Abstract
Tat is a critical viral transactivator essential for human immunodeficiency virus (HIV) gene expression. Activation involves binding to an RNA stem-loop structure and recruitment of the positive transcription elongation factor b. Tat also induces the remodeling of a single nucleosome in the HIV promoter. However, the mechanism of this remodeling has remained unclear. Knockdown of INI-1 and BRG-1, two components of the SWI/SNF chromatin-remodeling complex, suppressed Tat-mediated transactivation. Cells lacking INI-1 (G401 and MON) or BRG-1 (C33A) exhibited defective transactivation by Tat that was restored upon INI-1 and BRG-1 expression, respectively. Tat was co-immunoprecipitated with several SWI/SNF subunits, including INI-1, BRG-1, and beta-actin. The SWI/SNF complex interacted with the integrated HIV promoter in a Tat-dependent manner. We also found that INI-1 and BRG-1 synergized with the p300 acetyltransferase to activate the HIV promoter. This synergism depended on the acetyltransferase activity of p300 and on Tat Lys(50) and Lys(51). In conclusion, Tat-mediated activation of the HIV promoter requires the SWI/SNF complex in synergy with the coactivator p300.
Highlights
Scription is initiated within this nucleosome-free region
RNA Interference-mediated Depletion of INI-1 and BRG-1 Compromises Tat Transactivation—To examine whether INI1-containing chromatin-remodeling complexes are involved in the transcriptional activation of the human immunodeficiency virus (HIV) promoter, we used a cell line containing a doxycycline-inducible short hairpin RNA against the INI-1 gene
Because our results indicated that the synergism between INI-1 and p300 in Tat activation of the long terminal repeat (LTR) depends on the acetyltransferase activity of p300, we tested the role of Tat Lys50 in SWI/SNF recruitment and LTR activation
Summary
Plasmids and Retroviral Vectors—The HIV LTR-luciferase reporter construct (pEV229) [6]; the cytomegalovirus (CMV)driven expression vectors for N-terminally FLAG-tagged wildtype Tat (pEV280) and mutant Tat(K50R/K51R) (pEV538) [6]; and SV40-driven C-terminally FLAG-tagged INI-1 [21], CMVdriven hemagglutinin-tagged BRG-1 [22], CMV-driven p300 [6], and ⌬p300 catalytic mutant [6] expression constructs have been described. 2 mg of whole cell protein lysate was incubated overnight with 20 l of M2-agarose beads (Sigma) in immunoprecipitation buffer at 4 °C on a rotator. For immunoprecipitation of the INI-1, BRG-1, and PKD-1 complexes, 3 g of polyclonal antibody was incubated overnight with 5 mg of Jurkat cell lysate at 4 °C and bound to 60 l of protein A-agarose beads for 2 h at 4 °C, followed by extensive washes with immunoprecipitation buffer. The BRG-1, INI-1, and PKD-1 complex-coated beads were incubated for 2 h with reticulocyte lysate-expressed 35S-labeled Tat, washed extensively with immunoprecipitation buffer, and analyzed by SDSPAGE and autoradiography. Transient transfections were carried out with 200 ng of LTR-luciferase reporter plasmid and expression vectors CMV-FLAG-Tat (5–30 ng), Rous sarcoma virus-FLAG-INI-1 (50 –500 ng), CMV-BRG-1 (50 ng), CMV-p300 (50 ng), and CMV-⌬p300 (50 ng) as indicated. Input and immunoprecipitated DNAs (10 l) were subjected to 33 PCR cycles with primers (5Ј-TTGCCTGTACTGGGTCTCTCTG-3Ј) and (5Ј-TCGCTTTCAAGTCCCTGTTCG-3Ј)
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