Dominant-Negative Histone H3 Lysine 27 Mutant Derepresses Silenced Tumor Suppressor Genes and Reverses the Drug-Resistant Phenotype in Cancer Cells Phillip H. Abbosh, 1 John S. Montgomery, 1 Jason A. Starkey, 2 Milos Novotny, 2 Eleanor G. Zuhowski, 3 Merrill J. Egorin, 3 Annie P. Moseman, 1 Adam Golas, 1 Kate M. Brannon, 1 Curtis Balch, 1 Tim H.M. Huang, 4 and Kenneth P. Nephew 1 1 Medical Sciences, School of Medicine; 2 Department of Chemistry, Indiana University, Bloomington, Indiana; 3 Department of Medicine, Division of Hematology-Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania; and 4 Human Cancer Genetics Program, Department of Molecular Virology, Immunology, and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio Abstract Histone modifications and DNA methylation are epigenetic phenomena that play a critical role in many neoplastic processes, including silencing of tumor suppressor genes. One such histone modification, particularly at H3 and H4, is methylation at specific lysine (K) residues. Whereas histone methylation of H3-K9 has been linked to DNA methylation and aberrant gene silencing in cancer cells, no such studies of H3- K27 have been reported. Here, we generated a stable cell line overexpressing a dominant-negative point mutant, H3-K27R, to examine the role of that specific lysine in ovarian cancer. Expression of this construct resulted in loss of methylation at H3-K27, global reduction of DNA methylation, and increased expression of tumor suppressor genes. One of the affected genes, RASSF1 , was shown to be a direct target of H3-K27 methylation–mediated silencing. By increasing DNA-platinum adduct formation, indicating increased access of the drug to target DNA sequences, removal of H3-K27 methylation resensitized drug-resistant ovarian cancer cells to the chemo- therapeutic agent cisplatin. This increased platinum-DNA access was likely due to relaxation of condensed chromatin. Our results show that overexpression of mutant H3-K27 in mammalian cells represents a novel tool for studying epigenetic mechanisms and the Histone Code Hypothesis in human cancer. Such findings show the significance of H3-K27 methylation as a promising target for epigenetic-based cancer therapies. (Cancer Res 2006; 66(11): 5582-91) Introduction Chromatin structure affects numerous diverse aspects of cell biology, ranging from genomic integrity and telomere maintenance to gene activation and silencing. Chromatin architecture can be influenced by a number of covalent alterations to protein and DNA, including histone modifications and DNA methylation. One histone modification, methylation of lysine, has recently been a subject of intense study, particularly as it relates to the transcriptional status of genes and chromatin structure. A repressive histone modifica- tion, methylation of H3-K27, is mediated by proteins in the Polycomb group (PcG) family of genes, originally identified as genes suppressing the development of extra sex combs in Drosophila melanogaster (1). PcG repressors form multiple, unique complexes to suppress gene expression (2–4). Specifically, PcG complexes containing the histone methyltransferase E(z) (in Drosophila ) or EZH2 (in humans) silence chromatin via methylation of H3-K27 (2, 5–7). EZH2 was recently shown to be overexpressed in advanced prostate (8) and breast (9) cancers. In normal fibroblasts, forced overexpression of EZH2 can induce transformation and increase invasiveness whereas EZH2 knockdown in cancer cells results in decreased proliferation (8, 9). Moreover, another PcG gene, SU(Z)12 , has been shown to be up-regulated in a number of human tumors and exists in a repressive complexes with EZH2 (10), further implicating PcG proteins and consequently H3-K27 methylation in the development of tumor phenotypes. Despite findings of altered methyltransferase activity in cancer, more direct studies of histone modifications are needed to categorically determine their precise role in tumorigenesis and other cellular events. The Histone Code Hypothesis is now widely accepted, due in large part to yeast genetic studies in which components of chromatin or chromatin-modifying enzymes have been manipulated (11, 12). However, because mammalian cells contain multiple copies of each histone and multiple (often redundant) chromatin-modifying enzymes, similar genetic approa- ches have proved much more difficult. Consequently, mammalian studies of histone methylation have been limited primarily to specific methyltransferase gene knockouts in mice (13, 14), which are technically difficult and time-consuming. Furthermore, even fewer studies have specifically addressed the status, causes, and consequences of histone methylation in cancer. In the present study, we sought to directly examine the role of H3-K27 methylation in cancer cells. By overexpressing a dominant- negative histone transgene incapable of being methylated (due to an arginine-for-lysine mutation, H3-K27R), we successfully reduced global levels of H3-meK27 in human ovarian cancer CP70 cells, a cell line highly resistant to the chemotherapeutic agent cisplatin (15). Removal of H3-meK27 in CP70 cells resulted in altered chromatin structure and composition, reexpression of tumor suppressor genes, and resensitization to cisplatin. Further, we show concordance between DNA methylation and H3-K27 methylation. Our data indicate that the mechanism of chemo- sensitization may be due to ‘‘loosened’’ chromatin, allowing increased interactions between DNA-damaging agents and DNA, in addition to changes in gene expression. Our results may provide Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Kenneth P. Nephew, Medical Sciences, School of Medicine, Indiana University, 302 Jordan Hall, 1001 East 3rd Street, Bloomington, IN 47405-4401. Phone: 812-855-9445; Fax: 812-855-4436; E-mail: knephew@indiana.edu. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-3575 Cancer Res 2006; 66: (11). June 1, 2006 5582 www.aacrjournals.org Research Article Research. on November 25, 2015. © 2006 American Association for Cancer cancerres.aacrjournals.org Downloaded from