Limbal Stem Cell Fate and Corneal Specific Enhancers

角膜缘干细胞命运和角膜特异性增强剂

• 批准号: 9039606
• 负责人: GEORGE L SEN
• 金额: $ 47.39万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9039606
• 关键词: Address; Affect; Animal Model; Biological; Biological Assay; Biology; Blindness; Cells; Cellular biology; ChIP-seq; Characteristics; Chromatin; Cornea; Cultured Cells; DNA; Development; Disease; Down-Regulation; EP300 gene; Enhancers; Epigenetic Process; Epithelial; Epithelial Cells; Epithelium; Etiology; Eye; Eye Development; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic; Genetic Variation; Genomics; Goals; Health; High-Throughput Nucleotide Sequencing; Histones; Human; Infection; Inflammation; Keratin; Knowledge; Mediating; Medical; Molecular; Mutation; Play; Predisposition; Reagent; Regulation; Regulatory Element; Reporter; Repression; Resources; Role; Skin; Specificity; Staging; Stem cells; Surface; Testing; Tissue-Specific Gene Expression; Tissues; To specify; Transplantation; Trauma; Vision; Visual impairment; Work; base; cell type; corneal epithelium; epigenetic regulation; genome-wide analysis; human disease; interdisciplinary approach; limbal; progenitor; stem cell biology; stem cell fate; tool; transcription factor; translational study

 DESCRIPTION (provided by applicant): The cornea is a specialized epithelium, which protects the eye and maintains transparency for normal vision. Diseases of the cornea or its stem cells (limbal stem cells) affects millions of people worldwide and result from a spectrum of etiologies, ranging from genetic defects to infection, inflammation, or trauma. Vision loss often occurs as the result of transformation of the transparent cornea into a skin-like epithelium. Recent work from our labs and others now reveal that this critical transition may be precipitated by the loss of PAX6 expression. Downregulation of PAX6 causes the loss of cornea features and gene expression and the ectopic activation of skin-like epithelium. Conversely, introduction of PAX6 into skin cells induces many cornea-like features. These studies indicate a critical role of PAX6 for maintaining cornea epithelium, but the mechanisms, which determine cornea vs. skin identity, are still unknown. Our hypothesis is that PAX6 contributes to cornea identity through tissue-specific DNA regulatory elements called enhancers. Enhancers are responsible for coordinating tissue-specific gene expression and for the development of specific cell types. Enhancers are also thought to be of considerable medical importance as genetic variation in and epigenetic regulation of enhancers is thought to play a major role in susceptibility to disease. Despite their biological and medical importance, little is known about the enhancers of the cornea and disease. Advances in chromatin biology and high-throughput sequencing provide new, powerful tools to identify tissue-specific enhancers of the cornea and have the potential to expand our knowledge of cornea disease. Here we propose to (1) identify enhancers, which distinguish cornea from skin epithelium via ChIP-seq; (2) determine how tissue-specific enhancers are regulated in the cornea by chromatin analysis and reporter studies in cultured cells, animal model transplants; and (3) determine the role of PAX6 in the regulation of tissue-specific enhancers. To accomplish these goals, an interdisciplinary approach is needed. The combined expertise and resources of two labs (eye, skin) provides an ideal path to identifying cornea- vs. skin-specific regulation and will result in a better understanding of how ectopic skin features become activated in the cornea. This major effort will result in the identification of genome-wide enhancers in the cornea, critical to stem cell biology and translational studies, and the generation of new genetic reagents to study cornea disease.

 描述（由申请人提供）：角膜是一种特殊的上皮细胞，保护眼睛并保持正常视力的透明度。角膜或其干细胞（角膜缘干细胞）疾病影响着全球数百万人，其病因多种多样，从遗传缺陷到感染、炎症或创伤。视力丧失通常是由于透明角膜转变为皮肤样上皮的结果。我们实验室和其他人最近的工作现在表明，这种关键的转变可能是由于PAX6表达的丧失而引起的。PAX6的下调导致角膜特征和基因表达的丧失以及皮肤样上皮的异位激活。相反，将PAX6引入皮肤细胞会诱导许多角膜样特征。这些研究表明PAX6在维持角膜上皮细胞中的关键作用，但决定角膜与皮肤身份的机制仍然未知。我们的假设是PAX6通过称为增强子的组织特异性DNA调控元件来促进角膜身份。增强子负责协调组织特异性基因表达和特定细胞类型的发育。增强子也被认为具有相当大的医学重要性，因为增强子的遗传变异和表观遗传调节被认为在对疾病的易感性中起主要作用。尽管它们在生物学和医学上很重要，但人们对角膜和疾病的增强剂知之甚少。染色质生物学和高通量测序的进展提供了新的、强大的工具来识别角膜的组织特异性增强子，并有可能扩大我们对角膜疾病的了解。在这里，我们建议（1）通过ChIP-seq识别将角膜与皮肤上皮区分开的增强子;（2）通过染色质分析和培养细胞、动物模型移植中的报告基因研究确定角膜中组织特异性增强子的调控方式;（3）确定PAX6在组织特异性增强子调控中的作用。为了实现这些目标，需要跨学科的方法。两个实验室（眼睛，皮肤）的专业知识和资源相结合，为识别角膜与皮肤特异性调节提供了理想的途径，并将更好地了解异位皮肤特征如何在角膜中被激活。这一重大努力将导致角膜中全基因组增强子的鉴定，这对干细胞生物学和翻译研究至关重要，并产生新的遗传试剂来研究角膜疾病。