Involvement of Histone H3 Lysine 9 (H3K9) Methyltransferase G9a in the Maintenance of HIV-1 Latency and Its Reactivation by BIX01294

Abstract

Elucidating the mechanism of human immunodeficiency virus, type 1 (HIV-1) provirus transcriptional silencing in latently infected cells is crucial for understanding the pathophysiological process of HIV-1 infection. It is well established that hypoacetylation of histone proteins by histone deacetylases is involved in the maintenance of HIV-1 latency by repressing viral transcription. Although histone methylation is involved in the organization of chromatin domains and plays a central epigenetic role in gene expression, the role of histone methylation in the maintenance of HIV-1 latency has not been clarified. Here we present evidence that histone H3 Lys 9 (H3K9) methyltransferase G9a is responsible for transcriptional repression of HIV-1 by promoting repressive dimethylation at H3K9 and for the maintenance of viral latency. We observed that G9a significantly inhibited basal, as well as, the induced HIV-1 gene expression by tumor necrosis factor-␣ or Tat. Mutant G9a, however, lacking the SET domain responsible for the catalytic activity of histone methyltransferase, did not show such an effect. When G9a expression was knocked down by small interfering RNA, HIV-1 replication was augmented from cells transiently transfected with a full-length HIV-1 clone. Moreover, a specific inhibitor of G9a, BIX01294, could reactivate expression of HIV-1 from latently infected cells such as ACH-2 and OM10.1. Furthermore, chromatin immunoprecipitation assays revealed the presence of G9a and H3K9 dimethylation on nucleosome histones in the vicinity of the HIV-1 long terminal repeat promoter. These results suggest that G9a is responsible for the transcriptional quiescence of latent HIV-1 provirus and provide a molecular basis for understanding the mechanism by which HIV-1 latency is maintained.

Epigenetic Regulation of HIV-1 Latency in Astrocytes

“…However, other regulators reported to be relevant in T cells do not seem to play a significant role in astrocytes. For example, BIX01294 induced HIV transcription in latently infected Jurkat (ACH-2) and promyelocyte (OM10.1) cell lines (35). In astrocytes, this specific inhibitor of a monomethyltransferase did not augment LTR activity, but specific inhibition of SU(VAR)3-9, a well-known H3K9 trimethyltransferase, by chaetocin caused a dramatic increase in LTR activity as well as proviral transcription.…”

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

“…Similarly, Suv39H1, another HKMT responsible for H3K9me3, has been found to be recruited to latent HIV promoter in microglial cells [180] , while in a different T-cell latency model, G9a, another HKMT responsible for H3K9 methylation), was found to be a determinant of proviral latency [181] . Moreover, the HKMT LSD1 is also recruited to the HIV promoter by the cofactor CTIP2 and establishes H3K9me3 to promote latency, rather than activation [178] .…”

Progress and challenges in the use of latent HIV-1 reactivating agents

“…SUV39H1 has been implicated in maintaining HIV latency in microglial cells through its interaction with CTIP-2 and HP1 γ [53] . Similarly, G9α was also proposed to be involved in the establishment of HIV latency [19] . Chaetocin belongs to the 3-6-epi-dithio-diketopiperazines, which have been suggested to participate in immunosuppression [55] and anti-inflammation [56] .…”

“…H3K9 trimethylation (H3K9me3), mediated by Suv39H1 [53] ; H3K27 trimethylation (H3K27me3) [54] and H3K9 dimethylation (H3K9me2), mediated by G9α [19] lead to HIV transcriptional silencing in different cell models, including peripheral blood mononuclear cells (PBMCs) from infected individuals. Therefore, HMTIs could also be used to purge HIV-1 latent reservoirs.…”

“…Latency can be maintained at transcriptional levels (eg, lack of transcriptional activators, condensed chromatin structure) or at post-transcriptional levels (eg, inhibition of nuclear RNA transport and inhibition of translation by microRNA), and it is generally assumed that maintenance at the transcriptional level is the case for the majority of latently infected cells. Molecular mechanisms targeting transcription consist of both trans-effects and cis-effects, including: (1) the chromatin environment around the integration site and transcriptional interference [17,18] ; (2) the epigenetic control of the HIV-1 promoter or the presence of repressive nucleosomes (nuc-0 and nuc-1) [19,20] ; (3) lack of crucial transcriptional factors, such as NF-κB or nuclear factor of activated T cells (NFAT) [21] , sub-optimal concentrations of transcriptional activators such as Tat [22,23] or the presence of transcriptional suppressors such as CTIP2 (COUPTF Interacting Protein 2) [24,25] , DSIF (DRB-Sensitivity Inducing Factor) [26] and NELF (Negative Elongation Factor) [27] ; (4) the sequestration of positive transcriptional elongation factor b (P-TEFb) in its inactive form with HEXIM [hexamethylene bisacetamide (HMBA)-induced protein 1] and 7SK snRNA (small nuclear RNA) [28][29][30] .…”