Background
Age-related cataract (ARC) is the leading cause of visual impairment among older adults [
1,
2]. It is widely accepted that senescence, heredity, and environment are the major contributing factors in ARC and that oxidative stress plays an important role in cataractogenesis [
3,
4]. However, the precise mechanism of ARC remains to be further elucidated.
Lens crystallins are the predominant structural proteins involved in the maintenance of lens clarity and refractive properties [
5,
6]. α-crystallins constitute 35 % of all crystallins [
7]. Its molecular chaperone-like activity protects other crystallins against thermal-induced inactivation or aggregation and allows the lens to resist the aging-induced deterioration of proteins [
8]; therefore it is thought to be critical for maintaining lens transparency. Our previous studies have shown, for the first time, that DNA methylation regulates gene expression in lens epithelial cells [
9,
10].
CRYAA undergoes epigenetic repression in the lens epithelia in nuclear ARC [
11]. However, the mechanism of
CRYAA DNA methylation remains to be explored.
Epigenetic modifications are post-transcriptional, reversible, and hereditable events that do not alter the genetic sequence. The epigenetic regulation of gene expression mainly includes DNA methylation, histone modification, and non-coding RNA [
12]. DNA methylation, mostly in the form of 5′-methylcytosine in CpG dinucleotides in the presence of DNA methyltransferases (DNMTs), often acts as a transcriptional repressor [
13]. It can directly prevent the binding of transcription factors or act through interactions with methyl-CpG-binding domain (MBD) proteins and histone deacetylases (HDACs), resulting in gene silencing [
14].
In the present study, we analyzed the impact of the methylation of CpG sites on transcription factors to explore the underlying mechanism of the down regulation of CRYAA in nuclear ARC. We also investigated the effect of DNA-demethylating agent Zebularine on the expression of CRYAA.
Discussion
Diverse DNA methylation patterns have recently been discovered in many ocular diseases, including glaucoma [
20], age-related macular degeneration (AMD) [
21], retinoblastoma [
22‐
25], uveal melanoma [
26‐
29], and cataract [
11,
30]. Our previous work demonstrated, for the first time, that DNA methylation down-regulates
CRYAA gene expression in lens epithelial cells in nuclear ARC [
9‐
11]. However, little is known about the precise mechanism of DNA methylation of
CRYAA. In this study, we showed that in lens epithelial cells, the methylation of the CpG site of the
CRYAA promoter decreased the DNA-binding capacity of transcription factor Sp1. Treatment with the DNA-demethylating agent Zebularine increased
CRYAA expression in a dose- dependent and time- dependent pattern. Overall, these findings suggest that the methylation of the CpG sites of the
CRYAA promotor directly affects transcription factor binding and is related to the epigenetic repression in nuclear ARC lenses. We have demonstrated the underlying mechanism of the DNA methylation of
CRYAA in HLE B-3 cells.
DNA methylation may be an important mechanism in the pathogenesis and progression of cataract. Critical enzymes involved in DNA methylation, such as DNMT1 and MeCP2, were found in human lens epithelial cells [
9,
10]. The down-regulation of
CRYAA via the hypermethylation of CpG islands in its promoter was found in nuclear ARC cases [
11]. It was also reported to be related to the earlier onset of dark nucleus in highly myopic patients [
31], as well as nuclear cataract formation after pars plana vitrectomy [
32]. It is established that the localized reduction of antioxidative capacity in the nuclear region of the lens results in increasing numbers of denatured proteins [
3]. Down-regulation via the promoter hypermethylation of
CRYAA reduces the expression of chaperones, which are able to bind to these denatured proteins and thus preserve the transparency of the lens [
6]. This may accelerate the oxidative modification of proteins in the nucleus, resulting in the pathogenesis of nuclear cataract [
31]. The dysfunction of Nrf2-dependent antioxidant protection via endoplasmic-reticulum-associated degradation and redox- balance alteration in the lens due to the demethylation of the CpG islands in the
Keap1 promoter is linked to diabetic cataracts and ARCs in both human lens epithelial cells and animal models [
30,
33,
34]. The DNA hypermethylation of the promoter region of the DNA repair gene
MGMT may regulate the down-expression of the gene and be involved in the development of ARC [
35]. In cortical ARC, the loss of functional
OGG1 via the base excision repair pathway results in oxidative DNA base damage [
36,
37]. Reduced
OGG1 expression was correlated with the hypermethylation of a CpG island of
OGG1 in ARC lenses [
38]. Pseudoexfoliation syndrome (PEX)-complicated cataracts also underwent epigenetic regulation. The susceptible PEX gene LOXL1 was hypermethylated in its promoter region and was down- regulated on the mRNA and protein level in Uighur PEX cataract patients [
39]. Together, these results suggest that many critical genes related to antioxidative capacity and DNA repair underwent epigenetic repression during the pathogenesis of ARC. In this study, we demonstrated that demethylation treatment with Zebularine increased
CRYAA expression level in a dose- dependent and time- dependent pattern.
The methylation status of critical CpG sites often conversely correlates with the transcriptional activity of promoters. Two modes exist that explain how the methylation of CpG sites interferes with transcription. Methylated CpG sites can directly interfere with the binding of transcription factors to their recognition sites or facilitate the binding of a family of methyl-binding proteins to their cognate DNA sequences [
40]. In this study, we demonstrated that the methylation of the CpG sites of the CRYAA promoter could directly interfere with the binding of transcription factor Sp1 to its recognition elements, which is related to gene repression. Our results were consistent with the previous findings. Clark SJ et al. [
41] discovered that
mCp
mCpG methylation could have a biological function in preventing Sp1 binding, thereby contributing to the subsequent abnormal methylation of CpG islands often observed in tumor cells. Zhu WG et al. [
16] demonstrated that hypermethylation around consensus Sp1-binding sites may directly reduce Sp1/Sp3 binding, leading to reduced p21
Cip1 expression in response to depsipeptide treatment. Zelko et al. [
42] found that CpG methylation attenuates Sp1 and Sp3 binding to the human extracellular superoxide dismutase promoter and regulates its cell-specific expression. Douet et al. [
43] provided the first direct evidence that CpG methylation of the
Abcc6 proximal promoter region regulates the binding of transcription factor Sp1 and participates in tissue-specific expression control in mice. Li et al. [
44] demonstrated that treatment with the demethylation agent 5-aza-2′-deoxycytidine markedly enhanced the binding affinity of Sp1/Sp3 to the promoter region and restored the expression of
CIDE-A gene in cells. This treatment was also effective in the restoration of the binding of Sp1 to the promoter, as well as
Keap1 expression, in an A549 cell line [
45]. In the current study, the excess wild-type competitors did not completely competed away the binding of SP1 in Lane 2. HLE B-3 nuclear extracts were able to bind both the labeled and unlabeled wild-type Sp1 probe. When excess unlabeled wild-type Sp1 probe (100 fold) was used to compete with the labeled wild-type probe, most Sp1 and other transcription factors binded to the unlabeled wild-type Sp1 probe. However, the binding capacity of Sp1 and the wild-type probe was high. The low concentration of labeled wild-type probe could still bind to some SP1. The result indicated that the binding capacity of the low concentration of wild-type probe with SP1 was higher than the normal concentration of the methylated probe. It further confirmed that the methylation of the CpG sites of the
CRYAA promoter influences the binding capacity of transcription factor Sp1. These results indicate that CpG methylation plays an important role in establishing and maintaining the tissue- and cell-specific transcription of genes through the direct regulation of Sp1 binding.
In this study, we did not investigate the possibility of de novo DNA methylation occurring. Further studies should focus on the pathway that affects DNA methylation. Performing bisulphite analysis to analyze the methylation status of the CpG sites around this SP1 binding site will further increase the significance of the study.
Acknowledgements
We would like to acknowledge Dr. Zhang Shujie and Dr. Hu Fangyuan, from Eye and ENT hospital of Fudan University, for their help in study design and qRT-PCR experiment.