Abstract
Purpose:
Methylation of CpG islands in the 5’-reuglatory elements plays a pivotal role in regulating gene expression. However, it is unknown whether methylation of CpG within exons and introns also contributes to the regulation of cell type-specific gene expression. In this study, we examined the age dependent CpG methylation of Krt12 alleles (keratin 12 gene) of Krt12WT (wild type) and Krt12Cre (knock-in) using bi-transgenic Krt12Cre/ROSA26mTmG mice.
Methods:
The dual ROSA26mTmG reporter mice are crossbred with Krt12-Cre knock-in mice line to obtain bi-transgenic Krt12Cre/WT/ROSA26mTmG and Krt12Cre/Cre/ROSA26mTmG in which the membrane bound floxed Tomato-red (mT) gene is deleted in corneal epithelial cells expressing Cre recombinase. Thus, corneal epithelial cells are labeled by EGFP. Corneal epithelial cells were harvested from Krt12Cre/WT/ROSA26mTmG and Krt12Cre/Cre/ROSA26mTmG mice at different ages, postnatal day 0 through 24 weeks. The corneal epithelial cells were separated by fluorescence-activated cell sorting. Genomic DNAs were extracted, followed by bisulfite sequencing between K12 promoter region and intron1.
Results:
The green fluorescence positive epithelial cells that express Krt12Cre allele were separated from those red fluorescence positive cells that did not express the Krt12Cre allele of Krt12Cre/WT/ROSA26mTmG mice by cell sorting. Results of bisulfite sequencing indicate that in adult mice the Krt12 alleles of conjunctival epithelial cells are highly methylated throughout 5’-reuulatory elements, exon1 and intron 1 in conjunctival epithelial cells. In contrast, the degree of methylation of adult corneal epithelial cells is greatly decreased in various strains of mice, e.g., C57BL/6Jc1, 129/SVJ and FVB/NJ. There is an age-dependent decreased methylation of Krt12 allele in corneal epithelial cells from mice older than 2 weeks. The green corneal epithelial cells that express Krt12Cre isolated from Krt12Cre/WT/ROSA26mTmG and Krt12Cre/Cre/ROSA26mTmG mice exhibit lower methylation of Krt12 alleles, whereas those of red-fluorescence positive corneal epithelial cells display high methylation in the same region. of s degree of methylation has using. Kcells and non- expressed cells were successfully separated by cin a fraction of GFP high/RFP low and RFP high/GFP low, respectively.
Conclusions:
Expression of Krt12 genes is associated with methylation of CpG islands in 5’ regulatory elements, exon1 and intron 1.
Keywords: 482 cornea: epithelium •
533 gene/expression •
539 genetics