Prdm12 Is Induced by Retinoic Acid and Exhibits Anti-Proliferative Properties Through the Cell Cycle Modulation of P19 Embryonic Carcinoma Cells
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CELL STRUCTURE AND FUNCTION 38: 197–206 (2013) © 2013 by Japan Society for Cell Biology Prdm12 Is Induced by Retinoic Acid and Exhibits Anti-proliferative Properties through the Cell Cycle Modulation of P19 Embryonic Carcinoma Cells Chia-Ming Yang1,2 and Yoichi Shinkai3* 1Institute for Virus Research, Kyoto University, Kyoto, Japan, 2Graduate School of Biostudies, Kyoto University, Kyoto, Japan, 3Cellular memory laboratory, Riken, Wako, Saitama, Japan ABSTRACT. The Prdm (PRDI-BF1-RIZ1 homologous) family is involved in cell differentiation, and several Prdms have been reported to methylate histone H3 by intrinsic or extrinsic pathways. Here, we report that Prdm12 recruits G9a to methylate histone H3 on lysine 9 through its zinc finger domains. Because of the expression of Prdm12 in the developmental nervous system, we investigated the role of Prdm12 on P19 embryonic carcinoma cells as a model system for neurogenesis. In P19 cells, Prdm12 is induced by Retinoic acid (RA). Overproduction of Prdm12 in P19 cells impairs cell proliferation and increases the G1 population accompanied by the upregulation of p27. In contrast, the knockdown of Prdm12 increases the number of cells in a suspension culture of RA-induced neural differentiation. Both the PR domain and zinc finger domains are required for the anti-proliferative activity of Prdm12. While the data in this study is based on in vitro models, the results suggest that Prdm12 is induced by the RA signaling in vivo, and may regulate neural differentiation during animal development. Key words: Prdm12, Histone methyltransferase, Retinoic acid, cell cycle Introduction It has been reported that the Prdm family is expressed dynamically in the nervous system during the development The Prdm family contains a PR (PRDI-BF1-RIZ1 homolo- of mice and zebrafish (Hohenauer and Moore, 2012). The gous) domain in the N-terminal which has a 20–30% function of the Prdms in neurogenesis remains unclear, sequence identity to the conserved catalytic SET (SU(VAR) however. In zebrafish embryos, Prdm1 is important for the 3-9, E(Z), trithorax) domains of the histone lysine methyl- development of neural crest and sensory neurons (Hernandez- transferase (HKMTase) superfamily (Völkel and Angrand, Lagunas et al., 2005). Our laboratory has previously 2007). This similarity suggests that the Prdm family may described the expression pattern of Prdm8 in the mouse possess HKMTase properties. In fact, some Prdms show in- embryonic nervous system (Komai et al., 2009). Another trinsic HKMTase activity (Prdm2, Prdm3, Prdm8, Prdm9, paper using a knockout mice model indicated that Prdm8 and Prdm16). In addition, Prdm1, Prdm5, and Prdm6 lack is involved in neural development (Ross et al., 2012). intrinsic HKMTase activity, but instead recruit G9a/Ehmt2/ Although Prdm12 is expressed in the mouse and zebrafish KMT1C, a strong mammalian histone H3 lysine 9 (H3K9) embryonic nervous system (Kinameri et al., 2008; Sun et methyltransferase, to mediate HKMTase activity (see Fog al., 2008), the role of Prdm12 remains unknown. et al., 2012 for a review). Another structural feature is that Retinoic acid (RA) is a metabolite of vitamin A (retinol), the Prdm family has multiple kruppel-type zinc finger (ZF) which controls neural differentiation and patterning (Maden, domains in the C-terminus involved in sequence-specific 2007). To investigate the process of neural differentiation DNA binding and protein-protein interactions (Völkel and in vitro, P19 embryonic carcinoma cells were used as a Angrand, 2007). model system. Treatment with RA in aggregating condition induces P19 cells to differentiate into neurons and glia *To whom correspondence should be addressed: Yoichi Shinkai, Cellular (Jones-Villeneuve et al., 1982). The upregulation of p27, a memory laboratory, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan. cyclin-dependent kinase inhibitor (CKI), has been shown to Tel: +81–048–467–9370, Fax: +81–3–048–462–4670 be involved in arresting cell cycle progression at G1 phase E-mail: [email protected] in this in vitro model (Gill et al., 1998). Furthermore, the Abbreviations: PR domain, PRDI-BF1-RIZ1 homologous domain; ZF domain, kruppel-type zinc finger domain; HKMTase, histone lysine overexpression of p27 could induce neural differentiation in methyltransferases; RA, retinoic acid. mouse neuroblastoma cells (Kranenburg et al., 1995). 197 C.-M. Yang and Y. Shinkai Since many Prdms possess HKMTase properties, we are plate for 24 h before transfection. Two micrograms of indicated interested in whether Prdm12 also possesses HKMTase plasmids were transfected into HEK293T cells using TransIT-LT-1 properties. In this study, we first demonstrate that Prdm12 reagent (Mirus Bio corp., Madison, WI, USA) or 4 μg of plasmids recruits G9a to methylate H3K9. Because the localization in were transfected into NIH3T3 and P19 cells using Lipofectamine the embryonic nervous system implies a potential function 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the in neurogenesis, we investigated the role of Prdm12 in the manufacturer’s instructions. For infection, GP2-293 cells were co- RA-induced neural differentiation of P19 cells. We discover transfected with a pSUPER.retro.puro vector containing the that Prdm12 is induced by RA in P19 cells and exhibits anti- shRNA against mouse Prdm12 and a pVSV-G vector. After 48 h, proliferative effects partially through the regulation of G1 the culture media was filtered with a 0.45-μm filter then used to phase in the cell cycle. Furthermore, ectopic Prdm12 infect P19 cells with polybrene (8 μg/ml). To establish stable cell increases the expression of p27 with both the PR and ZF lines, cells were treated with 1 μg/ml puromycin for five days. domains being necessary for its function. In vitro histone methyltransferase (HMTase) assay Materials and Methods HMTase assays was performed as described (Tachibana et al. 2001), with some modifications. Briefly, 10 μl of reaction mixture μ Plasmids and cloning containing 2 g of core histones, immunoprecipitated enzymes, and 125 nCi of S-adenosyl-[methyl-14C]-L-methionine in assay Full-length Prdm12 cDNA was amplified by PCR from an EST buffer (50 mM Tris, pH 8.5, 5 mM DTT) were incubated for 1 h at clone (image #6734548) purchased from the IMAGE consortium. 30°C. Proteins were separated by 15% SDS-PAGE and visualized For the generation of expression plasmids, amplified Prdm12 by coomassie brilliant blue staining. Detection of methyl-14C was cDNA was inserted into pEGFP-C2 (Clontech, Mountair View, performed by using a BAS-5000 imaging analyzer (Fuji Film). CA, USA), pGEX4T-1 (GE Healthcare, Little Chalfont, UK), or pCAG-FLAG-IRES-Puro vectors at specific restrict enzyme sites Immunoprecipitation and immunocytochemistry in frame with the indicated tag. To make Prdm12 deletion and point mutants, mutated fragments were created by PCR amplifica- After 48 h of transfection (or at the harvesting times indicated), tion and were subcloned into a pCAG-FLAG-IRES-Puro vector. cells were lysed in lysis buffer containing 20 mM Hepes, pH 7.5, Glutathione S-transferase (GST)-tagged H3N, mutants, and GST- 420 mM NaCl, 1.5 mM MgCl2, 0.1% NP-40, and protease inhibi- G9aSET have been described earlier (Tachibana et al. 2001). tor cocktail (Nacalai Tesque, Kyoto, Japan). Supernatants were Three oligonucleotides of short hairpin RNAs (shRNA) targeting collected by centrifugation then incubated with 0.5–1 μg antibody the Prdm12 gene were designed and synthesized from Sigma (St. overnight at 4°C and subsequently isolated by protein G-agarose Louis, MO, USA) then inserted into a pSUPER.retro.puro vector (GE Healthcare). Immunoprecipitants were washed extensively (OligoEngine, Seattle, WA). Details for all the primers and restric- with lysis buffer then used in western blot or in vitro HMTase tion enzyme sites are listed in the supplemental information assays. For immunocytochemistry, cells were fixed and immuno- (Table. SI). stained as previous described (Komai et al. 2009). Cell culture and differentiation Antibodies HEK293T, NIH3T3, and P19 cells were cultured in Dulbecco’s To generate rabbit anti-Prdm12 polyclonal antibodies, cDNA frag- Modified Eagle Medium (DMEM, Nissui Pharmaceutical Co. ments corresponding to residues N43–F230 of mouse Prdm12 Ltd.) supplemented with 10% fetal bovine serum (FBS), 100 μg/ were subcloned into pGEX-4T-1, then the fusion protein was puri- ml streptomycin, and 100 U/ml penicillin (Gibco, Carlsbad, CA, fied and injected into a rabbit (Hokudo Co., Ltd., Sapporo, Japan). USA) at 37°C in a 5% CO2 atmosphere. To induce neural differen- The antibody was affinity-purified from the antiserum. The com- tiation, 1×106 P19 cells were cultured on 10 cm bacteria grade mercially available primary antibodies used were as follows: mouse dishes for aggregation in DMEM supplemented with 10% FBS and monoclonal antibodies to FLAG M2 (F3165; Sigma); GFP (11 814 1 μM RA (solved in 99.9% EtOH; Sigma). After 96 h, cells were 460 001; Roche, Mannheim, Germany); G9a (A8620A, Perseus trypsinized then transferred to poly-L-lysine (Sigma) coated tissue Proteomics Inc., Tokyo, Japan); GLP (#422, Perseus Proteomics culture dishes at a density of 1×105 cells/ml in 10% FBS DMEM Inc.); Neuronal Class III β-Tubulin (Tuj1, MMS-435P; (Covance, without RA. After 24 h, the medium was changed to 0.5% FBS Princeton, USA); p27 (K25020; BD Biosciences, San Jose, CA); DMEM to induce neural differentiation for another 96 h. All media α-tubulin (T-5168, Sigma) and normal rabbit IgG (sc-2027;