Viral Oncoproteins Target the DNA Methyltransferases

WA Burgers1,2,6, L Blanchon3,6, S Pradhan4, Y de Launoit5, T Kouzarides1 and FFuks 1,3

1Wellcome Trust/CRUK Gurdon Institute, University of Cambridge, Cambridge, UK; 2Faculty of Health Sciences, Division of Medical Virology, University of Cape Town Medical School, Cape Town, South Africa; 3Laboratory of Cancer Epigenetics, Faculty of Medicine, Free University of Brussels, Brussels, Belgium; 4New England Biolabs, MA, USA and 5UMR 8161, CNRS Institut Pasteur de Lille, Universite´s de Lille 1 et 2, Institut de Biologie de Lille, Lille, Cedex, France

Small DNA tumour viruses have evolveda number of to replicate their genomes. The adenovirus oncoprotein mechanisms to drive nondividing cells into S phase. Virally E1A possesses multiple functions, and can affect gene encoded oncoproteins such as adenovirus E1A and human transcription, induce DNA synthesis, immortalize cells papillomavirus (HPV) E7 can bindan array of cellular and inhibit differentiation (Frisch and Mymryk, 2002). proteins to override proliferation arrest. The DNA E1A associates with a large number of cellular proteins methyltransferase Dnmt1 is the major mammalian in infected or transformed cells, including the coacti- enzyme responsible for maintaining CpG methylation vators and histone acetyltransferases CBP/p300, the patterns in the cell following replication. One of the tumour-suppressor Rb, as well as sequence-specific hallmarks of tumour cells is disrupted DNA methylation transcription factors. E1A has several conserved regions patterns, highlighting the importance of the proper which mediate most of its functions, designated con- regulation of DNA methyltransferases in normal cell served region 1 (CR1, residues 40–80), CR2 (121–139) proliferation. Here, we show that adenovirus 5 E1A and and CR3 (140–188). The immortalization and transfor- HPV-16 E7 associate in vitro and in vivo with the DNA mation functions of E1A are mediated by CR1 and methyltransferase Dnmt1. Consistent with this interac- CR2, and these regions correspond with the interaction tion, we findthat E1A andE7 can purify DNA sites of Rb and CBP/p300 (Frisch and Mymryk, 2002). methyltransferase activity from nuclear extracts. These Human papillomaviruses (HPVs) are another group associations are direct and mediated by the extreme of small DNA tumour viruses, which are of particular N-terminus of E1A andthe CR3 zinc-finger domain of E7. importance as infection with the high risk human types Furthermore, we findthat a point mutant at leucine 20 of (such as HPV16 and 18) can lead to anogenital cancers. E1A, a residue known to be critical for its transformation E7 is the major transforming protein of human functions, is unable to bindDnmt1 andDNA methyl- papillomaviruses and shares sequence homology with transferase activity. Finally, both E1A andE7 can E1A. E7 targets the Rb family to overcome prolifera- stimulate the methyltransferase activity of Dnmt1 in vitro. tion arrest, by sequestering Rb from E2Fcomplexes Our results provide the first indication that viral onco- (McCance, 2005). proteins bindandregulate Dnmt1 enzymatic activity. DNA methyltransferases are responsible for the These observations open up the possibility that this methylation of cytosine in mammals and play a role in association may be usedto control cellular proliferation gene silencing. Dnmt1 is the main ‘maintenance’ pathways andsuggest a new mechanism by which small methyltransferase, restoring the methylation pattern on DNA tumour viruses can steer cells through the cell cycle. newly replicated DNA (Fuks, 2005). Aberrant methyla- Oncogene (2007) 26, 1650–1655. doi:10.1038/sj.onc.1209950; tion patterns are increasingly being recognized as an published online 18 September 2006 important and frequent event in cancers (Robertson, 2001; Jones and Baylin, 2002). Tumours and trans- Keywords: E1A; E7; Dnmt1; DNA methyltransferase; formed cell lines exhibit abnormal methylation of CpG viral oncoproteins islands and frequent inactivation of tumour suppressor genes. Several lines of evidence point to an increase in Dnmt1 activity being an important step in carcinoge- nesis. Overexpression of Dnmt1 in mouse fibroblasts Small DNA tumour viruses such as adenovirus and leads to transformation (Wu et al., 1993). Dnmt1 has papillomaviruses require viral and cellular host proteins been reported to be required for maintaining cellular transformation by fos (Ordway et al., 2005) and a Correspondence: Dr FFuks,Laboratory of Cancer Epigenetics, number of studies have reported elevated mRNA levels Faculty of Medicine, Free University of Brussels, 808 route de Lennik, and Dnmt1 activity in cancers (Robertson, 2001). Brussels 1070, Belgium. Although it remains to be established how increased E-mail: [email protected] 6These authors contributed equally to this work. levels of Dnmt1 may contribute to hypermethylation of Received 13 April 2006; revised 4 July 2006; accepted 5 July 2006; CpG islands and tumour development, inhibition of published online 18 September 2006 Dnmt1 has been shown to suppress tumorigenesis E1A and E7 target Dnmt1 WA Burgers et al 1651 in vitro and in vivo (Laird et al., 1995). These studies a transfected point to a role for increases in Dnmt1 activity playing an important part in CpG methylation-mediated cellular - - + Myc-PCNA transformation. + + + HA-E1A Viral regulation of Dnmt1 has been reported. SV40 Input large T antigen upregulates Dnmt1 RNA and protein + - - Myc-Dnmt1 levels post-transcriptionally, leading to a 5- to 15-fold increase in methyltransferase activity and an increase 50 kD in genomic methylation (Slack et al., 1999), and HA-E1A Dnmt1 acts as a downstream effector of the oncogenic programme of large T antigen. Similarly, HIV-1 infection leads to the upregulation of Dnmt1 expression 1234 and activity, resulting in global DNA hypermethylation IP ab: Myc with subsequent methylation of the IFN-g promoter West. ab: HA (Mikovits et al., 1998). Furthermore, an increase in DNA methylation at a particular promoter has been b Dnmt1 binding observed in cells infected with a range of tumorigenic 1 40 80 120 140 188 289 E1A 13S + viruses, such as HTLV-1, EBV and SV-40 (de Bustros CR1 CR2 CR3 et al., 1988). However, none of these previous studies E1A 1-90 + have demonstrated a direct association between parti- E1A 121-204 - cular viral proteins and Dnmt1. E1A 127-204 - - In the present study, we sought to determine whether E1A 140-204 viral oncoproteins can target Dnmt1 directly. We find E1A ∆2-36 - that adenovirus 5 E1A and HPV-16 E7 bind to Dnmt1 and precipitate DNA methyltransferase activity. These E1A R2 * + interactions are direct and map to the extreme E1A H3 * + N-terminus of E1A and to the zinc-finger CR3 region E1A L20 * - of E7. We also find that leucine 20 of E1A, known to play a key role in the ability of the virus to transform c GST-E1A cells, is required for the association of E1A with Dnmt1 2-36

as well as with DNA methyltransferase activity. Finally, ∆ both E1A and E7 upregulate the methyltransferase Input GST E1A 13S E1A 1-90 E1A 121-204 E1A 127-204 E1A 140-204 E1A E1A R2 E1A H3 E1A L20 activity of Dnmt1 in vitro. These results reveal that IVT-Dnmt1 Dnmt1 is a novel cellular target for E1A and E7 and uncover a novel mechanism whereby E1A and E7 may 132104956711 8 transform cells. * * * ** * * * Coomassie * To investigate whether adenovirus E1A interacts * * with Dnmt1, a co-immunoprecipitation approach was employed. HA-tagged Ad5 E1A 12S (HA-E1A) and Figure 1 E1A interacts with Dnmt1 through its extreme Myc-tagged, full-length Dnmt1 (Myc-Dnmt1) were N-terminal region. (a) E1A co-immunoprecipitates with Dnmt1 from transfected cell extracts. 293T cells were transfected with transfected into 293T cells. Cells were lysed, immuno- HA-Ad5 E1A (expressing HA-tagged E1A 12S) alone or together precipitation was carried out using an anti-Myc antibody, with either Myc-Dnmt1 (expressing Myc-tagged full-length Dnmt1, and precipitates were subjected to Western blot analysis Myc-Dnmt1) or pMyc-PCNA (Myc-tagged PCNA, Myc-PCNA) with an anti-HA antibody. Figure 1 (lane 2) indicates as a control, as indicated. After 48 h, cells were lysed in IPH buffer (Brenner et al., 2005) and whole-cell extracts were then precipitated that E1A associates specifically with Dnmt1 in vivo. with anti-Myc antibody (9E10, Roche) and the presence of E1A in We next wished to define the sequences within E1A the immunoprecipitates was visualized using anti-HA antibody that are required for the association with Dnmt1. (3F10, Roche), as described (Vire et al., 2006). (b) Schematic glutathione S-transferase (GST)-pulldown experiments representation of E1A with the deletions and point mutants used in indicate that the E1A N-terminus (amino acids 1–90) GST-pulldowns. (c) In vitro translated (IVT) Dnmt1 was incubated with the indicated GST fusions. The reactions were subjected to is needed for the association with in vitro translated GST-pulldown assays as described (Deplus et al., 2002). Bottom Dnmt1, whereas E1A CR2 or CR3 are not (Figure 1c, panel: Coomassie-stained gel showing the input of GST proteins lanes 3–7). Of note, Dnmt1 is known to associate with used. Rb (Pradhan and Kim, 2002) and Rb itself is a target of E1A (Whyte et al., 1988). We observed, however, that the major Rb binding site within CR2 (the LXCXE et al., 2002), is essential for the interaction with Dnmt1 motif) does not seem to contribute to the association of (Figure 1c, lane 8). The use of well-defined single point E1A with Dnmt1 (Figure 1c, compare lanes 5 and 6). mutants within this region of E1A shows that mutation We further found that the extreme N-terminus of E1A of leucine 20 specifically disrupts the binding to Dnmt1 (amino acids 2–36), which is involved in transformation, (Figure 1c, lanes 9–11). Amino acid 20 is critical for cell growth control and transcriptional regulation immortalization as well as transformation (Wang et al., (Wang et al., 1993; Sang and Giordano, 1997; Boyd 1993; Sang and Giordano, 1997; Boyd et al., 2002).

Oncogene E1A and E7 target Dnmt1 WA Burgers et al 1652 a These results were conﬁrmed by means of an in vitro DNA methyltransferase enzymatic assay. In this assay, 4 liquid scintillation counting is used to monitor the incorporation of [3H]-S-adenosyl-L-methionine into a synthetic 33-bp hemimethylated oligonucleotide. As 3 shown in Figure 2a we found that GST-E1A associates speciﬁcally, through residues 1–90, with DNA methyltransferase activity from nuclear extracts. Moreover, 2 mutation of leucine 20 of E1A strongly impairs association with DNA methyltransferase activity (Figure 2b). 1 E1A interacts directly with several histone acetyltransferases, and it has been reported that E1A has the ability to regulate the acetyltransferase activity of CBP/

Relative DNA metyltransferase activity (fold) DNA metyltransferase Relative 0 GST 13S 12S 1-90 121-204 127-204 140-204 p300 both positively (Ait-Si-Ali et al., 1998) and negatively (Chakravarti et al., 1999). This prompted GST E1A 123456 7 us to determine whether the association of E1A with Dnmt1 could modulate its enzymatic activity. To this * * * * Coomassie * * * end, baculovirus-expressed Dnmt1 was preincubated with GST-E1A and the reactions were subjected to West. blot: Input DNA methyltransferase assays. As depicted in Figure 3, anti-Dnmt1 incubation of Dnmt1 with GST-E1A signiﬁcantly stimulates DNA methyltransferase activity as compared b to GST alone (lanes 1 and 2). Region 1–90 of E1A 3

Relative DNA metyltransferase activity (fold) DNA metyltransferase Relative 0 GST 12S L20 H3 R2 GST E1A

12345 activity (fold) DNA methyltransferase Relative 0 Coomassie ** ** Dnmt1 ++++ * E1A West. blot: Input Eluted GST GST E1A E1A 1-90 127-204 anti-Dnmt1 1234 Figure 2 E1A associates with DNA methyltransferase activity and * * leucine 20 is important for enzymatic binding. (a) E1A precipitates Coomassie * DNA methyltransferase activity through residues 1–90. Equivalent * amounts of GST or GST-E1A fusion proteins bound to Sepharose West. blot: beads were incubated with HeLa nuclear extract, washed and Input assayed for DNA methyltransferase activity as described (Fuks anti-Dnmt1 et al., 2000). Activity is shown as c.p.m. of S-adenosyl-L- (methyl-3H)-methionine incorporated into a synthetic 33-bp hemi- Figure 3 E1A upregulates the methyltransferase activity of methylated oligonucleotide substrate (upper strand: GAT meCG- Dnmt1. We expressed and puriﬁed baculovirus human Dnmt1 as meC meCGA TGmeC GmeCG AAT meCGmeC GAT meCGA described previously (Pradhan et al., 1999). Baculovirus-expressed TGmeC GAT; lower strand: ATC GCA TCG ATC GCG ATT Dnmt1 was preincubated for 10 min on ice with increasing CGC GCA TCG GCG ATC). Values are normalized to back- concentrations (1 and 5 mg) of eluted and the indicated dialysed ground controls lacking substrate. (b) Point mutation within leucine GST proteins. Activity is shown as c.p.m. of S-adenosly-L- 20 of E1A strongly impaired association with DNA methyltransfe- (methyl-3H)-methionine incorporated into a hemimethylated oligo- rase activity. For panels (a) and (b): middle panels show Coomassie- nucleotide substrate. Bottom panel: Western blotting following the stained gel with the input of GST proteins; bottom panels represent enzymatic assays using anti-Dnmt1 (Pradhan and Kim, 2002) Western-blotting following the enzymatic assays using anti-Dnmt1 indicates that the E1A-mediated enzymatic stimulations (lanes 2 (Pradhan and Kim, 2002). The results from (a) and (b) are the and 3) are not simply due to stabilizing Dnmt1. The results are the average of at least three independent experiments with s.d. average of at least three independent experiments with s.d.

Oncogene E1A and E7 target Dnmt1 WA Burgers et al 1653 upregulates DNA methyltransferase activity as effi- interact when they are coexpressed in mammalian cells. ciently as full-length E1A, whereas another region of The contacts between Dnmt1 and E7, as well as E1A, E1A (residues 127–204) is unable to do so (Figure 3). are direct since GST-E7 and GST-E1A produced in Western blotting of Dnmt1 following DNA methyl- Escherichia coli bind efficiently to baculovirus-expressed transferase assays shows that the observed enzymatic Dnmt1 (Figure 4b). Next, we performed DNA methyl- stimulation is not simply due to stabilizing the Dnmt1 transferase assays to determine whether E7 could purify protein (Figure 3, bottom panel). Thus, E1A can DNA methyltransferase activity from nuclear extracts. stimulate the methyltransferase activity of Dnmt1 and As shown in Figure 4c, GST-E7 precipitates significant residues 1–90 of E1A are essential to mediate this effect. levels of DNA methyltransferase activity compared As HPV E7 shares sequence homology to E1A, it was to GST alone. Deletion analysis in GST-pulldowns of interest to determine whether E7 could also target indicates that the association of E7 with Dnmt1 is Dnmt1. Figure 4a indicates that Dnmt1 and HPV-16 E7 mediated by residues 62–84 encompassing the CR3

a transfected c 6 Flag-E7 + +

Input + - Myc-Dnmt1 4 Myc-Dnmt1 174 kD 123

IP ab: Flag /West. ab: Myc activity (fold) 2

b Relative DNA methyltransferase Relative 0 GST GST-E7 Input GST GST- E1A 12S GST-E7 Recombinant 12 Dnmt1 Coomassie * * West. blot: Input West. ab: Dnmt1 anti-Dnmt1

d Dnmt1 e 4 binding 1 98 E7 + CR1 CR2 CR3 3 E7H2 * + E7C24 * + 2 E7 25-98 +

E7 39-98 + activity (fold) E7 62-98 + 1 E7 84-98 - GST-E7 Relative DNA methyltransferase Relative 0 DNMT1 ++ IVT Input GST E7 E7H2 E7C24 E7 25-98 E7 39-98 E7 62-98 E7 84-98 Eluted GST GST GST-E7 Dnmt1 12 Coomassie * * 12 3456789 West. blot: Input * * * Coomassie * * * * * anti-Dnmt1

Figure 4 HPV-16 E7 associates with Dnmt1 in vitro and in vivo and can upregulate its DNA methyltransferase activity. (a) HPV16 E7 co-immunoprecipitates with Dnmt1. 293 T cell were transfected with pcDNA3-E7-F(Flag-E7),Myc-Dnmt1 and empty expression vectors as indicated. Cell extracts were precipitated with anti-Flag (M2, Kodak) and the presence of Myc-Dnmt1 in the immunoprecipitates was visualized by Western blot analysis using anti-Myc (3F10, Roche). (b) Dnmt1 binds E7 and E1A directly. The indicated GST proteins were incubated with baculovirus-expressed Dnmt1, followed by Western blotting using anti-Dnmt1. (c)E7 associates with DNA methyltransferase activity. Equivalent amounts of GST or GST-E7 were incubated with HeLa nuclear extract and assayed for DNA methyltransferase activity as described previously (Fuks et al., 2000). Middel panel: Coomassie-stained gel shows the input of GST fusion proteins used. Bottom panel: Western analysis using anti-Dnmt1 following enzymatic assays. Error bars represent s.d. (d) The indicated GST-E7 fusion proteins were tested in a GST-pulldown assay for binding to in vitro translated and 35S-radiolabelled full-length Dnmt1 (IVT-Dnmt1). (e) E7 stimulates DNA methyltransferase activity. Baculovirus-expressed Dnmt1 was preincubated as described in Figure 3 with eluted and dialysed GST protein alone or GST-E7. Samples were assayed for DNA methyltransferase activity as described previously (Fuks et al., 2000). The results show the average of three independent experiments with s.d.

Oncogene E1A and E7 target Dnmt1 WA Burgers et al 1654 zinc-finger region (Figure 4d), which is known to contri- to be elucidated. One mechanism could be related to the bute to E7 transformation functions (McCance, 2005). stimulation of the methyltransferase activity of Dnmt3a Deletion of the CR1 and CR2 regions as well as by Dnmt3L, a regulator of imprinting. In that situation, point mutations of residue at position 2 or within the recent biochemical studies suggest that Dnmt3L induces Rb-binding motif of E7 (residue C24) did not alter a conformational change in Dnmt3a that opens the the interaction with Dnmt1 (Figure 4d). Interestingly, active site of the enzyme and promotes binding of DNA constructs lacking the CR1 region appear to bind more and the S-adenosyl-L-methionine (Adomet). In other strongly than wild-type E1A. We speculate that this may words, Dnmt3L seems to act as a substrate exchange be due to conformation changes within the truncated factor that accelerates DNA and Adomet binding to E1A proteins, allowing the binding region within the Dnmt3a, thereby stimulating its enzymatic activity C-terminus greater access to Dnmt1. (Gowher et al., 2005). Using a similar approach as for E1A, the effect of The stimulation of Dnmt1 activity shown in the HPV-16 E7 on the DNA methyltransferase activity of present study may mimic the increase in DNA Dnmt1 was investigated. As shown in Figure 4e, GST- methyltransferase activity as a result of elevated levels E7 significantly upregulates the enzymatic activity of of Dnmt1 observed with other viral proteins and that baculovirus-expressed Dnmt1 while equivalent amounts observed in some cancers. Our data, however, represent of GST-alone have no effect on Dnmt1 activity. a much more direct link between viral proteins and Following DNA methyltransferase assays, we per- Dnmt1, where the activity of the enzyme is stimulated formed Western blotting of Dnmt1. Interestingly, directly. This increased activity may lead to similar Dnmt1 appears to be partially stabilized by E7, cellular consequences of aberrant methylation of the suggesting a possible mechanism for how E7 stimulates genome followed by cellular transformation as a result the enzymatic activity of the protein (Figure 4e, bottom of tumour suppressor gene silencing. panel). Thus, in addition to E1A, E7 also targets Dnmt1, and this interaction appears to involve the C-terminal CR3 zinc-finger domain of E7. Furthermore, Acknowledgements E7 also upregulates the enzymatic activity of Dnmt1, and this targeting thus represents a conserved feature of We thank B Moran for the kind gift of E1A constructs. WAB these viral oncoproteins. was supported by a scholarship from the National Research In summary, we have shown that the viral oncopro- Foundation of South Africa. LB was supported by the FNRS teins E1A and E7 bind directly to Dnmt1 using and the ‘Fondation pour la Recherche Me´ dicale’. SP was supported by NEB. FF is a ‘Chercheur Qualifie´ du FNRS’. sequences involved in their transformation functions. This work was funded by a programme grant from the Cancer In addition, we found that E1A as well as E7 can Research Campaign to TK and by grants from the ‘Fe´ de´ ration upregulate the DNA methyltransferase activity of Belge contre le Cancer’, the FNRS, ‘FB Assurances’, Dnmt1. The molecular basis underlying the stimulation and ‘ARC de la Communaute´ Française de Belgique’ to of Dnmt1’s enzymatic activity by E1A and E7 remains YdL and FF.

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