Tax relieves transcriptional repression by promoting histone deacetylase 1 release from the human T-cell leukemia virus type 1 long terminal repeat

Abstract

Expression of human T-cell leukemia virus type 1 (HTLV-1) is regulated by the viral transcriptional activator Tax. Tax activates viral transcription through interaction with the cellular transcription factor CREB and the coactivators CBP/p300. In this study, we have analyzed the role of histone deacetylase 1 (HDAC1) on HTLV-1 gene expression from an integrated template. First we show that trichostatin A, an HDAC inhibitor, enhances Tax expression in HTLV-1-transformed cells. Second, using a cell line containing a single-copy HTLV-1 long terminal repeat, we demonstrate that overexpression of HDAC1 represses Tax transactivation. Furthermore, a chromatin immunoprecipitation assay allowed us to analyze the interaction of transcription factors, coactivators, and HDACs with the basal and activated HTLV-1 promoter. We demonstrate that HDAC1 is associated with the inactive, but not the Tax-transactivated, HTLV-1 promoter. In vitro and in vivo glutathione S-transferase-Tax pull-down and coimmunoprecipitation experiments demonstrated that there is a direct physical association between Tax and HDAC1. Importantly, biotinylated chromatin pull-down assays demonstrated that Tax inhibits and/or dissociates the binding of HDAC1 to the HTLV-1 promoter. Our results provide evidence that Tax interacts directly with HDAC1 and regulates binding of the repressor to the HTLV-1 promoter.

FIG. 1.

Treatment with TSA enhances Tax mRNA and protein levels in HTLV-1 cells. After treatment with TSA (200 ng/ml), mRNA from C81 cells was prepared at various time points as described in Materials and Methods. Whole-cell extracts were prepared after 24-h treatment with TSA. (A) Tax mRNA levels were analyzed by RT-PCR. GAPDH was included as an internal control. PCR products were separated on 2% agarose. (B) Western blot assay of the Tax protein level. The GAPDH protein level was included as a control. C81 cell extracts were separated on 4-to-20% gels and transferred to polyvinylidene difluoride membranes, and Tax levels were determined by Western blot analysis with an anti-Tax monoclonal antibody.

FIG. 2.

Overexpression of HDAC1 represses Tax transactivation of the integrated HTLV-1 LTR reporter in vivo. (A) β-Gal activity from a single-copy HTLV-1 LTR-lacZ reporter in pA-18G-BHK-21 cells transfected with a Tax plasmid (50 ng) (lanes 2 and 4 to 6) and an HDAC plasmid (50, 100, and 200 ng; lanes 4, 5, and 6, respectively). Lane 3 contains 200 ng of HDAC1. Cotransfection of HDAC1 represses Tax-mediated transactivation. Data presented are the mean ± standard deviation (n = 3). Lanes 4 to 6 are significantly different (P < 0.01) from results in lane 2. (B) Cotransfection of HDAC1 did not alter Tax expression. Western blot assays of Tax levels in whole-cell extracts from BHK cells transfected with Tax (50 μg) and HDAC1 (200 μg) plasmids.

FIG. 3.

HDAC1 interacts with Tax directly in vivo and in vitro. (A) Left: Western blot assay of HDAC1 and Tax levels in the immunoprecipitates following incubation of purified Flag-tagged HDAC1 and Tax proteins and immunoprecipitation with anti-Flag antibody. Right: GST-HDAC1 pull-down experiment. Tax protein was detected by anti-Tax antibody by Western blotting. Lane 2, GST control; lane 3, GST-HDAC1 pull-down; lane 1, input representing one-third of the amount added to the binding reaction mixture. (B) GST-Tax associates with HDAC1 from HeLa cell extract. Western blot analysis of HDAC1 levels from GST-Tax pull-down after incubation of HeLa cell nuclear extract with GST-Tax (lane 5) and GST control (lane 4). (C) HDAC1 interacts with Tax in vivo, as shown in Western blot assays of Tax levels in immunoprecipitates from C81 nuclear extract with an anti-HDAC1 antibody (lane 3) or a control IgG (lane 2).

FIG. 4.

Tax localizes in discrete nuclear foci. Tax-expressing pA-18G-BHK-21 cells were generated through retroviral transduction. Tax-expressing cells (A) and control cells (B) were subjected to a direct immunofluorescence assay using an anti-Tax antibody. Each panel in a series is of the same field of view. DIC, differential interference contrast.

FIG. 5.

Presence of HDAC1 on the HTLV-1 LTR. (A) Tax-expressing pA-18G-BHK-21 cells were generated through retroviral transduction, and Tax-expressing cells (right panel) were positive for in situ β-Gal staining. Tax⁻ cells (left panel) were all negative for the staining. The ChIP assay was performed using chromatin either from Tax⁺ or Tax⁻ pA-18G-BHK-21 cells. (B) Tax, CREB, CBP, and RNAP II associated with the HTLV-1 LTR in Tax-expressing cells. ChIP assays were performed using anti-Tax, CREB, CBP, and RNAP II antibodies on extract from Tax⁺ or Tax⁻ pA-18G-BHK-21 cells. (C) Enhanced histone H3 and H4 acetylation of the LTR in the Tax-expressing cells as shown in a ChIP assay of acetylated histone H3 and H4 in the LTR or control β-globin gene promoter from Tax⁻ or Tax⁺ pA-18G-BHK-21 cells. (D) HDAC1 dissociated from activated HTLV-1 LTR. The ChIP assays were performed in Tax⁻ or Tax⁺ pA-18G-BHK-21 cells using anti-HDAC1 (lanes 2 and 4) or a control IgG (lanes 1 and 3), as indicated. PCR products from the LTR template and β-globin promoter were separated on 2% agarose gel and visualized by ethidium bromide staining.

FIG. 5.

Presence of HDAC1 on the HTLV-1 LTR. (A) Tax-expressing pA-18G-BHK-21 cells were generated through retroviral transduction, and Tax-expressing cells (right panel) were positive for in situ β-Gal staining. Tax⁻ cells (left panel) were all negative for the staining. The ChIP assay was performed using chromatin either from Tax⁺ or Tax⁻ pA-18G-BHK-21 cells. (B) Tax, CREB, CBP, and RNAP II associated with the HTLV-1 LTR in Tax-expressing cells. ChIP assays were performed using anti-Tax, CREB, CBP, and RNAP II antibodies on extract from Tax⁺ or Tax⁻ pA-18G-BHK-21 cells. (C) Enhanced histone H3 and H4 acetylation of the LTR in the Tax-expressing cells as shown in a ChIP assay of acetylated histone H3 and H4 in the LTR or control β-globin gene promoter from Tax⁻ or Tax⁺ pA-18G-BHK-21 cells. (D) HDAC1 dissociated from activated HTLV-1 LTR. The ChIP assays were performed in Tax⁻ or Tax⁺ pA-18G-BHK-21 cells using anti-HDAC1 (lanes 2 and 4) or a control IgG (lanes 1 and 3), as indicated. PCR products from the LTR template and β-globin promoter were separated on 2% agarose gel and visualized by ethidium bromide staining.

FIG. 5.

Presence of HDAC1 on the HTLV-1 LTR. (A) Tax-expressing pA-18G-BHK-21 cells were generated through retroviral transduction, and Tax-expressing cells (right panel) were positive for in situ β-Gal staining. Tax⁻ cells (left panel) were all negative for the staining. The ChIP assay was performed using chromatin either from Tax⁺ or Tax⁻ pA-18G-BHK-21 cells. (B) Tax, CREB, CBP, and RNAP II associated with the HTLV-1 LTR in Tax-expressing cells. ChIP assays were performed using anti-Tax, CREB, CBP, and RNAP II antibodies on extract from Tax⁺ or Tax⁻ pA-18G-BHK-21 cells. (C) Enhanced histone H3 and H4 acetylation of the LTR in the Tax-expressing cells as shown in a ChIP assay of acetylated histone H3 and H4 in the LTR or control β-globin gene promoter from Tax⁻ or Tax⁺ pA-18G-BHK-21 cells. (D) HDAC1 dissociated from activated HTLV-1 LTR. The ChIP assays were performed in Tax⁻ or Tax⁺ pA-18G-BHK-21 cells using anti-HDAC1 (lanes 2 and 4) or a control IgG (lanes 1 and 3), as indicated. PCR products from the LTR template and β-globin promoter were separated on 2% agarose gel and visualized by ethidium bromide staining.

FIG. 6.

Tax competes with HDAC1 binding to the LTR. Biotinylated LTR chromatin templates were incubated with purified Tax protein and HeLa cell nuclear extract as described in Materials and Methods. After incubation and washing, proteins were eluted from beads and analyzed by SDS-PAGE and Western blotting using antibody against HDAC1. (A) Schematics of the 4xTRE and −52 chromatin templates. (B) Tax inhibits HDAC1 association with the 4xTRE chromatin template. Different levels of Tax protein were incubated with either 4xTRE chromatin or a control −52 chromatin. (C) Tax promotes HDAC1 dissociation from the 4xTRE chromatin template. The asterisk indicates that Tax protein was added after incubation of HeLa extract with chromatin template.

FIG. 7.

Model of HTLV-1 LTR activation by Tax. Tax relieves transcriptional repression. On the nonactivated LTR, HDAC1 is associated with the template, probably through its interaction with cellular proteins bound to the LTR. In the presence of Tax, HDAC1 is released from the LTR through its interaction with Tax. Tax increases CREB association on the LTR, and the Tax/CREB/DNA complex recruits p300/CBP and RNAP II to activate transcription.