Recruitment of chromatin-modifying enzymes by CTIP2 promotes HIV-1 transcriptional silencing
2007; Springer Nature; Volume: 26; Issue: 2 Linguagem: Inglês
10.1038/sj.emboj.7601516
ISSN1460-2075
AutoresCéline Marban, Stella Suzanne, Samuel Dequiedt, Stéphane de Walque, Lætitia Redel, Carine Van Lint, Dominique Aunis, Olivier Rohr,
Tópico(s)HIV/AIDS Research and Interventions
ResumoArticle24 January 2007free access Recruitment of chromatin-modifying enzymes by CTIP2 promotes HIV-1 transcriptional silencing Céline Marban Céline Marban INSERM unité 575 Pathophysiology of Nervous System, Centre de Neurochimie, Strasbourg, France Search for more papers by this author Stella Suzanne Stella Suzanne INSERM unité 575 Pathophysiology of Nervous System, Centre de Neurochimie, Strasbourg, France Search for more papers by this author Franck Dequiedt Franck Dequiedt Cellular and Molecular Biology Unit, Faculty of Agronomy, Gembloux, Belgium Search for more papers by this author Stéphane de Walque Stéphane de Walque Laboratory of Molecular Virology, Institute for Molecular Biology and Medicine (IBMM) University of Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Laetitia Redel Laetitia Redel INSERM unité 575 Pathophysiology of Nervous System, Centre de Neurochimie, Strasbourg, France Search for more papers by this author Carine Van Lint Carine Van Lint Laboratory of Molecular Virology, Institute for Molecular Biology and Medicine (IBMM) University of Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Dominique Aunis Dominique Aunis INSERM unité 575 Pathophysiology of Nervous System, Centre de Neurochimie, Strasbourg, France Search for more papers by this author Olivier Rohr Corresponding Author Olivier Rohr INSERM unité 575 Pathophysiology of Nervous System, Centre de Neurochimie, Strasbourg, France IUT Louis Pasteur de Schiltigheim, Université de Strasbourg, France Search for more papers by this author Céline Marban Céline Marban INSERM unité 575 Pathophysiology of Nervous System, Centre de Neurochimie, Strasbourg, France Search for more papers by this author Stella Suzanne Stella Suzanne INSERM unité 575 Pathophysiology of Nervous System, Centre de Neurochimie, Strasbourg, France Search for more papers by this author Franck Dequiedt Franck Dequiedt Cellular and Molecular Biology Unit, Faculty of Agronomy, Gembloux, Belgium Search for more papers by this author Stéphane de Walque Stéphane de Walque Laboratory of Molecular Virology, Institute for Molecular Biology and Medicine (IBMM) University of Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Laetitia Redel Laetitia Redel INSERM unité 575 Pathophysiology of Nervous System, Centre de Neurochimie, Strasbourg, France Search for more papers by this author Carine Van Lint Carine Van Lint Laboratory of Molecular Virology, Institute for Molecular Biology and Medicine (IBMM) University of Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Dominique Aunis Dominique Aunis INSERM unité 575 Pathophysiology of Nervous System, Centre de Neurochimie, Strasbourg, France Search for more papers by this author Olivier Rohr Corresponding Author Olivier Rohr INSERM unité 575 Pathophysiology of Nervous System, Centre de Neurochimie, Strasbourg, France IUT Louis Pasteur de Schiltigheim, Université de Strasbourg, France Search for more papers by this author Author Information Céline Marban1, Stella Suzanne1,‡, Franck Dequiedt2,‡, Stéphane de Walque3, Laetitia Redel1, Carine Van Lint3, Dominique Aunis1 and Olivier Rohr 1,4 1INSERM unité 575 Pathophysiology of Nervous System, Centre de Neurochimie, Strasbourg, France 2Cellular and Molecular Biology Unit, Faculty of Agronomy, Gembloux, Belgium 3Laboratory of Molecular Virology, Institute for Molecular Biology and Medicine (IBMM) University of Bruxelles (ULB), Gosselies, Belgium 4IUT Louis Pasteur de Schiltigheim, Université de Strasbourg, France ‡These authors contributed equally to this work *Corresponding author. INSERM unité 575 Pathophysiology of Nervous System, Centre de Neurochimie, 5 rue Blaise Pascal, 67084 Strasbourg, France. Tel.: +33 388 45 66 01; Fax: +33 388 60 07 08; E-mail: [email protected] The EMBO Journal (2007)26:412-423https://doi.org/10.1038/sj.emboj.7601516 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction After entry into the target cell and reverse transcription, HIV-1 genes are integrated into the host genome. It is now well established that the viral promoter activity is directly governed by its chromatin environment (reviewed in Van Lint, 2000). Nucleosomes are precisely positioned at the HIV-1 promoter (Verdin et al, 1993; Van Lint et al, 1996). Nuc-1, a nucleosome located immediately downstream of the transcriptional initiation site directly impedes LTR activity. Epigenetic modifications and disruption of Nuc-1 are a prerequisite to activation of LTR-driven transcription and viral expression (for review, see Van Lint, 2000). The compaction of chromatin and its permissiveness for transcription are directly dependent on the post-translational modifications of histones such as acetylation, methylation, phosphorylation and ubiquitination (reviewed in Fischle et al, 2003). Euchromatin, a relaxed state of chromatin, is often associated with active transcription. On the other hand, heterochromatin, a compacted and more structured chromatin environment, is repressive for transcription. In contrast to productively infected cells, latently infected cells frequently harbor HIV-1 genomes integrated in heterochromatic structures, which allows viral persistence of silenced integrated proviruses (Jordan et al, 2003). These observations might at least partially explain how the virus can escape the host immune response and current therapeutic tools (Finzi et al, 1997; Pierson et al, 2000). Understanding the molecular mechanisms underlying HIV-1 transcriptional silencing is thus a major challenge in the fight against AIDS. HP1α is associated with transcriptionally inactive chromatin. At heterochromatic sites, HP1 binds methylated lysine 9 of histone H3 where it promotes gene silencing (Bannister et al, 2001). Transition from an active to inactive transcriptional state implies a series of ordered recruitment of histone-modifying enzymes. This is exemplified for K9/H3 methylation and HP1 recruitment, which requires the stepwise recruitment of histone deacetylase (HDAC) (to first remove the acetyl group) and histone methyltransferase (HMT) activities. HIV-1 gene transcription has been shown to be activated by trichostatin A (TSA) treatment, and several transcription factors bound to the viral LTR recruit class I or II HDAC (Van Lint et al, 1996; Coull et al, 2000; Williams et al, 2006). However, to date, the mechanisms associated with the establishment of a heterochromatic environment at the HIV-1 promoter remains unclear. COUP-TF interacting protein 2 (CTIP2) is a recently cloned transcriptional repressor that can associate with members of the COUP-TF family (Avram et al, 2000). This cofactor is expressed in the brain and the immune system (Leid et al, 2004). By regulating both differentiation and survival of thymocytes, CTIP2 is necessary for T-lymphocyte development (Wakabayashi et al, 2003). In the brain, CTIP2 plays a key role in the development of corticospinal motor neuron axonal projections to the spinal cord (Arlotta et al, 2005). Recently, we reported that CTIP2 inhibits HIV-1 replication in human microglial cells (Rohr et al, 2003; Marban et al, 2005). Microglial cells constitute the central nervous system (CNS)-resident macrophages. They are the main HIV-1 target cells in the brain, and because they are long lived and relatively protected by the blood–brain barrier, they constitute an important reservoir of viruses. Recently, brain macrophages have been described as latently HIV-1-infected cellular reservoirs (Barber et al, 2006). It is now clear that the long-lived reservoirs of HIV-1 can persist for years in the presence of HAART. However, contrary to CD4+ T-lymphocyte reservoirs (reviewed in Marcello, 2006), information on the virus state within macrophages and microglial cells is very limited. Here, we report that CTIP2 inhibits HIV-1 gene transcription by recruiting a chromatin-modifying complex and by establishing a heterochromatic environment at the HIV-1 promoter in microglial cells. Understanding HIV-1 silencing in the cellular reservoirs is actually the major challenge to viral eradication. Thereby, CTIP2, HDAC and HMT recruitments might uncover new therapeutic opportunities. Results CTIP2 associates with TSA-sensitive HDAC activities Our previous studies (Rohr et al, 2003; Marban et al, 2005) suggested that CTIP2 may repress HIV-1 transcription through association with HDAC activities in microglial cells. To decipher whether TSA-sensitive HDACs participate in CTIP2 repressive function, we examined the effect of TSA, a compound known to inhibit class I and II but not class III HDACs, on CTIP2-mediated repression of HIV-1 promoter in microglial cells (Figure 1A). TSA treatment and CTIP2 knockdown stimulated LTR-driven transcription in microglial cells. Interestingly, CTIP2 knockdown and TSA treatment strongly synergyized in HIV-1 transcriptional activation, suggesting an interaction of CTIP2 and TSA-sensitive HDACs (Figure 1A). Of note, control and HDAC3 shRNAs did not activate LTR-driven transcription, alone or in cooperation with TSA treatment (Supplementary Figure 1A). The specificity of CTIP2 knockdown and TSA cooperation in HIV-1 transcription was assessed by testing other cellular and viral promoters. No significant cooperation was observed in the modulation of the tested promoters (Supplementary Figure 1B). To test whether CTIP2 could associate with HDAC activity, FLAG-tagged CTIP2 was immunoprecipitated from HEK cell extracts and associated HDAC activity was assessed. As shown in Figure 1B, CTIP2 associated with robust HDAC activity. By comparison, the relative amount of HDAC activity associated with CTIP2 was about nine-fold higher than that observed with the control immunoprecipitation. Interestingly, TSA totally abolished CTIP2-associated HDAC activity, demonstrating that this activity was due to class I or II HDACs. 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). Download figure Download PowerPoint CTIP2 associates with HDAC1 and HDAC2 Based on the above findings, we next tested the presence of HDAC1–3 in the material associated with CTIP2 (Figure 1C). Western blot analysis revealed that CTIP2 specifically associated with HDAC1 and HDAC2 but not with HDAC3. Association of CTIP2 with HDAC1/2 in microglial cells was confirmed by immunofluorescence confocal microscopy (Supplementary Figure 2). Altogether, these results demonstrate that CTIP2 associates with an active HDAC complex. Several multiproteic complexes containing HDAC1 and HDAC2 have been described, such as Sin3, NuRD or Co-REST. To further investigate the nature of CTIP2-associated HDAC complex, Western blot analysis was performed on affinity-purified CTIP2-associated complex from HEK 293T cell extracts. Although Sin3, NuRD and Co-REST were present in CTIP2-expressing cells, no interaction was detected with CTIP2 (data not shown). This suggests that CTIP2 may not be involved in any of the HDAC1/2-containing complexes identified to date. The HDAC-interacting domain of CTIP2 is located in its N-terminus To delineate the CTIP2 region mediating HDAC interaction, we performed HDAC activity assays on affinity-purified materials from cells expressing truncated mutants of CTIP2 (Figure 2A). The N-terminal part of CTIP2 (aa 1–354) was sufficient to recruit 80% of the HDAC activity associated with the full-length CTIP2 (Figure 2A, compare columns 5 and 3). Interestingly, neither the 145–434 nor the 350–813 region is associated with significant HDAC activity, suggesting that aa 1–145 of CTIP2 are responsible for HDAC recruitment. 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. Download figure Download PowerPoint To verify the correlation between CTIP2-associated HDAC activity and the presence of HDAC1/2, we performed Western blot analysis of the material associated with the various CTIP2 mutants (Figure 2B). As expected, the full-length and the N-terminal part of CTIP2 associated with endogenous HDAC1 and HDAC2. HDAC1 and HDAC2 cooperate with CTIP2 to repress HIV-1 gene transcription and viral replication To decipher how HDAC1 and HDAC2 participate in CTIP2-repressive function, we examined their effect on CTIP2-mediated repression of chromatin-integrated HIV-1 promoter. TZM-bl cells, which contain a chromatin-integrated LTR, were cotransfected with expression vectors for CTIP2, HDAC1 or HDAC2, as indicated. Overexpression of HDAC1 and HDAC2 alone did not significantly affect LTR-driven transcription (Figure 3A). Moreover, CTIP2-mediated repression was further enhanced by coexpression of HDAC1 or HDAC2. Similar results were obtained in microglial cells transfected with an episomal LTR-LUC reporter (Figure 3B). Of note, a mutant of CTIP2 deficient in HDAC1/2 binding (i.e. 145–434 mutant) did not cooperate with HDAC1/2 (Supplementary Figure 3A). To validate the biological relevance of our finding, we next investigated the effect of knocking down CTIP2, HDAC1 and HDAC2 on HIV-1 LTR-driven transcription. Knockdown of HDAC1 or HDAC2 had very little to no impact on HIV-1 transcription, whereas knockdown of CTIP2 increased LTR transcriptional activity, both on integrated and on episomal HIV-1 LTR (Figure 3C and D). However, simultaneous knockdown of CTIP2 and HDAC1 or HDAC2 led to further increase HIV-1 transcription. To determine the impact of CTIP2-mediated recruitment on HIV-1 replication, microglial cells were transfected with the HIV-1 pNL4-3 vector together with expression vectors for CTIP2, HDAC1 and HDAC2, as indicated (Figure 3E). Forty-eight hours post-transfection, soluble p24 capsid protein was assessed in the culture medium by ELISA. As expected, overexpression of HDAC1 or HDAC2 did not affect HIV-1 replication significantly. In contrast, overexpression of CTIP2 was associated with a dramatic inhibition of HIV-1 replication, which was further enhanced by coexpression of HDAC1 or HDAC2 (Figure 3E). To further evaluate the functional cooperation of endogenous CTIP2 and HDAC1/2 enzymes, we quantified p24 production in HIV-1-infected microglial cells that had been knocked down for CTIP2, HDAC1 or HDAC2, alone or in combination, as indicated (Figure 3F). HDAC1 or HDAC2 knockdown only slightly stimulated HIV-1 replication. In contrast, knockdown of CTIP2 stimulated HIV-1 production up to 60-fold, thus confirming the repressive function of CTIP2 in microglial cells. More importantly, simultaneous knockdown of CTIP2 together with either HDAC1 or HDAC2 enhanced viral production up to 170-fold (Figure 3F). Altogether, these observations demonstrate a functional cooperation between CTIP2 and HDAC1/2 in HIV-1 gene silencing. 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). Download figure Download PowerPoint Association of CTIP2 with the HIV-1 proximal promoter induces recruitment of HDAC1 and HDAC2 and local histone H3 deacetylation We previously reported that CTIP2 was recruited to the HIV-1 promoter by the cellular transcription factor Sp1 (Marban et al, 2005). To determine whether endogenous CTIP2, HDAC1 and HDAC2 proteins are associated at the viral promoter, microglial cells were infected with an NL4.3-env− HIV-1 virus and recruitment of CTIP2, Sp1, HDAC1 and HDAC2 was analyzed by chromatin immunoprecipitation (ChIP) (Figure 4A). CTIP2 and Sp1 were found at the proximal region of the HIV-1 promoter (PCR1) (Figure 4A). In addition, both HDAC1 and HDAC2 were detected bound at the same region of the promoter. As a control, no binding of CTIP2, Sp1, HDAC1 or HDAC2 was observed for the control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter (data not shown). We next investigated whether HDAC1 and HDAC2 are recruited to the HIV-1 promoter by CTIP2. As expected, CTIP2 associated specifically with the LTR proximal region, but not with the Nuc-1-binding region (Figure 4B). Overexpression of CTIP2 increased recruitment of HDAC1 and HDAC2 to the Sp1-binding site (Figure 4B, panels α-HDAC1 and α-HDAC2, PCR1) and to the Nuc-1-binding region (Figure 4B, panels α-HDAC1 and α-HDAC2, column PCR2) of the LTR. As a control, overexpression of the N-terminal truncated 145–434 proteins did not increase HDAC1 and HDAC2 binding to the viral promoter (Supplementary Figure 3E). 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. Download figure Download PowerPoint We next assessed the impact of HDAC1/2 recruitment at the HIV-1 promoter by CTIP2 on local acetylation levels. For this purpose, we examined the acetylation of histone H3 at the Nuc-1-binding region and as a control at the proximal promoter region (Figure 4B, PCR2 and PCR1 respectively). No Ac/H3 was detected on the HIV-1 proximal promoter in the absence of CTIP2, whereas acetylated histones H3 were observed at the HIV-1 Nuc-1-binding region and at the constitutively active GAPDH promoter (Supplementary Figure 3C). Interestingly, overexpression of CTIP2 resulted in decreased levels of histone H3 acetylation at the HIV-1 Nuc-1-binding region (Figure 4B, panel α-Ac/H3, PCR2). As a control, no enrichment of Ac/H3 was observed at the proximal promoter (Figure 4B, panel α-Ac/H3, PCR1). These results suggest that CTIP2 targets HDAC1 and HDAC2 to the HIV-1 gene promoter to establish a chromatin environment detrimental to transcription. This conclusion was supported by the CTIP2-mediated repression of LTR-driven transcription in 293T cells (Figure 4C). To further explore the link between CTIP2 and association of HDAC1 and HDAC2 with the viral promoter, we performed ChIP experiments in a context of HIV-1-infected control and CTIP2 knockdown microglial cells. As expected, reduction of endogenous CTIP2 levels correlated with decreased binding at the HIV-1 proximal promoter region (Figure 4D, panel α-CTIP2). As control, the same amount of Sp1 (Figure 4D, panel α-Sp1) was found at the proximal promoter region in control and CTIP2 knockdown cells. Surprisingly, whereas HDAC2 binding at the viral promoter was reduced in CTIP2 knockdown cells (Figure 4D, panel α-HDAC2), HDAC1 recruitment was enhanced (Figure 4D, panel α-HDAC1). Together with the results from previous studies (Doetzlhofer et al, 1999), this observation suggests that displacement of the CTIP2-repressing complex may allow additional HDAC1 protein to interact via Sp1 with the viral promoter. To further explore the role of CTIP2 in HIV-1 gene transcription, we quantified the initiated and the elongated transcripts in the context of HIV-1-positive microglial cells, knocked down for CTIP2. As shown in Figure 4E, the relative amount of HIV-1-initiated transcripts was eight times increased in CTIP2 knockdown cells compared to control cells. CTIP2 promoted accumulation of initiated transcripts but did not significantly increase elongated transcripts under these experimental conditions (Figure 4E). CTIP2 associates with histone methyltransferase SUV39H1 We have previously reported that CTIP2 promotes HP1α recruitment to the HIV-1 promoter (Marban et al, 2005). In heterochromatic environments, HP1α associates with the methylated form of histone H3 lysine 9 (Bannister et al, 2001). As methylation of H3/K9 is typically mediated by SUV39H1, we logically tested whether CTIP2 is associated with SUV39H1. As shown in Figure 5, endogenous SUV39H1 was copurified with immunoprecipitated FLAG-tagged CTIP2 (Figure 5A, lane 1). In addition, deletion analysis revealed that the central 145–434 domain (Figure 5A, lane 3) but not the N- and C-terminal domains (Figure 5A, lanes 2 and 4) of CTIP2 was sufficient to mediate interaction with SUV39H1. The association of SUV39H1 with CTIP2 was further tested in vitro (Figure 5B). Pull-down experiments with in vitro-translated CTIP2 showed that 2–5% of CTIP2 proteins specifically interacted with GST-SUV39H1 in vitro (Figure 5B, column 3). Finally, confocal microscopic observations revealed colocalization of CTIP2 and SUV39H1 in the nuclei of microglial cells (Supplementary Figure 4). 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). Download figure Download PowerPoint CTIP2 cooperates with SUV39H1 to repress HIV-1 gene transcription and viral replication To further characterize the significance of CTIP2/SUV39H1 interaction, we performed luciferase assays on cellular extracts from TZM-bl cells containing an integrated LTR-LUC construct (Figure 5C) and from microglial cells transfected with an episomal LTR-LUC reporter (Figure 5D). The effects of CTIP2 and SUV39H1 were assessed after overexpression of each protein, independently or in combination. Overexpression of SUV39H1 alone had no effect on LTR-driven transcription (Figure 5C and D, column 2). However, it dramatically increased CTIP2-mediated repression of the HIV-1 promoter (Figure 5C and D, column 4). As a control, SUV39H1 had no effect on the aa 1–354 truncation CTIP2 mutant, which is unable to interact with SUV39H1 (Supplementary Figure 3B). These results suggest a functional cooperation between CTIP2 and SUV39H1 in the context of chromatinized HIV-1 promoter in microglial cells. Next, similar experiments were performed using shRNA constructs targeting SUV39H1 and CTIP2 (Figures 5E and F). As expected knockdown of CTIP2 and SUV39H1 independently or in combination stimulated transcription from genome-integrated as well as episomal HIV-1 LTR. The functional relevance of CTIP2/SUV39H1 to HIV-1 replication was next examined. For this purpose, CTIP2 and increasing amounts of SUV39H1 were expressed in pNL4-3-transfected microglial cells (Figure 5G). Production of p24 HIV-1 capsid was unaffected by overexpression of SUV39H1 (Figure 5G). However, in the presence of CTIP2, SUV39H1 further enhanced CTIP2-mediated repression of viral replication, confirming a functional cooperation between both proteins. As expected, the functional cooperation between CTIP2 and SUV39H1 was also observed in a similar experimental setting using knockdown of CTIP2 and SUV39H1 (Figure 5H). CTIP2-mediated recruitment of SUV39H1 to the HIV-1 proximal promoter induces K9/H3 methylation and HP1 association K9/H3 methylation is the ultimate epigenetic modification necessary for HP1 association and polymerization. To determine whether endogenous CTIP2, SUV39H1 and HP1 proteins are associated with the viral promoter in the context of infected microglial cells, cells were infected with a vesicular stomatostatis virus (VSV)-pseudotyped HIV-1 NL4.3-env− virus 24 h before being processed for ChIP experiments (Figure 6A). The presence of CTIP2, HDAC2 and HDAC1 at the viral promoter (Figure 4A) correlated with the recruitment of SUV39H1 and HP1 proteins (Figure 6A). To refine these observations, ChIP experiments using anti-trimethyl-K9/H3, anti-SUV39H1 and anti-HP1 antibodies were performed in the presence or absence of CTIP2 overexpression in LTR-LUC-transfected 293T cells (Figure 6B). The control GAPDH gene promoter, the LTR Nuc-1-binding region (Figure 6B, PCR2 columns) and the LTR proximal region (Figure 6B, PCR1 columns) were analyzed by quantitative real-time PCR. 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 infecte
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