Role of CTCF in EGF_Induced Corneal Epithelial Growth

描述（由申请人提供）：本项目的总体目标是确定人类角膜上皮（CE）细胞在自我更新和伤口愈合的遗传过程中如何对生长因子和环境应激作出反应的表观遗传机制。我们发现，EGF/应力诱导的Erk和NF？B信号通路随后增加/降低这些细胞中表观遗传因子CTCF的水平。我们的初步数据表明，CTCF介导的染色质重塑在人类CE细胞调节眼睛特异性Pax 6和17个识别的迁移相关基因之间的启动子区域的相互作用，这反过来控制CE增殖，分化和伤口愈合。因此，基于CTCF活性变化幅度的对EGF/应激的更大响应导致Pax 6和影响CE伤口愈合的迁移相关基因的染色质重塑改变。我们相信，本研究的结果将在提供CE伤口愈合和其他眼部疾病的表观遗传学机制方面产生重大影响，因为我们研究：1）CTCF介导的染色质重塑以鉴定EGF/应激刺激对角膜疾病的表观遗传学效应; 2）Pax 6启动子连接的染色质相互作用以特别关注眼睛特异性基因;和3）迁移相关基因表达和功能以理解CE伤口愈合的基本机制。我们的中心假设是，EGF和应力诱导的染色质重塑需要CTCF组装Pax 6和相关基因在一组协调表达，以确定CE细胞迁移和增殖的自我更新和伤口愈合。我们相信，这些新的研究将确定涉及CE自我更新和伤口愈合的基因表达的表观遗传调控的重要机制。我们提出了三个目标：1）确定CTCF如何控制Pax 6 P1启动子和Pax 6变体在LS/P和CE细胞中的表达。我们建议确定：P1转录本的功能作用是什么;以及CTCF如何控制P1启动子活性。我们还将确定P0/P1启动子的Pax 6转录变体如何在角膜中差异表达。 2)研究CTCF如何介导Pax 6及其相关基因参与的染色质重塑。我们将鉴定在CTCF介导的染色质重塑中与Pax 6基因相互作用的迁移相关基因，并确定所鉴定基因内的功能相互作用。3)确定EGF/应激诱导的CTCF活性改变对CE迁移和伤口愈合的影响。我们将展示：在CTCF活性缺失的情况下染色质如何相互作用;诱导所鉴定的基因的表达的变化;以及伤口愈合是否受到CTCF介导的染色质重塑的改变的影响。通过实现这些联合研究的目标，我们将提供新的机制，促进对角膜上皮自我更新和伤口愈合中LS/P和CE细胞功能的表观遗传调节的生长因子/应激诱导的影响的理解。

DESCRIPTION (provided by applicant): The overall objective of this project is to define the epigenetic mechanisms of how human Corneal Epithelial (CE) cells respond to growth factors and environmental stresses in the genetic processes of self-renewal and wound healing. We found that EGF/stress-induced activation of the Erk and NF?B signaling pathways subsequently increase/decrease levels of the epigenetic factor CTCF in these cells. Our preliminary data shows that CTCF mediates chromatin remodeling in human CE cells to regulate interactions in the promoter regions between eye-specific Pax6 and 17 identified migration-associated genes, which in turn control CE proliferation, differentiation and wound healing. Thus, larger responses to EGF/stress based on the magnitude of changes in CTCF activities result in altered chromatin remodeling of Pax6 and migration-associated genes that affect CE wound healing. We believe that results of this study will have a significant impact in providing the epigenetic mechanisms underlying CE wound healing and other eye diseases, as we investigate: 1) CTCF- mediated chromatin remodeling to identify the epigenetic effects of EGF/stress stimulation on corneal diseases; 2) Pax6 promoter-linked chromatin interactions to particularly focus on eye specific genes; and 3) migration-associated gene expression and function to understand fundamental mechanisms of CE wound healing. Our central hypothesis is that EGF- and stress-induced chromatin remodeling requires CTCF to assemble Pax6 and associated genes in a group for coordinated expression to determine CE cell migration and proliferation in self-renewal and wound healing. We believe that such novel studies will identify important mechanisms involving epigenetic regulation of gene expression in CE self-renewal and wound healing. We propose three aims: 1) to define how CTCF controls the Pax6 P1 promoter and expression of Pax6 variants in LS/P and CE cells. We propose to determine: what the functional role of the P1 transcripts is; and how CTCF controls P1 promoter activity. We will also determine how Pax6 transcript variants from P0/P1 promoters are differentially expressed in the cornea. 2) To investigate how CTCF mediates chromatin remodeling involving Pax6 and associated genes. We will identify migration-associated genes interacting with Pax6 gene in CTCF-mediated chromatin remodeling and determine functional interactions within the identified genes. 3) To determine EGF/stress-induced CTCF activity alteration on CE migration and wound healing. We will show: how chromatin interacts in the absence of CTCF activity; what changes in expression of the identified genes are induced; and whether wound healing is affected by alterations of CTCF-mediated chromatin remodeling. By achieving the goals of these combined studies, we will provide novel mechanisms that advance understanding of growth factor/stress-induced effects on epigenetic regulation of LS/P and CE cell functions in corneal epithelial self-renewal and wound healing.