c-Myc and Sp1 contribute to proviral latency by recruiting histone deacetylase 1 to the human immunodeficiency virus type 1 promoter

Abstract

Histone deacetylase (HDAC) inhibitors such as valproic acid (VPA) induce the expression of quiescent proviral human immunodeficiency virus type 1 (HIV-1) and may deplete proviral infection in vivo. To uncover novel molecular mechanisms that maintain HIV latency, we sought cellular mRNAs whose expression was diminished in resting CD4(+) T cells of HIV-1-infected patients exposed to VPA. c-Myc was prominent among genes markedly downregulated upon exposure to VPA. c-Myc expression repressed HIV-1 expression in chronically infected cell lines. Chromatin immunoprecipitation (ChIP) assays revealed that c-Myc and HDAC1 are coordinately resident at the HIV-1 long terminal repeat (LTR) promoter and absent from the promoter after VPA treatment in concert with histone acetylation, RNA polymerase II recruitment, and LTR expression. Sequential ChIP assays demonstrated that c-Myc, Sp1, and HDAC1 coexist in the same DNA-protein complex at the HIV promoter. Short hairpin RNA inhibition of c-Myc reduces both c-Myc and HDAC1 occupancy, blocks c-Myc repression of Tat activation, and increases LTR expression. These results expand the understanding of mechanisms that recruit HDAC and maintain the latency of HIV-1, suggesting novel therapeutic approaches against latent proviral HIV infection.

Figures

FIG. 1.

Expression of c-Myc in latently HIV-1-infected patient cells and HeLa-CAT-CD4 cells after VPA treatment. (A) c-Myc expression is significantly decreased upon exposure to VPA (P value for a change of >1.5-fold is <0.01). (B) In the resting CD4+ T cells of four HIV-infected patients, c-Myc RNA expression levels uniformly and substantially decreased following VPA exposure (red box). c-Myc RNA expression was unaffected by 20 U/ml IL-2 and upregulated modestly in some patients' cells following mitogen activation. (C) Decreased c-Myc protein expression 48 h after treatment with VPA. HeLa-CAT-CD4 cells or resting CD4+ T cells isolated from HIV-infected, highly active antiretroviral therapy-treated donors were treated with the amount of VPA indicated. The Western blot shows protein expression of c-Myc, HDAC1, the histone demethylase Dnmt3a, and GAPDH.

FIG. 2.

Overexpression of c-Myc negatively regulates HIV-1 LTR transcription. (A) c-Myc blunts Tat-activated LTR expression in HeLa-CAT-CD4 cells. pCMV vector, pCMV-Tat, pGCN-c-Myc, or pGCN-c-Myc plus pCMV-Tat were transfected into HeLa-CAT-CD4 cells. CAT reporter gene expression was assayed by real-time PCR after 48 h. RNA PCR demonstrates that GAPDH (loading control) and transfected Tat expression levels are equivalently unaffected by c-Myc. Relative CAT reporter gene expression was normalized to a value of 1.0 for pCMV-transfected cells. Bars indicate standard errors of the means for three independent experiments. P values are given for comparisons with vector control. n.s., not significant. (B) Dose-dependent inhibition of Tat activation by c-Myc. LTR expression was monitored in the presence of increased ratios of pGCN-c-Myc to pCMV-Tat (1:1, 1:2, 1:3, and 1:4). At the highest dose of c-Myc vector, Tat activation was repressed 5.7-fold (P < 0.001).

FIG. 3.

Recruitment of c-Myc to the HIV-1 LTR region. (A) Binding of c-Myc, HDAC1, acetylated H4, and RNA polymerase II to the Nuc-1 region of the HIV-1 LTR in HeLa-CAT-CD4 cells. VPA exposure resulted in significantly decreased occupancy of HDAC1 and c-Myc, increased occupancy of RNA polymerase II and acetylated histone H4 at the Nuc-1 region, and an increase in only RNA polymerase II detection downstream (DS) of the initiation region. Amplified products were quantitated by real-time PCR. The relative binding of indicated proteins to the HIV-1 LTR region was measured after normalization with that of input DNA and IgG. (B) Binding of c-Myc and HDAC1 to HIV-1 LTR in latently HIV-1-infected J89 lymphocytic cells. VPA exposure again resulted in significantly decreased occupancy of HDAC1, c-Myc, acetylated histone H4, and RNA polymerase II near Nuc-1 and little change at the downstream site. Amplified products were quantitated by real-time PCR. The relative binding of indicated proteins to the HIV-1 LTR region was measured after normalization with that of input DNA and IgG. Assays were performed in triplicate (or more), and the significance of changes was tested by a two-sided t test. P values are <0.05 for all changes of at least twofold and <0.01 for all changes of at least threefold.

FIG. 4.

c-Myc and HDAC1 are found in the same complex at the HIV LTR initiator region. Sequential ChIP assays were performed after an initial IP with anti-c-Myc. Protein-DNA complexes were detected near the Nuc-1 region after a second IP with anti-c-Myc or anti-HDAC1 but not in the downstream (DS) region and not with isotype IgG or antibody (Ab) against the methyltransferase Dnmt3a. Real-time PCR quantitation of DNA IP, as detailed in Materials and Methods, is indicated.

FIG. 5.

Sp1 mediates c-Myc regulation of HIV-1 LTR expression. Sp1 sites are required for c-Myc-mediated repression, and Sp1 and c-Myc coexist within a complex at the LTR. (A) Expression plasmids pCMV-empty vector, pCMV-Tat, and pcDNA-c-Myc and the wild-type (wt) pILIC-CAT, pSpA-CAT, and pNFA-CAT reporter plasmids were cotransfected into HeLa cells using Lipofectamine 2000 reagent. CAT expression was analyzed by real-time RT-PCR. Tat-activated LTR RNA expression is displayed, with normalization to the cells transfected with pCMV-Tat for each of the reporter assays. PCR products for the GAPDH loading control and transfected Tat are shown. Error bars indicate standard errors of the means for three independent experiments. (B) Sequential ChIP assays were performed after an initial IP with anti-Sp1. A protein-DNA complex near Nuc-1 was recovered after a second IP with anti-Sp1, anti-c-Myc, or anti-HDAC1 (P < 0.01) but not at the downstream region or with isotype IgG or antibody (Ab) against the methyltransferase Dnmt3a. Real-time PCR quantitation of DNA IP, as detailed in Materials and Methods, is indicated.

FIG. 6.

c-Myc occupancy at the HIV-1 LTR and its repression of Tat activation require Sp1. (A) Specific shRNA-mediated knockdown of Sp1 protein expression. HeLa-CAT-CD4 cells were transfected with pKD-sp-v1 plasmids directed against Sp1 or pKD-Neg-con-v1 (control) plasmids. Sp1, c-Myc, and GAPDH protein levels were assessed by immunoblotting. (B) Reduced occupancy of Sp1, c-Myc, and HDAC1 at the LTR initiator region upon Sp1 depletion. ChIP assays were performed after Sp1 knockdown, and changes in binding were quantitated by real-time PCR. (C) Blockade of c-Myc inhibition of Tat-mediated HIV-1 transcription following Sp1 knockdown. As expected, Tat activation is less efficient after Sp1 knockdown, but a 20-fold increase of RNA expression over the basal level was still measured. However, c-Myc expression had no effect of Tat activation after Sp1 knockdown. PCR products for the GAPDH loading control and transfected Tat are shown. Error bars indicate standard errors of the means for three independent experiments. n.s, not significant. (D) Specific shRNA-mediated knockdown of c-Myc protein expression. (E) Similarly, occupancy of c-Myc and HDAC1 is reduced at the LTR initiator region upon c-Myc depletion. (F) shRNA in the J89 Jurkat T-cell line directed against c-Myc depletes c-Myc protein levels and results in the significant upregulation of HIV-1 LTR expression. (G) Knockdown of c-Myc in J89 cells results in the significant upregulation of HIV LTR expression.

FIG. 7.

Model of convergent recruitment of HDAC1 to maintain HIV-1 latency. Proviral latency is maintained, in part, by the action of several transcription factors that recruit HDAC1 to the HIV-1 LTR, preserving the deacetylation of histones H3 and H4. This state is disrupted by deacetylase inhibition, which simultaneously decreases the occupancy of repressors, acetylates histones, and facilitates the action of activator factors.