DNA Demethylation and Muller Glia Reprogramming During Retina Regeneration

描述（由申请人提供）：与哺乳动物不同，斑马鱼能够再生受损的视网膜并恢复失去的视力。这种再生能力取决于米勒神经胶质细胞（MG），它通过去分化、增殖并最终分化再生新的神经元和神经胶质细胞，经历多次身份转变，从而对视网膜损伤做出反应。尽管 MG 可以被诱导在受损的哺乳动物视网膜中增殖，但它们并不表现出多能性，并且很少能再生受损的神经元。因此，了解驱动斑马鱼 MG 重编程为多能性的机制可能会提出从哺乳动物 MG 产生多能祖细胞的新策略。最近的研究表明，MG 重编程伴随着基因表达程序的激活，这些程序与胚胎干细胞和视网膜祖细胞中的作用相似。我们假设驱动 MG 去分化和多能性的遗传程序是由 DNA 甲基化控制的。在动物中，DNA 甲基化主要发生在 CpG 二核苷酸处，并控制转录调控过程，如印记、X 染色体失活、转座子沉默和基因活性的稳定沉默。靠近基因编码区的 DNA 甲基化与基因沉默相关。重要的是，DNA甲基化的变化与早期发育过程中发生的基因表达程序的激活和抑制相关，并伴随着体细胞的重新编程以产生诱导多能干细胞。基因组甲基化在关键位置的擦除和重建很可能 伴随着基因表达的变化，驱动 MG 去分化和多能性，以及随后新的视网膜细胞类型的再生。在这里，我们建议识别在视网膜再生过程中经历甲基化变化的 MG 基因组区域，并确定这些变化是否与之前使用微阵列技术表征的基因表达变化相关。此外，我们建议检验以下假设：去分化 MG 中的 DNA 去甲基化是由 Apobec2a 和 2b 蛋白驱动的主动过程。

DESCRIPTION (provided by applicant): Unlike mammals, zebrafish are able to regenerate a damaged retina and restore lost sight. This regenerative ability depends on Muller glia (MG) that respond to retinal injury by undergoing multiple shifts in identity as they dedifferentiate, proliferate, and finally differentiate to regenerate new neurons and glia. Although MG can be coaxed to proliferate in the injured mammalian retina, they do not exhibit multipotency and only rarely regenerate damaged neurons. Therefore understanding the mechanisms driving zebrafish MG reprogramming to mutlipotency may suggest novel strategies for generating multipotent progenitors from mammalian MG. Recent studies suggest that MG reprogramming is accompanied by activation of gene expression programs that are similar to those acting in embryonic stem cells and retinal progenitors. We hypothesize that genetic programs driving MG dedifferentiation and multipotency are controlled by DNA methylation. In animals, DNA methylation predominantly occurs at CpG dinucleotides and controls transcriptional regulatory processes like imprinting, X-chromosome inactivation, transposon silencing, and stable silencing of gene activity. Methylation of DNA proximal to gene-coding regions is correlated with gene silencing. Importantly, changes in DNA methylation have been correlated with the activation and suppression of gene expression programs that take place during early development and accompany the reprogramming of somatic cells to yield induced pluripotent stem cells. It is likely that erasure and reestablishment of genomic methylation, at key locations, accompanies the gene expression changes that drive MG dedifferentiation and multipotency and subsequently the regeneration of new retinal cell types. Here we propose to identify regions of the MG genome that are undergoing methylation changes during retina regeneration and determine if these changes correlate with gene expression changes that have previously been characterized using microarray technology. In addition, we propose to test the hypothesis that DNA demethylation in dedifferentiating MG is an active process driven by Apobec2a and 2b proteins.