Dataset Information

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

ABSTRACT:

Background

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.

Methods

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.

Results

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.

Conclusion

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.

SUBMITTER: Tsui S

PROVIDER: S-EPMC5008733 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Publications

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

Tsui Shanli S Wang Jie J Wang Ling L Dai Wei W Lu Luo L

PloS one 20160901 9

<h4>Background</h4>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.<h4>Methods</h4>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 ...[more]

PMID: 27583466

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Project description:BackgroundAltered dosage of the transcription factor PAX6 causes multiple human eye pathophysiologies. PAX6⁺/⁻ heterozygotes suffer from aniridia and aniridia-related keratopathy (ARK), a corneal deterioration that probably involves a limbal epithelial stem cell (LESC) deficiency. Heterozygous Pax6(+/Sey-Neu) (Pax6⁺/⁻) mice recapitulate the human disease and are a good model of ARK. Corneal pathologies also occur in other mouse Pax6 mutants and in PAX77(Tg/-) transgenics, which over-express Pax6 and model human PAX6 duplication.Methodology/principal findingsWe used electron microscopy to investigate ocular defects in Pax6⁺/⁻ heterozygotes (low Pax6 levels) and PAX77(Tg/-) transgenics (high Pax6 levels). As well as the well-documented epithelial defects, aberrant Pax6 dosage had profound effects on the corneal stroma and endothelium in both genotypes, including cellular vacuolation, similar to that reported for human macular corneal dystrophy. We used mosaic expression of an X-linked LacZ transgene in X-inactivation mosaic female (XLacZ(Tg/-)) mice to investigate corneal epithelial maintenance by LESC clones in Pax6⁺/⁻ and PAX77(Tg/-) mosaic mice. PAX77(Tg/-) mosaics, over-expressing Pax6, produced normal corneal epithelial radial striped patterns (despite other corneal defects), suggesting that centripetal cell movement was unaffected. Moderately disrupted patterns in Pax6⁺/⁻ mosaics were corrected by introducing the PAX77 transgene (in Pax6⁺/⁻, PAX77(Tg/-) mosaics). Pax6(Leca4/+), XLacZ(Tg/-) mosaic mice (heterozygous for the Pax6(Leca4) missense mutation) showed more severely disrupted mosaic patterns. Corrected corneal epithelial stripe numbers (an indirect estimate of active LESC clone numbers) declined with age (between 15 and 30 weeks) in wild-type XLacZ(Tg/-) mosaics. In contrast, corrected stripe numbers were already low at 15 weeks in Pax6⁺/⁻ and PAX77(Tg/-) mosaic corneas, suggesting Pax6 under- and over-expression both affect LESC clones.Conclusions/significancePax6⁺/⁻ and PAX77(Tg/-) genotypes have only relatively minor effects on LESC clone numbers but cause more severe corneal endothelial and stromal defects. This should prompt further investigations of the pathophysiology underlying human aniridia and ARK.

Project description:Corneal integrity, transparency and vision acuity are maintained by corneal epithelial cells (CECs), which are continuously renewed by corneal limbal stem cells (LSCs). Deficiency of CECs and/or LSCs is associated with numerous ocular diseases. Paired box (PAX)6 is an eye development-associated transcription factor that is necessary for cell fate determination and differentiation of LSCs and CECs. In the present study, the PAX6 gene was introduced into adipose-derived rat mesenchymal stem cells (ADMSCs) to investigate whether PAX6-transfected cells were able to transdifferentiate into corneal-like epithelial cells and to further verify whether the cells were suitable as a cell source for corneal transplantation. The ADMSCs were isolated from the bilateral inguinal region of healthy Sprague Dawley rats. The characteristics of ADMSCs were identified using flow cytometric analysis. After subculture, ADMSCs underwent transfection with recombinant plasmid containing either PAX6-enhanced green fluorescent protein (EGFP) complementary (c)DNA or EGFP cDNA (blank plasmid group), followed by selection with G418 and determination of the transfection efficiency. Subsequently, the morphology of the ADMSCs and the expression profiles of corneal-specific markers CK3/12 and epithelial-specific adhesion protein were determined. E-cadherin was detected using immunofluorescence staining and western blot analysis at 21 days following transfection. An MTT cell proliferation and a colony formation assay were performed to assess the proliferative activity and clonogenicity of PAX6-transfected ADMSCs. Finally, the PAX6-expressing ADMSCs were transplanted onto the cornea of a rabbits with limbal stem cell deficiency (LSCD). At 21 days after transfection, the ADMSCs with PAX6 transfection exhibited a characteristic flagstone-like appearance with assembled corneal-like epithelial cells, and concomitant prominent expression of the corneal-specific markers cytokeratin 3/12 and E-cadherin. Furthermore, the proliferation and colony formation ability of PAX6-overexpressing ADMSCs was significantly retarded. The transplantation experiment indicated that PAX6-reprogramed ADMSCs attached to and replenished the damaged cornea via formation of stratified corneal epithelium. Taken together, these results suggested that conversion of ADMSCs into corneal-like epithelium may be driven by PAX6 transfection, which makes ADMSCs a promising cell candidate for the treatment of LSCD.

Project description:BackgroundEpigenetic factors, such as microRNAs, are important regulators in the self-renewal and differentiation of stem cells and progenies. Here we investigated the microRNAs expressed in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, and their role in corneal epithelium.Methodology/principal findingsHuman LPC epithelia was extracted for small RNAs or dissociated for CEPC culture. By Agilent Human microRNA Microarray V2 platform and GeneSpring GX11.0 analysis, we found differential expression of 18 microRNAs against central corneal (CC) epithelia, which were devoid of CEPCs. Among them, miR-184 was up-regulated in CC epithelia, similar to reported finding. Cluster miR-143/145 was expressed strongly in LPC but weakly in CC epithelia (P = 0.0004, Mann-Whitney U-test). This was validated by quantitative polymerase chain reaction (qPCR). Locked nucleic acid-based in situ hybridization on corneal rim cryosections showed miR-143/145 presence localized to the parabasal cells of limbal epithelium but negligible in basal and superficial epithelia. With holoclone forming ability, CEPCs transfected with lentiviral plasmid containing mature miR-145 sequence gave rise to defective epithelium in organotypic culture and had increased cytokeratin-3/12 and connexin-43 expressions and decreased ABCG2 and p63 compared with cells transfected with scrambled sequences. Global gene expression was analyzed using Agilent Whole Human Genome Oligo Microarray and GeneSpring GX11.0. With a 5-fold difference compared to cells with scrambled sequences, miR-145 up-regulated 324 genes (containing genes for immune response) and down-regulated 277 genes (containing genes for epithelial development and stem cell maintenance). As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin β8 (ITGB8) expression in both human corneal epithelial cells and primary CEPCs.Conclusion/significanceWe found expression of miR-143/145 cluster in human corneal epithelium. Our results also showed that miR-145 regulated the corneal epithelium formation and maintenance of epithelial integrity, via ITGB8 targeting.

Dataset Information

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

Background

Methods

Results

Conclusion

Publications

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

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