DNA demethylation is a driver for chick retina regeneration

Abstract

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.

Transcriptome profiling of embryonic retinal pigment epithelium reprogramming

“…In agreement with our previous study, we observed increased expression of GADD45Β in the rRPE, and it is possible that GADD45B regulates DNA methylation to facilitate the epigenetic reprogramming of RPE (Supplementary File S2) [23]. As noted prior, and in agreement with previous studies, the neural retinal progenitor marker VSX2 was identified as one of the top up-regulated DEGs in rRPE (Figure 3b) [18].…”

“…The limited plasticity observed in mammals stands in contrast to organisms such as urodele amphibians and the chick embryo (Gallus gallus), which have the ability to fully regenerate their retina after damage [4,[12][13][14]. The embryonic chick can regenerate its retina between embryonic day (E) 4-4.5 (Hamburger & Hamilton stages [23][24][25] by reprogramming RPE cells into retinal progenitors that are capable of generating all major retinal cell types [4,12,[15][16][17]. RPE reprogramming in the chick has been characterized at the molecular level in 3 distinct phases [18].…”

“…RPE cells can be reprogrammed in vitro at later stages (HH Stage 28-31) by using combined treatment with FGF2 and TGFβ inhibitors, suggesting that signaling by TGFβ family members also exert an active role in promoting RPE differentiation [9, 22]. More recently, we demonstrated that the epigenetic landscape of RPE is dynamic during reprogramming, including histone modifications (H3K27me3/H3K4me3) and changes in DNA methylation profile, and that overexpression of DNA demethylation enzyme TET3 is sufficient to reprogram RPE [23]. Despite efforts to characterize the various pathways which govern RPE identity, there has not been a study to date characterizing the global changes in gene expression which occur during embryonic RPE reprogramming.…”

“…The intimacy of the RPE with surrounding tissues such as extraocular mesenchyme and retina has impeded efforts to profile the in-situ transcriptome associated with RPE reprogramming. Compounding this problem, we previously demonstrated that retinectomy leads to rapid changes in the transcriptome and epigenetic profile of the injured RPE cells, underscoring the need for tissue isolation techniques which do not introduce injury signals that may confound the analysis [18, 23]. In order to avoid the use of dissociation protocols that could mask crucial processes, such as those influenced by cell-ECM interactions, we performed L aser C apture M icrodissection followed by bulk RNA - seq uencing (LCM-RNAseq).…”

DNA methylation during development and regeneration of the annelidPlatynereis dumerilii

“…Our current hypothesis is therefore that Decitabine delays regeneration through its hypomethylating effect and therefore that DNA methylation may be required for proper regeneration in P. dumerilii . This hypothesis is at odds with what has been described in vertebrates in which, in contrast, demethylation has been suggested to be a driver of regeneration, in the axolotl limb [37], zebrafish fin [36] and the chick retina [80]. Additional experiments will be required to further test the requirement of DNA methylation in P. dumerilii regeneration and identify the involved mechanisms.…”