KDM8, a 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 modifications of histone tails play an impor- Results tant role in regulating transcription, and aberration of these JMJD5/KDM8 Is a H3K36me2 Demethylase. Our initial experiments processes leads to carcinogenesis. Methylated histone 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 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 -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 | | JmjC | cell cycle | transcription H3K36me1 nor methylation was altered, indicating the specificity of the enzyme. Immunoblot analysis of acid-extracted egulation of gene expression through posttranslational modi- 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, , 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 , a new- Additionally, MS analysis using a truncated recombinant KDM8 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 (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.

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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).

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Fig. 2. KDM8 is overexpressed in tumors and required for breast cancer cell proliferation. A quantitative examination of KDM8 expression in cancer cells was performed. (A) Western blot analysis showed significantly higher expression of endogenous KDM8 in a panel of breast cancer cell lines compared to primary HMECs. (B) Immunohistochemistry KDM8 staining of malignant breast cancer tumors (M) and patient-matched adjacent normal tissues (N). (C) Higher- magnification view of respective malignant and normal IHC staining of KDM8. (Scale bar, 10 μm.) (D) Intensity of staining was scored on a scale of lowest (0) to highest (3+). (E)(i) Loss of KDM8 contributes to a decrease in cell growth. Two KDM8 knockdown clones (shKDM8 no. 1, no. 5) or control cells (shControl) were maintained in culture for 5 d. Cell numbers were counted at indicated time points in triplicate. Growth data presented are means ± SD. from three independent experiments. Immunoblotted lysates showed decreased expression of KDM8 in knockdown lines (Upper). (ii) Enzymatic activity is required for MCF7 pro- liferation. KDM8 WT or catalytic inactive mutant (H321A) expression was induced (Dox) and cell growth was examined by counting cells for 6 d. Overexpression of KDM8 WT increased cell proliferation compared to noninduced control, whereas induction of mutant decreased cell proliferation. (F) Flow cytometry analysis of KDM8 shRNA MCF7 cells stained with PI identified 40% of total cells arrested in G2/M phase compared with 20% in control shRNA cells.

Because KDM8 was found to be overexpressed in cancer cells, KDM8, as induction of KDM8 WT but not catalytic mutant we focused on its potential role in cell proliferation. To examine stimulated cell growth (Fig. 2E, ii). Flow cytometric analysis of its role in cell growth, stable KDM8 knock-down cell lines were propidium iodide (PI)–stained cells revealed that cells accumu- generated by lentiviral-mediated expression of KDM8-targeting lated and arrested in G2/M as evidenced by an increase in the shRNAs in MCF7 (sh-KDM8-MCF7-1 and -5). To control for fraction of cells in G2/M from 20% to 40% in shRNA-Control- potential off-target effects of the KDM8 shRNAs, two stable cell MCF7 and shRNA-KDM8-MCF7 cells, respectively (Fig. 2F). lines with independent target sequences were generated. KDM8 Taken together, these results indicated that KDM8 is required silencing was validated by immunoblot analysis and the effects for G2/M phase cell cycle progression. on proliferation were monitored by standard cell counting with a hemocytometer. The shControl cells and shKDM8-MCF7s JMJD5/KDM8 Is Enriched in the Coding Region of Cyclin A1. To define were plated in six-well dishes and the cell proliferation was ex- the mechanism by which KDM8 regulates the cell cycle, KDM8 amined for 5 d. Cell numbers were counted every day from three target genes were identified by performing comprehensive gene independent wells. The results demonstrated that stable KDM8 expression profiling in combination with promoter tiling array knockdown resulted in a significant inhibition of growth of analysis for the detection of genomic recruitment sites. MCF7 MCF7 cells. At the end of the 5 d, shRNA-KDM8-MCF7 exhib- cells and MCF7 cells engineered for conditional expression of ited a fourfold decrease in growth compared to control shRNA WT KDM8 or KDM8-H321A were used in these experiments. cell lines (Fig. 2E, i). Further analyses indicated that the effects analysis was performed on the gene expression on MCF7 cell growth were dependent on enzymatic activity of data to identify differentially regulated genes that favor cell cycle

Hsia et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 progression and were selectively up-regulated by induced ex- examine whether KDM8 also affects cyclin A1 at the protein level, pression of WT KDM8. Our initial gene expression screening MCF10A cells were transfected with siRNA targeting KDM8 and revealed that several genes encoding cyclins and cyclin-de- immunoblotted for expression of KDM8, cyclin A, and H3K36me2. pendent kinases were up-regulated in response to KDM8 over- Consistent with the gene expression data, immunoblot analysis expression and down-regulated by KDM8-H321A (Fig. S3). In revealed that transient knockdown of KDM8 in cells exhibited parallel, binding sites for endogenous KDM8 were mapped using lowered levels of cyclin A protein, and increased levels of ChIP-on-chip analysis with promoter tiling arrays that provide 10 H3K36me2 (Fig. 3D). Taken together, these data strongly indicate kb of coverage for each promoter region spanning 7.5 kb up- that KDM8 is capable of up-regulating cyclin A1 transcription by stream through 2.45 kb downstream of the transcription start demethylating H3K36me2 located in the cyclin A1 coding region. sites (GeneChip Human Promoter 1.0R Arrays; Affymetrix). The results indicated that KDM8 is recruited to the cyclin A1 JMJD5/KDM8-Induced H3K36me2 Demethylation Leads to Increased locus. Importantly, cyclin A1 is required for progression through H3/H4 Acetylation and Cyclin A1 Transcription. In contrast to its func- ′ the G2/M phase of the cell cycle (9, 10). Of particular interest, tion as a classic activating mark in the 3 region of genes (11), the KDM8 binding sites were found primarily within the coding re- H3K36me2 chromatin mark acts as a transcriptional suppressor gion of this gene with the strongest ChIP-enrichment identified when located in the coding region (12), where it acts to suppress in exon 2, with surprisingly little binding to the promoter region spurious transcription events. Having observed KDM8 demethy- (Fig. 3A). To confirm the ChIP-on-chip cyclin A1 binding results, lation of H3K36me2 in vivo, we speculated that KDM8 targeted quantitative PCR was performed using endogenous KDM8 ChIP this methyl mark at the coding region to enhance the transcription DNA and primer sets designed to encompass the proximal pro- of cyclin A1. ChIP analysis performed with inducible KDM8 moter regions (primer sets A, B) and exon 2 (primer set C). This MCF7 cells demonstrated that KDM8 and its H321A inactive ChIP analysis demonstrated that exon 2 sequences were selectively mutant form were enriched along exon 2 of cyclin A1 but not the – enriched in anti-KDM8 chromatin immunoprecipitates (i.e., with promoter region (Fig. 4, bars 1 4, and Fig. S4). Additional ChIP respect to a control IgG ChIP) whereas sequences in the promoter analysis examining the presence of H3K36me2 at this coding re- fi gion revealed that it was greatly diminished in the presence of were not. In conclusion, KDM8 was recruited speci cally to the A coding region of the cyclin A1 gene (Fig. 3B). In agreement with our KDM8 WT, yet enhanced with H321A expression (Fig. 4 ,bars 5–8). ChIP analysis of HDAC1 enrichment, a transcriptionally re- initial microarray screening, corroborative quantitative RT-PCR fi data with induced KDM8 MCF7 cells showed 1.8-fold increase in pressive chromatin modi er (13) that is recruited by the H3K36me2 cyclin A1 expression over the noninduced controls (Fig. 3C). To mark (14), showed that during KDM8 overexpression, HDAC1 decreased by 50% (Fig. 4A,bars9–10), whereas H321A over- expression resulted in a threefold increase in HDAC1 recruitment (Fig. 4A,bars11–12). Further ChIP analysis also revealed that the A B induction of KDM8 led to enhanced H3 and H4 acetylation (Fig. 4A,bars13–14, 17–18), further supporting inhibition of HDAC1 recruitment. Finally, endogenous KDM8 knockdown led to in- creased levels of the H3K36me2 mark along with HDAC1 re- cruitment (Fig. 4B), which resulted in a significant reduction of cyclin A1 expression in MCF7 cells (Fig. 4C). These data establish a plausible relationship among KDM8-mediated H3K36me2 demethylation, reduced HDAC1 recruitment, and the transcrip- tional activation of cyclin A1. C D Discussion Identification of histone demethylases revealed the dynamic re- versibility of epigenetic methylation marks (15–18). A complete understanding of the functional and spatial differences of histone lysine methylation has yet to be revealed. Among the various methylated lysine residues, H3K36me2 may be one of the least studied epigenetic marks. Although the tri- and monomethylated forms of H3K36 have been shown to be transcriptionally active marks (19), H3K36me2 in the coding region is typically defined as a transcriptional silencing histone modification and consid- ered to influence transcript elongation (20, 21). Fig. 3. KDM8 increases cyclin A1 gene and protein expression through Although our in vivo studies conclusively demonstrated that A binding in the coding region. ( ) Endogenous KDM8 recruitment sites in full-length KDM8 can demethylate H3K36me2 in breast cancer MCF7 cells were mapped using ChIP-on-chip analysis. KDM8-associated DNA fi cells, in vitro demethylation results were only successful when was enriched by ChIP with a KDM8-speci c antibody and subsequently an- – – alyzed with Affymetrix Human Promoter 1.0R tiling arrays as described in SI using an N-terminal truncated (101 416 aa) KDM8 mutant. We Materials and Methods. CisGenome software was used for peak detection speculate that the lack of full-length enzymatic activity in vitro and annotation of genomic regions bound by KDM8. The figure demon- may be caused by either an autoinhibitory domain of the protein strates that endogenous KDM8 is recruited to the cyclin A1 coding region or the requirement of a cofactor protein, whose interaction alters and most prominently to exon 2. The binding profile along the CCNA1 locus KDM8 protein conformation (Fig. S1B). Because the N-terminal is depicted based on magnitude of enrichment at each location, which is mutants worked in vitro, we hypothesize that any potential reg- signified by the height of each bar. Location of primer pairs are indicated in fi B fi ulatory modi cations to KDM8 that direct its histone demethy- the panel by letters and black bars. ( ) Quantitative real-time PCR con rm- lase activity must occur within the first 101 amino acids. As ing the binding of endogenous KDM8 in the cyclin A1 gene. (C) Quantitative fi RT-PCR analysis of cyclin A1 in Dox-inducible Flag-KDM8/H321A MCF7 cells. previously seen in other histone modi ers (22, 23), these results Induction of KDM8 increased cyclin A1 expression. (D) Immunoblotting further suggest that interactions with cofactor proteins or post- analysis of MCF10A cells transfected with KDM8 siRNA and scramble siRNA translational modifications may regulate KDM8 demethylase shows that knockdown of KDM8 leads to a decrease in cyclin A expression activity. Our model is shown in Fig. S5. Preliminary analysis of and increases the amount of H3K36me2. the KDM8 amino acid sequence predicts a nuclear hormone

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Fig. 4. KDM8 mediated demethylation of H3K36me2 leads to a decrease in HDAC1 recruitment in the cyclin A1 coding region. (A) ChIP analysis was per- formed to examine KDM8 effects on H3K36me2 and HDAC1 recruitment to the coding region of the cyclin A1 gene with inducible cells. ChIP assays were performed with indicated antibodies. The results were normalized to IgG controls and shown as an average fold change in enrichment. Data are presentedas a mean ± SD from three independent experiments. (B). ChIP analysis was performed similar to A in the MCF7 cells that had stable knockdown of KDM8 with the indicated antibodies. (C). Immunoblot analysis of KDM8 knockdown MCF7 cells. The lysates from the KDM8 knockdown and control cells were probed with the antibodies as indicated.

receptor binding motif LXXL located in the N-terminal region. 250) was used for the antigen. KDM8 antibody was validated by immun- Additional preliminary studies have indicated that certain his- blotting bacculovirus-infected Sf9 cell lysates. Briefly, IHC staining on breast tone methyltransferases such as SUV39H1 and SETDB1 interact tumor tissue array (cat no. BR804), consisting of 40 patient cases (80 cores) i with KDM8 in the N-terminal region, indicating complex regu- matched with normal patient tissue, were analyzed by two methods: ( ) ii lation of KDM8. software scanning analysis using ScanScope and ( ) staining intensity scoring by a panel of Biomax pathologists. Scores range from lowest (0) to highest In this study, we demonstrated that KDM8 plays a critical role (3+) intensity. in the regulation of cell cycle by activating the cyclin A1 locus via demethylation of H3K36me2. Interestingly, KDM2B, another Histone Demethylation Assay. Histone peptides were incubated with Flag- H3K36me2 demethylase, also has been shown to regulate cell KDM8, Flag-KDM8 H321A, GST-KDM8 101-C, GST-KDM8 101-C H321A in cycle progression (24). Although the specific mechanism used by histone demethylase buffer [20 mM Tris-HCL, pH 7.3, 150 mM NaCl, 50 μM

these two demethylases and the target genes affected may be Fe(NH)4(SO4)2,1mMα-ketoglutarate, 2 mM ascorbic acid] at 37 °C for 2 to different, both results point to the significance of the H3K36me2 4 h. Purified proteins used in this study are shown in Fig. S6. Peptides were epigenetic mark in cell cycle regulation. commercially purchased from Millipore or custom synthesized by Thermo- Fisher Scientific. Reactions were analyzed by MALDI-TOF MS. Materials and Methods Generation of Inducible KDM8/H321A Cell Lines. MCF7 cell line expressing Tet- Immunofluorescence Microscopy and Quantification. KDM8 Dox-inducible repressor, MCF7-pTR-7 (25), was a gift from Xin-Bin Chen (University of MCF7 cells were cultured for 72 h after induction. Dox-induced and unin- California, Davis). Lentivirus carrying an inducible expression cassette of duced cells were then mixed and spotted onto coverslips, fixed with 4% KDM8 or KDM8 H321A was prepared by transfecting packaging plasmids formaldehyde, permeabilized in 0.1% Triton X-100/PBS solution, and blocked (Invitrogen) with pLTRE-Flag-KDM8 or pLTRE-Flag-KDM8H321A plasmid us- with 1% BSA/PBS solution. Detailed staining and quantification methods are ing Lipofectamine 2000. The MCF7-pTR-7 cells were transduced with described in SI Materials and Methods. recombinant lentivirus and cells were selected with puromycin for 2 to 3 wk. ChIP Assays. ChIP assays were performed following the University of California Immunohistochemistry. IHC was performed and analyzed by Biomax by using Davis Genome Center ChIP protocol (http://www.genomecenter.ucdavis.edu/ laboratory-generated KDM8 antibody. Purified GST-KDM8 (residues 150– farnham). The primary antibodies used in this study were as follows: KDM8,

Hsia et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 FLAG (Sigma), HDAC1, H3K36me2, acetylated , and acetylated ACKNOWLEDGMENTS. We thank Tony Martinez, Harryl Martinez, and Ryan histone H4. These antibodies were obtained from Millipore. The secondary R. Davis for their expert technical assistance and consultation for experiments; rabbit anti-mouse IgG and goat anti-mouse IgG were purchased from MP Dr. William Jewell at the University of California Davis Campus MS facility for Biomedicals. The nonspecific rabbit IgG and mouse IgG used as negative assistance; Dr. Ming-Daw Tsai and Dr. Pang-Hung Hsu for the initial phase of controls in the ChIP assays were purchased from Alpha Diagnostics. substrate analysis; Mel Campbell for revising the manuscript; and Dr. Xin-bin Chen (University of California School of Veterinary Medicine) for the TR7- MCF7 cell line. This work was supported by the Auburn Cancer Endowment siRNA Transfection. siRNA negative control scramble sequence and sequences Fund and contributions from the University of California Davis Cancer Center ′ targeting KDM8 were designed (targeting 3 UTR) and purchased through In- Genomics and Expression Resource (supported by Cancer Center Support tegrated DNA Technologies. The standard protocol for siRNA provided by IDT was Grant 2 P30 CA93373; principal investigator, Ralph de Vere White). This work followed. Briefly, cells were transfected with 10 nM of siRNA duplex sequences was also supported by a University of California Davis Health Science grant (to using Trifectin reagent, purchased from IDT. Cells were collected and prepared Y.I.), National Institutes of Health Grants DK52659, CA114575, and CA150179; after 72 h. Duplex sequences were as follows: siKDM8, 3′UTR, 5′-CGCUGUCA- Department of Defense (DoD) Idea Award PC093350 (to H.-J.K.); and a DoD CUGAUCCCAAUUACUCT-3′;3′-CGGCGACAGUGACUAGGGUUAAUGAGA-5′. postdoctoral training award (to M.R.P.).

1. Klose RJ, Kallin EM, Zhang Y (2006) JmjC-domain-containing proteins and histone 14. Joshi AA, Struhl K (2005) Eaf3 chromodomain interaction with methylated H3-K36 demethylation. Nat Rev Genet 7:715–727. links histone deacetylation to Pol II elongation. Mol Cell 20:971–978. 2. Klose RJ, et al. (2006) The transcriptional repressor JHDM3A demethylates trimethyl 15. Shi Y, et al. (2004) Histone demethylation mediated by the nuclear amine oxidase histone H3 lysine 9 and lysine 36. Nature 442:312–316. homolog LSD1. Cell 119:941–953. 3. Whetstine JR, et al. (2006) Reversal of histone lysine trimethylation by the JMJD2 16. Tsukada Y, et al. (2006) Histone demethylation by a family of JmjC domain-containing family of histone demethylases. Cell 125:467–481. proteins. Nature 439:811–816. 4. Iwase S, et al. (2007) The X-linked mental retardation gene SMCX/JARID1C defines 17. Agger K, et al. (2007) UTX and JMJD3 are histone H3K27 demethylases involved in Cell – a family of histone H3 lysine 4 demethylases. 128:1077 1088. HOX gene regulation and development. Nature 449:731–734. Cell 5. Li B, Carey M, Workman JL (2007) The role of chromatin during transcription. 128: 18. Chang B, Chen Y, Zhao Y, Bruick RK (2007) JMJD6 is a histone arginine demethylase. – 707 719. Science 318:444–447. 6. Wissmann M, et al. (2007) Cooperative demethylation by JMJD2C and LSD1 promotes 19. Wang Z, et al. (2008) Combinatorial patterns of histone acetylations and androgen receptor-dependent gene expression. Nat Cell Biol 9:347–353. in the human genome. Nat Genet 40:897–903. 7. Shi Y, Whetstine JR (2007) Dynamic regulation of histone lysine methylation by 20. Li B, et al. (2009) Histone H3 lysine 36 dimethylation (H3K36me2) is sufficient to demethylases. Mol Cell 25:1–14. recruit the Rpd3s histone deacetylase complex and to repress spurious transcription. J 8. Suzuki T, Minehata K, Akagi K, Jenkins NA, Copeland NG (2006) Tumor suppressor Biol Chem 284:7970–7976. gene identification using retroviral insertional mutagenesis in Blm-deficient mice. 21. Suganuma T, Workman JL (2008) Crosstalk among histone modifications. Cell 135: EMBO J 25:3422–3431. 604–607. 9. Yang R, et al. (1999) Functions of cyclin A1 in the cell cycle and its interactions with 22. Ketel CS, et al. (2005) Subunit contributions to histone methyltransferase activities of transcription factor E2F-1 and the Rb family of proteins. Mol Cell Biol 19:2400–2407. fl Mol Cell Biol – 10. Liu D, Liao C, Wolgemuth DJ (2000) A role for cyclin A1 in the activation of MPF and y and worm polycomb group complexes. 25:6857 6868. G2-M transition during meiosis of male germ cells in mice. Dev Biol 224:388–400. 23. Montgomery ND, et al. (2005) The murine polycomb group protein Eed is required for Curr Biol – 11. Bannister AJ, et al. (2005) Spatial distribution of di- and tri-methyl lysine 36 of histone global histone H3 lysine-27 methylation. 15:942 947. H3 at active genes. J Biol Chem 280:17732–17736. 24. He J, Kallin EM, Tsukada Y, Zhang Y (2008) The H3K36 demethylase Jhdm1b/Kdm2b 12. Carrozza MJ, et al. (2005) Histone H3 methylation by Set2 directs deacetylation of regulates cell proliferation and senescence through p15(Ink4b). Nat Struct Mol Biol coding regions by Rpd3S to suppress spurious intragenic transcription. Cell 123: 15:1169–1175. 581–592. 25. Scoumanne A, Chen X (2007) The lysine-specific demethylase 1 is required for cell 13. Yang XJ, Seto E (2008) The Rpd3/Hda1 family of lysine deacetylases: From bacteria proliferation in both p53-dependent and -independent manners. J Biol Chem 282: and yeast to mice and men. Nat Rev Mol Cell Biol 9:206–218. 15471–15475.

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