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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 favours hSET1 and WDR5 recruitment at the HIV-1 proximal promoter. (A) ChIP experiments were performed on HEK 293 T cells transfected with the HIV-1 LTR-LUC episomal vector in the presence 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 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. Specific enrichments in the HIV-1 proximal promoter region were quantified by real-time PCR. (B) HEK 293 T cells were transfected with the pLTR-LUC (1–789) or with the pLTR-LUC (1–789) mut Sp1 vector 48 h before being subjected to ChIP experiments with the indicated antibodies. Input and immunoprecipitated DNAs were quantified by real-time PCR using primers targeting the Sp1-binding sites region of the HIV-1 promoter. The amounts of immunoprecipitated material were normalized to the input DNA and presented relative to the non specific control IgG. (C) Microglial cells were infected with the VSV-pseudotyped pNL4.3-Env virus 24 h before being subjected to ChIP experiments with the indicated antibodies. Specific enrichments in the HIV-1 proximal promoter were quantified by real-time PCR. Enrichments are presented relative to the non specific IgG values set at 1.
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gkr857-F7: LSD1 favours hSET1 and WDR5 recruitment at the HIV-1 proximal promoter. (A) ChIP experiments were performed on HEK 293 T cells transfected with the HIV-1 LTR-LUC episomal vector in the presence 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 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. Specific enrichments in the HIV-1 proximal promoter region were quantified by real-time PCR. (B) HEK 293 T cells were transfected with the pLTR-LUC (1–789) or with the pLTR-LUC (1–789) mut Sp1 vector 48 h before being subjected to ChIP experiments with the indicated antibodies. Input and immunoprecipitated DNAs were quantified by real-time PCR using primers targeting the Sp1-binding sites region of the HIV-1 promoter. The amounts of immunoprecipitated material were normalized to the input DNA and presented relative to the non specific control IgG. (C) Microglial cells were infected with the VSV-pseudotyped pNL4.3-Env virus 24 h before being subjected to ChIP experiments with the indicated antibodies. Specific enrichments in the HIV-1 proximal promoter were quantified by real-time PCR. Enrichments are presented relative to the non specific IgG values set at 1.

Mentions: The epigenetic mark H3K4me3 has been shown to be associated with LSD1 recruitment (20,21). This association results from the interaction of LSD1 with a methyltransferase complex containing WDR5 and hSET1 (20,21). ChIP experiments performed with cells over-expressing LSD1 confirmed an increased recruitment of hSET1 and WDR5, two members of the hCOMPASS complex (Figure 7A, blue columns 4 and 5) to the HIV-1 promoter, together with an increased H3K4 trimethylation. Inversely, knocking-down endogenous LSD1 decreased hSET1 and WDR5 association to the viral promoter and H3K4 trimethylation (Figure 7A pink columns). ChIP experiments performed with the wt-LTR-Luc or the Sp1-mutated LTR-Luc reporter constructs further confirmed that hSET1, WDR5 and LSD1 are recruited concomitantly to the proximal Sp1-binding sites of the viral promoter. Indeed, the abrogated association of LSD1 to the Sp1-mutated LTR (Figure 7B lane 2 column versus column) correlated with a reduced recruitment of both hSET1 and WDR5 (Figure 7B, columns 3 and 4). From these results, we hypothesized that HIV-1 reactivation in microglial cells could be associated with a release of LSD1 and an alongside reduced recruitment of hSET1 and WDR5 to the HIV-1 promoter. In agreement with these results, PMA treatment released LSD1, WDR5 and hSET1 from the viral promoter of HIV-1 infected cells (Figure 7C, columns 2, 4 and 5) and decreased H3K4 trimethylation (Figure 7C column 3). Taken together, our data suggest that LSD1-associated increase of H3K4 trimethylation at the HIV-1 proximal promoter region might be linked to hSET1 and WDR5 recruitment.Figure 7.


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 favours hSET1 and WDR5 recruitment at the HIV-1 proximal promoter. (A) ChIP experiments were performed on HEK 293 T cells transfected with the HIV-1 LTR-LUC episomal vector in the presence 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 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. Specific enrichments in the HIV-1 proximal promoter region were quantified by real-time PCR. (B) HEK 293 T cells were transfected with the pLTR-LUC (1–789) or with the pLTR-LUC (1–789) mut Sp1 vector 48 h before being subjected to ChIP experiments with the indicated antibodies. Input and immunoprecipitated DNAs were quantified by real-time PCR using primers targeting the Sp1-binding sites region of the HIV-1 promoter. The amounts of immunoprecipitated material were normalized to the input DNA and presented relative to the non specific control IgG. (C) Microglial cells were infected with the VSV-pseudotyped pNL4.3-Env virus 24 h before being subjected to ChIP experiments with the indicated antibodies. Specific enrichments in the HIV-1 proximal promoter were quantified by real-time PCR. Enrichments are presented relative to the non specific IgG values set at 1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3300010&req=5

gkr857-F7: LSD1 favours hSET1 and WDR5 recruitment at the HIV-1 proximal promoter. (A) ChIP experiments were performed on HEK 293 T cells transfected with the HIV-1 LTR-LUC episomal vector in the presence 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 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. Specific enrichments in the HIV-1 proximal promoter region were quantified by real-time PCR. (B) HEK 293 T cells were transfected with the pLTR-LUC (1–789) or with the pLTR-LUC (1–789) mut Sp1 vector 48 h before being subjected to ChIP experiments with the indicated antibodies. Input and immunoprecipitated DNAs were quantified by real-time PCR using primers targeting the Sp1-binding sites region of the HIV-1 promoter. The amounts of immunoprecipitated material were normalized to the input DNA and presented relative to the non specific control IgG. (C) Microglial cells were infected with the VSV-pseudotyped pNL4.3-Env virus 24 h before being subjected to ChIP experiments with the indicated antibodies. Specific enrichments in the HIV-1 proximal promoter were quantified by real-time PCR. Enrichments are presented relative to the non specific IgG values set at 1.
Mentions: The epigenetic mark H3K4me3 has been shown to be associated with LSD1 recruitment (20,21). This association results from the interaction of LSD1 with a methyltransferase complex containing WDR5 and hSET1 (20,21). ChIP experiments performed with cells over-expressing LSD1 confirmed an increased recruitment of hSET1 and WDR5, two members of the hCOMPASS complex (Figure 7A, blue columns 4 and 5) to the HIV-1 promoter, together with an increased H3K4 trimethylation. Inversely, knocking-down endogenous LSD1 decreased hSET1 and WDR5 association to the viral promoter and H3K4 trimethylation (Figure 7A pink columns). ChIP experiments performed with the wt-LTR-Luc or the Sp1-mutated LTR-Luc reporter constructs further confirmed that hSET1, WDR5 and LSD1 are recruited concomitantly to the proximal Sp1-binding sites of the viral promoter. Indeed, the abrogated association of LSD1 to the Sp1-mutated LTR (Figure 7B lane 2 column versus column) correlated with a reduced recruitment of both hSET1 and WDR5 (Figure 7B, columns 3 and 4). From these results, we hypothesized that HIV-1 reactivation in microglial cells could be associated with a release of LSD1 and an alongside reduced recruitment of hSET1 and WDR5 to the HIV-1 promoter. In agreement with these results, PMA treatment released LSD1, WDR5 and hSET1 from the viral promoter of HIV-1 infected cells (Figure 7C, columns 2, 4 and 5) and decreased H3K4 trimethylation (Figure 7C column 3). Taken together, our data suggest that LSD1-associated increase of H3K4 trimethylation at the HIV-1 proximal promoter region might be linked to hSET1 and WDR5 recruitment.Figure 7.

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