Epigenetic regulation of lens fiber cell differentiation: The role of DNA methyla

描述（由申请人提供）：晶状体纤维细胞分化涉及基因表达的协调变化。Pax6、FoxE3、E-cadherin等基因在晶状体上皮细胞中表达，而在晶状体纤维细胞中表达下调。相反，许多其他基因，包括水孔蛋白0、2-和3-晶体蛋白以及晶状体特异性珠状丝蛋白CP49和纤维蛋白在晶状体上皮细胞中不表达，但在晶状体纤维细胞中被激活。近年来，人们越来越清楚，染色体DNA的表观遗传修饰（DNA或组蛋白的可逆共价修饰）在基因调控中起着重要作用。其中研究最好的表观遗传修饰是胞嘧啶甲基化在DNA的CpG二核苷酸。哺乳动物细胞中DNA的甲基化是由Dnmt1完成的，Dnmt1负责通过细胞分裂维持表观遗传甲基化，Dnmt3a和Dnmt3b负责在发育过程中产生从头甲基化变化。启动子DNA甲基化与基因的转录抑制有关。尽管转录因子参与晶状体发育的知识丰富，很少知道晶状体纤维细胞分化的表观遗传调控。最近的证据表明，Dnmt1和Dnmt3的活性是斑马鱼晶状体发育所特别需要的。我们假设启动子甲基化和去甲基化之间的平衡是晶状体纤维细胞正常分化所必需的，而这种平衡将需要维持甲基化和新生甲基化活性的活性。我们提出，维持甲基化将需要阻止晶状体上皮中与纤维细胞分化相关的基因的表达，而在纤维细胞分化过程中，需要重新甲基化来抑制晶状体上皮基因的表达。我们将在晶状体谱系缺乏维持或新生甲基化酶的小鼠中使用条件遗传策略研究DNA甲基化在纤维细胞分化中的作用。我们将使用组织学，免疫学和高通量下一代测序策略来全面研究DNA甲基化如何影响晶状体纤维细胞分化。

DESCRIPTION (provided by applicant): Lens fiber cell differentiation involves a coordinated change in gene expression. A number of genes including Pax6, FoxE3 and E-cadherin are expressed in lens epithelial cells but down-regulated in lens fiber cells. Conversely many other genes including Aquaporin0, 2- and 3-crystallins and lens-specific beaded filament proteins CP49 and filensin are not expressed in lens epithelial cells but are turned on in lens fiber cells. In recent years it has become increasingly clear that epigenetic modification (reversible covalent modification of DNA or histone proteins) of chromosomal DNA plays a major role in gene regulation. Among the best studied epigenetic modifications is methylation of cytosine in CpG dinucleotides in DNA. Methylation of DNA in mammalian cells is accomplished by Dnmt1, which is responsible for maintaining epigenetic methylation through cell divisions, and Dnmt3a and Dnmt3b, which are responsible for creating de novo methylation changes during development. Promoter DNA methylation is associated with transcriptional repression of genes. Despite the wealth of knowledge of transcription factors involved in lens development, very little is known about the epigenetic regulation of lens fiber cell differentiation. Recent evidence suggests that Dnmt1 and Dnmt3 activity are specifically required for lens development in zebrafish. We hypothesize that the balance between promoter methylation and demethylation is required for proper lens fiber cell differentiation, and that this balance will require the activity of both maintenance and de novo methylation activity. We propose that maintenance methylation will be required to prevent the expression of genes associated with fiber cell differentiation in the lens epithelium and that de novo methylation will be required to repress the expression of lens epithelial genes during fiber cell differentiation. We will investigate the role of DNA methylation in fiber cell differentiation using both conditional genetic strategies in mice lacking maintenance or de novo methylases in the lens lineage. We will use histological, immunological and high throughput next generation sequencing strategies to comprehensively investigate how DNA methylation influences lens fiber cell differentiation. PUBLIC HEALTH RELEVANCE: Recent experiments demonstrating that epigenetic reprogramming can convert differentiated cell types into pluripotent stem cells makes clear the critical importance for understanding the epigenetic regulation of the differentiated phenotype. We propose that the lens represents a unique opportunity to understand how epigenetic DNA methylation regulates differentiation. This understanding will be important not only for lens development, but for a global understanding of how to manipulate differentiated states, which is critical for the generation of patient-specific stem cells in medicine.