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.

中文翻译：

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DNA 去甲基化是小鸡视网膜再生的驱动力。

细胞重编程重置表观遗传景观，以驱动转录程序和细胞身份的变化。视网膜切除后，胚胎鸡可以在 FGF2 存在的情况下通过视网膜色素上皮 (RPE) 重新编程来再生完整的神经视网膜。在本研究中，我们系统地分析了具有重编程能力的雏鸡受伤前以及重编程不同阶段的 RPE。除了RPE重编程过程中与表观遗传修饰相关的基因表达发生变化外，我们还观察到与二价染色质（H3K27me3/H3K4me3）相关的组蛋白标记以及DNA去甲基化过程的中间体（包括5hmC和5caC）的动态变化。通过全基因组亚硫酸氢盐测序 (WGBS) 对甲基化组进行综合分析，证实了 DNA 甲基化模式的广泛重排，包括在与染色质组织和成纤维细胞生长因子产生相关的基因启动子处发现的差异甲基化区域 (DMR)。我们还发现 Tet 甲基胞嘧啶双加氧酶 3 (TET3) 是 DNA 去甲基化和视网膜再生的重要因子，能够在缺乏外源 FGF2 的情况下重新编程 RPE。总之，我们证明 RPE 重编程早期的损伤会触发染色质的全基因组动态变化，包括二价染色质和 DNA 甲基化。在 FGF2 存在的情况下，这些动态修饰在形成新视网膜的过程中得到进一步维持。我们的研究结果表明，主动 DNA 去甲基化是一个重要的过程，可用于消除表观遗传障碍，从而使哺乳动物的视网膜再生。

缩写

bp：碱基对；DMR：差异甲基化区域；DMC：差异甲基化胞嘧啶；GFP：绿色荧光蛋白；PCR：聚合酶链式反应。TET：10-11易位；RPE：视网膜色素上皮。