Dynamic control of H3K27 methylation 1, 2, 3
is central to gene silencing in development,4, 5, 6, 7
stem-cell pluripotency,9, 10
So far, only the mammalian EZH2 of the Polycomb repressive complex 2 (PRC2) is known to catalyze this important epigenetic histone modification.14, 15
H3K27 methylation as a viral strategy to regulate host transcription has not been reported. Viruses recruit chromatin-associated transcription proteins for maintenance of their genome and replication. For example, the papillomavirus E2 protein binds Brd4, tethering the viral genome to mitotic chromosomes to ensure persistence of viral episomes in infected cells.16
The adenovirus E1A protein interacts with the retinoblastoma protein p130 to silence E2F-responsive genes in quiescent cells.17
The HIV trans
-activator Tat recruits histone acetyltransferases for chromatin acetylation and remodeling for transcriptional activation of the provirus.18, 19
Despite the ability of viruses to recruit cellular histone-modifying enzymes to facilitate viral transcription, no viral enzymes have been identified that directly modify host histones and modulate transcription. Of the large family of SET domain HKMTases,20, 21
a small subset of SET proteins is encoded by viruses and bacteria.22, 23
The cellular functions of these proteins are unknown but their presence in viruses raises questions about whether they are able to modify histones and, if so, how this would affect the host cells. To address these questions, we have characterized SET domain proteins from chlorella viruses.
We first focused on the function of vSET, a SET domain protein encoded by PBCV-1, which is a large double-stranded DNA virus that replicates in the unicellular, eukaryotic green algae Chlorella
The 330-kb PBCV-1 genome has 366 non-overlapping protein-encoding genes and 11 tRNA genes.24
Despite its large genome, PBCV-1 lacks an RNA polymerase gene, suggesting that it is dependent on the host transcription machinery. Notably, PBCV-1 infection causes a rapid inhibition of total RNA synthesis in Chlorella
cells, achieving 60–80% inhibition by 20–40 min after infection (). PBCV-1 transcripts can be detected 5–10 min after infection, indicating that transition from host to virus transcription occurs rapidly.24
These results led us to postulate that a factor(s) packaged in the virion may be responsible for this rapid inhibition of host RNA synthesis. vSET is a possible candidate as it methylates H3K27, which has been linked to PRC-mediated gene silencing in eukaryotes.1, 2, 3
Presence of vSET in PBCV-1 virions
To investigate vSET function, we generated an anti-vSET antibody to immunoprecipitate vSET from PBCV-1 infected Chlorella cell extracts. vSET is present in mature virions and a small amount was detected as early as 10 min after infection of Chlorella cells, presumably, released from PBCV-1 (). Expression of vSET was markedly enhanced 120 min after infection. Pretreatment of isolated virus particles with trypsin, which removes any exogenous vSET contamination on the virus surface, had no effect on viral infectivity or the amount of vSET detected in PBCV-1 particles (). The presence of vSET in PBCV-1 virions was confirmed by Q-TOF mass spectrometry (data not shown). By comparing western blots from a known number of virus particles with blots containing different concentrations of vSET, we estimated that four vSET molecules are packaged per virion (data not shown). Furthermore, vSET in the disrupted virions possess H3K27 methylation activity (), which indicates that PBCV-1 virions contain active vSET.
To detect vset
transcripts during PBCV-1 replication, RNA from infected Chlorella
cells was hybridized with the vset
gene. The probe hybridized to an RNA of about 1.8 kb, beginning approximately 60 min after infection (). This transcript is larger than expected for a 119-residue protein and may be a bicistronic transcript encoding the co-linear genes a609l
(approximately 1.2 kb) and a612l
(approximately 0.4 kb). The latter gene encodes vSET. This result is consistent with vSET expression occurring about 120 min after infection ().24
vSET adopts a core β-barrel structure,25, 26
a fold that is conserved in eukaryotic SET domain HKMTases.21
To test whether vSET functions as a bona fide
HKMTase in Chlorella
cells, we cloned histone H3 from Chlorella
NC64A and found that it has high sequence identity with human H3 (). We measured vSET methylation activity using both the nucleosome and individual core histones and confirmed its activity on full-length free H3 and H3 within the nucleosome, but not on other core histones (). To identify vSET methylation site(s) in H3, we expressed a series of GST–H3 peptides (residues 1–57) with Arg substitutions at Lys methylation sites: Lys 4, Lys 9, Lys 27, Lys 36 and Lys 37 (Supplementary Information, Table S2
). The purified GST–H3 peptides produced two bands when subjected to SDS–PAGE, which corresponded to an intact H3 peptide of residues 1–57 (plus SGRIVTD from the expression vector) and a truncated H3 of residues 1–55, as confirmed by MALDI-TOF mass spectrometry (). The methylation assay showed that vSET only methylated H3 with an intact Lys 27 site (). Using site- and state-specific antibodies against Lys-methylated histone H3 (Supplementary Information, Figure S1a, b
), we confirmed that vSET catalyses H3K27 dimethylation (H3K27me2) and, to a much less extent, mono- and trimethylation. No methylation occurred at Lys 4, Lys 9 or Lys 36 sites in H3 (). These results are consistent with the kinetic analyses of vSET methylation of H3K27 peptide, showing that monomethylation and mono- to dimethylation are very rapid, whereas di- to trimethylation is about 10 times slower than mono- to dimethylation (Supplementary Information, Figure S1c, d
). Finally, we confirmed that histone H3 is the major substrate of vSET in an in vitro
methylation assay using heat-inactivated extracts from HeLa cells depleted of EZH2 by RNA interference (RNAi) and uninfected Chlorella
cells (Supplementary Information, Figure S2a–d
). Collectively, these results establish that vSET is a H3K27-specific methyltransferase.
Methylation of Chlorella NC64A histone H3K27 by vSET
We next assessed the effects of PBCV-1 infection on H3K27 methylation in Chlorella cells. Western blot analysis showed that H3K27me1 was unchanged, whereas H3K27me2 increased markedly as early as 30 min after infection, and H3K27me3 was slightly enhanced (). This observation correlates with in vitro vSET state-specific methylation activity at H3K27 (). Immunofluoresence showed that vSET was present in the nucleus of Chlorella cells after PBCV-1 infection (), and vSET colocalized with the H3K27me2 (). These results confirm that vSET is a bona fide HKMTase and causes increased H3K27me2 in Chlorella cells during virus replication.
Technical difficulties in manipulating the PBCV-1 genome and genetic transformation of the host Chlorella
cells prevented us from conducting a cellular study of vSET in its native host. But given the highly conserved H3 sequence and H3K27 methylation in eukaryotes, we reasoned that mammalian cells could serve as a suitable model system to study the biological function of vSET, which has enzymatic activity similar to that of EZH2 (ref. 3
If vSET participates in early suppression of host transcription, it must move to the host nucleus on viral infection. To assess whether a solvent-exposed KRMR motif in vSET (residues 85–88, see ) functions as a nuclear localization signal (NLS), NIH-3T3 cells were transfected with a GFP–vSET or a triple mutant in which KR(M)R was changed to AA(M)A. Fluorescence microscopy showed that wild-type GFP–vSET was localized to the nucleus, whereas the triple mutant remained in the cytoplasm (), confirming that the KR(M)R motif is an NLS in vSET. vSET H3K27 methylation activity was not compromised in this mutant (Supplementary Information, Figure S3a–c
). A classical NLS sequence is absent in most eukaryotic SET domains, except for yeast SET1, which has an RRIV motif located at a site analogous to the KRMR motif in vSET.
HKMT activity of SET proteins from chlorella viruses
Nuclear localization and H3K27 methylation activity of vSET
H3K27 methylation in eukaryotes is catalyzed by the EZH2 of PRC2 and recruits the PRC1 complex, leading to gene silencing.1, 14
To investigate whether vSET can methylate H3K27 in mammalian cells, we knocked down EZH2
using the RNAi method in HeLa cells. When treated with EZH2
-specific siRNA, EZH2 expression was reduced by more than 90%, compared with control cells (). EZH2
knockdown caused a reduction of SUZ12, a component of PRC2, as well as near complete loss of H3K27me2 and H3K27me3, and a slight reduction of H3K27me1. No significant changes were observed with RING1 of PRC1, H3K4, H3K9, and H3K36 dimethylation. These results confirm that EZH2 is responsible for H3K27 di- and trimethylation in HeLa cells. vSET treatment of the nuclear extract from EZH2
knockdown HeLa cells restored H3K27me2 and H3K27me3, and resulted in a slight reduction of H3K27me1, possibly because of its conversion to H3K27me2 and H3K27me3 by vSET (). Similar results were obtained when tetracycline was used to induce vSET expression in the EZH2
knockdown HeLa cells (). Thus, these results demonstrate that vSET can mimic EZH2 to methylate H3K27 in mammalian cells.
We next examined the effect of vSET methylation activity on gene transcription in a luciferase-based reporter assay. In this assay, we first transfected the 293T cells with vSET fused to a Gal4 DNA-binding domain (DBD) and a luciferase reporter gene encoding HSV-tk
promoter (plus Gal4 DNA-binding sites). Wild-type vSET repressed luciferase gene expression by about 95%, whereas the inactive mutants Y105A or Y105F had no vSET gene-silencing ability (). Notably, expression of a Flag- or HA-tagged vSET in the 293T cells caused repression of approximately 60%, compared with the vectors containing only Flag- or HA-tag, or inactive mutant Y105F (). The lesser repressive activity of Flag- or HA-tagged vSET on host transcription, compared with that of vSET–Gal4-DBD may be due, at least in part, to the presence of H3K27ac and/or H3S28p in host cells (), which would prevent H3K27 methylation by vSET 26
(). In a luciferase-based assay, we also observed that Flag–vSET co-transfected with HIV-Tat in 293T cells (Supplementary Information, Figure S4a
) or HeLa cells (Supplementary Information, Figure S4b
) repressed transcription of the HIV LTR
confirming repression of chromatin-mediated transcription by vSET.
Repression of gene transcription by vSET H3K27 methylation activity
To examine the biological consequence of H3K27 methylation by vSET, we assessed Polycomb group protein occupation at the H3K27me site on tetracycline-induction of vSET in the EZH2
knockdown HeLa cells. Although their protein levels were unaltered, vSET induction enhanced occupation of the PRC1 complex protein CBX8, but not CBX4 and CBX7 at the H3K27me2 and H3K27me3 sites (). This CBX8/H3K27me2/3 association is probably facilitated by the chromodomain of CBX8, which binds methyl-Lysine.27, 28
Interestingly, a search of the SMART database reveals that a virus PBCV-1 encoded protein (accession number NP_049022) seems to contain the chromodomain as in CBX8. Therefore, we conclude that vSET induces transcription repression by H3K27me, and facilitates accumulation of the PRC1 complex at the H3K27me site.
Using a quantitative RT–PCR assay, we examined whether vSET methylation activity can modulate Polycomb target genes.29
The analysis was performed in HeLa cells on five representative Polycomb target genes HOXA7
, and three general housekeeping genes GAPDH
. The effect on the three housekeeping genes was negligible; however EZH2
knockdown by siRNA caused a 2.5-fold increase in transcription of HOXA9
, and a 7.5-fold increase in HOXA7 expression (). The increase in the Polycomb target gene expression was reversed by tetracycline-induced expression of vSET to a level similar to or slightly higher than that of the control cells. Moreover, vSET induction in the normal HeLa cells did not alter the expression level of the five HOX genes when compared to the controls.
We next characterized vSET-dependent transcriptional repression of the HOXA7
promoter in luciferase gene expression and chromatin immunoprecipitation (ChIP) assays. Induction of vSET effectively repressed luciferase expression at the HOXA7
promoter in both EZH2
siRNA-treated and untreated HeLa cells (; Supplementary Information, Figure S4c
). The ChIP analysis confirmed that vSET specifically di- and tri-methylates H3K27 at the HOXA7
promoter, which recruits CBX8 of the PRC1 and causes gene repression (). We obtained similar results for the characterization of vSET-mediated transcription repression of HIV-Tat-dependent LTR
promoter at the nucleosome Nuc2 (Supplementary Information, Figure S4d, e
). These results establish that vSET can modulate transcription of Polycomb target genes through its H3K27 methylation activity.
We then examined the effect of vSET expression on the cell cycle. Flow cytometric analysis indicated that vSET caused cell accumulation at the G2/M phase in transiently transfected NIH-3T3 cells, whereas active-site mutants E100A and Y105A were less effective (). The vSET effect on the G2/M phase in the cell-cycle was confirmed in the HeLa cells that were stably co-transfected with vSET in a tetracycline-controlled expression vector (). Collectively, our data indicate that vSET H3K27 methylation activity results in the recruitment of the Polycomb repressive complexes and modulation of their target genes, leading to transcription repression and accumulation of cells at the G2/M phase.
To determine whether vSET is unique to viral PBCV-1 or is present in other chlorella viruses, we hybridized genomic DNA from 36 other chlorella viruses with a vset gene probe. Thirty-one of these viruses hybridized to the probe (). The probe did not hybridize to DNA from 5 viruses or the host DNA. However, a vset gene was identified in 3 of these 5 viruses using low stringency hybridization. The vSET proteins from these three viruses, NY-2A, NY-2B and MA-1D, have 85% amino acid similarity to PBCV-1 vSET, including the conserved active-site residues in the SET domain family (). We suspect the remaining two viruses, NYs-1 and IL-5-2S1, also have a vSET gene but we did not examine them further.
Using histones H2A, H2B, H3 and H4 as substrates, we established that the SET proteins from these three viruses selectively methylate H3 (data not shown). As with vSET, these viral SET proteins only methylated a H3 peptide containing residues 15–30 but not one containing H3 residues 1–20 (). They showed little methyltransferase activity towards H3 peptide that was previously dimethylated at Lys 27 or phosphorylated at Ser 28, suggesting a preference for the H3K27 site. The selective methylation at H3K27 was confirmed using GST–H3 (residues 1–57), in which these viral SET proteins had robust methylation activity on wild-type H3 but diminished activity on the K27R mutant (). These results establish that SET proteins from the chlorella viruses possess selective H3K27 methylation activity, and suggest that vSET function in host chromatin modification and transcription repression is conserved among the chlorella viruses.
Histone modifications provide epigenetic control of gene transcription. Viruses recruit host proteins to either integrate their genomes into host chromatin 16, 17
or to facilitate viral transcription and replication.18, 19
Here, we show for the first time that a virus regulates the host transcriptional machinery through direct modifications of chromatin. Our study reveals that vSET is packaged in the PBCV-1 virion and can directly methylate host H3K27, an important epigenetic modification in eukaryotes that is functionally linked to Hox
gene silencing,4, 5, 30, 31
and stem-cell pluripotency.9, 10
Our results establish that vSET H3K27 methylation promotes the recruitment of the PRC1 complex through a molecular interaction between CBX8 and di- and/or trimethylated H3K27. This interaction results in modulation of Polycomb target genes, transcription repression and cell accumulation at the G2/M phase of the cell cycle in continuously dividing cells. Our study supports the view that vSET proteins, which are encoded by all chlorella viruses tested, function to repress transcription in infected chlorella cells. Collectively, our findings suggest a unique and powerful mechanism by which some viruses commandeer the host transcription machinery through direct modifications of histones and in this way govern host chromatin-mediated cellular processes.