CTCF-Mediated and Pax6-Associated Gene Expression in Corneal Epithelial Cell-Specific Differentiation

2016-09-01 00:01:41

PLOS ONE; 1 September 2016:

Shanli Tsui, Jie Wang, Ling Wang, Wei Dai, Luo Lu



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.


Cell 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.


Our 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.


Our 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.


Corneal epithelial layer integrities are maintained by continuous processes of self-renewal and wound healing. Both the self-renewal and wound-healing processes are affected by stimulation of growth factors and environmental stresses that activate cellular signaling pathways and transcription factors to switch the stimulatory signals to genetic responses. For an example, the effect of EGF on suppressing eye-specific Pax6 transcription in proliferation of corneal epithelial cells is regulated through activation of an epigenetic regulator termed CCCTC binding factor (CTCF). CTCF is a highly conserved zinc finger (ZF) protein in mammalian cells to epigenetically control cellular physiological processes. CTCF regulates DNA imprinting, X chromosome inactivation and transcriptional control of various gene expressions, including p19ARF, p16INK4a, PIM-1, PLK, BRCA1, p53, p27, Ecadherine, E2F1, TERT and PAX6. 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. Genome-wide analyses have revealed that CTCF is able to bind tens of thousands of DNA sites using different combinations of its eleven zinc fingers involving intra- and inter-chromatin interactions. Emerged evidence indicates that CTCF plays a master role in genomic spatial organization and mediates these extensive long- range intra- and inter-chromatin interactions. One of the most significant epigenetic roles in chromatin remodeling of CTCF is to mediate environmental signals and cooperatively introduce cell-type specific inter-chromatin interactions leading to specific gene expression. In corneal epithelial and retinoblastoma cells, CTCF controls PAX6 transcription by interacting with a repressor element located in the 5’-flanking region upstream from the PAX6 P0 promoter. This interaction suppresses PAX6 transcription by blocking an ectoderm enhancer (EE) located approximately -3.5 kb upstream from the P0 promoter. However, important epigenetic question, concerning whether CTCF-mediated chromatin remodeling affecting interactions of PAX6 with other cell differentiation-related genes in eye-specific expression, is still unresolved.

Homeobox transcription factor Pax6 is an important member in the PAX family and plays a critical role in eye and neuronal development in both vertebrates and invertebrates. PAX6 is expressed essentially in all ocular structures, including the cornea, iris, lens and retina. PAX6 plays important roles in promoting corneal epithelial and neuron apoptosis. Regulation of PAX6 gene transcription is highly conserved during evolution. In most species, PAX6 transcription is regulated via two promoters, P0 and P1. There is a highly conserved transcriptional control element termed ectoderm enhancer (EE) that is located approximately -3.5 kbp upstream from the P0 promoter. It has shown that a repressor element composed of 80-bp nuclear acids that is located about 1.2 kb upstream from the P0 promoter of PAX6 gene. It contains five functional CCCTC motifs in this region. CTCF regulates PAX6 activities in response to growth factor and stress stimulation. For instance, epidermal growth factor (EGF)-induced suppression of Pax6 expression by CTCF is required in corneal epithelial proliferation. In contrast, ultraviolet (UV) stress inhibits CTCF expression and minimizes CTCF DNA binding activity to the repressor element in the PAX6 gene. In transgenic mice over-expressing CTCF, PAX6 is decreased its expression resulting in retardation of embryonic ocular development including the cornea, lens and retina. In addition, DNA methylation plays a role in CTCF-controlled PAX6 expression during mouse ES cell differentiation to further provide the regulatory mechanism of PAX6 in early stage ES and progenitor cells. In the present study, the effect of CTCF-mediated chromatin remodeling on PAX6 and differentiation-associated genes was investigated to demonstrate how eye-specific PAX6 interacts in the promoter regions with differentiation-associated genes, such as RCN1 and ADAM17 during corneal epithelial cell differentiation. Taken together, as one of the important chromatin architecture organizers, CTCF participates in gene transcriptional regulation in corneal epithelial cells. Our data reveal a novel mechanism involving CTCF-mediated chromatin remodeling that regulates interactions between eye-specific PAX6 gene and differentiation-associated genes to modulate corneal epithelial differentiation.

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Key Words

Cell Differentiation | Cornea | Chromatin | Epithelial Cells | Gene Expression | Promoter Regions | Gene Regulation | Cell Binding