The HPV E6 oncoprotein targets histone methyltransferases for modulating specific gene transcription

Oncogene (2012) 31, 2335–2349 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE The HPV E6 oncoprotein targets histone methyltransferases for modulating specific gene transcription C-H Hsu1, K-L Peng1,2, H-C Jhang1, C-H Lin1,3,4, S-Y Wu5, C-M Chiang5, S-C Lee3, WCY Yu6 and L-J Juan1,2,3 1 Genomics Research Center, Academia Sinica, Taipei, Taiwan, ROC; 2Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan, ROC; 3Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC; 4Institute of Cancer Research, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan, ROC; 5Simmons Comprehensive Cancer Center, Departments of Pharmacology and Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA and 6National Institute of Cancer Research, National Health Research Institutes, Miaoli County, Taiwan, ROC Expression of viral proteins causes important epigenetic changes leading to abnormal cell growth. Whether viral proteins directly target histone methyltransferases (HMTs), a key family enzyme for epigenetic regulation, and modulate their enzymatic activities remains elusive. Here we show that the E6 proteins of both low-risk and high-risk human papillomavirus (HPV) interact with three coactivator HMTs, CARM1, PRMT1 and SET7, and downregulate their enzymatic activities in vitro and in HPV-transformed HeLa cells. Furthermore, these three HMTs are required for E6 to attenuate p53 transactivation function. Mechanistically, E6 hampers CARM1- and PRMT1-catalyzed histone methylation at p53-responsive promoters, and suppresses the binding of p53 to chromatinized DNA independently of E6-mediated p53 degradation. p53 pre-methylated at lysine-372 (p53K372 monomethylation) by SET7 protects p53 from E6-induced degradation. Consistently, E6 downregulates p53K372 mono-methylation and thus reduces p53 protein stability. As a result of the E6-mediated inhibition of HMT activity, expression of p53 downstream genes is suppressed. Together, our results not only reveal a clever approach for the virus to interfere with p53 function, but also demonstrate the modulation of HMT activity as a novel mechanism of epigenetic regulation by a viral oncoprotein. Oncogene (2012) 31, 2335–2349; doi:10.1038/onc.2011.415; published online 3 October 2011 Keywords: HMT; chromatin; HPV; E6; p53 Introduction Human papillomaviruses (HPVs) cause cervical carcinoma and are associated with several other human cancers such as lung cancer (Chen et al., 2004; Ganguly Correspondence: Professor L-J Juan, Genomics Research Center, Academia Sinica, 128, Academia Rd, Sec. 2, Nankang, Taipei 115, Taiwan, ROC. E-mail: Received 15 November 2010; revised 19 July 2011; accepted 11 August 2011; published online 3 October 2011 and Parihar, 2009). They are small viruses with a doublestranded 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 nonhistone 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 E6 inhibits distinct HMTs C-H Hsu et al 2336 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). Whether functions of HMTs can be modulated by viral proteins remains mysterious. In the current study, we take p53, a well-known target of E6, as an example to illustrate how E6 modulates the activities of HMTs CARM1, PRMT1 and SET7 for controlling gene-specific transcription. We found that E6 at the physiological concentration downregulated p53 transcriptional activity and stability through inhibition of the arginine-specific HMTs CARM1 and PRMT1, and the lysine-specific HMT SET7. As these HMTs are importa (...truncated)