental time points. We are integrating multiple omics datasets to unravel the molecular mechanisms governing human retinogenesis, specifically focusing on the determination of cell fate during differentiation of iPSCs. 3-D chromatin organization of human retina and trabecular meshwork To elucidate the spatiotemporal dynamics of genomic architecture and its relationship with gene expression patterns during development, we generated high-resolution Hi-C map and concurrent transcriptome profiles of differentiating human retinal organoids chronologically. We demonstrate progressive and developmental stage-specific alterations in DNA topology including genomic compartments, topologically associated domains (TADs), and DNA loops that correlate to cell type-restricted gene transcription, bridging the gap in the retinal developmental studies of the relationship between genome topology and cell specification. WE are exploring how genome topology and epigenomic marks are altered in trabecular meshwork cells (TMCs) derived from healthy donor eyes to annotate regulatory landscape and 3D genome organization of genes expressed in TMCs with potential role in primary open-angle glaucoma. Transcriptome and epitranscriptome landscapes of human retina/macula We generated transcriptomic profiles of 185 postmortem control and AMD macula samples. Using only control samples for reference transcriptome, we identified 12,975 protein coding and 955 noncoding genes. Cis-eQTL analysis identified 5,959 eGenes and enrichment of best eQTL showed enrichment of secretory and mitochondrial genes. To determine the epitranscriptome landscape in human retina, we employed ultra-high performance liquid chromatographytandem mass spectrometry to detect RNA modifications in human retina. We identified Dihydrouridine and m3C as the most abundance RNA modifications and observed that 18 RNA modifications are significantly changed during human retina aging or AMD. How genetic variations affect aging of human retina? We evaluated the transcriptome of 192 postmortem human retinas during aging. After QC, we extracted as much variance due to aging and sex effect in the noisy data and studied differential gene expression in linear and categorical age. Currently, we are expanding the sample size from our previously published dataset to gain more power in evaluating our targets. In parallel, we are performing QC on genotype and methylation datasets to study quantitative trait loci and genetic regulations in non-coding variants. Characterizing the gene regulatory pathways in mouse retina in response to adaptation to diet We have undertaken studies to delineate molecular pathways through which the retina responds to nutritional inputs such as high and low glycemic index diets, high fat and Mediterranean diets, and supplements like AREDS and select B-vitamins. We have used an epigenome and transcriptome focused approach to identify gene regulatory networks and benchmarked the retina alongside key organs like the liver. We detected gene expression changes in the retina following HGI diet for 1-month; however, greater changes were apparent in pathways like energy metabolism, inflammation, and retinal disease genes after long-term HGI diet (12-months). In comparison, the livers immediate reworkings involved lipid metabolism and inflammation, and long-term adaptations featured switching to lipogenesis through overexpression of Srebf1 and Elovl genes. Our observations suggest that retinal adaptations to lifestyle are time-of-exposure dependent. Furthermore, we found notable differences in DNA methylation and transcriptional signatures of male and female mice retinas. Preliminary analyses indicate a sex-dependent retinal response to nutritional habits. Mechanistic studies of retinal aging and impact of metabolism on gene regulation We have investigated aging-dependent transcriptional changes in mouse retinal cells at single-cell resolution, with a goal to understand cumulative molecular aberrations that might contribute to late-onset visual decline. Additionally, we are assessing the impact of sex and age on the transcriptome dynamics of the retina. We are deciphering the role of 3D chromatin organization in molding the transcriptional landscapes during aging of the mouse retina. We are linking retinal glucose metabolism and epigenome changes via histone lactylation. Alterations in lactate levels impact the development of histone lactylation in the retina, potentially playing various roles via chromatin regulation. Histone Kla is predominantly present in active promoter regions within the retina and its distribution resembles H3K27ac. We have also explored the occurrence of histone lactylation in the developing and aging mouse retina through immunohistochemical analysis. Cell communication and synaptogenesis We are examining functions of TAFA3, a CC-chemokine like ligand of neurexin 3, Ackr1 and Eph10 in mouse retina using knockout mouse models. Evolution of rods and nocturnal vision We are investigating the evolution of rod photoreceptors by examining Maf proteins in vertebrate evolution. We are also studying the evolution of retinal cell types in 108 species using RNA-seq data, analyzing homologs of established cell-type markers and developing a tool to compare gene expression evolution in nocturnal and diurnal birds' retina.