The role of DNA methylation in lens fiber cell terminal differentiation

描述（由申请人提供）：DNA胞嘧啶残基的甲基化导致表观遗传转录抑制，异常甲基化在人类癌症中起作用。在发育过程中，DNA甲基化是细胞类型特异性基因表达模式在终末分化过程中形成的机制。然而，DNA甲基化在终末分化和器官发生中的具体作用迄今仅在极少数细胞类型中得到研究。这部分是由于缺乏DNA甲基化所需基因的敲除小鼠的早期胚胎致命性。与小鼠模型不同，斑马鱼在两个关键的表观遗传调节因子，DNA甲基转移酶1 （dnmt1）和泛素样，含有PHD和RING Finger Domains 1 （uhrf1）中发生突变，可以存活到胚胎后期，此时许多复杂的器官（包括眼睛）已经形成。我利用这些突变的斑马鱼品系来研究DNA甲基化在脊椎动物晶状体发育中的作用。晶状体仅由增生性上皮细胞和终末分化晶状体纤维两种细胞类型组成，是研究基因调控的理想组织。尽管如此，目前对DNA甲基化在晶状体发育中的作用知之甚少。我的初步数据显示，Dnmt1和Uhrf1功能的丧失导致白内障和晶状体形态异常，晶状体纤维紊乱和凋亡。dnmt1和uhrf1突变体的基因组DNA甲基化水平降低到野生型水平的25%，支持DNA甲基化是正常晶状体发育所必需的模型。这项研究的目的是确定DNA甲基化如何在晶状体纤维末端分化过程中沉默基因。具体目的1：确定晶状体上皮细胞基因是否因DNA甲基化而沉默，晶状体上皮细胞基因在晶状体纤维末端分化过程中下调。特异性目的2：确定dnmt1和uhrf1突变斑马鱼晶状体缺陷是否由组蛋白H3K9三甲基化缺陷引起。提出的实验是新颖的，因为它们将开始阐明DNA甲基化介导的基因沉默在晶状体形成过程中的作用和机制。这一主题与理解白内障形成的机制有关，它将增加我们对DNA甲基化失控如何导致癌症的理解。

DESCRIPTION (provided by applicant): Methylation of DNA cytosine residues results in epigenetic transcriptional repression, and aberrant methylation plays a role in human cancers. During development, DNA methylation is a mechanism whereby cell-type specific gene expression patterns are set during terminal differentiation. However, the specific role of DNA methylation in terminal differentiation and organogenesis has so far been studied in very few cell types. This is due in part to the early embryonic lethality of knockout mice lacking genes required for DNA methylation. Unlike mouse models, zebrafish with mutations in two key epigenetic regulators, DNA Methyltransferase 1 (dnmt1) and Ubiquitin-like, Containing PHD and RING Finger Domains 1 (uhrf1), survive to late embryonic stages, at which time many complex organs (including the eye) have formed. I have taken advantage of these mutant zebrafish lines to study the role of DNA methylation in development of the vertebrate lens. Consisting of only two cell types: proliferative epithelial cells and terminally differentiated lens fibers, the lens is an ideal tissue in which to study gene regulation. Despite this, little is currently known about the role of DNA methylation in lens development. My preliminary data show that loss of Dnmt1 and Uhrf1 function leads to cataracts and morphologically abnormal lenses that contain disorganized and apoptotic lens fibers. Methylation of genomic DNA in dnmt1 and uhrf1 mutants is reduced to 25% of wild type levels, supporting a model in which DNA methylation is required for normal lens development. The goal of the proposed research is to determine how DNA methylation functions to silence genes during lens fiber terminal differentiation. This will be determined with the following specific aims: Specific Aim 1: To determine whether lens epithelial cell genes, which are downregulated during lens fiber terminal differentiation, are silenced by DNA methylation. Specific Aim 2: To determine whether the lens defects in dnmt1 and uhrf1 mutant zebrafish are caused by deficient histone H3K9 tri-methylation. The experiments proposed are novel in that they will begin to elucidate the role and mechanism of DNA methylation-mediated gene silencing in the process of lens formation. This topic has relevance as a mechanism for understanding cataract formation, and it will increase our understanding of how deregulated DNA methylation can contribute to cancer.