Epigenetic Regulation of Keratin 12 Gene Expression

Abstract

Purpose:: Keratin 12 (K12) is one of the most dominantly and specifically expressed genes in corneal epithelial cells and is also known to play an important roll in the maintenance of these cells. Although several cis- and trans-acting factors were reported to be possibly involved in the transcriptional regulation of this gene, the mechanism behind how this gene is specifically expressed in corneal epithelial cells is not yet unveiled. As a result of our preliminary scanning of the K12 genomic sequence in a view of CpG dinucleotide distribution, the CpG-rich region, which is the so-called CpG island and is known to be frequently associated with epigenetic regulation, was identified at exons 4-6 of this gene. The goal of this study is to investigate whether expression of this gene is epigenetically regulated.

Methods:: 5’ RACE was performed to determine the authentic transcription initiation site (TIS) of the K12 gene. Genomic DNA was extracted from immortalized corneal epithelial cell lines (HCE-T) and 4 different types of normal stratified epithelial cells including those of the cornea, conjunctiva, oral mucosa, and skin. Five samples of each cell type (20 samples total) were utilized for this study, each unique sample being obtained from a separate individual. These DNAs were treated with sodium bisulfite and subjected to bisulfite sequencing or primer extension assay to determine the methylation status of the K12 promoter (-2000 ~ +550) and its exonic/intronic CpG island (+3500 ~ +4200). K12 gene expression was comparatively measured by RT-PCR using RNAs extracted from HCE-T cells before and after treatment with an HDAC inhibitor (Trichostatin A) or a demethylating agent (5-aza-2’-deoxycytidine).

Results:: A striking difference was found in the methylation status of the K12 genome between corneal epithelial cells and the other 3 types of epithelial cells. Specifically, the promoter region from 350 bases upstream to 500 bases downstream of the TIS was significantly demethylated in corneal epithelial cells compared with the other 3 types of stratified epithelial cells. The exonic/intronic CpG island was also prominently demethylated in corneal epithelial cells compared with the other 3 types of stratified epithelial cells. However, a promoter region further upstream (-350 ~ -2000) was highly methylated in all 4 types of epithelial cells. RT-PCR revealed that the HDAC inhibitor and the demethylating agent significantly increased K12 gene expression.

Conclusions:: Genomic methylation status appears to be involved in the specific gene expression of K12.

Keywords: cornea: epithelium • gene/expression