Abstract
BackgroundThe purpose of the study is to elicit the epigenetic mechanism involving CCCTC binding factor (CTCF)-mediated chromatin remodeling that regulates PAX6 gene interaction with differentiation-associated genes to control corneal epithelial differentiation.MethodsCell cycle progression and specific keratin expressions were measured to monitor changes of differentiation-induced primary human limbal stem/progenitor (HLS/P), human corneal epithelial (HCE) and human telomerase-immortalized corneal epithelial (HTCE) cells. PAX6-interactive and differentiation-associated genes in chromatin remodeling mediated by the epigenetic factor CTCF were detected by circular chromosome conformation capture (4C) and ChIP (Chromatin immunoprecipitation)-on-chip approaches, and verified by FISH (Fluorescent in situ hybridization). Furthermore, CTCF activities were altered by CTCF-shRNA to study the effect of CTCF on mediating interaction of Pax6 and differentiation-associated genes in corneal epithelial cell fate.ResultsOur results demonstrated that differentiation-induced human corneal epithelial cells expressed typical corneal epithelial characteristics including morphological changes, increased keratin12 expression and G0/G1 accumulations. Expressions of CTCF and PAX6 were suppressed and elevated following the process of differentiation, respectively. During corneal epithelial cell differentiation, differentiation-induced RCN1 and ADAM17 were found interacting with PAX6 in the process of CTCF-mediated chromatin remodeling detected by 4C and verified by ChIP-on-chip and FISH. Diminished CTCF mRNA with CTCF-shRNA in HTCE cells weakened the interaction of PAX6 gene in controlling RCN1/ADAM17 and enhanced early onset of the genes in cell differentiation.ConclusionOur results explain how epigenetic factor CTCF-mediated chromatin remodeling regulates interactions between eye-specific PAX6 and those genes that are induced/associated with cell differentiation to modulate corneal epithelial cell-specific differentiation.
Highlights
Corneal epithelial layer integrities are maintained by continuous processes of self-renewal and wound healing
During corneal epithelial cell differentiation, differentiation-induced RCN1 and ADAM17 were found interacting with PAX6 in the process of CCCTC binding factor (CTCF)-mediated chromatin remodeling detected by 4C and verified by Chromatin immunoprecipitation assay (ChIP)-on-chip and Fluorescence in situ hybridization (FISH)
Diminished CTCF mRNA with CTCF-shRNA in human telomerase-immortalized corneal epithelial (HTCE) cells weakened the interaction of PAX6 gene in controlling RCN1/ADAM17 and enhanced early onset of the genes in cell differentiation
Summary
Corneal epithelial layer integrities are maintained by continuous processes of self-renewal and wound healing. The effect of EGF on suppressing eye-specific Pax transcription in proliferation of corneal epithelial cells is regulated through activation of an epigenetic regulator termed CCCTC binding factor (CTCF) [9] [10]. Activities of CTCF insulator-function are often located between the boundaries of gene enhancers and promoters to regulate the chromatin’s effect on flanking regions, which is often modified by a DNA methylation (CH3)-sensitive process [15,16,17]. In corneal epithelial and retinoblastoma cells, CTCF controls PXA6 transcription by interacting with a repressor element located in the 5’-flanking region upstream from the PAX6 P0 promoter. The purpose of the study is to elicit the epigenetic mechanism involving CCCTC binding factor (CTCF)-mediated chromatin remodeling that regulates PAX6 gene interaction with differentiation-associated genes to control corneal epithelial differentiation
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