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Gene silencing in HIV-1 latency by polycomb repressive group.

Kim HG, Kim KC, Roh TY, Park J, Jung KM, Lee JS, Choi SY, Kim SS, Choi BS - Virol. J. (2011)

Bottom Line: The expression levels for four core histone proteins (H2A, H2B, H3 and H4) and HDACs (HDAC1-8) in NCHA cells were not significantly different from those in their parental cells.In addition, more ubiquitylation at histone H2A was detected in NCHA cells.Our results suggest that tri-methylation of histone H3K27 and H2A ubiquitylation via polycomb group protein may play a crucial role in epigenetic silencing accounting for HIV-1 latency in NCHA cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of AIDS, Center for Immunology and Pathology, Korea National Institute of Health, Chung-buk, Republic of Korea.

ABSTRACT

Background: The persistence of latently human immunodeficiency virus-1 (HIV-1) infected cellular reservoirs in resting CD4+ T cells is a major obstacle to HIV-1 eradication. The detailed mechanism of HIV-1 latency remains unclear. We investigated histones and their post-translational modification associated with HIV-1 latency in novel HIV-1 latently infected cell lines established previously, NCHA cells.

Methods: To examine histones and their modification linked with HIV-1 latency, the expression profiles for core histone proteins and histone deacetylases (HDACs) in NCHA cells were characterized by RT-PCR, ELISA, and western blot. The levels of histone acetylation and methylation at histone H3 Lys9 (H3K9) and Lys27 (H3K27) in HIV-1 latently infected cells were analyzed by western blot and chromatin immunoprecipitation-sequencing (ChIP-seq).

Results: The expression levels for four core histone proteins (H2A, H2B, H3 and H4) and HDACs (HDAC1-8) in NCHA cells were not significantly different from those in their parental cells. Histone H3K9 and H3K27 acetylations in NCHA cells showed no difference in parental and NCHA cells, whereas the levels of di- and tri-methylation were increased in NCHA cells. The expression of EED which is a component of polycomb repressive complex 2 (PRC2), and BMI1 and RING2 which are constituents of PRC1, were upregulated in NCHA cells. In addition, more ubiquitylation at histone H2A was detected in NCHA cells.

Conclusions: Our results suggest that tri-methylation of histone H3K27 and H2A ubiquitylation via polycomb group protein may play a crucial role in epigenetic silencing accounting for HIV-1 latency in NCHA cells.

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Related in: MedlinePlus

Induction of HIV-1 gene silencing by polycomb group proteins. A) Expression levels of EED, one of PRC2 components, were measured by western blot. Thirty ug of nuclear extract were also used. The antibodies were indicated on the right. B) The expression levels of polycomb group proteins, BMI1 and RING2, were examined by western blot. The right panel showed coimmunoprecipitation experiment, immunoprecipitated with RING2 antibody and blotted with BMI1 antibody. C) Ubiquitylation levels of all cell lines were investigated by western blot. Using 30 ug of nuclear extract and antibody against ubiqutylated histone H2A. Anti-laminB was used as a loading control.
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Figure 3: Induction of HIV-1 gene silencing by polycomb group proteins. A) Expression levels of EED, one of PRC2 components, were measured by western blot. Thirty ug of nuclear extract were also used. The antibodies were indicated on the right. B) The expression levels of polycomb group proteins, BMI1 and RING2, were examined by western blot. The right panel showed coimmunoprecipitation experiment, immunoprecipitated with RING2 antibody and blotted with BMI1 antibody. C) Ubiquitylation levels of all cell lines were investigated by western blot. Using 30 ug of nuclear extract and antibody against ubiqutylated histone H2A. Anti-laminB was used as a loading control.

Mentions: To understand HIV-1 latency related with gene silencing by polycomb group protein complex, we examined the expression levels of polycomb group proteins in NCHA cells. Firstly, the expression level of EED, which is part of PRC2 that recognizes histone H3K27 and/or H3K9 residues for histone methylation, was investigated by western blot using an antibody specific to EED. As shown in Figure 3A, the expression of EED was upregulated in NCHA cells. Secondly, the expression levels of BMI1 and RING2, which are components of PRC1 that recognizes and binds to PRC2, were also increased in the nuclear fraction of NCHA cells compared with their parent cells and the interaction between these two proteins was confirmed by coimmunoprecipitation (Figure 3B). Finally, ubiquitylation levels of NCHA cells on histone H2A were also enriched compared with the parent cells (Figure 3C). These results suggest that PRC-mediated repressive H3K27 methylation and H2A ubiquitylation may contribute to HIV-1 gene silencing in NCHA cells.


Gene silencing in HIV-1 latency by polycomb repressive group.

Kim HG, Kim KC, Roh TY, Park J, Jung KM, Lee JS, Choi SY, Kim SS, Choi BS - Virol. J. (2011)

Induction of HIV-1 gene silencing by polycomb group proteins. A) Expression levels of EED, one of PRC2 components, were measured by western blot. Thirty ug of nuclear extract were also used. The antibodies were indicated on the right. B) The expression levels of polycomb group proteins, BMI1 and RING2, were examined by western blot. The right panel showed coimmunoprecipitation experiment, immunoprecipitated with RING2 antibody and blotted with BMI1 antibody. C) Ubiquitylation levels of all cell lines were investigated by western blot. Using 30 ug of nuclear extract and antibody against ubiqutylated histone H2A. Anti-laminB was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3094299&req=5

Figure 3: Induction of HIV-1 gene silencing by polycomb group proteins. A) Expression levels of EED, one of PRC2 components, were measured by western blot. Thirty ug of nuclear extract were also used. The antibodies were indicated on the right. B) The expression levels of polycomb group proteins, BMI1 and RING2, were examined by western blot. The right panel showed coimmunoprecipitation experiment, immunoprecipitated with RING2 antibody and blotted with BMI1 antibody. C) Ubiquitylation levels of all cell lines were investigated by western blot. Using 30 ug of nuclear extract and antibody against ubiqutylated histone H2A. Anti-laminB was used as a loading control.
Mentions: To understand HIV-1 latency related with gene silencing by polycomb group protein complex, we examined the expression levels of polycomb group proteins in NCHA cells. Firstly, the expression level of EED, which is part of PRC2 that recognizes histone H3K27 and/or H3K9 residues for histone methylation, was investigated by western blot using an antibody specific to EED. As shown in Figure 3A, the expression of EED was upregulated in NCHA cells. Secondly, the expression levels of BMI1 and RING2, which are components of PRC1 that recognizes and binds to PRC2, were also increased in the nuclear fraction of NCHA cells compared with their parent cells and the interaction between these two proteins was confirmed by coimmunoprecipitation (Figure 3B). Finally, ubiquitylation levels of NCHA cells on histone H2A were also enriched compared with the parent cells (Figure 3C). These results suggest that PRC-mediated repressive H3K27 methylation and H2A ubiquitylation may contribute to HIV-1 gene silencing in NCHA cells.

Bottom Line: The expression levels for four core histone proteins (H2A, H2B, H3 and H4) and HDACs (HDAC1-8) in NCHA cells were not significantly different from those in their parental cells.In addition, more ubiquitylation at histone H2A was detected in NCHA cells.Our results suggest that tri-methylation of histone H3K27 and H2A ubiquitylation via polycomb group protein may play a crucial role in epigenetic silencing accounting for HIV-1 latency in NCHA cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of AIDS, Center for Immunology and Pathology, Korea National Institute of Health, Chung-buk, Republic of Korea.

ABSTRACT

Background: The persistence of latently human immunodeficiency virus-1 (HIV-1) infected cellular reservoirs in resting CD4+ T cells is a major obstacle to HIV-1 eradication. The detailed mechanism of HIV-1 latency remains unclear. We investigated histones and their post-translational modification associated with HIV-1 latency in novel HIV-1 latently infected cell lines established previously, NCHA cells.

Methods: To examine histones and their modification linked with HIV-1 latency, the expression profiles for core histone proteins and histone deacetylases (HDACs) in NCHA cells were characterized by RT-PCR, ELISA, and western blot. The levels of histone acetylation and methylation at histone H3 Lys9 (H3K9) and Lys27 (H3K27) in HIV-1 latently infected cells were analyzed by western blot and chromatin immunoprecipitation-sequencing (ChIP-seq).

Results: The expression levels for four core histone proteins (H2A, H2B, H3 and H4) and HDACs (HDAC1-8) in NCHA cells were not significantly different from those in their parental cells. Histone H3K9 and H3K27 acetylations in NCHA cells showed no difference in parental and NCHA cells, whereas the levels of di- and tri-methylation were increased in NCHA cells. The expression of EED which is a component of polycomb repressive complex 2 (PRC2), and BMI1 and RING2 which are constituents of PRC1, were upregulated in NCHA cells. In addition, more ubiquitylation at histone H2A was detected in NCHA cells.

Conclusions: Our results suggest that tri-methylation of histone H3K27 and H2A ubiquitylation via polycomb group protein may play a crucial role in epigenetic silencing accounting for HIV-1 latency in NCHA cells.

Show MeSH
Related in: MedlinePlus