Epigenetic Regulation of Werner Syndrome Gene in Age-Related Cataract

Abstract

Purpose. To examine the promoter methylation and histone modification of WRN (Werner syndrome gene), a DNA repair gene, and their relationship with the gene expression in age-related cataract (ARC) lens. Methods. We collected the lenses after cataract surgery from 117ARC patients and 39 age-matched non-ARC. WRN expression, DNA methylation and histone modification around the CpG island were assessed. The methylation status of Human-lens-epithelium cell (HLEB-3) was chemically altered to observe the relationship between methylation and expression of WRN. Results. The WRN expression was significantly decreased in the ARC anterior lens capsules comparing with the control. The CpG island of WRN promoter in the ARC anterior lens capsules displayed hypermethylation comparing with the controls. The WRN promoter was almost fully methylated in the cortex of ARC and control lens. Acetylated H3 was lower while methylated H3-K9 was higher in ARC anterior lens capsules than that of the controls. The expression of WRN in HLEB-3 increased after demethylation of the cells. Conclusions. A hypermethylation in WRN promoter and altered histone modification in anterior lens capsules might contribute to the ARC mechanism. The data suggest an association of altered DNA repair capability in lens with ARC pathogenesis.

Figure 1

Relative expression of mRNA and protein levels of WRN in anterior lens capsules of control and ARC. (a) RT-PCR analysis of the expression of WRN in control (n = 20) and ARC-C (n = 20), ARC-N (n = 20), and ARC-P (n = 20). Values represent mean ± SD. ^∗ P < 0.01. (b) WRN protein levels in control (n = 9) and ARC-C (n = 9), ARC-N (n = 9), and ARC-P (n = 9) anterior lens capsules were detected using Western blotting. Relative WRN protein level to GAPDH is presented as mean ± SD. ^∗ P < 0.01.

Figure 2

Methylation status at region 3 in WRN promoter in anterior lens capsules and cortex of control and ARC. (a) The positions of CpG islands within WRN promoter. In the following panels, each row of circles represents a single clone. Open and close circles represent unmethylated and methylated CpG sites, respectively. (b) Methylation status of region 3 in WRN promoter in anterior lens capsules of control (n = 20) and ARC-C (n = 20), ARC-N (n = 20), and ARC-P (n = 20). The ARC group displayed hypermethylation compared to the control group. ^∗ P < 0.01. (c) Methylation status of region 3 in WRN promoter in lens cortex of control and ARC lens. There were no significant statistical differences between the ARC group and the control group. P > 0.05.

Figure 3

Histone modifications of the WRN promoter in anterior lens capsules of control and ARC. In anterior lens capsules, compared with the control group (n = 10), ChIP analysis revealed that acetylated H3 levels were lower in ARC-C (n = 10), ARC-N (n = 10), and ARC-P (n = 10). At the same time, methylated H3-K9 was increased. ^∗ P < 0.01.

Figure 4

Relative methylation status and expression of protein levels of WRN in HLEB-3 after treatment with 5-aza-dC. In the following panels, each row of circles represents a single clone. Open and close circles represent unmethylated and methylated CpG sites, respectively. (a) After treatment with 5-aza-dC, the methylation rate of WRN in HLEB-3 was decreased. (b) Protein expression of WRN in the untreated control cells and in cells after treatment with 5-aza-dC. Relative WRN protein level to GAPDH is presented as mean ± SD. ^∗ P < 0.01.