Research Article: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis-Elements in GC-Rich Region of TATA-Less Gene Promoter

Date Published: May 16, 2012

Publisher: Public Library of Science

Author(s): Dhirendra P. Singh, Biju Bhargavan, Bhavana Chhunchha, Eri Kubo, Anil Kumar, Nigar Fatma, Ben C.B. Ko.


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.

Partial Text

Gene expression is transcriptionally regulated through the interaction between trans-acting factors and the cis-elements of gene-specific promoters. A nuclear protein discovered relatively recently, LEDGF/p75 affects a variety of functions involved with cellular survival and development of cellular abnormalities [1], [2], [3], [4]. The functions of LEDGF/p75 protein are related to its expression level. Studies in cultured cell lines as well as in vivo have shown that overexpression of LEDGF/p75 gives growing cells a selective survival advantage by blocking death pathways [5], [6], [7]. LEDGF/p75 provides cytoprotection by acting as a transregulator of stress-associated genes such as Hsp27, -25, and -90, and αB-crystallin [8]. However, overexpression of LEDGF/p75 has been shown to be involved in subcutaneous angiogenesis and lymphangiogenesis of ovarian carcinoma tumors [9], and aberrant expression of LEDGF/p75 has been reported in 61% of prostate tumors [10]. These studies suggest that the level of LEDGF/p75 expression can determine the fate of cells in various cellular microenvironments.

LEDGF/p75 is a nuclear protein expressed in many cell types [2], [6], [7]. It enhances cellular survival by increasing the expression of stress-associated genes and small heat shock proteins (hsps) through interacting stress response (STRE) and heat shock protein gene response elements (HSE) in these genes [2], [5], [6], [19], acting as a transcription factor. Recent evidence revealed that DNA binding activity of LEDGF/p75 is not limited to STRE or HSE in the stress-associated genes, but LEDGF/p75 also binds to supercoiled DNA [22] and active chromatin markers as well as RNA polymerase II, and is associated with transcriptional activity of the transcriptional unit [21], [22]. LEDGF/p75 gene inactivation has been shown to result in perinatal mortality and complex phenotype abnormalities [1]. LEDGF/p75’s diverse and dynamic patterns of expression which account for its diverse mode of biological action are now well recognized. Less well understood are the processes by which expression levels of LEDGF/p75 are regulated. In the present study, we observed that LEDGF/p75 expression was modulated in hLECs isolated from eye lenses of different ages, and the expression pattern of LEDGF/p75 was well correlated with Sp1 expression levels (Fig. 1). Our studies of Sp1 overexpression and inhibition of Sp1 activity by artemisinin and Sp1 shRNA revealed that the modulation in expression of LEDGF/p75 protein or mRNA depended upon abundance of Sp1 expression (Fig. 1). These results argue that Sp1 can be a regulator of LEDGF/p75 transcription. Sp1, a C2H2 zinc finger-containing factor, is a constitutively expressed protein that naturally engages in transregulating various TATA-less or TATA box-containing gene promoters. The promoter regions of these human genes are usually GC-rich, and, by definition, these genes are expressed ubiquitously [42], [43].