Modulation of the Brd4/P-TEFb interaction by the human T-lymphotropic virus type 1 tax protein - PubMed (original) (raw)
Modulation of the Brd4/P-TEFb interaction by the human T-lymphotropic virus type 1 tax protein
Won-Kyung Cho et al. J Virol. 2007 Oct.
Abstract
Positive transcription elongation factor (P-TEFb), which is composed of CDK9 and cyclin T1, plays an important role in cellular and viral gene expression. Our lab has recently demonstrated that P-TEFb is required for Tax transactivation of the viral long terminal repeat (LTR). P-TEFb is found in two major complexes: the inactive form, which is associated with inhibitory subunits 7SK snRNA and HEXIM1, and the active form, which is associated with, at least in part, Brd4. In this study, we analyzed the effect of Brd4 on human T-lymphotropic virus type 1 (HTLV-1) transcription. Overexpression of Brd4 repressed Tax transactivation of the HTLV-1 LTR in a dose-dependent manner. In vitro binding studies suggest that Tax and Brd4 compete for binding to P-TEFb through direct interaction with cyclin T1. Tax interacts with cyclin T1 amino acids 426 to 533, which overlaps the region responsible for Brd4 binding. In vivo, overexpression of Tax decreased the amount of 7SK snRNA associated with P-TEFb and stimulates serine 2 phosphorylation of the RNA polymerase II carboxyl-terminal domain, suggesting that Tax regulates the functionality of P-TEFb. Our results suggest the possibility that Tax may compete and functionally substitute for Brd4 in P-TEFb regulation.
Figures
FIG. 1.
Brd4 inhibits Tax transactivation of HTLV-1 LTR. (A) HeLa cells at a confluence of 80% were cotransfected with 200 ng of HTLV-1 LTR-Luc, 100 ng of RSV-lacZ, Tax (10 or 20 ng), or Brd4 (100, 200, or 400 ng) using Fugene 6 transfection reagent (Roche). The cells were incubated for 24 h, cell extracts were prepared, and the luciferase activity was analyzed. Western blot analysis of Tax (Tab 172) or Brd4 was performed to check protein expression. (B) HeLa cells were transfected with 200 ng of HIV-LTR-Luc, 100 ng of RSV-lacZ, and 0, 200, or 400 ng of Brd4. After 24 h, cell lysates were prepared and assayed for luciferase activity. (C) HeLa cells were transfected with 200 ng of HTLV-1 LTR-Luc, 100 ng of RSV-lacZ and 0, 200, and 400 ng of Brd4 (lanes 1 to 3) or 200 ng of HTLV-1 LTR-Luc, 100 ng of RSV-lacZ, 10 ng of Tax and 0, 100, 200, or 400 ng of Brd4 (lanes 4 to 7). The cells were incubated for 24 h, cell extracts were prepared, and the luciferase activity was analyzed. (A to C) To correct for transfection efficiency, all luciferase values were adjusted using RSV-LacZ. Graphs represent the average luciferase activity from three independent experiments with the standard deviation. The panel below the graph shows Western blot analysis using Tax or Brd4 antibody.
FIG. 2.
Tax inhibits the interaction of Brd4 and CDK9 in vitro. A total of 200 ng of P-TEFb and 100 ng of Brd4 purified proteins were incubated with 0, 200, or 400 ng of His-tagged Tax protein as indicated above the figure. After incubation, the reaction mixtures were immunoprecipitated with CDK9 antibody, and immunoblot analysis was performed with Tax, CDK9, cyclin T1, or Brd4 antibody to detect protein-protein interaction.
FIG. 3.
Tax interacts with cyclin T1. (A) His-tagged Tax (200 ng) and Brd4 (200 ng) proteins were incubated in binding buffer, immunoprecipitated with Tax antibody, and then analyzed by Western blot analysis with Brd4 and Tax antibodies. (B) Portions (600 ng) of GST, GST-CDK9, or GST-cyclin T1 were incubated with 400 ng of His-Tax protein in binding buffer. After incubation, the reaction mixtures were immunoprecipitated by using Tax antibody, followed by Western blot analysis with Tax, CDK9, or cyclin T1 antibody. The amounts of purified GST, GST-CDK9, or GST-cyclin T1 used for binding assay were equivalent, as determined by Gel-code staining (*, nonspecific band from overloading of lane 3).
FIG. 4.
Tax interacts with two domains of cyclin T1. (A and B) Portions (600 ng) of GST, GST-cyclin T1, or GST-cyclin T1 fragments were incubated with 400 ng of purified His-Tax protein. After incubation the mixtures were subjected to GST pull-down with glutathione beads, and Western blot analysis was performed using Tax antibody. (C) Portions (200 or 400 ng) of His-tagged Tax protein were incubated with 200 ng of Brd4 and 200 ng of GST 426-533. GST-bound proteins were precipitated with glutathione beads, and immunoblot analysis was performed with Brd4, Tax, or GST antibody to detect protein-protein interaction. Equivalent amounts of GST fusion proteins were added to the reactions as determined by Gel-code staining.
FIG. 5.
Tax interacts with P-TEFb in vivo and inhibits the interaction of Brd4 with P-TEFb. (A) Nuclear extracts from Molt4 or C81 cells were immunoprecipitated with Tax antibody and then immunoblotted with CDK9, cyclin T1, and Tax antibodies. (B) Nuclear extracts from C81 cells were immunoprecipitated with control immunoglobulin G, CDK9, CDK7, or Tax antibody and then immunoblotted with CDK9, cyclin T1, Tax, or CDK7 antibody. (C) HeLa cells were transfected with either a control or a Tax expression plasmid. At 24 h after transfection, nuclear extracts were prepared and immunoprecipitated with CDK9 antibody. Proteins present in the immunoprecipitates were analyzed by Western blot analysis with Tax, Brd4, and CDK9 antibodies. (D) HeLa cells were infected with Ad-GFP or Ad-Tax at an MOI of 100. At 24 h postinfection, the interaction of Tax and Brd4 with CDK9 was assayed by immunoprecipitation with CDK9 antibody and Western blot analysis with Tax, Brd4, and CDK9 antibodies. (E) Nuclear extracts from HeLa cells transfected with control or Tax expression plasmid were immunoprecipitated with control IgG, Brd4, or CDK9 antibody. Western blot analysis was then performed with CDK9 antibody.
FIG. 6.
Tax stimulates activity of P-TEFb in vivo. pA-18G-BHK-21 cells (A) or HeLa cells (B) were infected at an MOI of 100 with Ad-GFP or Ad-Tax. At 12 h postinfection, total cell extracts were prepared and analyzed by Western blot analysis with antibodies specific for Pol II CTD phospho-Ser2 (H5), Pol II CTD phospho-Ser5 (H14), RNA Pol II (8WG16), Tax, or actin. (C) HeLa cells were transfected with a total of 6 μg of plasmid DNA containing 0, 2, or 6 μg of Tax expression plasmid and 6, 4, or 0 μg of pcDNA3 plasmid. At 24 h posttransfection, nuclear extracts were prepared and immunoprecipitated with CDK9 antibody. Total RNAs from the immunoprecipitates were isolated and used for quantitative reverse transcription-PCR using primers specific for 7SK snRNA or U1 snRNA (upper panel). Total nuclear lysates were used for RNA isolation, and quantitative reverse transcription-PCR for 7SK and U1 snRNA was performed (bottom panel). CDK9 immunoprecipitates and nuclear extracts used for immunoprecipitation were also analyzed by Western blot analysis with Tax and CDK9 antibodies.
FIG. 7.
Model for competitive interaction of Tax and Brd4 with the cellular P-TEFb complex. Our results demonstrate that Tax interacts with P-TEFb through interaction with the cylin T1 subunit. The interaction of Tax with P-TEFb inhibits the interaction of Brd4, suggesting that there may be two LMW “active” fractions of P-TEFb in HTLV-1 Tax-expressing cells. Overexpression of Tax causes a decrease in the level of the HMW 7SK snRNA/HEXIM1/P-TEFb complex, with a concomitant increase in P-TEFb activity, as measured by the level of Pol II CTD phosphor-Ser2.
References
- Akagi, T., H. Ono, and K. Shimotohno. 1995. Characterization of T cells immortalized by Tax1 of human T-cell leukemia virus type 1. Blood 86**:**4243-4249. -PubMed
- Bex, F., and R. B. Gaynor. 1998. Regulation of gene expression by HTLV-1 Tax protein. Methods 16**:**83-94. -PubMed
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