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D. P. Singh, E. Kubo, Y. Takamura, K. Ishihara, R. Tulsawani, N. Fatma; LEDGF Expression in Human Lens Epithelial Cells is Transcriptionally Regulated by the SP1 Response Elements. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2612.
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© ARVO (1962-2015); The Authors (2016-present)
The transcription factor LEDGF fills its pro-survival, cytoprotective function by upregulating the transcription of small heat shock protein (shsp) genes. LEDGF is enriched in human and mice lens epithelial cells (LECs), but the factor(s) regulating its expression are largely unknown. We investigated the key transacting factor, SP1, and its cis-acting elements, GC box(es), controlling/regulating LEDGF expression in human LECs.
A Web-based computer analysis (MatInspector, Genomatix) was used to spot out putative transcription factor binding sites. 5’-flanking sequence spanning from -1289 to +35 bps of LEDGF gene was subcloned into pCAT-basic vector. Deletion mutants -1007, -316, -175, -132, -65 and -35 were prepared by PCR and cloned into p-CAT-basic vector with common 3’ end (+35bps). Gel-shift and supershift assays, mutational analysis and CAT assay were used to characterize transcriptional protein(s) that physically and functionally bind to DNA element(s) of LEDGF promoter. pCMV-SP1 eukaryotic construct was engineered to overexpress SP1 in the LECs and assess its ability in regulating LEDGF promoter. Western blot and real-time PCR were used to monitor expression levels of transcriptional protein SP1 and LEDGF in LECs.
Bioinformatic analysis of LEDGF promoter disclosed that LEDGF promoter near the transcription start site was devoid of TATA and CAAT boxes, but bore a highly GC-rich area with three SP1 binding elements (GC boxes), and this region is evolutionary well conserved in mammals. Western analysis and RT-PCR showed expression of SP1 in LECs, while overexpression of SP1 enhanced the expression of LEDGF mRNA and protein. Gel-mobility and supershift assay revealed that SP1 in nuclear extracts of LECs binds to the SP1 sites. Transactivation experiments with deletion mutants and point mutational screening of LEDGF promoter indicated that two SP1 response elements sites, -55 to -46 and -160 to-151 are essential for activation of LEDGF gene, while an SP1 site at position -99 to -109 represses the promoter activity. Co-transfection studies demonstrated that SP1 is a transcriptional regulator of LEDGF.
Findings provide first insight into the transcriptional regulation of LEDGF expression by SP1 response elements, and may provide clues to controlling LEDGF expression, which is essential for cellular survival.
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