We have previously shown that the parathyroid hormone-related protein (PTHrP) promoter contains binding sites for transcription factors Ets1 and Sp1 and that human T-cell lymphotropic virus type I (HTLV-I) Tax cooperates with Ets1 to transactivate the PTHrP P2 promoter. Using the yeast two-hybrid interaction system, we now provide evidence that Tax interacts with Ets1. Moreover, a double mutation (D22A,C23S) in the Tax protein that abrogated the Tax/Ets1 interaction also inhibited the Tax/Ets1 cooperative effect, suggesting that the interaction between Tax and Ets1 is important for transactivation of the PTHrP promoter. In coimmunoprecipitation assays, we find that Tax facilitates the interaction between Ets1 and Sp1, forming a ternary complex. When the Sp1 site in the PTHrP promoter was mutated, the Tax/Ets1 cooperative effect was dramatically decreased. This suggests that Sp1 plays an important role in the Ets1-dependent Tax transactivation of the PTHrP P2 promoter. Finally, we demonstrate that Gal4-Tax is a strong activator of the Gal PTHrP promoter, implying that Tax contributes directly to the transcriptional activation of the promoter. We propose a model in which the Tax/Ets1 cooperative effect on the PTHrP P2 promoter is based on the ability of Tax, Ets1, and Sp1 to form a ternary complex on the template DNA. Tax facilitates the interaction of Ets1/Sp1 and participates directly in the transcription initiation process.
We have used a yeast two-hybrid system to show that human papillomavirus E7 proteins can form oligomeric complexes in vivo. The carboxyl-terminal cysteine-rich metal-binding domain is critical for this activity although amino-terminal sequences also contribute to oligomerization. Our experiments also reveal that E7 possesses an intrinsic transcription activation activity in yeast, which resides in the amino terminus of the protein.
Using quantitative RNase protection assays, we have monitored the appearance of mRNAs generated during lytic infection of tightly synchronized murine cells by the autonomous parvovirus minute virus of mice. Our results demonstrate that transcripts from the P4 promoter can be detected prior to those from the P39 promoter, providing direct evidence for a temporal order of expression between the two parvovirus promoters.
The Tax protein of human T-cell lymphotropic virus type 1 (HTLV-1) is a 40-kDa transcriptional activator which is critical for HTLV-1 gene regulation and virus-induced cellular transformation. Tax is localized to the DNA through its interaction with the site-specific activators cyclic AMP-responsive element-binding protein, NF-B, and serum response factor. It has been suggested that the recruitment of Tax to the DNA positions Tax for interaction with the basal transcriptional machinery. On the basis of several independent assays, we now report a physical and functional interaction between Tax and the transcription factor, TFIIA. First, Tax was found to interact with the 35-kDa (␣) subunit of TFIIA in the yeast two-hybrid interaction system. Importantly, two previously characterized mutants with point mutations in Tax, M32 (Y196A, K197S) and M41 (H287A, P288S), which were shown to be defective in Tax-activated transcription were unable to interact with TFIIA in this assay. Second, a glutathione-S-transferase (GST) affinity-binding assay showed that the interaction of holo-TFIIA with GST-Tax was 20-fold higher than that observed with either the GST-Tax M32 activation mutant or the GST control. Third, a coimmunoprecipitation assay showed that in HTLV-1-infected human T lymphocytes, Tax and TFIIA were associated. Finally, TFIIA facilitates Tax transactivation in vitro and in vivo. In vitro transcription studies showed reduced levels of Tax-activated transcription in cell extracts depleted of TFIIA. In addition, transfection of human T lymphocytes with TFIIA expression vectors enhanced Taxactivated transcription of an HTLV-1 long terminal repeat-chloramphenicol acetyltransferase reporter construct. Our study suggests that the interaction of Tax with the transcription factor TFIIA may play a role in Tax-mediated transcriptional activation.Human T-cell lymphotropic virus type 1 (HTLV-1) is associated with adult T-cell leukemia and the degenerative neuromuscular disease tropical spastic paraparesis/HTLV-1-associated myelopathy (28,66,72,108). HTLV-1 encodes a 40-kDa protein, Tax, which transforms rodent fibroblasts (90), immortalizes normal human T cells (34), and can induce a leukemialike disease in transgenic mice (36). Tax is critical for HTLV-1 gene regulation (44,68,75,85) and has been shown to modulate the expression of a number of cellular genes which include genes encoding cytokines (interleukin-1 [IL-1], IL-2, IL-3, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor) (35,37,60,61,65,83,100,103), genes encoding cytokine receptors (IL-2 receptor ␣) (14,15,43,52,76), protooncogenes (c-fos, c-myc) (2, 20, 23-25), and genes encoding lymphotoxin (69, 70), parathyroid hormone-related protein (18, 98), and major histocompatibility complex class I proteins (77). The deregulation of these Tax-responsive cellular genes may play an important role in HTLV-1 transformation.The HTLV-1 Tax protein is able to modulate gene expression by several independent mechanisms. Tax can induce the nuclear localization of the...
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