Recruitment of chromatin-modifying enzymes by CTIP2 promotes HIV-1 transcriptional silencing

Abstract

Following entry and reverse transcription, the HIV-1 genome is integrated into the host genome. In contrast to productively infected cells, latently infected cells frequently harbor HIV-1 genomes integrated in heterochromatic structures, allowing persistence of transcriptionally silent proviruses. Microglial cells are the main HIV-1 target cells in the central nervous system and constitute an important reservoir for viral pathogenesis. In the present work, we show that, in microglial cells, the co-repressor COUP-TF interacting protein 2 (CTIP2) recruits a multienzymatic chromatin-modifying complex and establishes a heterochromatic environment at the HIV-1 promoter. We report that CTIP2 recruits histone deacetylase (HDAC)1 and HDAC2 to promote local histone H3 deacetylation at the HIV-1 promoter region. In addition, DNA-bound CTIP2 also associates with the histone methyltransferase SUV39H1, which increases local histone H3 lysine 9 methylation. This allows concomitant recruitment of HP1 proteins to the viral promoter and formation of local heterochromatin, leading to HIV-1 silencing. Altogether, our findings uncover new therapeutic opportunities for purging latent HIV-1 viruses from their cellular reservoirs.

Figure 1

Interactions of TSA-sensitive HDAC1 and HDAC2 with CTIP2. (A) Microglial cells were transfected with the episomal LTR-LUC vector in the presence or absence of 4 μg of pshRNA-CTIP2 vector. Cells were untreated or treated with 450nM TSA for 24 h. Two days post-transfection, LUC activities were measured and expressed relative to the value obtained with the empty vector. The knockdown efficiency of shRNA construction was controlled by Western blot (Supplement Figure 5A). Control shRNAs are also presented (Supplement Figure 1A). (B, C) HEK 293T cells were transfected with the indicated pFLAG-CTIP2 expression vector and the empty vector as control. Immunoprecipitated complexes were tested for HDAC activities (B) and for the presence of HDAC1, HDAC2 and HDAC3 by Western blot (C).

Figure 2

CTIP2 associates with HDAC1 and HDAC2 via its N-terminal domain. (A, B) HEK 293T cells were transfected with the indicated pFLAG-CTIP2 constructs and the empty vector as control. Cells extracts were normalized for the quantities of overexpressed FLAG-CTIP2 proteins and endogenous HDAC1 and HDAC2. Immunoprecipitated complexes were tested for HDAC activities (A) and for the presence of HDAC1, HDAC2 and FLAG-CTIP2 proteins by Western blot (B). (B) Input controls for HDAC1, HDAC2 (columns 1–5) and FLAG-CTIP2 construct expression (α-FLAG panel) are presented. A schematic CTIP2 linear structure is also drawn for a better visualization of CTIP2 domains.

Figure 3

HDAC1 and HDAC2 cooperate with CTIP2 to repress HIV-1 gene transcription and viral replication. (A, C) TZM-bl cells were transfected with the indicated plasmids. Two days post-transfection, LUC activities were measured and expressed relative to the value obtained with the empty vector. (B, D) Microglial cells were transfected with the episomal LTR-LUC and the indicated vectors. LUC activities were measured 2 days post-trasnfection and expressed relative to the value obtained with LTR-LUC alone. DNA quantities were normalized with the corresponding empty vector. (E, F) Microglial cells were transfected with pNL4-3 and the indicated vectors. Culture supernatants were analyzed for p24 Gag contents 48 h post-transfection. The knockdown efficiency of shRNA constructions was controlled by Western blot (Supplement Figure 5A).

Figure 4

Association of CTIP2 with the HIV-1 proximal promoter induces local H3 deacetylation with concomitant recruitment of HDAC1 and HDAC2. (A, D) Microglial cells (A) and CTIP2 knockdown cells (D) were infected with the VSV-pseudotyped pNL4.3-env⁻ virus 24 h before being subjected to ChIP experiments with the indicated antibodies. As a control, immunoprecipitations were performed in the absence of antibody (Ab control). Input (1/1000) and immunoprecipitated DNAs were quantified by real-time PCR using PCR1 LTR-specific oligonucleotides. The amount of immunoprecipitated material was normalized to the input DNA (A) and fold enrichments were normalized to the nonspecific enrichment in the GAPDH DNA (D). (B) ChIP experiments were performed on HEK 293T cells transfected with the HIV-1 LTR-LUC episomal vector in the presence or absence of the FLAG-CTIP2 expression vector as indicated. Cells were subjected to ChIP assays with the indicated antibodies. Specific enrichments in HIV LTR regions were quantitated by real-time PCR with the PCR1, PCR2 and GAPDH oligonucleotides. Results were normalized to enrichment in nonspecific GAPDH DNA. Results are representative of three independent experiments. (C) LTR-LUC-transfected HEK 293T cells were subjected to LUC activity quantification in the presence or absence of overexpressed CTIP2. (E) Initiated and elongated HIV-1 gene transcripts were quantitated by real-time RT–PCR in HIV-1-infected control and CTIP2 knockdown microglial cells. PCR quantifications target the HIV-1 TAR (initiation) and the HIV-1 Tat (elongation) regions. Results are presented relative to the initiated transcripts in control cells and normalized to β-actin copies.

Figure 5

CTIP2 cooperates and associates with HMT SUV39H1 via its central domain. (A) HEK 293T cells were transfected with the indicated pFLAG-CTIP2 constructs. Cell extracts were normalized for the quantities of overexpressed FLAG-CTIP2 proteins and endogenous SUV39H1. Complexes immunoprecipitated with the anti-SUV39H1 antibodies or the control non-immune serum were immunodetected for the presence of FLAG-CTIP2 proteins by Western blot. (B) GST pull-down assays were performed with ³⁵S-labelled CTIP2 protein incubated with GST (column 2) or GST-SUV39H1 fusion proteins (column 3). Approximately 10% of the total ³⁵S-labelled proteins obtained were loaded as input control (column 1). (C, E) TZM-bl cells were transfected with the indicated vectors. Two days post-transfection, LUC activities were measured and expressed relative to the value obtained with the empty vector. (D, F) Microglial cells were transfected with the indicated vectors. LUC activities were measured 2 days post-transfection and expressed relative to value obtained with the LTR-LUC alone taken as 1. DNA quantities were normalized with the corresponding empty vector. (G, H) Microglial cells were transfected with the HIV-1 pNL 4-3 vector and the indicated vectors. Two days post-transfection, culture supernatants were analyzed for p24 Gag contents. The knockdown efficiency of shRNA constructions was controlled by Western-blot (Supplement Figure S3).

Figure 6

CTIP2-mediated recruitment of SUV39H1 to the viral LTR promotes K9/H3 methylation and HP1 recruitments. (A, C) Microglial (A) and CTIP2 knockdown microglial cells were infected with the VSV-pseudotyped pNL4.3-env⁻ virus 24 h before being subjected to ChIP experiments with the indicated antibodies. As a control, immunoprecipitations were performed in the absence of antibody (Ab control). Input and immunoprecipitated DNA was subjected to real-time PCR using the PCR1 (A) or PCR2 and PCR3 (C) LTR-specific oligonucleotides. The amount of immunoprecipitated material was normalized to the input DNA (A) and the fold enrichments were normalized to enrichment in the nonspecific GAPDH promoter (C). (B) ChIP experiments were performed on HEK 293T cells transfected with the HIV-1 LTR-LUC episomal vector in the presence or absence of the FLAG-CTIP2 expression vector as indicated. Specific enrichments in HIV LTR regions obtained with the indicated antibodies were quantified by real-time PCR with the PCR1, PCR2 and GAPDH oligonucleotides. Results were normalized to enrichment in nonspecific GAPDH DNA. Results are representative of three independent experiments.

Figure 7

Transcriptional HIV-1 activation is accompanied by a decreased recruitment of CTIP2 and HP1 to the viral proximal promoter in the chromosomal context of integrated proviruses. ChIP assays were used to detect association of TBP, Sp1, CTIP2, HP1α, HP1β, HP1γ, triMeK9/H3 or SUV39H1 with the HIV-1 promoter proximal region (PCR1) (A), the Nuc-1-binding region (PCR2) (B), the Nuc-2-binding region (PCR3) (C) or the GAPDH promoter (D). U1 cells were mock-treated (−) or treated (+) with PMA (100 nM) for 1 h (A–D) and 20–80 min for time-ChIP assays (E). The amount of immunoprecipitated material was normalized to the input DNA. (E) U1 cells were mock-treated or treated with PMA (100 nM) for the indicated times and the proteins were crosslinked with formaldehyde for 10 min and DNA sheared. The crosslinked protein/DNA complexes were immunoprecipitated with the indicated antibodies or with a purified IgG as negative control. The protein/DNA crosslinks were reversed and the purified DNA was amplified and quantified by real-time PCR using the PCR1 primer. The amount of immunoprecipitated material was normalized to the input DNA.

Figure 8

Model for CTIP2-mediated establishment of heterochromatin environment to HIV-1 gene promoter and viral silencing. CTIP2 first recruits HDAC1 and HDAC2 enzymes to deacetylate H3 histones located at the viral promoter and particularly Nuc-1 H3 histones. After H3 deacetylation, CTIP2 recruits SUV39H1 to methylate Nuc-1 histone H3 lysine 9. This last H3 modification allows HP1 binding and polymerization, heterochromatin formation and HIV-1 silencing.