Genetic and epigenetic regulation of retinal development, aging and evolution

National Eye Institute

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Abstract

Molecular Mechanisms of NRL-mediated Transcriptional Regulation We have demonstrated that NRL undergoes phosphorylation, glycosylation, and sumoylation. These PTMs can alter NRL structure and localization, thus affecting NRL interaction with DNA and protein. We recently reported the phosphorylation of NRL at S117 by CK2 and its potential to modulate NRL-mediated rhodopsin (Rho) promoter activity. Working with Jerome Roger (a former fellow, now faculty in Paris), we have demonstrated that GSK3 regulates NRL phosphorylation, which affect NRL stability (in preparation). To examine putative phospho-sites by mass spectrometry (MS), we will design and use synthetic peptides for all 34 predicted phospho-sites of NRL. Given that NRL has limited tryptic peptides available for PTM analysis, we have developed a novel multi-enzyme parallel reaction monitoring (ME-PRM) strategy and validated S6, S38 and S50 phospho-sites. Many serine and threonine phosphorylation sites in NRL are also putative O-glycosylation sites. Interconversion of phosphorylation and glycosylation proteoforms may contribute to NRL stability, nuclear localization, protein-protein interactions, and transcriptional activity. Moreover, we suspect differential PTM states of NRL mediate its function during retinal development and aging. NRL interactome NRL interacts with CRX and its target NR2E3 to control expression of photoreceptor genes. Using retinal lysate of various species and using co-immunoprecipitation (Co-IP), GST-pull down assays, MS, and yeast two-hybrid (Y2H), we identified and validated several RNA binding proteins including splicing factors and R-loop binding proteins, focusing on potential role of NRL with RNA resolvases, such as DHX9 and DDX5. Furthermore, we validated NRL’s interaction with two bZIP proteins, ATF4 and BACH1, by proximity ligation assays, among others. These proteins likely produce heterodimers with each other, bind to overlapping CREs (as indicated by Cut&Tag assays), and modulate expression of rod genes; shRNA knockdown studies in mouse retina by electroporation are being completed (manuscript in preparation). Moreover, the validated interaction between NRL and MeCP2, which binds to methylated DNA, initiated another study into the crosstalk between DNA methylation and transcription in rod cells within the retina during development and aging. NRL structure-function analysis While we have reported the crystal structure of CRX-homeodomain bound to rhodopsin Ret4 element, resolving the structure of NRL is difficult due to the intrinsically disordered region (IDR) on the N-terminal. A precise molecular structure of NRL will allow us to define functions of PTMs, interaction surfaces and human mutations, which in turn can assist in targeting specific NRL residues for precision therapies. Therefore, we have implemented genetic code expansion to incorporate fluorescent or tetrazine-reactive unnatural amino acids (uAAs), such as L-ANAP or BCNK, respectively, into NRL. This expansion will enable quantitative, site-specific labeling for MS, small angle X-ray scattering (SAXS), cryo-EM and/or Raman, investigate whether these shifts lead to functionally distinct conformational state(s) and explore structure and stability of NRL complexes with CREs. NRL condensates and gene regulation in rods Biomolecular condensates are membraneless organelles formed by dynamic assembly of proteins and nucleic acids. The IDRs play an important role in the generation of these condensates, spatiotemporal organization of chromatin structure for quantitatively precise gene regulation. We have employed STED (Stimulated Emission Depletion) microscopy and ACC-Seq (Assay for Chromatin-bound Condensates by exploratory Sequencing) to resolve NRL-associated ribonucleoprotein complexes at nanoscale and genome-wide levels. Our findings reveal distinct patterns of NRL distribution and co-localization within the rod nuclei. NRL forms punctate structures with NONO in transcriptionally active regions, partial overlaps with MeCP2 suggesting roles in chromatin organization, and associates with H3K27Ac-marks. Understanding the role of PTMs in the NRL IDR will also determine how formation and function of these condensates may be regulated. NRL-mediated regulation of TAFA3 and EphA10 expression When developing the Nrl-knockout mouse model, we saw increased expression of the chemokine-like molecule TAFA3 and EphA10. Using a knock–in mouse line expressing mCherry, we localized TAFA3 to cones and a subset of OFF cone bipolar cells. We also uncovered TAFA3 interaction with Neurexin 3a, which is likely its receptor. Tafa3-KO mice that we generated show a decrease in photopic and flicker ERG indicating a decline in cone function and changes in visual acuity. EphA10, an ephrin and a receptor tyrosine kinase, responds to extracellular cues to facilitate signal transduction. We generated EphA10 knockout mice that demonstrated enhanced photopic b-wave at 3-mon age and augmented Muller glia morphology in synaptic layers. We have generated two knock-in mouse lines harboring mCherry in the 5’ or 3’ UTR to localize EphA10 in retinal and brain neurons. Regulation of NRL expression in the retina and beyond Though we have demonstrated Nrl expression in the developing mouse brain, we focused on Nrl expression in retinal rod photoreceptors. However, recent studies have demonstrated Nrl is aberrantly expressed in certain subsets of medulloblastoma, a common pediatric brain cancer. This has prompted us to further investigate Nrl expression in the brain as well. Therefore, we generated, by CRISP/Cas9, a knock-in mouse line in which Nrl is expressed in tandem with a tdTomato reporter and iCre recombinase that can be used for lineage tracing and delineated the expression of Nrl in specific cell types of the central and peripheral nervous system during development. We are now identifying Nrl-expressing neurons in the cortex and cerebellum by single cell transcriptomic analysis followed by immunohistochemistry. Tegshee has identified a Nrl transcript variant specific to the brain, and the knock-in line is being used to delete CREs that are needed for tissue-specific Nrl expression. We have generated human iPSC lines with dual fluorescent reporters knocked in at OTX2 and NRL loci and established the retina and brain organoid differentiation systems. We plan to use a live imaging system to track fluorescence gene expression during differentiation to monitor the NRL expression window and conduct comprehensive “omics” analyses to complement studies in mouse. Chromatin dynamics and regulatory network guiding human retina development Our goals are to produce comprehensive genomic regulatory maps of the developing and mature human retina, especially photoreceptors. Specifically, we plan to elucidate cis-regulatory elements (CREs) and DNA looping in healthy and disease retina, identify hubs that co-regulate expression of retinal genes, and uncover chromatin contacts of variants (QTLs) associated with aging, AMD, glaucoma, and retinopathies. Marchal developed a computational tool that generated a high-resolution genome topology map of the human retina by integrating our HiC and epigenome data with that from the Cherry lab. Our analysis uncovered how 3D chromatin contacts and CREs confer new interpretations to known genetic variants for retinal diseases and direct links to gene regulation. Our data imply that 3D genomic topology establishes the spatial landscape for lineage commitment in a well-orchestrated temporal manner. Photoreceptor differentiation and NRL To understand how NRL affects transcription via the altering the chromatin landscape, we introduce WT human NRL via AAV7m8 into iPSC-derived retinal organoids expressing the NRL L75Pfs mutation. We are evaluating, at multiple levels (e.g., by introducing NRL mutations), disruptions to the rod gene regulatory network and decipher the relative dominance of epigenome versus NRL in rod development. Neuronal cell fate determination Taking advantage of clearly defined periods in organoid development and availability of molecular markers and tagged iPSC lines (fluorescent markers knocked-in at OTX2 and NRL loci), we have begun to assemble large “omics” datasets (bulk and scRNA-seq, scATAC-seq, Hi-C, and Cut&Tag for multiple histone marks and key transcription factors). We hope to complete this part within this year and then start analyses and integration. Noise and disorder in differentiation Biological systems rely on dynamic interactions, thus are inherently noisy. Multifunctionality of proteins and variability in protein interactions based on extracellular cues cannot be described in a linear function. Therefore, novel complex network analysis is required to decipher complex biological systems and create models foreshadowing biological responses. Our new postdoc will use potential energy models to decipher dynamics of complex systems, such as developing retinal organoids, using single cell datasets we and others have produced. Hamiltonian and Lagrangian mechanics have been used in physics for centuries allowing description of complex systems including non-linear, lossy and noisy ones. Evolution of Nrl, rods and nocturnal vision We are investigating the evolution of rod photoreceptors by examining Maf proteins across vertebrate species. We are also studying the evolution of retinal cell types in 108 species using RNA-seq data, focusing on the homologs of established cell-type markers and developing a tool to compare gene expression and signatures in nocturnal and diurnal birds' retina. We have produced expression constructs of Nrl homologs from different chordates for sub-retinal injection experiments in Nrl Ko mice to test cross- species functional similarities. These studies will help us to elucidate the origin of Nrl from ancestral homologs. scRNA-seq of aging mouse retinas uncovers two rod subpopulations and differential response of rod pathway to aging Single cell RNA-seq analysis of mouse retina before and after rod depletion at different ages uncovered two distinct subpopulations of rod photoreceptors – one enriched for phototransduction genes and the other for those associated with synaptic transmission. Re-analyses of data from other single cell studies validated our findings. Dynamic age-dependent changes in expression of rods and rod bipolar genes correlate well with rod dysfunction documented at advancing age. Influence of age was evident on other retinal cells as well. Chromatin landscape provides a rationale for gene expression changes during rod photoreceptor aging We have integrated multi-omics data to identify age-associated functional changes in mouse photoreceptor chromatin. We observe that rod chromatin undergoes a global compaction during aging. Locally, chromatin 3D reorganization occurs predominantly in accessible and transcribed regions, associated with active histone marks. Local changes in chromatin landscape and transcription are enriched for and within changes in chromatin topology. The strongest transcription changes present notable alterations in chromatin organization and landscape at genes associated with AMD and Alzheimer disease. Finally, we uncover a strong increase of the expression of a pseudogene encoding a histone acetyl transferase (HAT) inhibition domain. This increase of expression parallels a global disruption of H3 acetylation in the photoreceptors during aging. Comprehensive studies on the Aging-Diet-Epigenome nexus Genetic and epigenetic changes during aging depend on intrinsic and extrinsic factors, such as biological sex and diet respectively. As previously mentioned, there are changes in chromatin topology, gene expression, and protein expression associated with aging. Moreover, studies have demonstrated that biological age-induced molecular changes include altered metabolic regulation and changes in diet in turn can alter gene expression. Metabolic products, such as S-adenosyl methionine and acetyl-CoA participate in epigenetic changes that alter gene expression. MS can now be used to identify metabolic substrate-induced epigenetic changes. Photoreceptors are metabolically active cells with high aerobic glycolysis and lactate production resulting in the identification of H3K18-lactylation (H3K18La). Our postdoc Gaur aims to identify and characterize the role of gene regulation by metabolites in retinas during aging. Changes in metabolism and the resulting metabolites depend on diet and genetic predisposition to how the diet is metabolized in various tissues, including the retina, liver, and pancreas. Studies have demonstrated the role of Mediterranean diet (MD) and dietary supplements in slowing AMD progression. We completed a pilot study comparing obesity-inducing Western diet (WD) with saturated fats and MD with healthy unsaturated fats together with the effect of AREDS vitamins and zinc in male and female C57BL/6J mice. Retina and liver to examine genetic, epigenetic, and preteomic changes. Serum was collected to examine lipid changes. In order to confirm a link between molecular changes and retinal function, a new study has been completed focusing on WD, MD, and caloric-restriction WD (CR) in males and females at different time points (0 wk, 2-wk, 3-mon, and 6 mon). The mice were evaluated for changes in weekly weight, daily food consumption, interaction with caretakers and cagemates, and home cage activity. Prior to tissue collection, mice underwent electroretinogram (ERG) and optical coherence tomography (OCT). Moreover, we implemented open field tests (OFT) to examine exploratory behavior and activity. Innate response to a new environment and novel visual cues in mice that may induce escape or freeze reactions are mediated by cortical and thalamic circuits, thus making the OFT optimal for assessing the link between behavior and vision. Upon sacrifice, we have flash frozen retina, brain, liver, pancreas, spleen, kidney, and heart for isolation of DNA, RNA, protein, and lipids for multiomic analysis. Retinal Extracellular Vesicles (EVs) in aging, disease and therapy EVs are heterogenous membranous structures released by cells carrying a variety of molecular cargo. Studies have demonstrated that EV number and cargo is altered during aging and in disease progression. In other diseases, EVs function as markers of disease and vehicles to deliver drugs and or molecular cargo to restore tissue homeostasis. Retinal EV research is still in its infancy, however, we have established a protocol to look enrich EVs from one or two mouse retinas that are sufficient for various studies. We aim to purify and characterize rod-derived EVs to examine changes to EV milieu during aging and disease with the purpose of generating therapeutic EVs that minimize off target effects.