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DP Singh, NP Fatma, P Sharma, K Hayakawa, LT Chylack, T Shinohara; Structural and Functional Organization of the Human Lens Epithelium-Derived Growth Factor (LEDGF) Gene Promoter . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2345.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:LEDGF, a stress-responsive transcriptional activator and survival factor, binds to heat shock and stress-related elements to activate stress related genes. LEDGF's gene exons bear PWWP, NLS, HTH and b-Zip domains, suggesting its role in gene regulations. We have characterized the functional organization of LEDGF promoter to understand the molecular mechanism of its expression. Method: 5'-flanking sequences, the first exon and intron, and part of second exon, spanning from -5149 to + 1076 bps, was subcloned into pCAT3-basic. Deletion mutants: 5046, 4126, 3020, 2522 were prepared with common 3'-ends (+1076bp). 3'-end of the 2522 CAT construct was deleted and the -1446 to +25 bps fragment was cloned in CAT vector. Similarly, the -1239, -1007, -510, -316, -175, and -100 constructs were engineered and CAT activity was assayed. Transcription start site (TSS) was determined with primer extension and product was analyzed on sequencing gels. Putative transcription binding sites were spotted with MalInspector (Genomatix). Results:Three transcriptional start sites were found, a major site (+1) residue A, and two minor sites, residues G (+35) and C (+55). Exon 1(372bps) is GC-rich with GC-rich, 487 bps; intron 1 suggesting that LEDGF gene transcription is tightly regulated. The result demonstrated the presence of an additional exon and intron and its gene comprised 16 exons and 15 introns. No TATA, CAAT boxes were adjacent to the TSS. 5' end of LEDGF gene revealed potentially putative (+) regulatory elements (e.g. AP1, HSE, and STRE, Interferon regulatory factor 1 and 2, E2F, Oct1 andothers). Deletion-analyses confirmed major regulatory elements are present within -500bp.Conclusion:The LEDGF gene has multiple regulatory elements. It also contains HSE and STRE elements suggesting, it may be self-regulatory. The presence of three TSS sites and unique regulatory elements in LEDGF promoter may provide a unique and specific mechanism for transcriptional regulation of LEDGF mRNA under different physiological conditions.
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