Activation of HIV transcription by the viral Tat protein requires a demethylation step mediated by lysine-specific demethylase 1 (LSD1/KDM1)

Abstract

The essential transactivator function of the HIV Tat protein is regulated by multiple posttranslational modifications. Although individual modifications are well characterized, their crosstalk and dynamics of occurrence during the HIV transcription cycle remain unclear.We examine interactions between two critical modifications within the RNA-binding domain of Tat: monomethylation of lysine 51 (K51) mediated by Set7/9/KMT7, an early event in the Tat transactivation cycle that strengthens the interaction of Tat with TAR RNA, and acetylation of lysine 50 (K50) mediated by p300/KAT3B, a later process that dissociates the complex formed by Tat, TAR RNA and the cyclin T1 subunit of the positive transcription elongation factor b (P-TEFb). We find K51 monomethylation inhibited in synthetic Tat peptides carrying an acetyl group at K50 while acetylation can occur in methylated peptides, albeit at a reduced rate. To examine whether Tat is subject to sequential monomethylation and acetylation in cells, we performed mass spectrometry on immunoprecipitated Tat proteins and generated new modification-specific Tat antibodies against monomethylated/acetylated Tat. No bimodified Tat protein was detected in cells pointing to a demethylation step during the Tat transactivation cycle. We identify lysine-specific demethylase 1 (LSD1/KDM1) as a Tat K51-specific demethylase, which is required for the activation of HIV transcription in latently infected T cells. LSD1/KDM1 and its cofactor CoREST associates with the HIV promoter in vivo and activate Tat transcriptional activity in a K51-dependent manner. In addition, small hairpin RNAs directed against LSD1/KDM1 or inhibition of its activity with the monoamine oxidase inhibitor phenelzine suppresses the activation of HIV transcription in latently infected T cells.Our data support the model that a LSD1/KDM1/CoREST complex, normally known as a transcriptional suppressor, acts as a novel activator of HIV transcription through demethylation of K51 in Tat. Small molecule inhibitors of LSD1/KDM1 show therapeutic promise by enforcing HIV latency in infected T cells.

Figure 1. In vitro acetylation and methylation assays using synthetic Tat peptides.

(A) In vitro methylation assays of Tat ARM peptides (aa 45–58). Unmodified, K50-acetylated or K51-methylated peptides were incubated with recombinant SET7/9/KMT7 and ³H-radiolabeled S-adenosyl-L-methionine (SAM). Peptides were separated by Tris-Tricine gel electrophoresis and visualized by autoradiography. A quantification of band intensities of three independent experiments is shown below. (B) In vitro acetylation assays of Tat ARM peptides. Unmodified, K50-acetylated or K51-methylated peptides were incubated with recombinant p300-HAT and ¹⁴C-acetyl coenzyme A. Peptides were processed as in A. A quantification of band intensities of three independent experiments is shown below.

Figure 2. MALDI-TOF mass spectrometric analysis of cellular Tat confirms K51 monomethylation.

(A) MALDI-TOF MS spectrum of digested peptides from Tat-FLAG (900-1,500 m/z) immunoprecipitated from J-Lat A2 cells activated with TNFα. The peptide ions (designated as A and B) further analyzed by MS/MS are indicated by m/z values and number of amino acid sequence. Their position within the Tat-FLAG molecule used in this study is indicated below. Please note that studies were performed with chymotrypsin to avoid restraints in trypsin-based cutting caused by modifications of lysines. However, some identified ARM peptides are shorter than anticipated based on the predicted size of chymotrypsin-digested peptides in Tat which is likely due to a contamination with some trypsin-like activity in the chymotrypsin preparations used in this experiment. (B) MALDI-TOF/TOF MS/MS spectra of peptide A ion (1084.681 m/z). (C) MALDI-TOF/TOF MS/MS spectra of peptide B ion (1197.724 m/z). In these spectra, identified fragment ions were denoted by the ion types, a, b, c, y according to the nomenclature by Roepstorff and Fohlman . The assignment of fragment ions to the amino acid sequence of the ARM region of the Tat molecule was inserted in each spectrum. The symbol * indicates methylated amino acid residue.

Figure 3. No detection of cellular acetylated/methylated Tat by newly generated Tat antibodies.

(A) Dot-blot analysis of ARM peptides using α-Me1K51 or α-AcK50/Me1K51 Tat antibodies. (B) Western blot analysis of synthetic Tat (aa 1–72) with α-Tat, α-Me1K51 Tat or α-AcK50/Me1K51 Tat antibodies. Of note, we have not succeeded so far to synthesize a Tat peptide of 101 aa length corresponding to the full-length Tat species encoded by two tat exons in cells. (C) Whole cell lysates from 293T cell transfected with FLAG-tagged Tat (aa 1–101) were subjected to immunoprecipitation with α-FLAG agarose. Purified proteins were analyzed by western blot analysis together with synthetic Tat proteins using the indicated antibodies.

Figure 4. LSD1/KDM1 demethylates mono-methylated K51 in Tat.

(A) Synthetic K51-mono-methylated Tat proteins were incubated with increasing amounts of recombinant LSD1/KDM1 (0, 0.5, 1, 2 µg) for 1 h at 37°C. Reaction products were analyzed by western blotting using α-Tat, α-Me1K51 Tat antibodies. (B) Purified histone proteins were subjected to the same procedure as in A and analyzed by α-Me2H3K4 or α-histone H3 antibodies. (C) Synthetic K51-mono-methylated Tat or K50-acetylated/K51-monomethylated Tat proteins were incubated with 1 µg of recombinant LSD1 as described in A. (D) Increase in K51-monomethylation of Tat in LSD1 shRNA-infected J-Lat A2 cells. Whole cell lysates isolated from J-Lat A2 cells infected with shRNAs directed against LSD1 or control shRNAs and stimulated with TNFα were analyzed by western blotting with indicated antibodies. Band intensities were quantified using ImageJ Software (NIH). R.I.: Relative intensities of bands as compared to the control-vector transduced cells (100%).

Figure 5. In vivo recruitment of LSD1 and CoREST to the HIV LTR.

(A) Co-immunoprecipitation of endogenous LSD1 and CoREST with Tat/FLAG and the Tat K51A mutant in transiently transfected 293T cells. (B) Chromatin immunoprecipitation analysis of LSD1, CoREST and HDAC1 in J-Lat A2 cells. A2 cells were stimulated with TNFα over night and chromatin immunoprecipitation was performed using α-LSD1, α-CoREST, α-HDAC1 or no antibodies followed by real-time RT-PCR with primers specific for the HIV LTR region. (C) Whole cell lysates isolated from A2 cells stimulated with TNFα over night were analyzed by western blotting using α-LSD1, α-tubulin or α-FLAG antibodies.

Figure 6. LSD1/KDM1 and CoREST activate HIV transcription.

(A) Lentiviral vectors expressing shRNAs against LSD1 (#1 and #2), luciferase or scrambled shRNAs were infected into J-Lat A2 cells for 5–10 days; cells were then stimulated with a low dose of TNFα (0.08 ng/ml) over night. Number of GFP+ cells was analyzed by flow cytometry and expressed as percent GFP+ cells as compared to luciferase-shRNA-infected A2 cells. The average (mean±SEM) of three independent experiments is shown. *corresponds to a p value <0.01. (B) Number of violet dye-negative cells was measured as a marker of cell viability by flow cytometry in cells described in (A). Of note, propidium iodide (PI) staining could not be performed in these cells because of the mCherry marker expressed by the LSD1 shRNA vector (C) Whole cell lysates from shRNA-expressing cells were analyzed by western blotting using α-LSD1 and α-tubulin antibodies. (D) Lentiviral vectors expressing shRNAs against CoREST (#1 and #2) or control shRNAs were infected into J-Lat A2 cells. The same experiment as in (A) was performed. The average (mean±SEM) of three independent experiments is shown. *corresponds to a p value <0.01. (E) Number of PI-negative cells was measured as a marker of cell viability by flow cytometry in cells described in (D). (F) Whole cell lysates from shRNA-infected A2 cells were analyzed by western blotting using α-CoREST and α-tubulin antibodies. (G) SiRNA-transfected HeLa cells (48 h after siRNA transfection) were re-transfected with an HIV LTR luciferase reporter construct and expression vectors for wildtype or K51A mutant Tat. In parallel, same amounts of EF-1α RL reporter constructs as used for Tat were transfected to monitor Tat expression. Luciferase or Renilla activities were measured 24 h after plasmid transfections. The relative differences in luciferase activity as compared to wildtype Tat transactivation were calculated. The average (mean±SEM) of three independent experiments is shown. *corresponds to a p value <0.01. (H) Whole cell lysates from siRNA-transfected HeLa cells were analyzed by western blotting using α-LSD1 and α-tubulin antibodies.

Figure 7. LSD1/KDM1 as a potential drug target in HIV transcription.

(A) J-Lat A2 cells were stimulated with 0.08 ng/ml of TNFα in the presence of increasing amounts of phenelzine (100, 300, 1000 or 3000 µM), DRB (0.3, 1, 3 or 10 µM) or DMSO as carrier control overnight. Numbers of GFP+ cells (marker of HIV LTR activity) as well as propidium iodide (PI)-negative cells (marker of cell viability) were analyzed by flow cytometry. The percentage compared to DMSO-treated control cells was calculated. The average of two independent experiments is shown. (B) Purified resting primary CD4+ T cells were infected at a high m.o.i. with infectious HIV-NL4-3 luciferase reporter virus. Three days after infection, cells were stimulated with α-CD3 and α-CD28 antibodies in the presence of phenelzine (100, 300, or 1000 µM), DRB (1 or 10 µM) or DMSO overnight followed by analysis for luciferase activity and PI uptake.

Figure 8. Model of LSD1/KDM1 action in HIV transcription.

Demethylation of Tat by the LSD1/KDM1/CoREST complex is a required new step during Tat transactivation of the HIV LTR. We propose that it may occur before acetylation of Tat by p300/KAT3B. Inhibition of LSD1/KDM1 by phenelzine blocks this demethylation step and inhibits Tat transactivation during reactivation from latency.