Molecular and Cellular Pathobiology Histone Lysine Methyltransferase SETD 8 Promotes Carcinogenesis by Deregulating PCNA Expression

Although the physiologic significance of lysine methylation of histones is well known, whether lysine methylation plays a role in the regulation of nonhistone proteins has not yet been examined. The histone lysine methyltransferase SETD8 is overexpressed in various types of cancer and seems to play a crucial role in S-phase progression. Here, we show that SETD8 regulates the function of proliferating cell nuclear antigen (PCNA) protein through lysinemethylation. We found that SETD8methylated PCNA on lysine 248, and either depletion of SETD8 or substitution of lysine 248 destabilized PCNA expression. Mechanistically, lysine methylation significantly enhanced the interaction between PCNAand theflap endonuclease FEN1. Loss of PCNAmethylation retarded the maturation of Okazaki fragments, slowed DNA replication, and induced DNA damage, and cells expressing a methylation-inactive PCNAmutant weremore susceptible to DNA damage. An increase ofmethylated PCNAwas found in cancer cells, and the expression levels of SETD8 and PCNA were correlated in cancer tissue samples. Together, our findings reveal a function for lysine methylation on a nonhistone protein and suggest that aberrant lysine methylation of PCNA may play a role in human carcinogenesis. Cancer Res; 72(13); 3217–27. 2012 AACR. Introduction Protein methylation is recently considered an important posttranslational modification and is predominantly found on lysine and arginine residues. Lysine methylation involves the addition of 1 to 3 methyl groups on the amino acid's e-amine group, to form mono-, di-, or tri-methyllysine. Its function is best understood in histones (1). With the exception of Dot1/ DOT1L, all histone lysine methyltransferases (HKMT) contain a SET domain of about 130 amino acids, and so far nearly 40 SET domain–containing HKMTs or potential HKMTs have been identified (2). While our knowledge of the physiologic functions of HKMTs is growing, their involvement in human diseases including cancer is still not well understood. Proliferating cell nuclear antigen (PCNA) is an evolutionally well-conserved protein found in all eukaryotic species from yeast to humans, as well as in archaea. PCNA functions are related to vital cellular processes such as DNA replication, chromatin remodeling, DNA repair, sister chromatid cohesion, and cell-cycle control (3). PCNA was originally reported as an antigen for autoimmune disease in patients with systemic lupus erythematosus, detected only in the proliferating cell populations (4). Thereafter, it was shown that expression levels of PCNA during cell cycle are differential and associated with proliferation and transformation (5, 6). In the following years, a number of experiments have been done to uncover the role of PCNA in DNA replication, and one of the first functions clarified was a sliding clamp for DNA polymerase d (7, 8). Meanwhile, the progress in the field not only strengthened the importance of PCNA, but also even placed PCNA at the crossroad of many essential pathways. Importantly, PCNA is posttranslationally modified in several ways, which affects its function. So far, it has been reported that PCNA is ubiquitinated, phosphorylated, acetylated, and even SUMOylated (3). One of the well-documented posttranslational modifications of PCNA is ubiquitination. In response to DNA damage, PCNA is monoubiquitinated at the lysine 164 residue by the E2 Ub– conjugated enzyme Rad6 and the E3 Ub ligase Rad18 (Rad6/ Rad18 complex; ref. 9). Rad18 not only binds to Rad6 andPCNA, but also to DNA (10). Thus, Rad18 recruits the ubiquitination machinery to the chromatin-bound target, PCNA. In addition Authors' Affiliations: Laboratory of Molecular Medicine, HumanGenome Center, Institute of Medical Science, The University of Tokyo; Laboratory for Biomarker Development, RIKEN, Minato-ku; Department of Surgery, Kyorin University School of Medicine, Mitaka; Division of Colorectal Surgery, National Cancer Center Hospital, Chuo-ku, Tokyo; Biomolecular Characterization Team, RIKEN, Wako, Saitama; Laboratory for Medical Informatics, RIKEN, Yokohama, Kanagawa, Japan; Departments of Genetics and Oncology, Cancer Research UK, Cambridge Research Institute, University of Cambridge, Cambridge; Institute of Ophthalmology, University College London, London, United Kingdom; and Section of Hematology/Oncology, The University of Chicago, Chicago, Illinois Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). M. Takawa and H. -S. Cho contributed equally to this work. Corresponding Author: Ryuji Hamamoto, Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5233; Fax: 81-3-5449-5123; E-mail: ryuji@ims.u-tokyo.ac. jp doi: 10.1158/0008-5472.CAN-11-3701 2012 American Association for Cancer Research. Cancer Research www.aacrjournals. org 3217 on May 1, 2017. © 2012 American Association for Cancer Research. cancerres.aacrjournals. org Downloaded from Published OnlineFirst May 3, 2012; DOI: 10.1158/0008-5472.CAN-11-3701