Petkova Tihomira D, Seigel Gail M, Otteson Deborah C
Department of Vision Science, College of Optometry, Houston, TX 77204-2020, USA.
Vision Res. 2011 Jan 28;51(2):260-8. doi: 10.1016/j.visres.2010.09.022. Epub 2010 Sep 25.
Understanding the mechanisms regulating expression of retinal ganglion cell (RGC) specific and axon-guidance genes during development and in retinal stem cells will be critical for successful optic nerve regeneration. Müller glia have some characteristics of retinal stem cells but in mammals have demonstrated limited potential to differentiate into RGCs. Chromatin remodeling through histone deacetylation and DNA methylation are a potential mechanism for silencing genes necessary for neuronal differentiation of glial cells. We investigated DNA methylation as a mechanism for regulating expression of mouse EphA5, one member of a large family of ephrin receptor genes that regulate patterning of the topographic connections of RGCs during visual system development. We analyzed spatial and age-related patterns of EphA5 promoter methylation by bisulfite sequencing and mRNA expression by quantitative RT-PCR in the mouse retina. The CpG island in the EphA5 promoter was hypomethylated in the retina and showed no change in overall methylation with age, despite a decline in EphA5 mRNA expression levels in the adult retina. In the nasal retina of post-natal day 0 mice, there was a modest, but statistically significant increase in methylation. Increased methylation corresponded with lower levels of receptor mRNA expression in the nasal retina. We cloned the EphA5 promoter and found that site-specific differences in methylation could preferentially activate or repress promoter activity in transient transfections of rat retinal progenitor cells (R28) using luciferase assays. In sphere cultures generated by EGF/FGF2 stimulation of conditionally immortalized mouse Müller glia (ImM10), EphA5 promoter was hypermethylated and EphA5 mRNA was not detected. Demethylation using 5-azadeoxycytidine (AzadC) resulted in a significant decrease of methylation of the EphA5 promoter and re-expression of the EphA5 mRNA. The inverse relationship between EphA5 promoter methylation and mRNA expression is consistent with a role for DNA methylation in modulating the spatial patterns of EphA5 gene expression in the retina and in silencing EphA5 expression in ImM10 cells. The robust up-regulation of EphA5 in ImM10 cells following demethylation suggests that modulation of chromatin structure may be a useful approach for promoting expression of silenced developmental genes and increasing the neurogenic potential of Müller glia.
了解在发育过程中以及视网膜干细胞中调节视网膜神经节细胞（RGC）特异性基因和轴突导向基因表达的机制，对于成功实现视神经再生至关重要。穆勒胶质细胞具有一些视网膜干细胞的特征，但在哺乳动物中，其分化为RGC的潜力有限。通过组蛋白去乙酰化和DNA甲基化进行的染色质重塑是使胶质细胞神经元分化所需基因沉默的一种潜在机制。我们研究了DNA甲基化作为调节小鼠EphA5表达的一种机制，EphA5是ephrin受体基因大家族的成员之一，在视觉系统发育过程中调节RGC地形连接的模式。我们通过亚硫酸氢盐测序分析了EphA5启动子甲基化的空间和年龄相关模式，并通过定量RT-PCR分析了小鼠视网膜中的mRNA表达。EphA5启动子中的CpG岛在视网膜中是低甲基化的，并且随着年龄的增长总体甲基化没有变化，尽管成年视网膜中EphA5 mRNA表达水平有所下降。在出生后第0天小鼠的鼻侧视网膜中，甲基化有适度但具有统计学意义的增加。甲基化增加与鼻侧视网膜中受体mRNA表达水平降低相对应。我们克隆了EphA5启动子，发现甲基化的位点特异性差异在使用荧光素酶测定法对大鼠视网膜祖细胞（R28）进行瞬时转染时可优先激活或抑制启动子活性。在通过表皮生长因子/成纤维细胞生长因子2刺激条件性永生化小鼠穆勒胶质细胞（ImM10）产生的球状体培养物中，EphA5启动子是高甲基化的，未检测到EphA5 mRNA。使用5-氮杂脱氧胞苷（AzadC）进行去甲基化导致EphA5启动子甲基化显著降低，EphA5 mRNA重新表达。EphA5启动子甲基化与mRNA表达之间的负相关关系与DNA甲基化在调节视网膜中EphA5基因表达的空间模式以及使ImM10细胞中EphA5表达沉默方面的作用一致。去甲基化后ImM10细胞中EphA5的强烈上调表明，染色质结构的调节可能是促进沉默的发育基因表达以及增加穆勒胶质细胞神经发生潜力的一种有用方法。
