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期间设定的机制。然而，DNA甲基化在终末分化和器官发生中的特定作用迄今为止在极少数细胞类型中进行了研究。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死亡。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（dnmt 1）和泛素样，含有PHD和RING指结构域1（uhrf 1）中发生突变，可以存活到胚胎晚期，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甲基化在脊椎动物透镜发育中的作用。透镜仅由两种细胞类型组成：增殖上皮细胞和终末分化的透镜纤维，是研究基因调控的理想组织。尽管如此，目前对DNA甲基化在透镜发育中的作用知之甚少。我的初步数据表明，Dnmt 1和Uhrf 1功能的丧失导致白内障和形态异常的晶状体，其中包含无序和凋亡的透镜纤维。dnmt 1和uhrf 1突变体中基因组DNA的甲基化降低到野生型水平的25%，支持DNA甲基化是正常透镜发育所需的模型。 这项研究的目的是确定DNA甲基化如何在透镜纤维末端分化过程中沉默基因。具体目的1：确定在透镜纤维末端分化过程中下调的透镜上皮细胞基因是否被DNA甲基化沉默。具体目的2：确定dnmt 1和uhrf 1突变斑马鱼的透镜缺陷是否由组蛋白H3 K9三甲基化缺陷引起。所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透镜形成过程中的作用和机制。这个主题作为理解白内障形成的机制具有相关性，它将增加我们对DNA甲基化失调如何导致癌症的理解。 公共卫生相关性：当人类透镜发育的正常过程由于基因突变或其他因素而出现错误时，就会导致白内障。本研究中使用的斑马鱼模型将白内障与DNA甲基化的表观遗传调控机制联系起来。目前对DNA甲基化在透镜发育中的作用知之甚少，这项研究的结果将提高对白内障形成的医学理解，并可能进一步改善治疗选择。除了透镜发育和白内障发生之外，异常的DNA甲基化在人类癌症中起作用，本研究的结果将提供另一种体内系统，通过该系统可以研究分化期间甲基化的组织特异性作用。

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. PUBLIC HEALTH RELEVANCE: When the normal process of lens development goes awry in humans due to genetic mutations or other factors, cataracts result. The zebrafish models utilized in this study link cataracts to the epigenetic regulatory mechanism of DNA methylation. Very little is currently known about the role of DNA methylation in lens development, and the results of this study will improve medical understanding of cataract formation, and may additionally lead to improved therapeutic options. Beyond lens development and cataractogenesis, aberrant DNA methylation plays a role in human cancers, and the results of this study will provide an additional in vivo system through which tissue- specific roles for methylation during differentiation can be studied.