Transcriptome Profiling of Embryonic Retinal Pigment Epithelium Reprogramming

The plasticity of human retinal pigment epithelium (RPE) has been observed during proliferative vitreoretinopathy, a defective repair process during which injured RPE gives rise to fibrosis. In contrast, following injury, the RPE of the embryonic chicken can be reprogrammed to regenerate neural retina in a fibroblast growth factor 2 (FGF2)-dependent manner. To better explore the mechanisms underlying embryonic RPE reprogramming, we used laser capture microdissection to isolate RNA from (1) intact RPE, (2) transiently reprogrammed RPE (t-rRPE) 6 h post-retinectomy, and (3) reprogrammed RPE (rRPE) 6 h post-retinectomy with FGF2 treatment. Using RNA-seq, we observed the acute repression of genes related to cell cycle progression in the injured t-rRPE, as well as up-regulation of genes associated with injury. In contrast, the rRPE was strongly enriched for mitogen-activated protein kinase (MAPK)-responsive genes and retina development factors, confirming that FGF2 and the downstream MAPK cascade are the main drivers of embryonic RPE reprogramming. Clustering and pathway enrichment analysis was used to create an integrated network of the core processes associated with RPE reprogramming, including key terms pertaining to injury response, migration, actin dynamics, and cell cycle progression. Finally, we employed gene set enrichment analysis to suggest a previously uncovered role for epithelial-mesenchymal transition (EMT) machinery in the initiation of embryonic chick RPE reprogramming. The EMT program is accompanied by extensive, coordinated regulation of extracellular matrix (ECM) associated factors, and these observations together suggest an early role for ECM and EMT-like dynamics during reprogramming. Our study provides for the first time an in-depth transcriptomic analysis of embryonic RPE reprogramming and will prove useful in guiding future efforts to understand proliferative disorders of the RPE and to promote retinal regeneration

DNA demethylation is a driver for chick retina regeneration

Cellular reprogramming resets the epigenetic landscape to drive shifts in transcriptional programmes and cell identity. The embryonic chick can regenerate a complete neural retina, after retinectomy, via retinal pigment epithelium (RPE) reprogramming in the presence of FGF2. In this study, we systematically analysed the reprogramming competent chick RPE prior to injury, and during different stages of reprogramming. In addition to changes in the expression of genes associated with epigenetic modifications during RPE reprogramming, we observed dynamic changes in histone marks associated with bivalent chromatin (H3K27me3/H3K4me3) and intermediates of the process of DNA demethylation including 5hmC and 5caC. Comprehensive analysis of the methylome by whole-genome bisulphite sequencing (WGBS) confirmed extensive rearrangements of DNA methylation patterns including differentially methylated regions (DMRs) found at promoters of genes associated with chromatin organization and fibroblast growth factor production. We also identified Tet methylcytosine dioxygenase 3 (TET3) as an important factor for DNA demethylation and retina regeneration, capable of reprogramming RPE in the absence of exogenous FGF2. In conclusion, we demonstrate that injury early in RPE reprogramming triggers genome-wide dynamic changes in chromatin, including bivalent chromatin and DNA methylation. In the presence of FGF2, these dynamic modifications are further sustained in the commitment to form a new retina. Our findings reveal active DNA demethylation as an important process that may be applied to remove the epigenetic barriers in order to regenerate retina in mammals. Abbreviations bp: Base pair; DMR: Differentially methylated region; DMC: Differentially methylated cytosines; GFP: Green fluorescent protein; PCR: Polymerase chain reaction. TET: Ten-eleven translocation; RPE: retinal pigment epithelium

Lens Epithelial Explants Treated with Vitreous Humor Undergo Alterations in Chromatin Landscape with Concurrent Activation of Genes Associated with Fiber Cell Differentiation and Innate Immune Response

Lens epithelial explants are comprised of lens epithelial cells cultured in vitro on their native basement membrane, the lens capsule. Biologists have used lens epithelial explants to study many different cellular processes including lens fiber cell differentiation. In these studies, fiber differentiation is typically measured by cellular elongation and the expression of a few proteins characteristically expressed by lens fiber cells in situ. Chromatin and RNA was collected from lens epithelial explants cultured in either un-supplemented media or media containing 50% bovine vitreous humor for one or five days. Chromatin for ATAC-sequencing and RNA for RNA-sequencing was prepared from explants to assess regions of accessible chromatin and to quantitatively measure gene expression, respectively. Vitreous humor increased chromatin accessibility in promoter regions of genes associated with fiber differentiation and, surprisingly, an immune response, and this was associated with increased transcript levels for these genes. In contrast, vitreous had little effect on the accessibility of the genes highly expressed in the lens epithelium despite dramatic reductions in their mRNA transcripts. An unbiased analysis of differentially accessible regions revealed an enrichment of cis-regulatory motifs for RUNX, SOX and TEAD transcription factors that may drive differential gene expression in response to vitreous

IHH enhancer variant within neighboring NHEJ1 intron causes microphthalmia anophthalmia and coloboma

Abstract Genomic sequences residing within introns of few genes have been shown to act as enhancers affecting expression of neighboring genes. We studied an autosomal recessive phenotypic continuum of microphthalmia, anophthalmia and ocular coloboma, with no apparent coding-region disease-causing mutation. Homozygosity mapping of several affected Jewish Iranian families, combined with whole genome sequence analysis, identified a 0.5 Mb disease-associated chromosome 2q35 locus (maximal LOD score 6.8) harboring an intronic founder variant in NHEJ1, not predicted to affect NHEJ1. The human NHEJ1 intronic variant lies within a known specifically limb-development enhancer of a neighboring gene, Indian hedgehog (Ihh), known to be involved in eye development in mice and chickens. Through mouse and chicken molecular development studies, we demonstrated that this variant is within an Ihh enhancer that drives gene expression in the developing eye and that the identified variant affects this eye-specific enhancer activity. We thus delineate an Ihh enhancer active in mammalian eye development whose variant causes human microphthalmia, anophthalmia and ocular coloboma. The findings highlight disease causation by an intronic variant affecting the expression of a neighboring gene, delineating molecular pathways of eye development