Genetic and epigenetic regulation of retinal development, aging and evolution

National Eye Institute

Investigators

Linked publications & trials

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 delineate the genes directly regulated by NRL and CRX during rod development, we have performed cut&run experiments using developing mouse retina. This analysis uncovered C-Jun as an early transcriptional target of NRL during rod photoreceptor development. We confirmed that NRL binds to the AP-1 sites located with the upstream promoter of Jun by EMSA and performed shRNA-mediated knockdown of Jun by electroporation. We also performed Cut&Run experiments to reveal the genomic occupancy of Jun in developing rods. 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. Preliminary studies suggest that fibroblast growth factor (FGF) signaling promotes phosphorylation of NRL and alters the activity of NRL. We are also purifying NRL transcriptional complexes to identify in vivo interactions to elucidate qualitative and quantitative changes in gene expression in response to photoreceptor aging and disease. To characterize novel and known interactions of NRL, we also developed a transgenic mouse that harbors a C-terminus peptide (Avi tag) on NRL for efficient affinity purification. This mouse also harbors the bacterial biotin transferase BirA that specifically adds a biotin group to the Avi tag. Preliminary data shows that the tag is correctly expressed and biotinylated in the retina, and it does not seem to affect rod development. DNA methylation valleys (DMVs) during the development of rod photoreceptors DNA methylation regulates gene expression programs and chromatin integrity during development and aging. We have identified DNA methylation valleys (DMVs) in rod photoreceptors that are absent in progenitor cells suggesting that they encompass regulatory regions important for rod-specific expression programs. To test the importance of these regions in rods, we removed DMVs in candidate regulatory elements of the rhodopsin gene in collaboration with the NEI genetic engineering core. We will study the rod phenotype in retinal organoids and mice lacking this region. To identify the mechanisms that allow unmethylation at DMVs, we are currently studing the genomic occupancy of the demethylases TET2 and TET3 and the Polycomb complex enzymes EZH1 and EZH2 by performing ChIP-qPCR experiments. Small open reading frame (smORF) peptides in the retina We were able to identify over 1800 peptides originating in known and novel ncRNAs in the mouse retinal proteome. By combing the proteome data with several additional Retinal NGS data (e.g RNA-seq and ChiP-seq of retinal TFs) and bioinformatic analysis (including conservation and functional prediction) we were able to identify a subset of 50 smORF peptides associated with RNA processing and/or translation. We have cloned about half of them and are currently analyzing their cellular localization, and potential molecular function. Cell communication and synaptogenesis We identified TAFA3 gene encoding a chemokine that could play a role in a novel receptor mediated cellular response. Using specific antibodies, we showed that this gene is expressed in cones as well as in a subset of cone bipolar cells. The Tafa3 project has moved forward by identifying and confirming its binding partner Neurexin 3 through co-IP experiments. Mapping genes associated with photoreceptor function We are using inbred mouse strains, the BXD mice, to study the basis of phenotypic variations in the retina. We have specifically focused on BXD32 and 3 other BXD strains because of changes in scotopic and/or photopic ERG response. Gene expression profiling and identification of eQTLs is in progress to identify underlying genetic defects and to gain insight into biological processes of photoreceptor development. Aging of rod photoreceptors We recently published a major review article concerning the impact of aging on the retina, with an emphasis on age-related molecular events that contribute to decline in visual function. Since advanced age is a major risk factor for common blinding eye diseases, we also discussed whether lifestyle habits including dietary changes can ameliorate retinal aging and consequently the clinical impact of age-related diseases such as AMD. To identify aging-dependent transcriptional changes of non-rod retinal neurons, we improved the retinal dissociation procedure and designed an optimized single-cell RNA sequencing protocol where rods were removed through CD73 cell surface maker-mediated cell depletion. Expression changes followed monotonic trajectories, gradually increasing or decreasing with time. Common transcriptionally dysregulated genes enriched for mitochondria, protein synthesis and stress-response (upregulated), and energy metabolism, axon growth and guidance, and synaptic transmission (downregulated). Cell type-specific aberrant expression was detected for genes with specialized functions such as the cone phototransduction cascade. To validate our results and obtain spatial gene expression, we will perform qRT-PCR and in situ hybridization. We also aims to identify the key components of the proteostasis network, and the glycation induced damage on the mitochondrial dynamics of aging photoreceptors. Genome regulatory architecture of the human retina To characterize the three-dimensional organization of the genome, we have generated the first high-resolution contact map of the human retina. We integrated our Hi-C data with epigenetic datasets to define retinal super-enhancers conserved across fetal development and identify transcription factor binding motifs enriched in accessible regulatory regions bound by NRL and/or CRX. Additionally, we integrated transcriptomic data to map long-range interactions between retina-enriched genes and distal enhancer regions and GWAS data to connect AMD variants to specific genes. 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. Nrl-like proteins are being cloned into vectors for retinal electroporation into Nrl-KO mice. This project will identify the earliest chordate lineage to express a protein capable of generating rods. We also 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. Using retinal organoids to study human retinal development To elucidate chromatin dynamics during retinal differentiation, high resolution Hi-C were performed on human retinal organoids at chronological differentiation stages. The genome topological structure varies dynamically with photoreceptor differentiation time at different levels. Interestingly, organoids with more mature photoreceptors show higher oxygen consumption rate than younger ones but their maximal respiration capacity are similar. Future work will be focus on integration of multi-omics data to explain the interaction network that instruct transcriptional programs in human retina.