title: Transcriptional protein Sp1 regulates LEDGF transcription by directly interacting with its cis-elements in GC-rich region of TATA-less gene promoter.
abstract: LEDGF/p75 interacts with DNA/protein to regulate gene expression and function. Despite the recognized diversity of function of LEDGF/p75, knowledge of its transregulation is in its infancy. Here we report that LEDGF/p75 gene is TATA-less, contains GC-rich cis elements and is transcriptionally regulated by Sp1 involving small ubiquitin-like modifier (Sumo1). Using different cell lines, we showed that Sp1 overexpression increased the level of LEDGF/p75 protein and mRNA expression in a concentration-dependent fashion. In contrast, RNA interference depletion of intrinsic Sp1 or treatment with artemisinin, a Sp1 inhibitor, reduced expression of LEDGF/p75, suggesting Sp1-mediated regulation of LEDGF/p75. In silico analysis disclosed three evolutionarily conserved, putative Sp1 sites within LEDGF/p75 proximal promoter (-170/+1 nt). DNA-binding and transactivation assays using deletion and point mutation constructs of LEDGF/p75 promoter-CAT revealed that all Sp1 sites (-50/-43, -109/-102 and -146/-139) differentially regulate LEDGF/p75. Cotransfection studies with Sp1 in Drosophila cells that were Sp1-deficient, showed increased LEDGF/p75 transcription, while in lens epithelial cells (LECs) promoter activity was inhibited by artemisinin. These events were correlated with levels of endogenous Sp1-dependent LEDGF/p75 expression, and higher resistance to UVB-induced cell death. ChIP and transactivation assays showed that Sumoylation of Sp1 repressed its transcriptional activity as evidenced through its reduced binding to GC-box and reduced ability to activate LEDGF/p75 transcription. As whole, results revealed the importance of Sp1 in regulating expression of LEDGF/p75 gene and add to our knowledge of the factors that control LEDGF/p75 within cellular microenvironments, potentially providing a foundation for LEDGF/p75 expression-based transcription therapy.
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, Nebraska, United States of America. dpsingh@unmc.edu
authors.forename: Dhirendra P
authors.initials: DP
authors.lastname: Singh
authors.forename: Biju
authors.initials: B
authors.lastname: Bhargavan
authors.forename: Bhavana
authors.initials: B
authors.lastname: Chhunchha
authors.forename: Eri
authors.initials: E
authors.lastname: Kubo
authors.forename: Anil
authors.initials: A
authors.lastname: Kumar
authors.forename: Nigar
authors.initials: N
authors.lastname: Fatma
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics; D004330:Drosophila; D020862:GC Rich Sequence; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism*; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000235:genetics* / Q000378:metabolism*; D035181:TATA-Box Binding Protein / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation; D055815:Young Adult
chemical_list: D004268:DNA-Binding Proteins; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; D016329:Sp1 Transcription Factor; D035181:TATA-Box Binding Protein; C119380:lens epithelium-derived growth factor