Genetic and epigenetic regulation of retinal development, aging and evolution

National Eye Institute

Investigators

Linked publications, trials & patents

Abstract

Gene Regulation in photoreceptors: Studies on NRL and retinal transcription factors The leucine zipper transcription factor NRL determines rod photoreceptor cell fate and regulates the expression of most rod genes by synergistic interactions with CRX and other proteins. To determine the genes directly regulated by NRL and CRX during photoreceptor development, we performed cut&run experiments with developing mouse retina. We have delineated the genomic occupancy of NRL in developing retinas and examined the role of another retinal transcription factor, c-Jun, as an early regulator for gene expression in rod photoreceptors. NRLs transcriptional activity is modulated by post-translational modifications. We are therefore purifying distinct NRL isoforms to identify specific amino acid residues that are targeted distinct signaling pathways. We are currently investigating the function of Casein Kinase 2, a kinase protein involved in protein phosphorylation, in modulating the activity of NRL. Role of NRL on RNA processing and RNA-mediated transcription regulation We have identified the interaction of NRL with several RNA binding proteins that participate in various aspects of RNA homeostasis. We therefore hypothesize that NRL has a role in regulating RNA processing and RNA-mediated transcriptional regulation in photoreceptors. We focused on the RNA/DNA helicase DHX9 that is associated with human disease and implicated in the regulation of R-loops during transcriptional elongation. R-loops are non-B DNA structures comprised of RNA-DNA hybrids with displaced single-stranded DNA that play key roles in cellular metabolism including transcriptional elongation and termination. Accumulation of R-loops can interfere with transcriptional elongation causing alterations in gene expression and genomic instability. We characterized the interaction of NRL and DHX9 in vivo and in vitro and determined that it is not mediated by RNA or DNA. We have performed knockdown of DHX9 in developing retinas by RNA interference and observed gene expression changes in genes that are regulated by NRL. NRL knockout retinas display higher levels of R-loops compared to wildtype suggesting that NRL has a role in regulating R-loops dynamics in rod photoreceptors. We are performing experiments to identify whether NRL binds to R-loops in vivo and to delineate the molecular mechanisms of R-loop regulation through DHX9 in the retina. This study will shed light into roles of NRL beyond transcriptional activation in the retina. Impact of three-dimensional organization of the genome on retina The spatiotemporal expression of genes is critical for ensuring proper retinal development and function. This expression is orchestrated, in part, by the three-dimensional organization of the genome in the nucleus. To elucidate chromatin dynamics during retinal development, high resolution Hi-C with epigenetic and transcriptomic data were performed on both adult human retina (publication in review) and pluripotent cell-derived retinal organoids (publication in prep). The genome topological structure varies dynamically with differentiation time, with the 3D genome structure reorganization highly correlated with retinal development. These projects have provided new insight into the genetic control of tissue-specific functions and serve as a valuable resource for the vision science community. To elucidate tissue specific gene expression and how non-coding genomic variants contribute to disease we generated the first high-resolution contact map of the human retina. We integrated our Hi-C data with epigenetic marks, retinal transcription factors, CTCF and super-enhancers (SEs). We identified 1325 SEs in human retina. The topologically associated domains (TADs) with central SEs depicted stronger insulation as compared to TADs with edge SEs. Merging eQTLs and GWAS with chromatin contacts identified corresponding genes linked to retinal neurodegeneration, and AMD/glaucoma respectively. Metabolic impact of histone acylation and transcription in aging retina Inspired by the discovery of histone lactylation in cancer, we hypothesized and anticipated the non-metabolic role of lactate in retina through lactate derived histone lactylation. It has been observed that retinal photoreceptors contain very high physiological lactate concentration ranging from 550 mM depending on species. Since the modifications use lactyl-coA as substrate, it is conceivable that metabolic stress such as aging could affect glucose metabolism and thus availability to the substrate which could result in an altered pattern of these histone modifications in aged mice. Our preliminarily results provided fundamental evidence for the occurrence of histone lactylation in the retina and photoreceptor cells and its potential regulation by aging-induced glucose metabolic changes. Mitochondria and metabolic defects leading to photoreceptor cell death To better understand the role of metabolism in retinal development, we have studied oxygen consumption of photoreceptor-derived retinal organoids. We have found that organoids with more mature photoreceptors demonstrate higher oxygen consumption compared to organoids with cells earlier in the developmental process. Future work will integrate metabolomic, genetic regulation, and transcriptomic data in a multi-omics framework to elucidate the interaction network that affect human retina. GATD3A is a mitochondrial protein similar to Parkinson-associated DJ-1/PARK7-like protein and is hypothesized to have deglycase activity, and as such, provide a protective role during metabolism. Using sub-cellular fractionation experiments, we showed that GATD3A is localized to the mitochondria, and to a lesser extent in the cytoplasm and the nucleus. The deglycase activity in the mitochondria may play a protective role in the removal of AGE products resulting from metabolic side reactions, thereby preserving mitochondrial function. To study its function in the mitochondria, and to test if exogenous dicarbonyls affect its deglycase activity, we performed in vitro experiments using fibroblasts derived from GATD3A knockout and wild type mice. Mitochondrial stress assays were performed using the Seahorse XF analyzer. We demonstrated that the glyoxal pretreated knockout cells had lower basal as well as uncoupled respiratory rates. We concluded that GATD3A is a relevant target for diseases involving AGE deposition. Cell communication and synaptogenesis We identified TAFA3 as CC-chemokine like ligand that could play a role in maintaining cellular homeostasis. We co-immunoprecipitated proteins interacting with TAF3A utilizing FLAG-tagged TAF3A proteins from knockin mice, and subsequently performed mass spectrometry analyses. We identified several candidate proteins including neurexin 3 which has been linked to age-related disorders. Further studies will elucidate the role of TAFA3 and neurexin 3 interactions in the retina. Evolution of rods and nocturnal vision We are investigating the evolution of rod photoreceptors by identifying Nrl-like proteins in early vertebrate evolutionary lineages from their whole retinal transcriptomes. We generated an in-solution probe set that efficiently captures the full phototransduction cascade from any species of bird, facilitating molecular analysis of vision evolution. By examining these sequences, we identified selection acting on phototransduction genes associated with the emergence of avian nocturnal vision and the specialization of high-speed vision in an order of neotropical birds.