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Dhirendra Singh, Bhavana Chhunchha, Biju Bhargavan, Eri Kubo, Nigar Fatma; Methylation of CpG Island in Aging Lens Epithelial Cells And During Oxidative Stress Affects Sp1 Binding And Activity in LEDGF Promoter. Invest. Ophthalmol. Vis. Sci. 2013;54(15):481.
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© ARVO (1962-2015); The Authors (2016-present)
Lens Epithelium-Derived Growth Factor (LEDGF) protects cells, and its downregulation leaves them vulnerable to stress. Oxidative stress and changes in DNA methylation status are found during aging. This study examined the effects of methylation of CGs nucleotides within and around Sp1 binding site(s) of CpG island on transcription of LEDGF and its biological response.
Prdx6-deficient (Prdx6-/-) lens epithelial cells (LECs) and Prdx6+/+ LECs or lenses from mice and human subjects of different ages were used to examine the expression of Sp1, LEDGF, DNA methyltransferases (DNMTs) and Hsps by real-time PCR and Western analyses. LECs exposed to H2O2, demethylating agent 5-AzaC, was used to examine LEDGF and Hsps expression. Reactive oxygen species (ROS) were monitored using H2DCF dye. Bisufite sequencing, methylation-specific PCR, ChIP with 5-methyl-cytosine antibody, and in vitro methylation of promoter with M.SssI methylase assessed LEDGF methylation status. LEDGF promoter-CAT reporter basic vector (-170/+35nts), plus point mutation at each of three Sp1 sites were made for transactivation assay. Gelshift assay were done with wildtype oligos consisting Sp1 sites and methylated oligos at GCs within or adjacent to Sp1 elements.
Compared to Prdx6+/+ LECs of identical age, Prdx6-/- LECs containing elevated ROS showed decreased expression of LEDGF and its target genes, Hsp27 and αB-crystallin, concordant with their decreased expression in aging Prdx6+/+ and hLECs. 5-AzaC treatment derepressed LEDGF and Hsps expression. DNMT1 expression and activity was reduced in aging and Prdx6-/- LECs, while DNMT3a and DNMT3b were increased, suggesting that sporadic, de novo methylation of LEDGF occurred due to DNMT3a and DNMT3b. Analysis of CAT activity of LEDGF promoter (170/+1) plasmid and ChIP assays in Prdx6-/- LECs showed that 5-AzaC functioned on Sp1 binding sites to induce LEDGF expression. Gelshift assay revealed that GCs methylation within and adjacent to the Sp1 elements significantly reduced Sp1-binding.
Our data suggest that de novo methylation of CGs around and within Sp1-element in the CpG island directly reduces Sp1 binding, leading to repression of LEDGF expression and cellular activity. Pharmacological manipulation of DNMTs may be possible for treating age- and oxidative stress-related diseases, including cataract.
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