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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 represses HIV-1 gene transcription and viral replication. (A) Microglial cells were transfected with the pNL-4.3 and the indicated vectors. Culture supernatants were analysed for p24 Gag contents 48 h post-transfection. (B and C) Microglial cells were transfected with the episomal LTR-LUC and the indicated vectors. DNA amounts were normalized in all transfection assays with pshRNA-Control or pcDNA3-FLAG control vectors. Luciferase activities were measured 2 days post-transfection and expressed relative to the value obtained with episomal LTR-LUC alone. (A and C) The knock-down efficiency of sh-RNA constructs (versus sh-control) has been controlled by western blot analysis.
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gkr857-F1: LSD1 represses HIV-1 gene transcription and viral replication. (A) Microglial cells were transfected with the pNL-4.3 and the indicated vectors. Culture supernatants were analysed for p24 Gag contents 48 h post-transfection. (B and C) Microglial cells were transfected with the episomal LTR-LUC and the indicated vectors. DNA amounts were normalized in all transfection assays with pshRNA-Control or pcDNA3-FLAG control vectors. Luciferase activities were measured 2 days post-transfection and expressed relative to the value obtained with episomal LTR-LUC alone. (A and C) The knock-down efficiency of sh-RNA constructs (versus sh-control) has been controlled by western blot analysis.

Mentions: The function of LSD1 in HIV-1 infected cells was investigated by using an LSD1 knock-down strategy. We co-transfected microglial cells with a complete HIV-1 infectious provirus (pNL-4.3) and with or without a shLDS1 expressing vector. The efficiency of the knock-down of LSD1 was checked by western blot (Figure 1A). As shown in Figure 1A, the knock-down of LSD1 was associated with a 6-fold increase in p24 production, which argues in favour of a repressive role of LSD1 in HIV-1 replication. We next investigated whether LSD1 has a direct impact on transcription of the HIV-1 genes since this protein is involved in the transcriptional regulation of many cellular genes. Microglial cells were transfected with the episomal LTR-Luc vector with or without the shLSD1 expressing vector in the absence (Figure 1B) or presence (Figure 1C) of Tat. In the absence of Tat, LSD1 repressed LTR transcriptional activity in a dose-dependent manner (Figure 1B columns 2 and 3). When Tat was expressed together with the shLDS1 vector, we observed a synergistic activation of LTR-driven transcription (Figure 1C column 4 compared to columns 2 and 3). Thus LSD1 inhibits HIV-1 replication as a result of transcriptional repression occurring at both the early Tat-independent and the late Tat-dependent steps.Figure 1.


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 represses HIV-1 gene transcription and viral replication. (A) Microglial cells were transfected with the pNL-4.3 and the indicated vectors. Culture supernatants were analysed for p24 Gag contents 48 h post-transfection. (B and C) Microglial cells were transfected with the episomal LTR-LUC and the indicated vectors. DNA amounts were normalized in all transfection assays with pshRNA-Control or pcDNA3-FLAG control vectors. Luciferase activities were measured 2 days post-transfection and expressed relative to the value obtained with episomal LTR-LUC alone. (A and C) The knock-down efficiency of sh-RNA constructs (versus sh-control) has been controlled by western blot analysis.
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gkr857-F1: LSD1 represses HIV-1 gene transcription and viral replication. (A) Microglial cells were transfected with the pNL-4.3 and the indicated vectors. Culture supernatants were analysed for p24 Gag contents 48 h post-transfection. (B and C) Microglial cells were transfected with the episomal LTR-LUC and the indicated vectors. DNA amounts were normalized in all transfection assays with pshRNA-Control or pcDNA3-FLAG control vectors. Luciferase activities were measured 2 days post-transfection and expressed relative to the value obtained with episomal LTR-LUC alone. (A and C) The knock-down efficiency of sh-RNA constructs (versus sh-control) has been controlled by western blot analysis.
Mentions: The function of LSD1 in HIV-1 infected cells was investigated by using an LSD1 knock-down strategy. We co-transfected microglial cells with a complete HIV-1 infectious provirus (pNL-4.3) and with or without a shLDS1 expressing vector. The efficiency of the knock-down of LSD1 was checked by western blot (Figure 1A). As shown in Figure 1A, the knock-down of LSD1 was associated with a 6-fold increase in p24 production, which argues in favour of a repressive role of LSD1 in HIV-1 replication. We next investigated whether LSD1 has a direct impact on transcription of the HIV-1 genes since this protein is involved in the transcriptional regulation of many cellular genes. Microglial cells were transfected with the episomal LTR-Luc vector with or without the shLSD1 expressing vector in the absence (Figure 1B) or presence (Figure 1C) of Tat. In the absence of Tat, LSD1 repressed LTR transcriptional activity in a dose-dependent manner (Figure 1B columns 2 and 3). When Tat was expressed together with the shLDS1 vector, we observed a synergistic activation of LTR-driven transcription (Figure 1C column 4 compared to columns 2 and 3). Thus LSD1 inhibits HIV-1 replication as a result of transcriptional repression occurring at both the early Tat-independent and the late Tat-dependent steps.Figure 1.

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