Viral oncoproteins target the DNA methyltransferases

Abstract

Small DNA tumour viruses have evolved a number of mechanisms to drive nondividing cells into S phase. Virally encoded oncoproteins such as adenovirus E1A and human papillomavirus (HPV) E7 can bind an array of cellular proteins to override proliferation arrest. The DNA methyltransferase Dnmt1 is the major mammalian enzyme responsible for maintaining CpG methylation patterns in the cell following replication. One of the hallmarks of tumour cells is disrupted DNA methylation patterns, highlighting the importance of the proper regulation of DNA methyltransferases in normal cell proliferation. Here, we show that adenovirus 5 E1A and HPV-16 E7 associate in vitro and in vivo with the DNA methyltransferase Dnmt1. Consistent with this interaction, we find that E1A and E7 can purify DNA methyltransferase activity from nuclear extracts. These associations are direct and mediated by the extreme N-terminus of E1A and the CR3 zinc-finger domain of E7. Furthermore, we find that a point mutant at leucine 20 of E1A, a residue known to be critical for its transformation functions, is unable to bind Dnmt1 and DNA methyltransferase activity. Finally, both E1A and E7 can stimulate the methyltransferase activity of Dnmt1 in vitro. Our results provide the first indication that viral oncoproteins bind and regulate Dnmt1 enzymatic activity. These observations open up the possibility that this association may be used to control cellular proliferation pathways and suggest a new mechanism by which small DNA tumour viruses can steer cells through the cell cycle.

Figure 1

E1A interacts with Dnmt1 through its extreme N-terminal region. (a) E1A co-immunoprecipitates with Dnmt1 from transfected cell extracts. 293T cells were transfected with HA-Ad5 E1A (expressing HA-tagged E1A 12S) alone or together with either Myc-Dnmt1 (expressing Myc-tagged full-length Dnmt1, Myc-Dnmt1) or pMyc-PCNA (Myc-tagged PCNA, Myc-PCNA) 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 with anti-Myc antibody (9E10, Roche) and the presence of E1A in the immunoprecipitates was visualized using anti-HA antibody (3F10, Roche), as described (Vire et al., 2006). (b) Schematic representation of E1A with the deletions and point mutants used in GST-pulldowns. (c) In vitro translated (IVT) Dnmt1 was incubated with the indicated GST fusions. The reactions were subjected to GST-pulldown assays as described (Deplus et al., 2002). Bottom panel: Coomassie-stained gel showing the input of GST proteins used.

Figure 2

E1A associates with DNA methyltransferase activity and leucine 20 is important for enzymatic binding. (a) E1A precipitates DNA methyltransferase activity through residues 1–90. Equivalent amounts of GST or GST-E1A fusion proteins bound to Sepharose beads were incubated with HeLa nuclear extract, washed and assayed for DNA methyltransferase activity as described (Fuks et al., 2000). Activity is shown as c.p.m. of S-adenosyl-l-(methyl-³H)-methionine incorporated into a synthetic 33-bp hemimethylated oligonucleotide substrate (upper strand: GAT meCG-meC meCGA TGmeC GmeCG AAT meCGmeC GAT meCGA TGmeC GAT; lower strand: ATC GCA TCG ATC GCG ATT CGC GCA TCG GCG ATC). Values are normalized to background controls lacking substrate. (b) Point mutation within leucine 20 of E1A strongly impaired association with DNA methyltransferase activity. For panels (a) and (b): middle panels show Coomassie-stained gel with the input of GST proteins; bottom panels represent Western-blotting following the enzymatic assays using anti-Dnmt1 (Pradhan and Kim, 2002). The results from (a) and (b) are the average of at least three independent experiments with s.d.

Figure 3

E1A upregulates the methyltransferase activity of Dnmt1. We expressed and purified baculovirus human Dnmt1 as described previously (Pradhan et al., 1999). Baculovirus-expressed Dnmt1 was preincubated for 10 min on ice with increasing concentrations (1 and 5 μg) of eluted and the indicated dialysed GST proteins. Activity is shown as c.p.m. of S-adenosly-l-(methyl-³H)-methionine incorporated into a hemimethylated oligonucleotide substrate. Bottom panel: Western blotting following the enzymatic assays using anti-Dnmt1 (Pradhan and Kim, 2002) indicates that the E1A-mediated enzymatic stimulations (lanes 2 and 3) are not simply due to stabilizing Dnmt1. The results are the average of at least three independent experiments with s.d.

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.