Involvement of histone H3 lysine 9 (H3K9) methyltransferase G9a in the maintenance of HIV-1 latency and its reactivation by BIX01294

Abstract

Elucidating the mechanism of human immunodeficiency virus, type 1 (HIV-1) provirus transcriptional silencing in latently infected cells is crucial for understanding the pathophysiological process of HIV-1 infection. It is well established that hypoacetylation of histone proteins by histone deacetylases is involved in the maintenance of HIV-1 latency by repressing viral transcription. Although histone methylation is involved in the organization of chromatin domains and plays a central epigenetic role in gene expression, the role of histone methylation in the maintenance of HIV-1 latency has not been clarified. Here we present evidence that histone H3 Lys(9) (H3K9) methyltransferase G9a is responsible for transcriptional repression of HIV-1 by promoting repressive dimethylation at H3K9 and for the maintenance of viral latency. We observed that G9a significantly inhibited basal, as well as, the induced HIV-1 gene expression by tumor necrosis factor-alpha or Tat. Mutant G9a, however, lacking the SET domain responsible for the catalytic activity of histone methyltransferase, did not show such an effect. When G9a expression was knocked down by small interfering RNA, HIV-1 replication was augmented from cells transiently transfected with a full-length HIV-1 clone. Moreover, a specific inhibitor of G9a, BIX01294, could reactivate expression of HIV-1 from latently infected cells such as ACH-2 and OM10.1. Furthermore, chromatin immunoprecipitation assays revealed the presence of G9a and H3K9 dimethylation on nucleosome histones in the vicinity of the HIV-1 long terminal repeat promoter. These results suggest that G9a is responsible for the transcriptional quiescence of latent HIV-1 provirus and provide a molecular basis for understanding the mechanism by which HIV-1 latency is maintained.

FIGURE 1.

Activation of HIV-1 by 5-aza-CdR. A, reactivation of the silenced HIV-1 gene in the latently infected cells by 5-aza-CdR. Latent HIV-1-infected ACH-2 and OM10.1 cells were incubated with 5-aza-CdR (0, 1, 5, or 15 μm) for 48 h. The cell lysates were analyzed for viral proteins by immunoblotting with the collected AIDS patients sera or G9a antibody. Positions of HIV-1 proteins are indicated on the right. The α-tubulin was used as an internal control. B, down-regulation of H3K9me2 by 5-aza-CdR. The experiments were similarly performed as in A except that the histone protein fraction was partially purified from these cells. The cell lysates were analyzed for the dimethylated histone H3 at Lys⁹ (H3K9me2) or trimethylated histone H3 at Lys⁹ (H3K9me3) by immunoblotting using specific antibodies. The unmodified H3 protein was used as a control.

FIGURE 2.

Repression of HIV-1 LTR gene expression by G9a overexpression. A, effects of G9a on gene expression from transiently transfected HIV-1 LTR. The expression plasmid pcDNA-G9a was co-transfected with the HIV-1 LTR-luc reporter construct (20) into CEM T cells. Amounts of G9a plasmid co-transfected were 2 and 8 μg/transfection. Extents of HIV-1 gene expression and the effects of G9a are shown in CEM cells at the basal level (left panel), upon TNF-α stimulation (middle panel), or upon co-transfecting the Tat-expressing plasmid pCMV-Tat at 2 μg/transfection (right panel). Upon TNF-α stimulation, cells were incubated with 3 ng/ml of TNF-α after 24 h of transfection and incubated for an additional 24 h. B, effects of G9a SET domain deletion on HIV-1 gene expression. CEM cells were transfected with HIV-1 LTR-luc together with the SET-defective G9a deletion mutant (G9aΔSET). The cells were harvested and luciferase activity was measured. Amounts of G9a, tagged with EGFP, and the dimethylated form of histone 3 at the Lys⁹ position were analyzed by Western blotting using specific antibodies. Each value shown is the fold-increase in the luciferase activity (mean ± S.D.) relative to the control transfection of three independent experiments.

FIGURE 3.

Effects of G9a knockdown. A, confirmation of the siRNA-mediated G9a knockdown. 293 cells were transfected with 100 nm siRNAs directed against either G9a or gfp (control) mRNAs. After 36 h of transfection, cells were lysed and levels of G9a and H3K9me2 were assessed by immunoblotting using specific antibodies. Afterward, the immunoblotted membrane was stripped and reprobed with anti-α-tubulin antibody. B, augmentation of HIV-1 gene expression by G9a depletion. 293 cells were transfected with HIV-1 LTR-luc concurrently with either G9a siRNA or its control. After 24 h of transfection, cells were either untreated or treated with 3 ng/ml of TNF-α and incubated for an additional 24 h. Cells were harvested and luciferase activity was measured. Each value is the fold increase in the luciferase activity (means ± S.D.) relative to the control transfection of three independent experiments. C, effects of G9a knockdown on HIV-1 viral replication. 293 cells were transfected with pNL4-3, a replication competent full-length HIV-1 clone, and either G9a siRNA or control siRNA (GFP). After 24 h of transfection, cells were either untreated (left panel) or treated (right panel) with 3 ng/ml of TNF-α and incubated for an additional 24 h. The culture supernatants were collected and subjected to HIV-1 p24 antigen level determination by ELISA. Each value shown is the fold increase in the HIV p24 level (means ± S.D.) relative to the control transfection of three independent experiments.

FIGURE 4.

Effects of G9a inhibitor BIX01294 on HIV-1 replication. A, activation of HIV-1 gene expression by BIX01294 (left) and restoration of the G9a-mediated repression of HIV-1 gene expression by BIX01294 (right). The HIV-1 LTR-luc plasmid was transfected into CEM cells, incubated for 24 h, treated with BIX01294 (0, 1.5, 5, or 10 μm) for another 24 h, and luciferase activity was measured. In the right panel, the HIV-1 LTR-luc and G9a plasmids were transfected into CEM cells, incubated for 24 h, treated with BIX01294 (0, 5 or 10 μm) for another 24 h, and the luciferase activity was measured. B, effects of BIX01294 on gene expression from HTLV-1, CMV, and MMTV promoters. As positive controls, each promoter was treated with known inducers. PMA, phorbol 12-myristate 12-acetate; Dex, dexamethasone. Experiments with MMTV-luc were performed with HeLa cells where the glucocorticoid receptor is expressed. For the data in A and B, each data point represents the mean ± S.D. of three independent experiments. C, activation of HIV-1 replication by BIX01294. Latently infected cell lines, ACH-2 and OM10.1, were incubated in the presence of various concentrations of BIX01294 for 48 h and both the culture supernatant and the cell lysate were measured for viral p24 antigen levels: upper panels, viral p24 proteins in the culture supernatant; middle panels, major viral proteins in the cytoplasm; and lower panels, the dimethylated histones, H3K9me2 and H3K27me2, in the nuclear histone fractions through immunoblotting using specific antibodies. Each value shown is the HIV p24 level (means ± S.D.) of three independent experiments.

FIGURE 5.

Presence of G9a and H3K9me2 in the proviral HIV-1 LTR revealed by ChIP assay. ChIP assays were carried out to detect the dimethylated histone H3 in the vicinity of HIV-1 proviral DNA in ACH-2 cells. ACH-2 cells were either treated or untreated with 10 μm BIX01294 (left panel) or 3 ng/ml of TNF-α (right panel) for 90 min and subjected to ChIP assays as described under “Experimental Procedures.” The final PCR was carried out to amplify the viral sequence from −176 to +61 within the viral LTR. The antibodies used in the ChIP assays are indicated on the left. Input DNA (input) represents 10% of total input chromatin DNA, whereas immunoprecipitation with non-immune IgG (“IgG”) served as a negative control. RNAP II, RNA polymerase II.

FIGURE 6.

Synergistic activation of HIV-1 replication by BIX01294 and SAHA or 5-aza-CdR. To investigate the mode of actions of these chemical compounds with distinct biochemical actions, the HIV-1 latently infected ACH-2 cells were treated with various combinations of SAHA, 5-aza-CdR, and BIX01294 for 48 h. The culture supernatants and the cell lysates were analyzed for p24 antigen levels by ELISA (upper panels) and detection of virus proteins by immunoblotting (lower panels), respectively. Each value shown is the HIV p24 level (means ± S.D.) of three independent experiments.