Abstract
Purpose:
We examined the functional connection between dysregulation of activity of the stress-inducible Lens Epithelial Derived Growth Factor (LEDGF) and aberrant epigenetic signaling, and restoration of LEDGF expression and activity by Sulforaphane (SFN), a naturally occurring isothiocyanate.
Methods:
Prdx6-deficient (Prdx6-/-) and Prdx6+/+ lens epithelial cells (LECs) or lenses from mice of different ages and human LECs were treated with SFN and exposed to different doses of UVB/H2O2 for different periods. Expression of LEDGF, DNA methyltransferases (DNMTs), histone deacetylases (HDACs), acetyl H3 and H2B, SUV39H1, trimethyl-H3K9 and heat shock proteins (hsps) mRNA and protein were assessed by q PCR and Western blot. Levels of reactive oxygen species (ROS) were quantified with H2DCFH-DA dye assay. Methylation status of LEDGF gene was analyzed by methylation specific PCR (MSP) bisulfite sequencing. ChIP analysis was used with acetyl-H3, acetyl-H3K9, acetyl 2B, Sp1, a transactivator of LEDGF and HDAC1, SUV39H1, di- and trimethyl-H3K9, 5-methyl cytosine antibodies to examine status of active and inactive chromatin and Sp1 on CpG island of LEDGF gene promoter (-170/-10). Effect of depletion of Sp1 (SiRNA) on LECs was monitored by MTS and TUNEL assays.
Results:
SFN treatment restored activity of LEDGF and its target genes hsp27/αB-crystallin, and cells had reduced ROS levels. SFN treatment significantly promoted cell viability and attenuated expression levels of DNMT1, DNMT3b and reduced site-specific CpG methylation. SFN also reduced expression levels of HDAC1, SUV39H1 and dimethyl-H3 and increased levels of active chromatin markers, acetyl H3/acetyl-H3K9 and acetyl-H2B. ChIP analysis of LEDGF promoter revealed that SFN increased the active chromatin markers acetyl-H3K9, acetyl-2B and Sp1 access at CpG island. In contrast the dimethyl-H3K9, SUV39H1 inactive chromatin markers were decreased at the site in dose-dependent fashion. SFN induced acetylation and reduced methylation, facilitating binding of LEDGF activator Sp1 to its site in LEDGF. Knockdown of Sp1 reduced SFN-induced upregulation of LEDGF mRNA and facilitated cell death.
Conclusions:
Our studies provide insights into SFN-mediated epigenetic upregulation of LEDGF and its target genes, hsps, and may open avenues for SFN/antioxidant-mediated prevention or delay of cataractogenesis.
Keywords: 739 transcription factors •
634 oxidation/oxidative or free radical damage •
533 gene/expression