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 (Wang et al., 2001; Nishioka et al., 2002). Likewise, SET7 methylates non-histone substrates, which include p53 (Pradhan et al., 2009). SET7-mediated mono-methylation of p53 at K372 (p53K372me1) increases p53 stability, likely through recruitment of the histone acetyltransferase Tip60 for p53 acetylation (Kurash et al., 2008). SET7-mediated p53 methylation also antagonizes the transcriptional repression by Smyd2-mediated p53 methylation (Huang et al., 2006).
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So far our data demonstrate that the E6-mediated downregulation of the HMT activities of CARM1 and PRMT1 has important function in vivo. Whether inhibition of SET7 by E6 observed in vitro (Figure 2) is also physiologically relevant was investigated. Again, p53 was used as a working model as p53 is one of the SET7 substrates and E6 targets. First we confirmed whether SET7, and presumably the resulting mono-methylation of p53 at lysine-372 (p53K372me1), stabilizes p53 protein level as reported earlier (Chuikov et al., 2004; Ivanov et al., 2007; Kurash et al., 2008). As observed in Supplementary Figure S11, without Adr treatment, knocking down SET7 (lane 3) or expressing methylase-dead SET7 (lane 4) affected neither p53 nor p21 protein level in U2OS cells, indicating that SET7 unlikely controls p53 stability and activity under normal conditions. By contrast, Adr induced p53 and p21 levels (compare lane 5 with lane 1), but failed to do so when SET7 was knocked down (lane 7) or the catalytically dead SET7 was included (lane 8), indicating that SET7 activity on p53 (p53K372me1) is required for p53 stabilization and downstream gene expression in response to genotoxic stresses. Subsequently, we investigated whether SET7 indeed contributes to the E6-mdiated repression of p53 function under DNA damage conditions. As shown in Figure 7a, the 16E6-mediated inhibition of p21 expression (compare lane 2 with lane 1) was greatly lost when SET7 was depleted from U2OS cells (compare lane 4 with lane 3), indicating that E6-mediated repression of p53 function depends on SET7. Consistently, E6 reduced SET7-mediated p53 methylation in DNA damage-induced cells. As shown in Figure 7b, p53K372me1 signal was abolished in U2OS cells expressing Flag-18E6 (lanes 1 and 2) and stimulated in HeLa cells depleted of 18E6 (lanes 3 and 4). p21 protein level was induced as well in HeLa cells without 18E6 (lane 4). We further addressed that SET7 was required to maintain Adr-induced p53 protein level in both U2OS and HeLa cells as p53 protein level decreased upon SET7 knockdown (Figure 7c, lanes 3 and 6). As expected, Adr only slightly enhanced the p53 level in HeLa cells, compared with that in U2OS cells (compare lane 5 with lane 2). SET7 depletion almost totally abolished the p53 protein level in HeLa cells (compare lane 6 with lane 4), whereas a certain portion of p53 was left upon SET7 depletion in U2OS cells (compare lane 3 with lane 1). These differential effects are likely due to the stable E6 expression in HeLa cells. In agreement with this idea, E6 knockdown in HeLa cells greatly recovered the p53 protein level (compare lane 7 with lane 5). These in vivo results strongly suggest that endogenous SET7 protects p53 from E6-mediated p53 degradation.
The current work presents the first evidence to show that HPV E6 is able to suppress the activities of both the arginine-specific HMTs, CARM1 and PRMT1, and the lysine-specific HMT, SET7. The inhibition is functionally significant in HPV-transformed cells as through this E6 downregulates the stability and transactivation function of p53. Figure 8 provides a model based on our current findings and reports by others. In HPV-non-infected normal cells (Figure 8a), DNA damage induces SET7-mediated p53 methylation and stabilization (Chuikov et al., 2004; Ivanov et al., 2007; Kurash et al., 2008), as well as p53-dependent recruitment of CARM1 and PRMT1 to p53-target gene promoters for transcriptional activation (An et al., 2004). In HPV E6-expressing cells (Figure 8b), E6 attenuates stress-induced p53 function at least through the following two distinct pathways: (1) E6 downregulates p53 protein stability depending on E6AP (Scheffner et al., 1990, 1993) or by inhibiting SET7-mediated p53 methylation at K372 (Figure 7). Given that p53 pre-methylated by SET7 resists E6-triggered degradation (Figure 7), inhibition of SET7 by E6 might prime E6-mediated p53 degradation. This also in part explains the fact that not all p53 is degraded in E6-expressing cells (Howie et al., 2009); (2) E6 can further target p53-responsive promoters (Figure 6) and downregulate the corresponding histone methylation mediated by the p53 coactivators CARM1 and PRMT1 (Figure 4), leading to reduced binding of p53 to chromatin (Figures 4 and 6) and loss of p53 transactivation (Figure 3). Together, a novel model is provided to show the dual roles of E6 in regulating p53 function through modulation of distinct HMT activity. We not only demonstrate the upstream regulatory mechanism leading to E6-mediated p53 degradation, but also reveal an alternative approach for E6 to shut down the function of the remaining p53 not degraded by E6. As CARM1, PRMT1, and SET7 are critical co-regulators not only for p53 (Lee and Stallcup, 2009; Pradhan et al., 2009) and E6 associates with these HMTs in p53-null H1299 cells (Supplementary Figure S12), E6-mediated HMT inhibition is expected to have a broader impact.
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.
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. 2ff7e9595c
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