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LSD1 cooperates with CTIP2 to promote HIV-1 transcriptional silencing.

Le Douce V, Colin L, Redel L, Cherrier T, Herbein G, Aunis D, Rohr O, Van Lint C, Schwartz C - Nucleic Acids Res. (2011)

Bottom Line: We have previously demonstrated that the cellular cofactor CTIP2 forces heterochromatin formation and HIV-1 gene silencing by recruiting HDAC and HMT activities at the integrated viral promoter.We show that recruitment of LSD1 at the HIV-1 proximal promoter is associated with both H3K4me3 and H3K9me3 epigenetic marks.Finally, our data suggest that LSD1-induced H3K4 trimethylation is linked to hSET1 recruitment at the integrated provirus.

View Article: PubMed Central - PubMed

Affiliation: University of Strasbourg, EA4438, Institute of parasitology, Strasbourg, France.

ABSTRACT
Microglial cells are the main HIV-1 targets in the central nervous system (CNS) and constitute an important reservoir of latently infected cells. Establishment and persistence of these reservoirs rely on the chromatin structure of the integrated proviruses. We have previously demonstrated that the cellular cofactor CTIP2 forces heterochromatin formation and HIV-1 gene silencing by recruiting HDAC and HMT activities at the integrated viral promoter. In the present work, we report that the histone demethylase LSD1 represses HIV-1 transcription and viral expression in a synergistic manner with CTIP2. We show that recruitment of LSD1 at the HIV-1 proximal promoter is associated with both H3K4me3 and H3K9me3 epigenetic marks. Finally, our data suggest that LSD1-induced H3K4 trimethylation is linked to hSET1 recruitment at the integrated provirus.

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CTIP2 recruitment on the HIV-1 proximal promoter requires LSD1. (A) ChIP experiments were performed on HEK 293 T cells transfected with the HIV-1 LTR LUC episomal vector in the presence of the pFLAG-LSD1, the pshRNA-LSD1 or the respective pcDNA3-FLAG and pshRNA-control vectors. Cells were subjected to ChIP assays with the indicated antibodies. Specific enrichments in the HIV-1 proximal promoter were quantified by real-time PCR targeting the Sp1-binding sites. Specific enrichments were calculated relative to the control IgG and relative enrichments in the context of LSD1 over-expression or LSD1 depletion were expressed relative to the value obtained with the pcDNA3-FLAG and the pshRNA-control vectors, respectively. Results were expressed relative to the value obtained with the episomal LTR-LUC plasmid co-transfected with the pcDNA3-FLAG or the pshRNA-control vectors. (B) Control and CTIP2 knocked-down microglial cells were infected with the VSV-pseudotyped pNL-4.3-Env virus 24 h before being subjected to ChIP experiments with the indicated antibodies. Specific enrichments were calculated relative to the control IgG and relative enrichments in the context of CTIP2 depletion were expressed relative to the value obtained with the control cells. Specific enrichments at the HIV-1 proximal promoter were quantified by real-time PCR targeting the LTR-Sp1-binding sites region.
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gkr857-F6: CTIP2 recruitment on the HIV-1 proximal promoter requires LSD1. (A) ChIP experiments were performed on HEK 293 T cells transfected with the HIV-1 LTR LUC episomal vector in the presence of the pFLAG-LSD1, the pshRNA-LSD1 or the respective pcDNA3-FLAG and pshRNA-control vectors. Cells were subjected to ChIP assays with the indicated antibodies. Specific enrichments in the HIV-1 proximal promoter were quantified by real-time PCR targeting the Sp1-binding sites. Specific enrichments were calculated relative to the control IgG and relative enrichments in the context of LSD1 over-expression or LSD1 depletion were expressed relative to the value obtained with the pcDNA3-FLAG and the pshRNA-control vectors, respectively. Results were expressed relative to the value obtained with the episomal LTR-LUC plasmid co-transfected with the pcDNA3-FLAG or the pshRNA-control vectors. (B) Control and CTIP2 knocked-down microglial cells were infected with the VSV-pseudotyped pNL-4.3-Env virus 24 h before being subjected to ChIP experiments with the indicated antibodies. Specific enrichments were calculated relative to the control IgG and relative enrichments in the context of CTIP2 depletion were expressed relative to the value obtained with the control cells. Specific enrichments at the HIV-1 proximal promoter were quantified by real-time PCR targeting the LTR-Sp1-binding sites region.

Mentions: We next asked whether LSD1 is required for CTIP2 recruitment to the HIV-1 promoter. To address this question, we performed additional ChIP experiments in the LSD1 over-expression or LSD1 knock-down contexts. As shown in Figure 6A, over-expression of LSD1 was associated with an increase of endogenous CTIP2 recruitment to the viral promoter. As expected, LSD1 knock-down decreased CTIP2 association with the viral promoter (Figure 6A). To further study LSD1 and CTIP2 recruitment at the HIV-1 promoter, we performed ChIP experiments with HIV-1 infected microglial cells expressing (control) or not CTIP2 (shCTIP2) (Figure 6B). As a control, we checked that CTIP2 is less recruited onto the HIV-1 proximal promoter in the infected shCTIP2 microglial cell line (Figure 6B column 2 compared to column 1) compared to the control cell line. Unexpectedly, knocking-down CTIP2 slightly increased LSD1 recruitment to the LTR (Figure 6B column 3). Moreover, this recruitment was correlated with an increased H3K4 trimethylation (Figure 6B column 4). These results suggest that LSD1 is required for CTIP2 recruitment to the HIV-1 proximal promoter.Figure 6.


LSD1 cooperates with CTIP2 to promote HIV-1 transcriptional silencing.

Le Douce V, Colin L, Redel L, Cherrier T, Herbein G, Aunis D, Rohr O, Van Lint C, Schwartz C - Nucleic Acids Res. (2011)

CTIP2 recruitment on the HIV-1 proximal promoter requires LSD1. (A) ChIP experiments were performed on HEK 293 T cells transfected with the HIV-1 LTR LUC episomal vector in the presence of the pFLAG-LSD1, the pshRNA-LSD1 or the respective pcDNA3-FLAG and pshRNA-control vectors. Cells were subjected to ChIP assays with the indicated antibodies. Specific enrichments in the HIV-1 proximal promoter were quantified by real-time PCR targeting the Sp1-binding sites. Specific enrichments were calculated relative to the control IgG and relative enrichments in the context of LSD1 over-expression or LSD1 depletion were expressed relative to the value obtained with the pcDNA3-FLAG and the pshRNA-control vectors, respectively. Results were expressed relative to the value obtained with the episomal LTR-LUC plasmid co-transfected with the pcDNA3-FLAG or the pshRNA-control vectors. (B) Control and CTIP2 knocked-down microglial cells were infected with the VSV-pseudotyped pNL-4.3-Env virus 24 h before being subjected to ChIP experiments with the indicated antibodies. Specific enrichments were calculated relative to the control IgG and relative enrichments in the context of CTIP2 depletion were expressed relative to the value obtained with the control cells. Specific enrichments at the HIV-1 proximal promoter were quantified by real-time PCR targeting the LTR-Sp1-binding sites region.
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Related In: Results  -  Collection

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gkr857-F6: CTIP2 recruitment on the HIV-1 proximal promoter requires LSD1. (A) ChIP experiments were performed on HEK 293 T cells transfected with the HIV-1 LTR LUC episomal vector in the presence of the pFLAG-LSD1, the pshRNA-LSD1 or the respective pcDNA3-FLAG and pshRNA-control vectors. Cells were subjected to ChIP assays with the indicated antibodies. Specific enrichments in the HIV-1 proximal promoter were quantified by real-time PCR targeting the Sp1-binding sites. Specific enrichments were calculated relative to the control IgG and relative enrichments in the context of LSD1 over-expression or LSD1 depletion were expressed relative to the value obtained with the pcDNA3-FLAG and the pshRNA-control vectors, respectively. Results were expressed relative to the value obtained with the episomal LTR-LUC plasmid co-transfected with the pcDNA3-FLAG or the pshRNA-control vectors. (B) Control and CTIP2 knocked-down microglial cells were infected with the VSV-pseudotyped pNL-4.3-Env virus 24 h before being subjected to ChIP experiments with the indicated antibodies. Specific enrichments were calculated relative to the control IgG and relative enrichments in the context of CTIP2 depletion were expressed relative to the value obtained with the control cells. Specific enrichments at the HIV-1 proximal promoter were quantified by real-time PCR targeting the LTR-Sp1-binding sites region.
Mentions: We next asked whether LSD1 is required for CTIP2 recruitment to the HIV-1 promoter. To address this question, we performed additional ChIP experiments in the LSD1 over-expression or LSD1 knock-down contexts. As shown in Figure 6A, over-expression of LSD1 was associated with an increase of endogenous CTIP2 recruitment to the viral promoter. As expected, LSD1 knock-down decreased CTIP2 association with the viral promoter (Figure 6A). To further study LSD1 and CTIP2 recruitment at the HIV-1 promoter, we performed ChIP experiments with HIV-1 infected microglial cells expressing (control) or not CTIP2 (shCTIP2) (Figure 6B). As a control, we checked that CTIP2 is less recruited onto the HIV-1 proximal promoter in the infected shCTIP2 microglial cell line (Figure 6B column 2 compared to column 1) compared to the control cell line. Unexpectedly, knocking-down CTIP2 slightly increased LSD1 recruitment to the LTR (Figure 6B column 3). Moreover, this recruitment was correlated with an increased H3K4 trimethylation (Figure 6B column 4). These results suggest that LSD1 is required for CTIP2 recruitment to the HIV-1 proximal promoter.Figure 6.

Bottom Line: We have previously demonstrated that the cellular cofactor CTIP2 forces heterochromatin formation and HIV-1 gene silencing by recruiting HDAC and HMT activities at the integrated viral promoter.We show that recruitment of LSD1 at the HIV-1 proximal promoter is associated with both H3K4me3 and H3K9me3 epigenetic marks.Finally, our data suggest that LSD1-induced H3K4 trimethylation is linked to hSET1 recruitment at the integrated provirus.

View Article: PubMed Central - PubMed

Affiliation: University of Strasbourg, EA4438, Institute of parasitology, Strasbourg, France.

ABSTRACT
Microglial cells are the main HIV-1 targets in the central nervous system (CNS) and constitute an important reservoir of latently infected cells. Establishment and persistence of these reservoirs rely on the chromatin structure of the integrated proviruses. We have previously demonstrated that the cellular cofactor CTIP2 forces heterochromatin formation and HIV-1 gene silencing by recruiting HDAC and HMT activities at the integrated viral promoter. In the present work, we report that the histone demethylase LSD1 represses HIV-1 transcription and viral expression in a synergistic manner with CTIP2. We show that recruitment of LSD1 at the HIV-1 proximal promoter is associated with both H3K4me3 and H3K9me3 epigenetic marks. Finally, our data suggest that LSD1-induced H3K4 trimethylation is linked to hSET1 recruitment at the integrated provirus.

Show MeSH
Related in: MedlinePlus