Versatile reporter systems show that transactivation by human T-cell leukemia virus type 1 Tax occurs independently of chromatin remodeling factor BRG1

Abstract

Potent activation of human T-cell leukemia virus type 1 (HTLV-1) gene expression is mediated by the virus-encoded transactivator protein Tax and three imperfect 21-bp repeats in the viral long terminal repeats. Each 21-bp repeat contains a cAMP-responsive-element core flanked by 5' G-rich and 3' C-rich sequences. Tax alone does not bind DNA. Rather, it interacts with basic domain-leucine zipper transcription factors CREB and ATF-1 to form ternary complexes with the 21-bp repeats. In the context of the ternary complexes, Tax contacts the G/C-rich sequences and recruits transcriptional coactivators CREB-binding protein (CBP)/p300 to effect potent transcriptional activation. Using an easily transduced and chromosomally integrated reporter system derived from a self-inactivating lentivirus vector, we showed in a BRG1- and BRM1-deficient adrenal carcinoma cell line, SW-13, that Tax- and 21-bp repeat-mediated transactivation does not require BRG1 or BRM1 and is not enhanced by BRG1. With a similar reporter system, we further demonstrated that Tax- and tumor necrosis factor alpha-induced NF-kappaB activation occurs readily in SW-13 cells in the absence of BRG1 and BRM1. These results suggest that the assembly of stable multiprotein complexes containing Tax, CREB/ATF-1, and CBP/p300 on the 21-bp repeats is the principal mechanism employed by Tax to preclude nucleosome formation at the HTLV-1 enhancer/promoter. This most likely bypasses the need for BRG1-containing chromatin-remodeling complexes. Likewise, recruitment of CBP/p300 by NF-kappaB may be sufficient to disrupt histone-DNA interaction for the initiation of transcription.

FIG. 1.

Schematic diagrams of lentivirus vectors SMPU-18x21-EGFP and SMPU-E-sel-EGFP. In the SMPU vectors, the majority of the human immunodeficiency virus gag coding region and the complete coding region for pol, env, and accessory genes have been removed. The 5′ U3 region is replaced by the CMV enhancer/promoter, and a deletion is made in the 3′ U3 region (ΔU3 R). The reporter gene to be transduced, the EGFP gene in this case, is driven by the 18x21 (A) or E-selectin (E-sel) promoter cassette (B). Upon introduction of the reporter vector into a target cell, the U5 region in the 5′ LTR and the ΔU3 region in the 3′ LTR of the vector mRNA are duplicated through reverse transcription. The only functional enhancer/promoter element in the integrated DNA is the Tax-responsive 18x21 promoter or the E-sel promoter.

FIG. 2.

The 18x21-EGFP reporter cassettes can be readily transduced by the SMPU vector and are highly Tax responsive. Four cell lines, MT4, PX1, HeLa, and Jurkat, were selected for transduction by SMPU-18x21-EGFP. MT4 is an HTLV-1-transformed human T-cell line that expresses Tax constitutively. PX1 is a fibroblast cell line derived from an LTR-tax transgenic mouse. PX1 also constitutively produces Tax. HeLa and Jurkat are cell lines unrelated to HTLV-1. Twenty-four hours before transduction, cells were seeded in six-well plates. The same amount of the reporter vector was then added to each cell line individually. EGFP expression was observed 48 h after transduction using an Olympus IX81 inverted fluorescence microscope. Panels on the bottom show fluorescence images of the transduced cells. Panels on the top are corresponding bright-field images. Cell lines used for panels A to F are as labeled. All were transduced with the SMPU-18x21-EGFP reporter. Cell lines in panels D and F were additionally transduced with LV-Tax, a lentivirus vector for HTLV-1 Tax.

FIG. 3.

Characterizations of the NF-κB reporter SMPU-E-sel-EGFP. SMPU-E-sel-EGFP vector particles were produced as described in Materials and Methods and analyzed similarly to the method described in the legend to Fig 2. Fluorescence images are shown on the left and the corresponding bright-field images on the right.

FIG. 4.

Transactivation of viral LTR and E-selectin promoter by Tax does not require BRG1. DNA transfections of SW-13 cells were carried out as described in Materials and Methods. The amounts of plasmids used in each transfection are as indicated. DNA transfection was done in triplicates. The averages of the luciferase reporter activities and standard deviations were calculated and plotted. The reporter and transactivators used for panels A, B, and C were Tax/LTR-Luc, Tax/E-sel-Luc, and Rta/K8-Luc, respectively.

FIG. 5.

Tax-mediated transactivation of chromosomally integrated 21-bp repeats does not require chromatin-remodeling factor BRG1 or BRM1. A total of 2.5 million SW-13 cells was seeded on a 10-cm plate to 30% confluence. Approximately 5 × 10⁶ infectious units of SMPU-18x21-EGFP was added to the culture medium together with 8 μg/ml polybrene. After 48 h, transduced SW-13 cells were trypsinzed and reseeded in a six-well plate at a density of approximately 4 × 10⁵ cells per well. Cells in each well were then transfected with pCMV-Tax (4 μg) and pREP7-BRG1 (4 μg) individually or in combination. Forty-eight hours later, cells were examined under a fluorescence microscope. Panels A to D represent SW-13 cells transduced with SMPU-18x21-EGFP vector only (labeled as “vector”) (A), cells similar to those in panel A but further transfected with pREP7-BRG1 (BRG1) (B), cells transfected with pCMV-Tax construct (Tax) (C), and cells cotransfected with both pCMV-Tax and pREP7-BRG1 (Tax+BRG1) (D). (E) The extent of EGFP expression of cells in panels A to D was determined by subjecting the cells to flow cytometry, followed by integrating the fluorescence signals (in arbitrary units) of all cells that have been sorted. The fluorescence signal corresponding to panel A (deemed background) was subtracted from the signal of each set in panels A to D, and the results were plotted. (F) Immunoblot analyses of cells in panels A to D. A rabbit polyclonal antibody against BRG1 (Santa Cruz Biotechnology, Inc.) and a mouse hybridoma Tax antibody 4C5 were used. Panels G and H show fluorescence images of an SW-13/18x21-EGFP stable cell line transduced with LV-Tax (G) and SMPU-SV40-Puro (H), respectively. (I) Cells were cotransduced with SMPU-18x21-EGFP and LV-Tax. Vimentin expression in SW-13 cells was detected by indirect immunofluorescence 48 h later. Cells expressing both vimentin and EGFP are marked with arrows.

FIG. 6.

Tax and NF-κB transactivates chromosomally integrated E-selectin promoter in the absence of BRG1 and BRM1. SW-13 cells were seeded in a six-well plate and transduced at a multiplicity of infection of 2 with SMPU-E-sel-EGFP reporter vector alone (untreated) (A). Forty-eight hours after transduction with the reporter vector, cells were concurrently cotransduced with LV-Tax vector (B) or treated with 20 ng/ml TNF-α (panel C). Both fluorescence and corresponding bright-field images of the cells are shown. (D) Flow cytometry of cells from panels A to C. Flow cytometry was carried out as detailed in Materials and Methods. (E) The relative levels of EGFP expression were determined as described in the legend to Fig. 5E, except the background fluorescence signal of untransfected and reporterless cells was subtracted.