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.

Epigenetic repression of LEDGF during UVB exposure by recruitment of SUV39H1 and HDAC1 to the Sp1-responsive elements within LEDGF promoter CpG island

“…9,40 LEDGF promoter region is evolutionarily conserved in different species and is devoid of TATA box and enriched with G/C bases containing three active Sp1-response elements, each of which differentially regulates LEDGF transcription. 9 Furthermore, we have found that all three Sp1-responsive elements were located within a CpG island present in the LEDGF promoter (current study).…”

“…9,40 LEDGF promoter region is evolutionarily conserved in different species and is devoid of TATA box and enriched with G/C bases containing three active Sp1-response elements, each of which differentially regulates LEDGF transcription. 9 Furthermore, we have found that all three Sp1-responsive elements were located within a CpG island present in the LEDGF promoter (current study). Based on recent studies suggesting that genes with CpG island can be under the control of epigenetic reprogramming, 41 we posit that the LEDGF promoter containing CpG island may be a target for chromatin remodeling during stress (current study).…”

“…exposure, while cells exposed to these oxidants for longer periods or at higher doses repress the LEDGF transcript. 8,9 These studies indicate that level of LEDGF expression is vital and can be governed at the transcriptional level.…”

“…50 Previously, we have shown that Sp1 specifically and differentially regulates human LEDGF gene transcription by directly binding to Sp1 sites present within the CpG island of LEDGF to regulate its transcription. 9 Additionally, we reported that Sp1 overexpression increases LEDGF mRNA and protein expression in cells, and these cells gain resistance against UVB, 9 a known inducer of etiopathogenesis-induced injury, including cataractogenesis.…”

Genetic association study of UCMA/GRP and OPTN genes (PDB6 locus) with Paget's disease of bone

“…We found that the presence of a G was consistent with TFBSs for VDR/RXR, Sp1 and PAX4 and the presence of an A was consistent with TFBSs for PPARG and HNF4 (Table 2). Since PAX4 has been reported to be strongly expressed in the retina of the rat [25] and SP1 has been involved in the regulation of genes in the lens [26], this putative regulation of OPTN by these TFs might be relevant. It is worth noting that OPTN is highly expressed in the brain, retina and skeletal muscle.…”