Elucidation of gene regulation mechanism by which corneal epithelial cells achieve their specific differeatiation status, especially those regarding to corneal epithelial cell-specific transcription factor

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Keratin 12 is a gene that is specifically expressed in corneal epithelial cells. In this study, we investigated whether such a tissue-specific expression is controlled by the genomic methylation status. First, we evaluated keratin 12 gene expression among various types of human keratinocyte cells. As has been reported by many researchers, keratin 12 was only expressed in in vivo corneal epithelial cells. Surprisingly and notably, subcultured primary human corneal epithelial cells (hCEC) and immortalized hCEC did not express keratin 12. We then treated the immortalized hCEC by 5-aza-cytidine, a demethylation agent and TSA, a histone deacetylase inhibitor to see whether such a tissue-specific gene expression of keratin 12 involved an epigenetic gene regulation mechanism. We subsequently discovered that those cells express keratin 12. Next, we performed bisulfite sequencing analysis to see the methylation status of keratin 12 among various kinds of in vivo keratinocyte cells. As a result, we found that keratin 12 was in the hypomethylated status in in vivo corneal epithelial cells, while other keratinocyte cells exhibited hypermethylation of keratin 12. Also, the subcultured primary hCEC (P6, P7, and P8) and the immortalized hCEC exhibited hypermethylation of keratin 12. We then tested the hypothesis that the culture process may affect the genomic methylation status. We performed repeated culture of hCEC from in vivo to P6 and subjected each of these cells to bisulfite sequencing analysis. We found that the keratin-12 methylation rate was gradually increased by the number of passage. Finally, we evaluated the methylation status of the limbal basal epithelial cells, which have been reported to contain the stem cells of the corneal epithelial cells: We found that keratin 12 was in the hypermethylated status in these cells, suggesting that keratin 12 may be demethylated when the limbal basal cells are differentiated into corneal epithelial cells.