Epigenetic regulation of polyomavirus JC

JC virus (JCV) is a human neurotropic polyomavirus and is the causative agent of progressive multifocal leukoencephalopathy, PML, which is a fatal demyelinating disease of the brain that involves the cytolytic destruction of oligodendrocytes by JCV replication. PML lesions are multiple foci of myelin loss, which cause debilitating neurological symptoms and are areas of demyelination in the brain containing oligodendrocytes with viral nuclear inclusion bodies and bizarre astrocytes, which are also productively infected by JCV. The common underlying feature of PML is a severe weakening of the immune system, especially HIV-1/AIDS. Even after the introduction of combination anti-retroviral therapies (cART), PML still remains a problematic disorder associated with HIV-1/AIDS [1]. Despite the rarity of PML, the high prevalence (66-92%) of antibodies in human sera against JCV indicates that exposure to the virus is very common and begins in childhood and continues into middle age [reviewed in [2]. After the primary infection virus persists in a latent state and further sequelae only occur in people with severe immunosuppression where viral reactivation leads to PML. Many important aspects of the JCV life cycle and the pathogenesis of PML remain unclear including the nature of the latent state, the mechanisms whereby it is maintained and the regulation of restoration of viral transcription/replication when virus reactivates and causes PML.

JCV is a circular double-stranded DNA virus of the Polyomaviridae family [3] that was isolated in 1971 from the brain of a patient with PML [4]. It has two protein coding regions, which coordinate the viral life cycle: the early and late coding regions. These are transcribed in opposite directions starting from the Non-Coding Control Region (NCCR), which lies between them [5]. The NCCR functions as the promoter for both the early and late coding regions and also contains the viral origin of DNA replication. A variety of cellular transcription factors, some being glial cell-specific and others ubiquitous, bind and regulate the NCCR and these cellular factors, together with the viral early gene product large T-antigen (T-Ag) facilitate the JCV life cycle [reviewed in [6]. For example, we have described a site (the KB element) that is located on the early side of the origin of replication and binds the transcription factors NF-κB and C/EBPβ [7] as well as NFAT4 [8]. Since these transcription factors are regulated by signal transduction pathways that are controlled by extracellular cytokines, we have suggested that control of the latency/reactivation of JCV may be regulated by cytokines acting through the KB element. We have found that cytokines including TNF-α and IL-1β stimulate JCV early and late transcription and that this is mediated through the KB element [9].

In addition to the binding of transcription factors, the expression of genes can be regulated by post-translational covalent modifications of chromatin itself, which is known as epigenetic regulation. DNA within the cell nucleus, including the circular episomal viral DNA in JCV-infected cells, is packaged into a dynamic complex of DNA and histones as well as other non-histone proteins and RNA. Changes in chromatin structure can regulate the degree of compactness of chromatin and its availability to the transcriptional machinery, thus modulating transcription of chromatin in vivo [10, 11]. A complex series of regulatory signals orchestrate the epigenetic status of chromatin including DNA methylation and histone acetylation. The association of DNA methylation with the silencing of gene expression is a well-established mechanism of eukaryotic transcriptional regulation [12]. Methylation of DNA is a post-replication process whereby cytosine residues in the dinucleotide sequence 5’-CG-3’ (CpG) are methylated. Experimentally, DNA methylation can be inhibited by 5-azacytidine (AZA), which can activate transcription of genes whose expression is suppressed by methylation. Typically, eukaryote genes have CpG islands rich in the CpGs located near the promoter [13]. However, the genome of JCV (strain Mad-1, [5]) is remarkably lacking in CpGs and contains only 6 CpGs in the 394 bp NCCR. In contrast to DNA methylation, histone acetylation is associated with transcriptional activation. Dynamic reversible acetylation of histones is a key part of the transcriptional process [10, 14]. Acetylation of histones is catalyzed by histone acetyltransferase enzymes (HAT) and removal by histone deacetylases (HDAC). Both HAT and HDAC act not only on histones but also on nonhistone proteins including certain transcription factors. Experimentally, HDACs can be inhibited by trichostatin A (TSA) or sodium butyrate (SB), which can activate gene expression by increasing histone acetylation.

In this study, we investigate a role for epigenetic modifications in the regulation of Mad-1 JCV. We found that TSA and SB but not AZA robustly stimulated JCV transcription. This effect was mediated by the JCV NCCR KB element, was additive with cotransfected NF-κB p65 and was inhibited by p65 siRNA. Thus JCV is regulated epigenetically by acetylation events involving NF-κB p65 operating at the KB element of the control region.

The state of latency/persistence is of central importance to the life cycle of JCV and when it is disrupted, the pathological events leading to PML ensue. While the site and molecular nature of viral latency/persistence are poorly understood, JCV is thought to persist in a number of organs including the kidney, bone marrow and brain [reviewed in [2, 20]. In the kidney, JCV has an archetype NCCR configuration [21] and is likely undergoing active asymptomatic replication at a low level or episodically in the epithelial cells of the kidney tubules as shown by continuous shedding of the same strains of JCV [22]. On the other hand, JCV detected in the brain has a neurotropic configuration [23, 24] and is likely to be within viral chromatin in a nonreplicating, nontranscribed state since JCV DNA can be detected but not expression of viral proteins [23]. Since, transcription of a given piece of DNA can be regulated by epigenetic modification to chromatin, we surmised that such regulation may occur for JCV.

Post-translational covalent modifications of chromatin, i.e., epigenetic changes, determine the openness of chromatin conformation and availability to the transcriptional apparatus, which can determine the level of gene expression of a region of DNA within the cell nucleus [10, 11]. The major determinants of the epigenetic status of chromatin are DNA methylation and histone acetylation. Thus, in our initial experiments, we inhibited DNA methylation with AZA or enhanced acetylation of histones with HDACi, which would be expected to increase transcription if it was restrained DNA methylation or lack of histone acetylation respectively. We found that both JCV early and late transcription were greatly stimulated by the HDACi TSA and SB but not by AZA (Figure 1) indicating the importance of protein acetylation in JCV regulation but no involvement of DNA methylation. Since typically, promoter regulation involves large promoter-proximal CpG islands [13] and JCV contains only 6 CpGs in the NCCR [5], this is perhaps not surprising. Importantly, since our JCV early and late reporter constructs contained many extraneous CpGs from the luciferase and vector regions of the plasmids, these may potentially interfere with our analyses and so it was important to verify our data using a CpG-free reporter plasmid background. We used plasmids based on pCpGL-basic, which completely lacks CpG dinucleotides [15] and these constructs gave essentially similar data (Figure 2A). For some earlier studies, we had generated some stably transfected JCV early and late clonal reporter cell lines from TC620 cells [9]. When we investigated the effects of the epigenetic reagents, we also obtained the same results (Figure 2B & C) as for the transient transfection experiments.

Analysis of mutant JCV promoters implicated the KB element of the NCCR in mediating the effect of histone deacetylation inhibition since mutations in the element (m1 and m2) abrogated the effect (Figure 3A) and the effect of p65 on a heterologous promoter containing the KB element was potentiated by sodium butyrate (Figure 3B). Further, the stimulation of JCV late transcription by p65 was potentiated by TSA (Figure 4A) and siRNA to p65 inhibited the stimulatory effect of TSA on early (Figure 5A) and late (Figure 5B) transcription. It should be noted that there is one other report of JCV activation by HDACi I the literature [25] using the MH1 strain of JCV. In this report, deletion and site-directed mutational analyses of TSA-mediated activation indicated the importance of the enhancer region and an Sp1 binding site upstream of the TATA box, which is not present in the Mad-1 JCV NCCR [26]. Thus, it is possible that the mechanism of transcriptional induction by TSA may vary between strains of JCV.

From the gel shift data in Figure 6, it can be seen that p65 binding is induced by p65 overexpression (as expected), or TNF-α treatment, which activates the NF-κB signaling pathway (also expected) or by TSA. The TSA induction indicates that increased acetylation of p65, histones in the chromatin at the NF-κB site or both is sufficient to recruit NF-κB binding. By performing chromatin immune precipitation (ChIP) assays, we were able to show that TSA induced acetylation of histone H3 on lysine-9 within the chromatin of the JCV NCCR (Figure 7) but the assay was not sensitive enough to reveal if TSA changed the acetylation status of p65 using either acetyl-p65-specific antibody or anti-p65 immunoprecipitation followed by Western blot with anti-acetyl-lysine antibody (data not shown). Similarly, no effect of p65 on histone acetylation within the chromatin of the JCV NCCR was observed in the absence of TSA (Figure 7B) indicating that either it does not occur or, if it does, it is below the level necessary to be detected by the ChIP assay.

Taken together, our data suggest the region of chromatin at the NF-κB-binding site is involved in the stimulation of Mad-1 JCV transcription by HDACi such as TSA. The mechanism of this effect is still under investigation but it is possible that acetylation of histones and/or p65 via the p300 transcriptional coactivators/acetyltransferases is involved. NF-κB p65 is regulated by acetylation by p300 and CBP acetyltransferases, which principally target lysines 218, 221 and 310 [17‐19]. Analysis of p65 mutants containing lys-to-arg substitutions indicates acetylation at K221 enhances DNA binding and impairs assembly with IκBα while acetylation at K310 is required for full p65 transcriptional activity [18]. In another study, data pointed to a role for K314/K315 in regulating p65 function [27]. As well as binding p65, the KB element binds C/EBPβ LIP [7] and NFAT4 [8], which act together with p65 to regulate JCV transcription. Hence, it is also possible that acetylation of these proteins is also involved in controlling transcription. C/EBPβ is functionally modified at several lysine residues but only K215/K216 is in the LIP domain [28]. There are no reports of NFAT4 acetylation but NFAT2 is acetylated [29]. In principal, acetylation of any of the three transcription factors, NF-κB p65, C/EBPβ and NFAT4, alone or in combination, may be responsible for activation of the JCV KB element by HDACi.

In conclusion, our data are consistent with a model where latent JCV is present in transcriptionally silent, deacetylated chromatin but can be activated by the action of transcription factors that act downstream from cytokines such as TNF-α and involve acetylation events. This is similar to latent HIV-1 provirus where marked transcriptional activation of the HIV-1 promoter also occurs in response to deacetylase inhibitors. Deacetylation events are an important mechanism of HIV-1 transcriptional repression during latency, whereas acetylation events are involved HIV-1 reactivation from latency [30]. Notably, HDACi (TSA and SB) synergized with both ectopically expressed p50/p65 and TNF-α treatment to activate the HIV-1 LTR [31]. While these findings with HIV could open new therapeutic strategies aimed at decreasing or eliminating the pool of latently HIV-infected reservoirs by forcing viral expression, our findings for JCV could open new therapeutic strategies for PML aimed at preventing viral expression and containing JCV in a latent state. Finally, at least ten human polyomaviruses are now known to exist [32] and it will be of interest to investigate if any of these are also regulated epigenetically.