Epigenetic regulation of polyomavirus JC
© Wollebo et al.; licensee BioMed Central Ltd. 2013
Received: 3 April 2013
Accepted: 21 August 2013
Published: 23 August 2013
Polyomavirus JC (JCV) causes the CNS demyelinating disease progressive multifocal leukoencephalopathy (PML), which occurs almost exclusively in people with immune deficiencies, such as HIV-1/AIDS patients. JCV infection is very common and usually occurs early in life. After primary infection, virus is controlled by the immune system but, rarely when immune function is impaired, it can re-emerge and multiply in the astrocytes and oligodendrocytes in the brain and cause PML. Thus a central question in PML pathogenesis is the nature of the molecular mechanisms maintaining JCV in a latent state and then allowing reactivation.
Since transcription can be regulated by epigenetic mechanisms including DNA methylation and histone acetylation, we investigated their role in JCV regulation by employing inhibitors of epigenetic events.
The histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate powerfully stimulated JCV early and late transcription while the DNA methylation inhibitor 5-azacytidine had no effect. Analysis of JCV mutants showed that this effect was mediated by the KB element of the JCV control region, which binds transcription factors NF-κB p65, NFAT4 and C/EBPβ and mediates stimulation by TNF-α. Stimulation of transcription by p65 was additive with TSA as was cotransfection with transcriptional coactivators/acetyltransferase p300 whereas depletion of endogenous p65 by RNA interference inhibited the effect of TSA. EMSA with a KB oligonucleotide showed p65 expression, TNF-α stimulation or TSA treatment each caused a gel shift that was further shifted by antibody to p65.
We conclude that JCV is regulated epigenetically by protein acetylation events and that these involve the NF-κB p65 binding site in the JCV control region.
KeywordsEpigenetic Acetylation Transcriptional regulation
Combination antiretroviral therapy
Histone deacetylase inhibitor
JC virus, polyomavirus JC
Noncoding control region
Progressive multifocal leukoencephalopathy
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
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 . 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 . 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  that was isolated in 1971 from the brain of a patient with PML . 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 . 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 . 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  as well as NFAT4 . 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 .
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 . 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 . However, the genome of JCV (strain Mad-1, ) 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.
JCV early and late transcription are inhibited by the histone deacetylase inhibitors (HDACi) trichostatin A (TSA) and sodium butyrate (SB) but not by the DNA methylation inhibitor 5-azacytidine (AZA)
Mapping of the region within the JCV NCCR responsible for TSA inducibility implicates the KB element
NF-κB p65 and TSA cooperate to stimulate JCV early and late transcription
Expression of p300 enhances JCV early and late transcription
Many transcription factors including p65 recruit the transcriptional coactivator p300, an acetyltransferase. p300 binds to p65 and catalyzes histone acetylation to open chromatin for the transcriptional machinery and acetylation of p65 itself to increase its activity [18, 19]. We found that expression of the JCV early and late transcription (Figure 4B) and acted cooperatively with p65 (Figure 4C).
Knockdown of p65 by RNA interference reduces the stimulation of JCV early and late transcription by TSA
TSA induces an EMSA gel shift with a KB region oligonucleotide that co-migrates with that induced by p65 expression and TNF-α treatment and is supershifted by antibody to p65
TSA induces acetylation of histone H3 on lys-9 detected be ChIP assay
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  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 . 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 . 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  and JCV contains only 6 CpGs in the NCCR , 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  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 . 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  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 . 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 . In another study, data pointed to a role for K314/K315 in regulating p65 function . As well as binding p65, the KB element binds C/EBPβ LIP  and NFAT4 , 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 . There are no reports of NFAT4 acetylation but NFAT2 is acetylated . 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 . Notably, HDACi (TSA and SB) synergized with both ectopically expressed p50/p65 and TNF-α treatment to activate the HIV-1 LTR . 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  and it will be of interest to investigate if any of these are also regulated epigenetically.
Cell culture and plasmids
Culture of the human TC620 oligodendroglioma cell line was performed as we have previously described . Stable clonal cell lines expressing luciferase under the control of the JCV early and late promoters were derived from TC620 as previously described . Reporter constructs, JCVE-LUC and JCVL-LUC contained the JCV promoter from the Mad-1 strain linked to the luciferase gene in the early and late orientations respectively . JCVE-LUC promoter mutants m1 and m2 contained mutations at two adjacent sites within the KB site of the early promoter and heterologous reporter plasmids, pBLCAT2-wt-kB and pBLCAT2-mt-kB contained wild-type and mutant KB elements respectively cloned into the CAT reporter plasmid pBLCAT2, which contains the constitutive Herpes simplex virus thymidine kinase (tk) promoter . New reporters were generated based on a CpG-free luciferase vector, a kind gift from Michael Rehli, University Hospital Regensburg, Germany  by cloning the Mad-1 JCV NCCR was cloned into the BglII site of pCpGL-basic (pCpGL-JCVE and pCpGL-JCVL). The expression plasmids pCMV-p65 and pCMV-LIP were described previously .
The following antibodies were used for Western blot: Rabbit polyclonal anti-p65 (c-20, sc-372, Santa Cruz Biotechnology Inc., Santa Cruz, CA) and mouse monoclonal anti-Grb2 (610111; BD Biosciences, San Jose, CA). For EMSA, rabbit polyclonal anti-p65 (c-20, sc-372X, Santa Cruz) was used and for ChIP, rabbit monoclonal to acetyl-histone H3 (K9) (C5B11, Cell Signaling Technology, Danvers MA).
Western blot assays were performed as previously described . Briefly, 50 μg of protein was resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with primary antibody (1/1000 dilution) and secondary antibody (1/10000 dilution). Bound antibody was detected with an ECL detection kit (Amersham, Arlington Heights, IL).
Transient transfection and reporter assays
Co-transfection of reporter plasmids and expression plasmids were performed as we have previously described [7, 9]. Briefly, TC620 cells were transfected with reporter constructs alone (0.5 μg) or in combination with the various expression plasmids for 48 h prior to. The total amount of transfected DNA was normalized with empty vector DNA. Treatment with epigenetic reagents was performed for 24 h prior to harvesting: SB – 0, 5 mM, 20 mM and 15 mM; TSA – 0, 0.2 μM, 0.4 μM and 1 μM; AZA – 0, 10 μM, 15 μM and 25 μM. Assays for luciferase and CAT were performed as previously described [7, 9]. For RNA interference experiments, 200 nmol of Smartpool siRNA or control nontargeting siRNA (Dharmacon, Lafayette, CO) against p65 were transfected into cells 48 hours prior to transfection with the reporter plasmid as we have previously described .
Gel shift assays
TC620 cells were transfected with p65 expression plasmid for 48 h, treated overnight with 250 nM TSA or treated 10 ng/ml TNF-α for 30 min and then harvested. Nuclear proteins were then extracted and 10 μg were incubated with 50,000 cpm of a γ-32P-labeled, double-stranded oligodeoxyribonucleotide probe as previously described . The probe that was used in these gel shift experiments corresponded to the KB element: κB: 5′-aaaacaagggaatttccctggcctc-3′ (nts 5052–5078, Mad-1 JCV, GenBANK # NC_001699).
Chromatin immune precipitation (ChIP) assay
TC620 cells were plated at a density of 1 × 106 cells/ml in 100 mm dishes and the next day transfected with 3 μg of JCVE-LUC plasmid. The next day, cells were treated with 0.5 μM TSA for 24 hours and ChIP assays performed using the ChIP assay kit (Upstate Cell Signaling Solutions) as we have previously described . Briefly, cells were cross-linked with formaldehyde and DNA sheared by sonication. After lysis, immunoprecipitation was performed with antibody to acetyl-histone H3 (Lys9) or nonimmune rabbit serum control as indicated. After DNA extraction, PCR was performed using the following primers which amplify a region spanning the entire JCV NCCR:
Forward: 5′-cctccctattcagcactttgtcc-3′ (Mad-1, 4989–5011).
Reverse: 5′-ggccagctggtgacaagcc-3′ (276–258).
PCR was 30 cycles (94°C for 30 s, 55°C for 30 s, 72°C for 30 s) then 72°C for 7 min.
We thank past and present members of the Center for Neurovirology for their insightful discussion and sharing of ideas and reagents. This study utilized services offered by the Comprehensive NeuroAIDS Center (CNAC NIMH Grant Number p30MH092177) at Temple University School of Medicine. This work was supported by Grant Number R01 AI077460 awarded by the NIH to MKW.
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