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.

Public Health Relevance

This project plans to investigate significant questions concerning how growth factors and environmental stresses affect human corneal epithelial self-renewal and wound healing. We will determine whether the epigenetic factor CTCF is responsible for growth factor- and stress-induced chromatin remodeling to regulate interactions between eye-specific Pax6 and cell migration-associated genes. All of these determinative factors are critical for corneal epithelial self-renewal and for improving the wound healing process in normal and disease stages.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Mckie, George Ann
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Tsui, Shanli; Wang, Jie; Wang, Ling et al. (2016) CTCF-Mediated and Pax6-Associated Gene Expression in Corneal Epithelial Cell-Specific Differentiation. PLoS One 11:e0162071
Restuccia, Agnese; Yang, Feikun; Chen, Changyan et al. (2016) Mps1 is SUMO-modified during the cell cycle. Oncotarget 7:3158-70
Tsui, S; Dai, W; Lu, L (2014) CCCTC-binding factor mediates effects of glucose on beta cell survival. Cell Prolif 47:28-37
Wang, Ling; Wu, Xiaolin; Shi, Ting et al. (2013) Epidermal growth factor (EGF)-induced corneal epithelial wound healing through nuclear factor ?B subtype-regulated CCCTC binding factor (CTCF) activation. J Biol Chem 288:24363-71
Wang, Jie; Wang, Yumei; Lu, Luo (2012) De-SUMOylation of CCCTC binding factor (CTCF) in hypoxic stress-induced human corneal epithelial cells. J Biol Chem 287:12469-79
Tsui, Shanli; Gao, Jie; Wang, Charles et al. (2012) CTCF mediates effect of insulin on glucagon expression. Exp Cell Res 318:887-95
Wang, Ling; Deng, Sophie X; Lu, Luo (2012) Role of CTCF in EGF-induced migration of immortalized human corneal epithelial cells. Invest Ophthalmol Vis Sci 53:946-51
Gao, Jie; Wang, Jie; Wang, Yumei et al. (2011) Regulation of Pax6 by CTCF during induction of mouse ES cell differentiation. PLoS One 6:e20954
Wang, Yumei; Lu, Luo (2011) Activation of oxidative stress-regulated Bcl-3 suppresses CTCF in corneal epithelial cells. PLoS One 6:e23984
Wang, Ling; Payton, Reid; Dai, Wei et al. (2011) Hyperosmotic stress-induced ATF-2 activation through Polo-like kinase 3 in human corneal epithelial cells. J Biol Chem 286:1951-8

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