Abstract
The Vpr (viral protein R) of human immunodeficiency virus, type 1, which is expressed during the late stage of the viral infection, has received special attention because of its ability to control transcription of the human immunodeficiency virus, type 1, long terminal repeat and to influence cell cycle progression. Here we demonstrate that Vpr has the ability to regulate transcription of the cyclin-dependent kinase inhibitor, p21(WAF1) (p21), one of the key regulators of the cell cycle, in human astrocytic cells. The results from transcription assays demonstrated that Vpr augments promoter activity of p21 through the GC-rich region located between nucleotides -84 and -74 with respect to the +1 transcription start site. Activation of p21 by Vpr required cooperativity of Sp1, which binds to the DNA sequence spanning -84 to -74. Results from bandshift assay revealed an increased level of Sp1 DNA binding activity in the presence of Vpr. Furthermore, Vpr was able to associate with Sp1 via the zinc finger domain located in the C-terminal region of Sp1. Functional studies revealed that the cooperativity between Vpr and Sp1 requires the zinc finger domain at the C terminus and the glutamine-rich domain at the N terminus of Sp1. Expression of p53 further enhanced the level of Vpr-Sp1-mediated transcription activation of p21 through the sequence spanning -84 to -74 and increased the DNA binding activity of Sp1 in the presence of Vpr. Results from glutathione S-transferase pull-down assay showed the association of Vpr with p53 in extracts containing Sp1. Altogether, the outcome of our functional and binding studies suggested that the physical interaction of Vpr with Sp1 and p53 could modulate transcriptional activity of p21.
Highlights
The cyclin-dependent kinase inhibitor, p21WAF1 (p21), arrests cell cycle by modulating the activity of cyclin-dependent kinases and regulates DNA methylation by interacting directly with proliferating cell nuclear antigen, a subunit of DNA polymerase, and prevents DNA synthesis [1,2,3,4]. p21 plays important roles in the control of cell senescence, apoptosis, and differentiation [5,6,7]
We have demonstrated that Vpr affects p21 functions through a physical interaction
We identified the mechanisms used by Vpr to regulate p21 gene expression and the involvement of p21 and Vpr in the regulation of HIV-1 LTR
Summary
The cyclin-dependent kinase inhibitor, p21WAF1 (p21), arrests cell cycle by modulating the activity of cyclin-dependent kinases and regulates DNA methylation by interacting directly with proliferating cell nuclear antigen, a subunit of DNA polymerase, and prevents DNA synthesis [1,2,3,4]. p21 plays important roles in the control of cell senescence, apoptosis, and differentiation [5,6,7]. P53, retinoic acid, vitamin D3, and interferon may utilize the distalspecific cis-acting DNA motifs that extend between positions Ϫ2280 and Ϫ120 to stimulate transcription of the p21 gene [14] The other regulators such as transforming growth factor , progesterone, phorbol esters, and phosphatase inhibitors mediate their effects on p21 gene expression via the proximal region of the promoter, which spans Ϫ120 to ϩ1 [15]. Sp1 belongs to a zinc finger family of transcription factors and was first identified based on its ability to interact with the GC-rich motif of SV40 regulatory sequences [17,18,19] This protein plays a critical role in many cellular events by regulating expression of several other genes, including early embryonic development and the maintenance of terminal cell differentiation [20]. We demonstrate that the interaction of Vpr and Sp1 can potentiate p53 to enhance transcription of p21 via the GC-rich motif
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