Introduction

Human papillomaviruses (HPVs) cause cervical carcinoma and are associated with several other human cancers such as lung cancer (Chen et al., 2004; Ganguly and Parihar, 2009). They are small viruses with a double-stranded circular DNA, which replicates in the nucleus of infected cells. Among the HPV proteins, the early proteins E6 and E7 attract most attention as they transform and immortalize cells (Ganguly and Parihar, 2009; McLaughlin-Drubin and Munger, 2009). One of the well-documented mechanisms by which HPV causes cervical carcinoma is E6-mediated degradation of the tumor suppressor p53 (Scheffner et al., 1990; Munger and Howley, 2002; Howley and Livingston, 2009). E6, complexed with the E6-associated protein (E6AP), functions as a ubiquitin ligase to perform p53 ubiquitination for destruction (Scheffner et al., 1990; Scheffner et al., 1993). Nevertheless, this is unlikely the only mechanism by which E6 transforms cells. Additional p53-independent functions/targets of E6 have been described (Howley, 2006; Howie et al., 2009). Several groups reported that E6 inhibits the histone acetyltransferase activity of the important coactivator p300 (Patel et al., 1999; Zimmermann et al., 1999; Thomas and Chiang, 2005).

To activate gene transcription from the condensed chromatin with DNA wrapped by histone and non-histone chromosomal proteins, transcriptional activators with sequence-specific DNA-binding activity will recruit coactivators to the regulatory sequence of a particular gene. These coactivators usually contain at least one of the following activities: as a scaffold protein, histone modification, ATP-dependent chromatin remodeling or the activity to regulate DNA methylation (Trojer and Reinberg, 2007). Histone modifiers can post-translationally modify histones as well as a growing list of non-histone proteins (Lee and Stallcup, 2009; Pradhan et al., 2009). The modifications on histones, alone or in combination, constitute an epigenetic language, which is believed to alter gene expression by changing the DNA–histone interaction, histone–protein interaction or by serving as signals to recruit other regulators (Strahl and Allis, 2000).

Many sites on histone tails can be modified by various histone modifiers. Up to date, the reported modifications include acetylation, methylation, phosphorylation, ubiquitination, sumoylation, ADP-ribosylation, proline isomerization, and so on (Kouzarides, 2007; Campos and Reinberg, 2009). Unlike histone acetylation, a hallmark of gene activation occurring exclusively on lysine, histone methylation is involved in both gene activation and repression, and takes place on both lysine and arginine. Arginine could be di-methylated either asymmetrically by type-I arginine histone methyltransferases (HMTs) or symmetrically by type-II HMTs (Wolf, 2009). An et al. (2004) showed that p53 recruits the type-I arginine HMTs CARM1 and PRMT1 to methylate histones at p53-responsive promoters and activate p53 downstream genes. Notably, CARM1 and PRMT1 coactivate and methylate many other proteins (Lee and Stallcup, 2009). By contrast, lysine can be mono-, di- or tri-methylated (Shukla et al., 2009). SET7 performs mono-methylation on lysine-4 of H3 to exert its coactivation function (Figure 3, the remaining p53 esca** from E6-mediated degradation was still able to activate transcription to 5.7-fold (compare lane 7 with lane 6). Results published by two other laboratories also support the same conclusion. First, Zimmermann et al. (1999) showed that the remaining p53 still retained 1/3 of the total p53 activity (Figure 7a in Zimmermann et al., 1999, compare bar 4 with bar 2). Second, Patel et al. (1999) demonstrated that the remaining p53 regained activity in the presence of the histone acetyltransferase p300 (Figure 8a in Patel et al., 1999, compare bar 5 with bar 4). Therefore, we believe that the remaining p53 is still active in transcription.

Distinct roles of CARM1, PRMT1 and SET7 in mediating p53 function

Although CARM1, PRMT1 and SET7 all interacted with p53, unlike PRMT1 and CARM1, SET7 did not enhance p53 transactivation function without DNA-damage insult (compare Figure 3 with Supplementary Figure S13). This is not surprising as SET7 activity is minimal unless being induced by DNA damage (Ivanov et al., 2007). In addition, only SET7 among these three HMTs methylates p53 under stress (Chuikov et al., 2004; Ivanov et al., 2007; Kurash et al., 2008; Figure 2, and data not shown). In agreement with previous reports (Chuikov et al., 2004; Ivanov et al., 2007; Kurash et al., 2008), we found that the enzymatic activity of SET7 was important for p53 stabilization and downstream gene expression in response to DNA-damage stress (Supplementary Figure S11). It is likely that SET7 methylates p53 and directly prevents p53 from being ubiquitylated as K372 is one of p53 ubiquitylation sites (Kruse and Gu, 2008). Alternatively, p53 methylation by SET7 might indirectly influence p53 protein stability by facilitating p53 acetylation. Indeed, SET7-mediated p53K372 methylation primes p53 acetylation on several sites in the C-terminus (Ivanov et al., 2007; Kurash et al., 2008), and p53 acetylation increases p53 stability by blocking the ubiquitination sites of p53 (Li et al., 2002). These mechanisms likely explain how p53 methylation by SET7 resists E6-dependent p53 degradation through E6AP/ubiquitin (Scheffner et al., 1990, 1993). Nevertheless, E6-triggered p53 degradation can rely on neither E6AP (Massimi et al., 2008) nor ubiquitin (Camus et al., 2007). Whether E6-mediated SET7 inhibition also contributes to the ubiquitin-independent pathway remains unknown.

Low-risk E6, which does not degrade p53, downregulates p53 function through HMTs

Although low-risk HPVs have no detectable cell transformation or immortalization activity, they are major causative agents of anogenital warts and recurrent respiratory papillomatosis (Lacey et al., 2006; Hu and Goldie, 2008; Pim and Banks, 2010). For example, almost 100% of anogenital warts are caused by HPV type-6 or type-11 (Pim and Banks, 2010). The mechanism by which low-risk HPVs cause abnormal cell growth is largely unknown. Consistent with the previous report in which low-risk E6 is shown to affect p53 function through inhibition of p300 (Thomas and Chiang, 2005) or destabilization of TIP60 (Jha et al., 2010), in the current study we further observe that, similar to the high-risk HPV E6, the low-risk E6 from HPV type-11 suppressed p53 transactivation function likely by targeting HMTs (Figures 2, 3c and 5, and Supplementary Figures S2 and S5). This strongly suggests that both low-risk and high-risk E6 proteins may induce abnormal cell growth by interfering with the function of p53 and HMTs.

Global impact of E6-mediated HMT inhibition

Given that (i) E6 interacts with CARM1, PRMT1 and SET7 regardless of the presence of p53 (Supplementary Figure S12); (ii) E6 directly inhibits the enzymatic activities of these HMTs (Figure 2); and (iii) these HMTs are involved in a variety of cellular functions by methylating a growing list of substrates, the p53-independent impact of E6-mediated HMT inhibition is expected. CARM1 and PRMT1 belong to the protein arginine methyltransferase family, which at least contains 10 members with homology within the active site (Lee and Stallcup, 2009). They participate in various cellular processes involved in signal transduction, cell proliferation, transcriptional regulation, chromatin remodeling, DNA repair, RNA processing, protein stability and nucleo-cytoplasmic trafficking (Pahlich et al., 2006; Pal and Sif, 2007; Lee and Stallcup, 2009; Wolf, 2009). Importantly, preventing PRMT1-mediated methylation disturbs the DNA-repair function of MRE11 and 53BP1 (Boisvert et al., 2005a, 2005b). PRMT1 is also critical in maintaining genome integrity (Yu et al., 2009). The lysine-specific HMT SET7 methylates a list of non-histone proteins besides p53. These include TAF10, p65 of nuclear factor-κB, DNA methyltransferase-1 (DNMT1) and estrogen receptor (ER) (Kouskouti et al., 2004; Subramanian et al., 2008; Ea and Baltimore, 2009; Esteve et al., 2009; Yang et al., 2009). SET7-mediated DNMT1 methylation decreases DNMT1 protein stability (Esteve et al., 2009). Note that defects in DNA repair and genome integrity are signs for cancer, and that DNMT1 is generally induced and stabilized in cancer cells to methylate the tumor-suppressor gene promoter for subsequent transcriptional suppression of the gene (Miremadi et al., 2007). Thus, loss of CARM1, PRMT1 or SET7 function by E6 might contribute to the oncogenic activity of E6.

Viral regulation of histone methylation

Several viral mechanisms to affect gene transcription involve modulation of histone methylation. These include recruitment of G9a, Suv39h1 to the viral major immediate-early promoter by the immediate-early-2 protein of the human cytomegalovirus (Reeves et al., 2006); removal of the HMT-associated cellular transcriptional regulator from DNA by adenovirus E1A (Ghosh and Harter, 2003); recruitment of the histone demethylase LSD1 by host cell factor-1 to immediate-early promoters of herpes simplex virus and varicella zoster virus (Liang et al., 2009); and direct expression of viral protein with HMT activity such as the vSET protein of paramecium bursaria chlorella virus-1 (Mujtaba et al., 2008). Together with our current study, the importance of HMT regulation in viral survival is highlighted.

Materials and methods

Cell line, DNA transfection and luciferase assay

The human cervix adenocarcinoma cell line, HeLa, the human osteosarcoma cell line, U2OS, and the human lung carcinoma cell line, H1299, were obtained from, and maintained as instructed by, the ATCC (Manassas, VA, USA). DNA transfections were performed by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. For ectopic protein expression, cells were harvested at 36∼72 h after transfection. For knockdown of endogenous proteins, cells were collected 72 h after transfection with 10 nM specific siRNA (Ambion, Austin, TX, USA). The sense sequence of si-SET7 is 5′-GGACCGCACUUUAUGGGAAtt-3′; of si-CARM1 is 5′-AGACAGAGCUACGACAUCAtt-3′; of si-PRMT1 is 5′-GCAACUCCAUGUUUCAUAAtt-3′; of si-18E6 is 5′-GAAAAACUUAGACACCUUAtt-3′; and of si-p53 is 5′-GUAAUCUACUGGGACGGAAtt-3′. Luciferase assays were performed as described previously (Juan et al., 2000). Briefly, transfections were performed at 80% confluence for H1299 and U2OS cells. Cells grown in six-well dishes were harvested 48 h after transfection. A 2- to 10-μl volume of each lysate was mixed with 50 μl of buffer (dual-luciferase reporter assay system; Promega, Madison, WI, USA) and measured for luciferase activities.

Plasmids

The following plasmids have been described previously: pcDNA3-F16E6 (Patel et al., 1999; Thomas and Chiang, 2005) and p513-16E66C/6S F47R (Nomine et al., 2006), which encode 16E66C/6S F47R and Flag-18E6, respectively; pcDNA 3.1-p53, which encodes wt p53 for mammalian expression (Ou et al., 2005); pRL-SV40 (Dual-Luciferase Reporter Assay System; Promega); p21-Luc (el-Deiry et al., 1993); pSG5HA-CARM1, pSG5HA-PRMT1, pSG5HA-CARM1 E267Q and pSG5HA-PRMT1 E153Q, encoding wt HA-CARM1, HA-PRMT1 and their methylase-dead mutants, respectively (Chen et al., 1999; Strahl et al., 2001; Lee et al., 2002, 2007); and pcDNA3.1-SET7 and SET7 H297A, encoding, respectively, wt SET7 and the methylase-dead mutant (Chuikov et al., 2004). Plasmids pRK5-11E6 and pRK5-18E6, which express Flag-HPV-11E6 and Flag-HPV-18E6, respectively, were constructed by inserting the corresponding cDNA fragments into the EcoRI and SalI sites of pRK5-Flag. Plasmids pET-29a11E6, pET-29a16E6 and pET29a18E6, which express His-tagged 11E6, 16E6 and 18E6, respectively, in Escherichia coli were constructed by inserting the corresponding cDNA fragments into the EcoRI and SalI sites of the pET-29a(+) vector (Novagen, Darmstadt, Germany). The plasmid encoding the p53 point mutant p53K372R was generated by using pcDNA 3.1-p53 as the template for in vitro site-directed mutagenesis (Stratagene, Santa Clara, CA, USA) according to the manufacturer's instructions.

In vitro methyltransferase assay

A 10-μg weight of core histones (Millipore, Basel, Switzerland or Roche, Basel, Switzerland) or a 5-μg weight of p53 made from baculovirus (Wang et al., 2003) was mixed with recombinant E6 protein purified from bacteria and recombinant CARM1, PRMT1 or SET7 (Upstate, Billerica, MA, USA, 14-575, 14-474 or 14-469, respectively), followed by methylation reactions at 30 °C for 30 min using 1 μl of S-adenosyl-L-[methyl-3H]-methionine ([3H]-SAM (1 mCi/ml; Amersham, Piscataway, NJ, USA) in HMT buffer (50 mM Tris–HCl (pH 8.0) and 0.5 mM dithiothreitol). The reaction mixtures were then separated by 15% sodium dodecyl sulfate (SDS)–PAGE and the gels were stained with Coomassie blue, dried and subjected to autoradiography. The non-isotope methyltransferase assays were performed by using unlabeled SAM to replace 3H-SAM. After in vitro methylation reaction, proteins were separated by 15% SDS–PAGE, followed by western blot analysis.

Western blotting, IP and DAPA

Cells were lysed on ice in ice-cold IP buffer (137 mM NaCl, 2.7 mM KCl, 7.7 mM NaH2PO4, 1.5 mM KH2PO4, 0.5% NP-40, 1 mM EDTA, 10% glycerol, 1 mM dithiothreitol) for IP or in DAPA buffer (137 mM NaCl, 2.7 mM KCl, 7.7 mM NaH2PO4, 1.5 mM KH2PO4, 0.1% NP-40, 1 mM EDTA, 10% glycerol, 1 mM dithiothreitol) for DAPA. Both buffers contain a mixture of protease inhibitors (Complete, EDTA-free, Protease Inhibitor Cocktail Tablets; Roche Molecular Biochemicals, Basel, Switzerland). Western blot and IP assays were performed as described by Hsu et al. (2004). Briefly, 4 mg of cell extracts from HeLa cells or 1 mg from U2OS cells were pre-cleaned with protein-A or protein-G beads for 30 min, and then incubated with the specific Ab at 4 °C overnight. Protein-A or protein-G-conjugated beads were added for an additional 2 h incubation. The beads were then collected by centrifugation and washed six times with IP buffer. For DAPA, the biotinylated DNA fragments were incubated with streptavidin beads (S-1638; Sigma, St Louis, MO, USA) at 4 °C overnight and washed three times with DAPA buffer. Subsequently, the DNA-conjugated beads were incubated with 2 mg of cell lysates and 10 μg of Poly dI–dC (Sigma; P4929) at 4 °C overnight. The beads were then collected by centrifugation and washed six times with DAPA buffer containing 0.5% NP-40. The pulled down complexes were then resolved by 15% SDS–PAGE and analyzed by western blotting.

Chromatin IP

ChIP assays were performed as described by Hsu et al. (2004), with modifications. The immunoprecipitated DNA fragments were extracted by using a PCR purification kit (Qiagen, Hilden, Germany; no. 28106) and analyzed by real-time PCR (Light Cycler 480; Roche). The amplifications were performed in a reaction volume of 20 μl containing 2 μl of immunoprecipitated material. The sequences of the p21 promoter-specific primers are 5′-GGCTGTGGCTCTGATTG-3′ (forward) and 5′-GAGGTCTCCTGTCTCCTAC-3′ (reverse). The sequences of the GADD45 promoter-specific primers are 5′-TAGAGTGTGGCTGGACTTT-3′ (forward) and 5′-TCACAGGGATCTCTTCCG-3′ (reverse). The sequences of primers recognizing the p53-unrelated region, approximately 2 kb downstream from the p53-response element on the GADD45 promoter (An et al., 2004), are 5′-GGAGTTGGAGTTGTCAGGAAAAAGGG-3′ (forward) and 5′-GGTTGTGGTCTTTCAGGCCTCCACACC-3′ (reverse).

Antibodies

The commercial Abs used for western blotting were against the following: β-tubulin (MAB1637; Millipore), actin (MAB1501; Millipore), Flag (M2; Sigma), HA (MMS-101R; Covance, Princeton, NJ, USA), CARM1 (A300-421A; Bethyl, Montgomery, TX, USA), PRMT1 (sc-13392; Santa Cruz, Santa Cruz, CA, USA), SET7 (04-805; Millipore), histone H3 (ab1791; Abcam), Asy-H3R17me2 (07-214; Millipore), histone H4 (ab10158), Asy-H4R3me2 (07-213; Millipore), p53 (DO-1) (sc-126; Santa Cruz), p53K372me1 (ab16033; Abcam, Cambridge, UK) and p21 (C-19; Santa Cruz). The commercial Abs for IP and ChIP were against the following: CARM1 (A300-421A; Bethyl, Figure 1a; and sc-5421; Santa Cruz, Figure 4b), PRMT1 (sc-13392; Santa Cruz), SET7 (04-805; Millipore), rabbit normal IgG (PP64B; Millipore), Ace-H3 (06-599; Millipore), Asy-H3R17me2 (07-214; Millipore), Ace-H4 (06-866; Millipore), Asy-H4R3me2 (07-213; Millipore), HPV E6 (sc-460 and sc-57835; Santa Cruz) and p53 (FL-393) (sc-6243; Santa Cruz). The p53K372me1 Ab used in Figure 7d for IP was generated by immunizing rabbits with the peptide NH2-CSRAHSSHLKSK(me1)KG-COOH. The Abs against Asy-H3R26me2 and Sym-H3R26me2 for IP were generated by immunizing rabbits with KLH-conjugated NH2-KVAR(me2)KSAPC-COOH. The synthetic peptides corresponding to the symmetric and asymmetric H3R26me2 were obtained from Open Biosystems (Huntsville, AL, USA).

In vitro transcription/translation-coupled degradation assay

The assay was performed as described (Nomine et al., 2006) with modifications. In vitro transcribed and translated proteins were expressed form pcDNA3.1-SET7, pcDNA3-F16E6, pcDNA3.1-p53, and pcDNA3.1-p53K372R by TNT system (Promega). p53 was then directly incubated with E6 in degradation buffer (25 mM Tris–HCl, pH 7.5, 100 mM NaCl, 2 mM dithiothreitol) at 28°C for 2 h, or first with SET7 and SAM at 30°C for 30 min and then E6. Reactions were terminated by adding SDS sample buffer and samples analyzed by western.

Reverse transcription–PCR

RNAs were extracted by using an RNA extraction kit (Qiagen; no. 74134), reversed-transcribed and analyzed by real-time RT–PCR (Light Cycler 480; Roche) using the QuantiTect SYBR Green reagent (Qiagen; no. 204243). The expression levels of the examined genes were normalized to actin expression. The sequences of primers amplifying p21, GADD45, 18E6 and the β-actin gene are as follows: p21: 5′-CTGGAGACTCTCAGGGTCGAAA-3′ (forward) and 5′-GATTAGGGCTTCCTCTTGGAGAA-3′ (reverse); GADD45: 5′-CCCGGACCTGCACTGCG-3′ (forward) and 5′-TCAGATGCCATCACCGT-3′ (reverse); 18E6: 5′-ATTAATAAGGTGCCTGCGG-3′ (forward) and 5′-CTCTATAGTGCCCAGCTATGTT-3′(reverse); and β-actin: 5′-CCCTGGACTTCGAGCAAGAGAT-3′ (forward) and 5′-AAGGTAGTTTCGTGGATGCCACA-3′ (reverse).