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Biju Bhargavan, Nigar Fatma, Eri Kubo, Bhavana Chhunchha, Dhirendra P. Singh; Epigenetic Repression of LEDGF Gene by UVB Stress Requires Histone H3 Lysine 9 Dimethylation Recruitment at CpG Island in Its Gene Promoter. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5288.
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Expression of the transcriptional protein lens epithelial derived growth factor (LEDGF) gives cells a survival advantage by blocking death pathway. We investigated the epigenetic regulation of the LEDGF gene in lens epithelial cells (LECs) exposed to UVB radiation, and showed that Histone H3 lysine 9 (H3-K9) dimethylation at non-methylated cytosine/guanine (CpGs) island adjacent to transcription start site is responsible for repression of LEDGF transcription, not DNA methylation.
Web-based CpGPlot program was used to spot CpG Island. Methylation status was measured by methylation specific PCR (MSP) with bisulfite sequencing and methylated DNA immunoprecipitation with 5-methylcytosine antibody (Ab) in LECs following UVB stress (40, 80 and 100J/m2) for variable periods. H3-K9 dimethylation and acetylation was monitored with ChIP assay. Deletion mutants of LEDGF promoter -316, -175, -132 and -65 with +35 bps were constructed by PCR in pCAT-basic vectors. Promoter activity was monitored with CAT ELISA. Histone acetyl transferase (HAT) activity was done by commercial kit. Western analysis and qPCR or RT-PCR measured expression of genes with their corresponding specific probes. In vitro methylation was determined with SsssI methylase treatment.
LEDGF promoter revealed two CpG islands spanning from -365/-259 and -205/+1029 bps, and later was naturally methylated at +590/+780. Transactivation assay in LECs with multiple exposures (40J/m2) or single higher exposure to UVB revealed significant repression of promoter activity. ChIP assay with Sp1 Ab revealed that the binding affinity of all three functional Sp1 sites at -170/-10 of CpG island was attenuated following decrease in total HAT activity. ChIP scanning and Western analysis also demonstrated that methylated-acetyl H3 (dimethyl-H3-K9) recruited to CpGs (-170/-10), which is followed by decreased expression of acetylated H3, and increased expression of methylated H3 (dimethylated H3-K9), histone deacetylase 1 (HDAC1) and DNMT (DNA methyltransferase) 3b in LECs exposed to multiple 40J/m2 or single higher radiation, demonstrating that acetylation of H3-K9 is essential for LEDGF’s active transcription.
Our data reveals novel regulation of epigenetic reprogramming and its enforced repression of Sp1 transactivation of LEDGF in UVB-stressed LECs. The finding may contribute to understanding the basis of many diseases associated with specific epigenetic modifications.
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