Limits...
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.

Show MeSH
LSD1 associates with CTIP2 and co-localizes with CTIP2 and Tat within nuclear structures. (A) HEK 293 T cells were transfected with the pFLAG-CTIP2, the pFLAG-LSD1 expression vectors or the control pCDNA3-FLAG vector. Complexes immunoprecipitated with the anti-FLAG antibody were immunodetected for the presence of FLAG-CTIP2, FLAG-LSD1, endogenous LSD1 and CTIP2 proteins by western blot as indicated. (B–D) Microglial cells were transfected with pTat-GFP or/and pRFP-CTIP2 as indicated and accessed for endogenous LSD1 immunodetection with primary anti-LSD1 antibodies. The primary immunocomplexes were revealed by CY3- or CY5-labeled secondary antibodies. Mask columns show the co-localized staining.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3300010&req=5

gkr857-F5: LSD1 associates with CTIP2 and co-localizes with CTIP2 and Tat within nuclear structures. (A) HEK 293 T cells were transfected with the pFLAG-CTIP2, the pFLAG-LSD1 expression vectors or the control pCDNA3-FLAG vector. Complexes immunoprecipitated with the anti-FLAG antibody were immunodetected for the presence of FLAG-CTIP2, FLAG-LSD1, endogenous LSD1 and CTIP2 proteins by western blot as indicated. (B–D) Microglial cells were transfected with pTat-GFP or/and pRFP-CTIP2 as indicated and accessed for endogenous LSD1 immunodetection with primary anti-LSD1 antibodies. The primary immunocomplexes were revealed by CY3- or CY5-labeled secondary antibodies. Mask columns show the co-localized staining.

Mentions: Our data strongly suggest a functional cooperation between LSD1 and CTIP2. We therefore investigated whether these proteins could interact physically. To this end, we performed FLAG-targeted immunoprecipitation experiments with nuclear extracts from cells expressing FLAG-LSD1 or FLAG-CTIP2 proteins. As shown in Figure 5A, FLAG-CTIP2 and FLAG-LSD1 co-immunoprecipitated with endogenous LSD1 and CTIP2 proteins, respectively, arguing for a physical interaction between these two proteins. We next investigated whether LSD1 co-localizes with Tat as previously shown for CTIP2 (30). Cells transfected with a RFP-CTIP2 expressing vector in the presence or not of GFP-Tat were examined for endogenous LSD1 localization using confocal microscopy. Endogenous LSD1 expression was observed in both the cytoplasm and the nucleus (Figure 5B, pictures 4 and 6). As previously described (13), nuclear expression of CTIP2 harboured ball-like structures (Figure 5B, pictures 7 and 9). As shown in Figure 5C (pictures 5–8), LSD1 and CTIP2 co-localized in the CTIP2-induced nuclear structures (30), suggesting that CTIP2 relocated LSD1 into these structures. Interestingly, GFP-Tat expression relocated LSD1 from the cytoplasm to the nucleus (Figure 5C, pictures 1–4). Finally, observations of the concomitant expressions of RFP-CTIP2 and GFP-Tat revealed co-localization of both proteins with LSD1 in the nucleus (Figure 5D). Staining of genomic DNA are presented in Figure 5B. Altogether, these results support that CTIP2 and LSD1 interact physically and that LSD1 is re-localized by CTIP2 and Tat in dense sub-nuclear structures.Figure 5.


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 associates with CTIP2 and co-localizes with CTIP2 and Tat within nuclear structures. (A) HEK 293 T cells were transfected with the pFLAG-CTIP2, the pFLAG-LSD1 expression vectors or the control pCDNA3-FLAG vector. Complexes immunoprecipitated with the anti-FLAG antibody were immunodetected for the presence of FLAG-CTIP2, FLAG-LSD1, endogenous LSD1 and CTIP2 proteins by western blot as indicated. (B–D) Microglial cells were transfected with pTat-GFP or/and pRFP-CTIP2 as indicated and accessed for endogenous LSD1 immunodetection with primary anti-LSD1 antibodies. The primary immunocomplexes were revealed by CY3- or CY5-labeled secondary antibodies. Mask columns show the co-localized staining.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr857-F5: LSD1 associates with CTIP2 and co-localizes with CTIP2 and Tat within nuclear structures. (A) HEK 293 T cells were transfected with the pFLAG-CTIP2, the pFLAG-LSD1 expression vectors or the control pCDNA3-FLAG vector. Complexes immunoprecipitated with the anti-FLAG antibody were immunodetected for the presence of FLAG-CTIP2, FLAG-LSD1, endogenous LSD1 and CTIP2 proteins by western blot as indicated. (B–D) Microglial cells were transfected with pTat-GFP or/and pRFP-CTIP2 as indicated and accessed for endogenous LSD1 immunodetection with primary anti-LSD1 antibodies. The primary immunocomplexes were revealed by CY3- or CY5-labeled secondary antibodies. Mask columns show the co-localized staining.
Mentions: Our data strongly suggest a functional cooperation between LSD1 and CTIP2. We therefore investigated whether these proteins could interact physically. To this end, we performed FLAG-targeted immunoprecipitation experiments with nuclear extracts from cells expressing FLAG-LSD1 or FLAG-CTIP2 proteins. As shown in Figure 5A, FLAG-CTIP2 and FLAG-LSD1 co-immunoprecipitated with endogenous LSD1 and CTIP2 proteins, respectively, arguing for a physical interaction between these two proteins. We next investigated whether LSD1 co-localizes with Tat as previously shown for CTIP2 (30). Cells transfected with a RFP-CTIP2 expressing vector in the presence or not of GFP-Tat were examined for endogenous LSD1 localization using confocal microscopy. Endogenous LSD1 expression was observed in both the cytoplasm and the nucleus (Figure 5B, pictures 4 and 6). As previously described (13), nuclear expression of CTIP2 harboured ball-like structures (Figure 5B, pictures 7 and 9). As shown in Figure 5C (pictures 5–8), LSD1 and CTIP2 co-localized in the CTIP2-induced nuclear structures (30), suggesting that CTIP2 relocated LSD1 into these structures. Interestingly, GFP-Tat expression relocated LSD1 from the cytoplasm to the nucleus (Figure 5C, pictures 1–4). Finally, observations of the concomitant expressions of RFP-CTIP2 and GFP-Tat revealed co-localization of both proteins with LSD1 in the nucleus (Figure 5D). Staining of genomic DNA are presented in Figure 5B. Altogether, these results support that CTIP2 and LSD1 interact physically and that LSD1 is re-localized by CTIP2 and Tat in dense sub-nuclear structures.Figure 5.

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