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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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LSD1 association with the HIV-1 proximal promoter induces local trimethylation of histone 3 lysines 4 and 9. (A) ChIP experiments were performed on microglial cells transfected with the pNL-4.3 provirus 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 Nuc-1 region. Specific enrichments were calculated relative to the control IgG and relative enrichments in the context of LSD1 over-expression or depletion were expressed relative to the value obtained with the pcDNA3-FLAG or the pshRNA-control vectors, respectively. (B) Mock-treated and PMA-treated U1 cells were subjected to ChIP experiments with the indicated antibodies. Specific enrichments in the HIV-1 proximal promoter were quantified by real-time PCR targeting the Nuc-1 region. The amounts of immunoprecipitated material were normalized to the input DNA and presented relative to the non specific IgG precipitation. (C) pcDNA3 and pFLAG-LSD1 transfected microglial cells were subjected to ChIP experiments with the anti-LSD1 antibody. Specific enrichment of the NF-kB and the Nuc-1 regions of the promoter and the Gag and Vpr intragenic regions are presented relative to the non specific enrichment obtained with the control IgG setted at 1. (D) pshRNA-control and pshRNA-LSD1 transfected microglial cells were subjected to ChIP experiments with the indicated antibodies. Specific enrichment of the SCN2A2, CEBPα and GAPDH promoters are indicated relative to the control IgG.
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gkr857-F2: LSD1 association with the HIV-1 proximal promoter induces local trimethylation of histone 3 lysines 4 and 9. (A) ChIP experiments were performed on microglial cells transfected with the pNL-4.3 provirus 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 Nuc-1 region. Specific enrichments were calculated relative to the control IgG and relative enrichments in the context of LSD1 over-expression or depletion were expressed relative to the value obtained with the pcDNA3-FLAG or the pshRNA-control vectors, respectively. (B) Mock-treated and PMA-treated U1 cells were subjected to ChIP experiments with the indicated antibodies. Specific enrichments in the HIV-1 proximal promoter were quantified by real-time PCR targeting the Nuc-1 region. The amounts of immunoprecipitated material were normalized to the input DNA and presented relative to the non specific IgG precipitation. (C) pcDNA3 and pFLAG-LSD1 transfected microglial cells were subjected to ChIP experiments with the anti-LSD1 antibody. Specific enrichment of the NF-kB and the Nuc-1 regions of the promoter and the Gag and Vpr intragenic regions are presented relative to the non specific enrichment obtained with the control IgG setted at 1. (D) pshRNA-control and pshRNA-LSD1 transfected microglial cells were subjected to ChIP experiments with the indicated antibodies. Specific enrichment of the SCN2A2, CEBPα and GAPDH promoters are indicated relative to the control IgG.

Mentions: To investigate whether LSD1 is recruited at the HIV-1 promoter in vivo, we performed ChIP assays using microglial cells transfected with the pNL-4.3 provirus. Over-expression of LSD1 was associated to an increase of H3K9 trimethylation (H3K9me3) and more surprisingly to an increase of H3K4 trimethylation (H3K4me3) (Figure 2A columns 3 and 4) in the proximal region of the HIV-1 promoter. In agreement, knocking-down endogenous LSD1 expression disfavoured H3K9 and H3K4 trimethylation (Figure 2A columns 3 and 4). Interestingly, knocking-down LSD1 in HIV-1 transfected microglial cells was associated with a strong increase of H3 global acetylation level (Figure 2A column 6) and with a stable amount of H3 histones associated to the viral promoter (Figure 2A column 5). Furthermore, knocking-down LSD1 strongly increased the recruitment of the RNA pol II to the HIV-1 promoter, thereby confirming the activated status of the viral promoter (Figure 2A column 7). From these results we speculated that HIV-1 reactivation in latently infected U1 cells could be associated with a release of LSD1 and a concomitant decrease of H3K9 and H3K4 trimethylation levels at the HIV-1 promoter. To test this hypothesis, histone methylation marks and LSD1 recruitment at the HIV-1 promoter were monitored in the latently infected U1 cell line after activation of viral gene transcription. As shown in Figure 2B, PMA (phorbol-12-myristate 13-acetate) treatment induced a release of the endogenous LSD1 from the viral promoter (column 2). Moreover, this phenomenon was associated with decreased trimethylation levels of H3K4 and H3K9 (columns 3 and 4 from Figure 2B, respectively). As expected, the release of the endogenous LSD1 following PMA treatment of the latently infected U1 cell line was associated with an increase of the global histone H3 acetylation level and with an increased RNA pol II recruitment to the HIV-1 promoter (Figure 2B columns 6 and 7). To verify whether LSD1 is specifically located at the promoter region of the HIV-1 genome, we performed several ChIP experiments with additional sets of primers hybridizing in adjacent regions of the viral genome. As shown in Figure 2C, LSD1 was only associated to the proximal promoter region (columns 1 and 2) and not with adjacent regions such as intragenic Gag or Vpr regions (columns 3 and 4). Moreover, the epigenetic marks associated with the loss of LSD1 we described above were also observed with other LSD1 regulated genes since we observed the same events with the LSD1-regulated gene CEBP alpha (Figure 2D, columns 5, 6 and 7), as previously described (35). In accordance with the literature (36), we showed that knock-down of LSD1 is correlated with an increase of H3K4me3 in the promoter region of LSD1 regulated genes such as SCN1A, SCN3A (data not shown) and SCN2A2 (Figure 2D columns 2, 3 and 4), suggesting that these LSD1-sensitive genes may be regulated by molecular mechanisms linked to the previously described enzymatic activity of LSD1. As a control, we showed that LSD1 is not associated to the promoter of the LSD1-insensitive gene GAPDH (Figure 2D). In addition, H3K4 trimethylation level at the GAPDH promoter was not sensitive to the modulation of LSD1 expression.Figure 2.


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)

LSD1 association with the HIV-1 proximal promoter induces local trimethylation of histone 3 lysines 4 and 9. (A) ChIP experiments were performed on microglial cells transfected with the pNL-4.3 provirus 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 Nuc-1 region. Specific enrichments were calculated relative to the control IgG and relative enrichments in the context of LSD1 over-expression or depletion were expressed relative to the value obtained with the pcDNA3-FLAG or the pshRNA-control vectors, respectively. (B) Mock-treated and PMA-treated U1 cells were subjected to ChIP experiments with the indicated antibodies. Specific enrichments in the HIV-1 proximal promoter were quantified by real-time PCR targeting the Nuc-1 region. The amounts of immunoprecipitated material were normalized to the input DNA and presented relative to the non specific IgG precipitation. (C) pcDNA3 and pFLAG-LSD1 transfected microglial cells were subjected to ChIP experiments with the anti-LSD1 antibody. Specific enrichment of the NF-kB and the Nuc-1 regions of the promoter and the Gag and Vpr intragenic regions are presented relative to the non specific enrichment obtained with the control IgG setted at 1. (D) pshRNA-control and pshRNA-LSD1 transfected microglial cells were subjected to ChIP experiments with the indicated antibodies. Specific enrichment of the SCN2A2, CEBPα and GAPDH promoters are indicated relative to the control IgG.
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gkr857-F2: LSD1 association with the HIV-1 proximal promoter induces local trimethylation of histone 3 lysines 4 and 9. (A) ChIP experiments were performed on microglial cells transfected with the pNL-4.3 provirus 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 Nuc-1 region. Specific enrichments were calculated relative to the control IgG and relative enrichments in the context of LSD1 over-expression or depletion were expressed relative to the value obtained with the pcDNA3-FLAG or the pshRNA-control vectors, respectively. (B) Mock-treated and PMA-treated U1 cells were subjected to ChIP experiments with the indicated antibodies. Specific enrichments in the HIV-1 proximal promoter were quantified by real-time PCR targeting the Nuc-1 region. The amounts of immunoprecipitated material were normalized to the input DNA and presented relative to the non specific IgG precipitation. (C) pcDNA3 and pFLAG-LSD1 transfected microglial cells were subjected to ChIP experiments with the anti-LSD1 antibody. Specific enrichment of the NF-kB and the Nuc-1 regions of the promoter and the Gag and Vpr intragenic regions are presented relative to the non specific enrichment obtained with the control IgG setted at 1. (D) pshRNA-control and pshRNA-LSD1 transfected microglial cells were subjected to ChIP experiments with the indicated antibodies. Specific enrichment of the SCN2A2, CEBPα and GAPDH promoters are indicated relative to the control IgG.
Mentions: To investigate whether LSD1 is recruited at the HIV-1 promoter in vivo, we performed ChIP assays using microglial cells transfected with the pNL-4.3 provirus. Over-expression of LSD1 was associated to an increase of H3K9 trimethylation (H3K9me3) and more surprisingly to an increase of H3K4 trimethylation (H3K4me3) (Figure 2A columns 3 and 4) in the proximal region of the HIV-1 promoter. In agreement, knocking-down endogenous LSD1 expression disfavoured H3K9 and H3K4 trimethylation (Figure 2A columns 3 and 4). Interestingly, knocking-down LSD1 in HIV-1 transfected microglial cells was associated with a strong increase of H3 global acetylation level (Figure 2A column 6) and with a stable amount of H3 histones associated to the viral promoter (Figure 2A column 5). Furthermore, knocking-down LSD1 strongly increased the recruitment of the RNA pol II to the HIV-1 promoter, thereby confirming the activated status of the viral promoter (Figure 2A column 7). From these results we speculated that HIV-1 reactivation in latently infected U1 cells could be associated with a release of LSD1 and a concomitant decrease of H3K9 and H3K4 trimethylation levels at the HIV-1 promoter. To test this hypothesis, histone methylation marks and LSD1 recruitment at the HIV-1 promoter were monitored in the latently infected U1 cell line after activation of viral gene transcription. As shown in Figure 2B, PMA (phorbol-12-myristate 13-acetate) treatment induced a release of the endogenous LSD1 from the viral promoter (column 2). Moreover, this phenomenon was associated with decreased trimethylation levels of H3K4 and H3K9 (columns 3 and 4 from Figure 2B, respectively). As expected, the release of the endogenous LSD1 following PMA treatment of the latently infected U1 cell line was associated with an increase of the global histone H3 acetylation level and with an increased RNA pol II recruitment to the HIV-1 promoter (Figure 2B columns 6 and 7). To verify whether LSD1 is specifically located at the promoter region of the HIV-1 genome, we performed several ChIP experiments with additional sets of primers hybridizing in adjacent regions of the viral genome. As shown in Figure 2C, LSD1 was only associated to the proximal promoter region (columns 1 and 2) and not with adjacent regions such as intragenic Gag or Vpr regions (columns 3 and 4). Moreover, the epigenetic marks associated with the loss of LSD1 we described above were also observed with other LSD1 regulated genes since we observed the same events with the LSD1-regulated gene CEBP alpha (Figure 2D, columns 5, 6 and 7), as previously described (35). In accordance with the literature (36), we showed that knock-down of LSD1 is correlated with an increase of H3K4me3 in the promoter region of LSD1 regulated genes such as SCN1A, SCN3A (data not shown) and SCN2A2 (Figure 2D columns 2, 3 and 4), suggesting that these LSD1-sensitive genes may be regulated by molecular mechanisms linked to the previously described enzymatic activity of LSD1. As a control, we showed that LSD1 is not associated to the promoter of the LSD1-insensitive gene GAPDH (Figure 2D). In addition, H3K4 trimethylation level at the GAPDH promoter was not sensitive to the modulation of LSD1 expression.Figure 2.

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