KDM8, a H3K36me2 histone demethylase that acts in the cyclin A1 coding region to regulate cancer cell proliferation Datsun A. Hsiaa, Clifford G. Teppera, Mamata R. Pochampallia, Elaine Y. C. Hsiaa, Chie Izumiyaa, Steve B. Huertaa, Michael E. Wrightb, Hong-Wu Chena, Hsing-Jien Kunga,1, and Yoshihiro Izumiyaa,c,1 aDepartment of Biochemistry and Molecular Medicine, University of California Davis School of Medicine, University of California Davis Cancer Center, Sacramento, CA 95817; bDepartment of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52242; and cDepartment of Dermatology, University of California Davis School of Medicine, University of California Davis Cancer Center, Sacramento, CA 95817 Edited* by James C. Wang, Harvard University, Cambridge, MA, and approved April 21, 2010 (received for review January 13, 2010) Localized chromatin modifications of histone tails play an impor- Results tant role in regulating gene transcription, and aberration of these JMJD5/KDM8 Is a H3K36me2 Demethylase. Our initial experiments processes leads to carcinogenesis. Methylated histone lysine resi- explored the potential histone demethylase activity of KDM8. To this dues, a key player in chromatin remodeling, are demethylated by end, WT KDM8 and KDM8-H321A (JmjC domain mutant)– the JmjC class of enzymes. Here we show that JMJD5 (now renamed inducible cell lines were generated using MCF7 breast cancer cells. KDM8), a JmjC family member, demethylates H3K36me2 and is By analogy to other JmjC histone demethylases, the H321A mutation required for cell cycle progression. Chromatin immunoprecipitation is predicted to knock out KDM8 enzymatic activity through disrup- assays applied to human genome tiling arrays in conjunction with tion of its ability to bind the ferrous ion and thus its putative deme- RNA microarray revealed that KDM8 occupies the coding region of thylase activity. Immunofluorescence as well as immunoblotting as- cyclin A1 and directly regulates transcription. Mechanistic analyses says were performed using methylation-specific antibodies to screen showed that KDM8 functioned as a transcriptional activator by for probable histone residue substrates. Immunofluorescence assay inhibiting HDAC recruitment via demethylation of H3K36me2, an revealed that 80% of KDM8-overexpressing cells exhibited a sub- CELL BIOLOGY epigenetic repressive mark. Tumor array experiments revealed stantial decrease in H3K36me2 staining. In contrast, a significant KDM8 is overexpressed in several types of cancer. In addition, increase of H3K36me2 methylation was detected in approximately loss-of-function studies in MCF7 cells leads to cell cycle arrest. These 60% of KDM8-H321A–expressing cells (Figs. 1 A and B). The result studies identified KDM8 as an important cell cycle regulator. also indicates that KDM8-H321A may act as a dominant negative for H3K36me2 demethylation. Significantly, the overall level of neither breast cancer | epigenetics | JmjC | cell cycle | transcription H3K36me1 nor H3K36me3 methylation was altered, indicating the specificity of the enzyme. Immunoblot analysis of acid-extracted egulation of gene expression through posttranslational modi- histones from the inducible cell lines further verified that over- Rfication of the core histones has increasingly shown to be of expression of KDM8, but not the enzymatically inactive H321A great importance, particularly in a cancer setting. Among the mul- mutant, demethylated H3K36me2 in vivo (Fig. 1C). The in vivo tiple types of histone modifications, histone methylation, once con- specificity of KDM8 toward H3K36me2 was further demonstrated by sidered irreversible, has quickly emerged to become a key epigenetic blotting with other commercially available antibodies against meth- mark in regulating many critical cellular functions. The recent dis- ylated lysine or arginines of histones H3 and H4, including anti- covery of histone demethylases has shed light on the reversibility of H3K36me1 and anti-H3K36me3; none revealed a decreased in- C A this chromatin mark and its effects on gene expression. Studies ex- tensity in the KDM8-overexpressing cell line (Fig. 1 and Fig. S1 ). ploring the JmjC (Jumonji C domain)–containing proteins, a new- Additionally, MS analysis using a truncated recombinant KDM8 protein consisting of the JmjC domain (101-C) or its equivalent class of histone demethylases (1–4), primarily identified their H321A mutant with H3K36me2 peptide was performed. These enzymatic activity at the promoters of specific target genes (5, 6). results revealed that WT 101-C truncated protein demethylates The JmjC domain–containing gene family encodes a wide range H3K36me2 directly, as indicated by the 14-Da shift from the origi- of the eukaryotic genome and is conserved in species spanning fi nal peptide mass. In contrast, no mass shift was observed when cat- from yeast to humans. Currently, most family members classi ed alytically inactive KDM8 101-C H321A mutant was used (Fig. 1D). as histone demethylases contain known histone-binding domains such as PHD and Tudor domains (7). JMJD5 (renamed KDM8) JMJD5/KDM8 Expression Is Critical for MCF7 Cancer Cell Proliferation. is a member of this extensive protein family that lacks recog- The biological functions and clinical significance of KDM8 in the nizable histone-binding domains and remains largely unexplored. context of breast cancer were next investigated. First, we com- Although one study speculated that KDM8 acts as a potential pared the expression of KDM8 in a panel of breast cancer cell tumor suppressor gene based on retrovirus insertional mutagen- lines to that of primary human mammary epithelial cells (HMECs) esis (8), no biological and molecular characterizations were de- by immunoblot analysis with a custom-generated rabbit polyclonal scribed in the report. We extensively examine and provide evidence that KDM8 possesses H3K36me2 demethylase activity and has the ability to Author contributions: D.A.H., M.R.P., H.-J.K., and Y.I. designed research; D.A.H., C.G.T., regulate cyclin A1 transcription in MCF7 breast cancer cells. We M.R.P., E.Y.C.H., C.I., S.B.H., and Y.I. performed research; D.A.H., E.Y.C.H., C.I., H.-W.C., and Y.I. contributed new reagents/analytic tools; D.A.H., C.G.T., M.R.P., M.E.W., H.-J.K., and found that KDM8 is recruited to cyclin A1 coding region bound Y.I. analyzed data; and D.A.H., C.G.T., H.-J.K., and Y.I. wrote the paper. H3K36me2 and demethylates this mark, resulting in increased The authors declare no conflict of interest. transcriptional activity. This finding is a departure from previous *This Direct Submission article had a prearranged editor. studies that showed that the majority of histone demethylases 1To whom correspondence may be addressed. E-mail: [email protected] or yizumiya@ exert their epigenetic effects at the promoters of genes. Addi- ucdavis.edu. tionally, we describe overexpression of KDM8 in breast cancer This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tumors as well as its requirement for MCF7 cell cycle progression. 1073/pnas.1000401107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1000401107 PNAS | May 25, 2010 | vol. 107 | no. 21 | 9671–9676 Downloaded by guest on October 2, 2021 A B C D Fig. 1. KDM8 H3K36me2 demethylation in MCF7 breast cancer cells. (A) KDM8 and H321A, an inactive enzymatic mutant, were induced to express as Flag fusion proteins. Indirect immunofluorescence with antibodies against Flag (red staining) and methylated H3K36me1, H3K36me2, or H3K36me3 (green staining) was used to analyze in vivo substrate specificity of KDM8. DAPI staining (blue) indicates nuclei location in each field. Cells overexpressing KDM8 (arrows) showed significant loss of H3K36me2 staining, which was dependent on the active JmjC domain, and not observed in the H321A overexpressed cells. (B) Quantitative analysis of Dox-induced KDM8/H321A MCF7 H3K36me2 stained cells from 10 random fields. Eighty percent of induced KDM8 cells showed diminished H3K36me2, whereas 60% of H321A-induced cells exhibited increases in H3K36me2 staining. Percentage of cells that exhibited no change in H3K36me2 is not shown. (C) Histones extracted from Dox-induced KDM8/H321A MCF7 were analyzed by Western blotting with antibodies against H3K36me2 and H3K9me3. A decreased signal in H3K36me2 was observed in KDM8 overexpressed cells (lane 2 vs. lane 1), which was dependent on the active JmjC domain (inactive mutant lanes 3–4). (D) MS analysis of observed in vitro KDM8 demethylase activity. Reactions using H3K36me2 peptide combined with either GST KDM8 101-C WT or GST-KDM8 101-C H321A show 14 Da shift in WT reactions only (asterisk). antisera specific to KDM8. Although KDM8 protein expression indicated that 97.5% of the tumor samples were intensely stained was very low in HMECs, all of the breast cancer cell lines, including for KDM8 expression (i.e., 3+ score) compared with 67.5% in MCF7, exhibited significantly higher expression (Fig. 2A). To ex- normal patient-matched tissues (Fig. 2D). Both scoring systems tend our results, immunohistochemistry (IHC) was conducted (computerized and manual) concluded that KDM8 is overex- using a human tissue microarray containing 40 individual breast pressed in tumor tissues compared to their nonmalignant coun- cancer samples and patient-matched adjacent normal tissue con- terparts. To extend these results and determine if KDM8 trols (Fig. 2 B and C). Consistent with the results obtained in breast overexpression is potentially a general aberration occurring in cancer cell lines, KDM8 was widely overexpressed in breast cancer, a tumor array membrane consisting of spotted tumor tumors. IHC staining was quantified via computerized scanning and normal cell lysates was probed for KDM8 protein expression. analysis and manual scoring by an expert panel of pathologists Multiple types of tumors including thyroid, adrenal, bladder, who scored the IHC results on a scale from 0 through 3+, with 3+ uterine, and liver exhibited overexpressed KDM8 protein in indicating the most intense staining. The summarized IHC data comparison to their respective normal tissue controls (Fig. S2). 9672 | www.pnas.org/cgi/doi/10.1073/pnas.1000401107 Hsia et al.