Gene silencing in HIV-1 latency by polycomb repressive group

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.

Figure 1

Histone and HDAC profiles in HIV-1 latently infected A3.01derived cells. A) Expression levels of four core histone proteins were investigated by western blot. Thirty ug of nuclear extract were loaded and immunoblotted using antibodies specific to H2A, H2B, H3 and H4. Anti-laminB was used as a loading control. B) The acetylation levels of four core histone proteins were quantified using the H2A-, H2B-, H3- and H4 -PathScan acetylated histone sandwich ELISA kit. Error bar represent the S.D. from the mean. C) mRNA levels of HDACs in each cell line were measured with reverse transcriptase PCR (RT-PCR). GAPDH was used as a control. D) Protein levels of HDAC1, HDAC2 and HDAC3 were investigated by western blot using 30 ug of nuclear extract. Anti-laminB was used as a loading control.

Figure 2

Gene silencing by polycomb group-proteins in NCHA cells for HIV-1 latency. A) Methylation levels of histone H3 on the specific lysine residues were measured by western blot. In each lane, 30 ug of nuclear extract were used and the antibodies were indicated on the right. Anti-laminB was used as a loading control. B) Global distributions of histone modifications were examined by ChIP-Seq. Promoters were the regions of 1 kb upstream and downstream of the transcription start site, and gene body was defined as the length from 1 kb downstream of the transcription start site to transcription end site and the remaining regions were intergenic regions. C) The H3K9me3 profile was shown on HIV-1 provirus genome. The X axis represents the HIV-1 genomic position and the Y axis indicates the normalized tag count which is calculated by total tag numbers detected in a 1 kb window divided by total sequenced tag number.

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.