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Transcriptome and DNA Methylome Signatures Associated with Retinal M¨Uller Glia Development, Injury Response, and Aging

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Transcriptome and DNA Methylome Signatures Associated with Retinal M¨Uller Glia Development, Injury Response, and Aging Retina Transcriptome and DNA Methylome Signatures Associated With Retinal M¨uller Glia Development, Injury Response, and Aging Siyuan Lin, Jingyi Guo, and Shuyi Chen State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China Correspondence: Shuyi Chen, State PURPOSE. The purpose of this study was to systematically characterize and correlate the Key Laboratory of Ophthalmology, transcriptome and DNA methylome signatures of mouse Muller¨ cells that may underlie the Zhongshan Ophthalmic Center, Sun development, physiological functions, and regeneration capacity of these cells. Yat-sen University, Guangzhou 510060, China; METHODS. Mouse Muller¨ cells under normal, injury, and aging conditions were sorted from [email protected]. Muller¨ cell–specific green fluorescent protein (GFP)-expressing mice. RNA sequencing was Submitted: April 17, 2019 used to sequence transcriptomes, and reduced representation bisulfite sequencing was used Accepted: September 22, 2019 to sequence DNA methylomes. Various bioinformatics tools were used to compare and correlate the transcriptomes and DNA methylomes. Citation: Lin S, Guo J, Chen S. Tran- scriptome and DNA methylome sig- RESULTS. Muller¨ cells express a distinct transcriptome that is in line with their retinal natures associated with retinal Muller¨ supporting roles and dormant retinogenic status. Injury changes the Muller¨ cell transcriptome glia development, injury response, dramatically but fails to stimulate the cell cycle machinery and retinogenic factors to the states and aging. Invest Ophthalmol Vis Sci. observed in early retinal progenitor cells (RPCs). Muller¨ cells exhibit a less methylated 2019;60:4436–4450. https://doi.org/ genome than that of early RPCs, but most regulatory elements for Muller¨ cell– and RPC- 10.1167/iovs.19-27361 specific genes are similarly hypomethylated in both Muller¨ cells and RPCs, except for a subset of Muller¨ cell–specific functional genes. Aging only subtly affects the transcriptome and DNA methylome of Muller¨ cells. CONCLUSIONS. Failure to reactivate the cell cycle machinery and retinogenic factors to necessary levels might be key barriers blocking Muller¨ cells from entering an RPC-like regeneration state. DNA methylation might regulate the expression of a subset of Muller¨ cell– specific functional genes during development but is likely not involved in restricting the regeneration activity of Muller¨ cells. Keywords: Muller¨ cells, transcriptome, DNA methylome, regeneration, cell cycle uller¨ cells are the primary glia of the retina and play to proliferate by downregulating the cell cycle inhibitor M essential supporting roles in maintaining the structural p27Kip1.5 This proliferation tendency of Muller¨ cells is exploited and physiological homeostasis of the retina. Muller¨ cell nuclei most effectively in lower vertebrates, such as zebrafish; Muller¨ are located in the midstratum of the inner nuclear layer of the cells in these species react to injuries with dedifferentiation, retina, but their cell bodies extend across the entire thickness extensive proliferation, and differentiation to all types of retinal of the retina and project numerous short lateral branches to neurons to repair the retina.6,7 However, the regeneration ensheath all nearby retinal neurons. This special radial structure activity of Muller¨ cells decreases to a negligible level in of Muller¨ cells allows them to intimately interact with retinal mammals.8 Nonetheless, the dramatic regeneration ability of neurons in both healthy and diseased conditions. The well- Muller¨ cells in lower vertebrates has inspired researchers to established functions of Muller¨ cells include maintaining the explore ways to use endogenous Muller¨ cells to regenerate ion and water homeostasis of the retinal microenvironment, retinal neurons in situ in mammals, with the ultimate goal of providing nutrition for retinal neurons, recycling neurotrans- developing regeneration methods for treating human retinal mitters and photopigments, protecting retinal neurons from degeneration diseases. Encouragingly, by overexpressing tran- oxidative stress, regulating retinal blood flow, and contributing to the blood–retinal barrier.1 Consistent with the essential scription factors (TFs) coupled with epigenetic manipulations functions of Muller¨ cells, depletion or malfunction of Muller¨ or signaling pathway stimulation, some retinal interneurons and cells causes severe disruption of retinal structure and visual photoreceptors have been successfully regenerated from 9–11 function, eventually leading to retinal degeneration.2–4 reprogrammed Muller¨ cells in situ in mice. However, thus Upon assaults to the retina (physical, chemical, or patho- far, only limited types of retinal neurons can be regenerated, logical), Muller¨ cells respond with a series of gliotic reactions, and retinal ganglion cells, the major cells damaged in glaucoma, including reduced potassium conductance and membrane which is the most prevalent retinal degeneration disease, seem depolarization, cellular hypertrophy, and upregulation of resistant to regeneration from Muller¨ cells. Therefore, efforts intermediate filaments, such as GFAP, nestin, and vimentin1 are needed to investigate the molecular barriers blocking the More interestingly, upon injury, Muller¨ cells exhibit a tendency regeneration capacity of Muller¨ cells in mammals and to Copyright 2019 The Authors iovs.arvojournals.org j ISSN: 1552-5783 4436 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from iovs.arvojournals.org on 10/01/2021 Transcriptome and DNA Methylome of Retinal Muller¨ Glia IOVS j October 2019 j Vol. 60 j No. 13 j 4437 develop more efficient protocols to regenerate various types of (Shanghai, China) for RNA extraction, library preparation, retinal neurons in situ. sequencing, and data analyses. The cDNA libraries were From a developmental perspective, Muller¨ cells share the constructed using the TruSeq Stranded Total RNA with Ribo- same progenitor as retinal neurons. Muller¨ cells and retinal Zero Gold kit (Illumina, San Diego, CA, USA), and sequenced neurons are all generated by retinal progenitor cells (RPCs) in a by HiSeq XTen (Illumina) on a 150 base pair (bp) paired-end highly ordered sequential differentiation process. Muller¨ cells run. A total of 6 to 8 3 107 reads were generated for each are the last cell type generated by RPCs toward the end of sample, and on average, 96% reads were mapped to the mouse retinogenesis.12,13 A number of gene expression analyses have genome. noted interesting overlaps of gene expression patterns between Muller¨ cells and RPCs, which partially explains the RNA-Seq Data Processing regeneration potential of this mature retinal cell type.14,15 RPCs change their competence to generate retinal cells during Before read mapping, clean reads were obtained from the raw retinogenesis such that early RPCs generate early-born retinal reads by removing the adaptor sequences and low-quality neurons, including retinal ganglion cells, horizontal cells, reads. The clean reads were then aligned to mouse genome amacrine cells, and cone photoreceptors, and they gradually (GRCm38/mm10) using HISAT2.19 HTseq20 was used to get transform to late RPCs to generate later-stage retinal cells, gene counts, and the fragments per kilobase per million including bipolar cells, rod photoreceptors, and Muller¨ mapped fragments (FPKM) method as used to normalize the cells.16,17 As the last cell type differentiated from late RPCs, gene expression. We applied the limma algorithm21 on the Muller¨ cells are closer to late RPCs than to early RPCs value of Log2(FPKMþ0.5) to filter differentially expressed genes molecularly,14 which may explain why Muller¨ cells are more (DEGs) under the following criteria: (1) fold change > 2or< prone to be reprogrammed to late-stage retinal neurons such as 0.5; (2) P value < 0.05, false discovery rate (FDR) < 0.05. For rods and bipolar cells.9,10 However, early RPCs proliferate more long noncoding RNA (lncRNA) cis prediction, we identified actively and possess the full potential to generate all types of genomic localization of lncRNAs and paired mRNAs that are retinal neurons. Exactly how Muller¨ cells are different from less than 10 kb upstream or downstream away from the early-stage RPCs at the genome-wide level awaits further lncRNA. Gene Ontology (GO) term analysis was performed investigation. using DAVID bioinformatics resources.22 For gene functional In this study, we systematically measured, compared, and association network analysis, the gene coexpression network correlated the transcriptomes and DNA methylomes of Muller¨ modeling algorithm was used23 based on the normalized cells and early RPCs, as well as the transcriptomes and DNA expression values of genes.24 We focused on cell type–specific methylomes of Muller¨ cells under injury and aging conditions, TFs and genes in enriched GO terms in each cell type. For each to explore the possible molecular mechanisms governing the pair of genes, we calculated the Pearson correlation and chose development, physiological functions, and regeneration capac- the significant correlation pairs (FDR < 0.05) to construct the ity of Muller¨ cells. network. Zebrafish high-throughput sequencing data were downloaded from Gene Expression Omnibus (GEO): zebrafish Muller¨ cells (SRR4241537, SRR4241538, SRR4241539), 36hpf METHODS zebrafish eyes (SRR5398205-SRR5398212), zebrafish
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