Oxidative Stress and Age-Dependent Expression of LEDGF in Eye Lens or Lens Epithelial Cells is Associated with Methylation of GC Box Elements in Its Gene Promoter

Abstract

Purpose: : Transcriptional protein lens epithelium-derived growth factor (LEDGF) overexpression protects against apoptosis. Oxidative stress evoked by reactive oxygen species (ROS) and concordant DNA methylation alterations are observed during aging. We investigated whether promoter methylation could account for downregulation of LEDGF expression in human lens epithelial cells (hLECs) facing oxidative stress or in aging human eye lenses.

Methods: : Human lenses of variable ages were obtained from the Lions Eye Bank (UNMC-NE). LEDGF expression was analyzed in hLECs exposed to variable concentrations of H₂O₂ (1-100μM) and aging lenses by real-time PCR and Western analysis. The Web-based CpGplot/CpGreport program was used to spot CpG Island. Methylation status of LEDGF promoter was examined by MSP- PCR, bisulfite sequencing and chromatin immunoprecipitation with 5-methyl cytosine antibody. In vitro methylation was determined with M.SssI methylase treatment. Deletion mutants of LEDGF promoter -316, -170, -132 and -65 with +35 bps were constructed by PCR in pCAT-basic vectors. Promoter activity was monitored with CAT-ELISA. 5-Aza-2'-deoxycytidine and temozolomide were used to evaluate effect of epigenetic regulation on LEDGF mRNA expression.

Results: : hLECs exposed to higher concentrations of H₂O₂ (100μM) and aging LECs/lenses (≥50 years) had decreased expression of LEDGF mRNA and protein with increased ROS and significantly reduced promoter activity. LEDGF promoter had two major CpG Islands spanning -365/-259 and -205/+1029bps, and later was naturally methylated at +590/+780. H₂O₂ exposure caused methylation at Sp1, LEDGF transcription regulator, binding sites (-170/-10), resulting in decreased binding of Sp1 to its cis-elements. Treatment of LECs with 5-Aza reversed methylation and upregulated LEDGF expression. In vitro methylated LEDGF-CAT constructs did not show promoter activity, while treatment with 5-Aza restored activity, suggesting DNA methylation at Sp1binding sites caused reduced promoter activity. Human lenses showed methylation status at the same region (-170/-10).

Conclusions: : Methylation at Sp1 cis-elements of the CpG Island near the transcriptional start site in LEDGF promoter is the mechanism of LEDGF repression in hLECs facing oxidative stress or in aging lenses.

Keywords: transcription factors • transcription • stress response