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Suguru Nakagawa, Ryo Nakaki, Satoru Yamagami, Tomohiko Usui, Hiroyuki Aburatani; Regulatory networks defining human corneal limbal epithelial cells identity through epigenomes.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2284.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal epithelial cells are unique type of non-keratinized epithelial cells arranged in an orderly fashion with a strong barrier function, and this is essential for vision by maintaining transparency for light transmission. However, the molecular mechanisms distinguishing human corneal limbal epithelial cells from other ectodermal derived cells have been largely unknown. In this study, we aimed to dissect regulatory landscape that orchestrate cornea-specific phenotype in human corneal limbal epithelial cells.
Primary cultivated human corneal limbal epithelial cells (CEpi) and two ectodermal derived cells: normal human epidermal keratinocytes (NHEK) and immortalized retinal pigment epithelial cells (ARPE19) were used.We performed global transcriptional analysis by messenger RNA-sequencing (mRNA-seq) using next generation sequencer (Illumina Hiseq 2500). To examine chromatin patterns associated with regulatory regions, we performed formaldehyde-assisted isolation of regulatory elements (FAIRE)-sequencing and chromatin immunoprecipitation (ChIP) sequencing using antibody recognizing histone 3 lysine 27 acetylation (H3K27ac) that marks active enhancer. To examine genome-wide PAX6 binding sites, we performed ChIP-seq using antibodies recognizing PAX6.
We identified active enhancers by FAIRE hyper sensitive sites with simultaneous enrichment of H3K27ac in CEpi, NHEK and ARPE19. Comparing CEpi active enhancers with those of NHEK and ARPE19, CEpi enhancers were divided into several groups. CEpi selective elements were located near corneal genes such as KRT12, KRT3 and PAX6. CEpi and NHEK common regions were located near genes that express in epithelial cells. Most of CEpi selective enhancers had CEpi selective PAX6 binding sites. The motif-based sequence analysis of CEpi selective regions and PAX6 binding sites revealed that these regions were concentrated around the motifs of PAX6 and stratified epithelium-related transcription factors.
Our findings suggest that PAX6 and stratified epithelium-related transcription factors act together to maintain cornea-specific phenotype, and these regulatory networks might be a therapeutic target for corneal surface diseases.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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