To effectively direct targeted repression, the class I histone deacetylases (HDACs) associate with many important regulatory proteins. In this paper we describe the molecular characterization of a member of the Jumonji domain 2 (JMJD2) family of proteins, and demonstrate its binding to both class I HDACs and the retinoblastoma protein (pRb). JMJD2 proteins are characterized by the presence of two leukemia-associated protein/plant homeodomain (LAP/PHD) zinc fingers, one JmjN, one JmjC (containing an internal retinoblastoma-binding protein 2 (RBBP2)-like sequence), and two Tudor domains. The first member of this group, JMJD2A, is widely expressed in human tissues and cell lines, and high endogenous expression of JMJD2A mRNA was found in several cell types, including human T-cell lymphotropic virus 1 (HTLV-1)-infected cell lines. JMJD2A and JMJD2B exhibit cell type-specific responses to the HDAC inhibitor trichostatin A. We show that the JMJD2A protein associates in vivo with pRb and class I HDACs, and mediates repression of E2F-regulated promoters. In HTLV-1 virus-infected cells, we find that JMJD2A binds to the viral Tax protein. Antibodies to JMJD2A recognize the native protein but also a half-sized protein fragment, the latter up-regulated in THP-1 cells during the G2/M phase of the cell cycle. The ability of JMJD2A to associate with pRb and HDACs and potentiate pRb-mediated repression of E2F-regulated promoters implies an important role for this protein in cell proliferation and oncogenesis. To effectively direct targeted repression, the class I histone deacetylases (HDACs) associate with many important regulatory proteins. In this paper we describe the molecular characterization of a member of the Jumonji domain 2 (JMJD2) family of proteins, and demonstrate its binding to both class I HDACs and the retinoblastoma protein (pRb). JMJD2 proteins are characterized by the presence of two leukemia-associated protein/plant homeodomain (LAP/PHD) zinc fingers, one JmjN, one JmjC (containing an internal retinoblastoma-binding protein 2 (RBBP2)-like sequence), and two Tudor domains. The first member of this group, JMJD2A, is widely expressed in human tissues and cell lines, and high endogenous expression of JMJD2A mRNA was found in several cell types, including human T-cell lymphotropic virus 1 (HTLV-1)-infected cell lines. JMJD2A and JMJD2B exhibit cell type-specific responses to the HDAC inhibitor trichostatin A. We show that the JMJD2A protein associates in vivo with pRb and class I HDACs, and mediates repression of E2F-regulated promoters. In HTLV-1 virus-infected cells, we find that JMJD2A binds to the viral Tax protein. Antibodies to JMJD2A recognize the native protein but also a half-sized protein fragment, the latter up-regulated in THP-1 cells during the G2/M phase of the cell cycle. The ability of JMJD2A to associate with pRb and HDACs and potentiate pRb-mediated repression of E2F-regulated promoters implies an important role for this protein in cell proliferation and oncogenesis. The tight confines of chromatin have necessitated the evolution of specialized chromatin remodeling complexes to achieve appropriate regulation of gene expression within the mammalian cell. One such mechanism that has recently been elucidated involves the acetylation of the histone tails within the nucleosome. The histone deacetylase (HDAC) 1The abbreviations used are: HDAC, histone deacetylase; pRb, retinoblastoma protein; LAP, leukemia-associated protein; PHD, plant homeodomain; aa, amino acid(s); GST, glutathione S-transferase; CMV, cytomegalovirus; EYFP, enhanced yellow fluorescent protein; HA, hemagglutinin; RT, reverse transcriptase; RPA, RNase protection assay; AD, activation domain; TK, thymidine kinase; TSA, trichostatin A; HTLV-1, human T-cell lymphotropic virus 1; RBBP, retinoblastoma-binding protein. family of proteins is involved with removing such modifications, and its members have been subgrouped into three classes depending upon their similarity to yeast proteins (1.Gray S.G. Ekström T.J. Exp. Cell Res. 2001; 262: 75-83Crossref PubMed Scopus (497) Google Scholar, 2.Gray S.G. Teh B.T. Curr. Mol. Med. 2001; 1: 401-429Crossref PubMed Scopus (83) Google Scholar). Since the isolation of the first HDAC many members of this family have been identified, currently consisting of at least 18 members (1.Gray S.G. Ekström T.J. Exp. Cell Res. 2001; 262: 75-83Crossref PubMed Scopus (497) Google Scholar, 2.Gray S.G. Teh B.T. Curr. Mol. Med. 2001; 1: 401-429Crossref PubMed Scopus (83) Google Scholar, 3.Gao L. Cueto M.A. Asselbergs F. Atadja P. J. Biol. Chem. 2002; 277: 25748-25755Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). Critical roles for these proteins have been identified in many aspects of cell function. The product of the retinoblastoma gene, pRb, has been shown to have multiple roles in cellular processes, including regulation of apoptosis and the cell cycle (4.Liu H. Dibling B. Spike B. Dirlam A. Macleod K. Curr. Opin. Genet. Dev. 2004; 14: 55-64Crossref PubMed Scopus (84) Google Scholar). pRb interacts with E2F family transcription factors and blocks their activity during the G0 and G1 phases of the cell cycle. E2F proteins activate the expression of many genes involved in cell cycle progression, including cyclins A and E, as well as other proteins and enzymes required for DNA replication (5.Stevaux O. Dyson N.J. Curr. Opin. Cell Biol. 2002; 14: 684-691Crossref PubMed Scopus (352) Google Scholar). Binding sites for E2F are found in the promoters of genes whose expression occurs at the G1/S transition, and repression of the promoters containing E2F-binding sites has been shown to involve binding of E2F/pRb pocket protein complexes to these sites. Previous studies have identified two distinct, although not mutually exclusive, mechanisms for the transcription repression function of pRb (5.Stevaux O. Dyson N.J. Curr. Opin. Cell Biol. 2002; 14: 684-691Crossref PubMed Scopus (352) Google Scholar, 6.Harbour J.W. Dean D.C. Nat. Cell Biol. 2000; 2: E65-E67Crossref PubMed Scopus (422) Google Scholar, 7.Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (963) Google Scholar). The first mechanism involves the direct interaction of pRb with the E2F transactivation domain, resulting in masking of this domain and blocking its ability to stimulate transcription (8.Ross J.F. Liu X. Dynlacht B.D. Mol. Cell. 1999; 3: 195-205Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The second mechanism is based on the ability of pRb to recruit chromatin remodeling proteins, such as HDACs, and assemble transcription repression complexes at E2F-regulated promoters (9.Meloni A.R. Smith E.J. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9574-9579Crossref PubMed Scopus (167) Google Scholar, 10.Magnaghi-Jaulin L. Groisman R. Naguibneva I. Robin P. Lorain S. Le Villain J.P. Troalen F. Trouche D. Harel-Bellan A. Nature. 1998; 391: 601-605Crossref PubMed Scopus (805) Google Scholar, 11.Luo R.X. Postigo A.A. Dean D.C. Cell. 1998; 92: 463-473Abstract Full Text Full Text PDF PubMed Scopus (839) Google Scholar, 12.Brehm A. Miska E.A. McCance D.J. Reid J.L. Bannister A.J. Kouzarides T. Nature. 1998; 391: 597-601Crossref PubMed Scopus (1080) Google Scholar). A separate series of experiments demonstrated that RBBP1, a known pRb interacting protein, acts as a bridge to recruit HDACs 1–3 to pRb-E2F complexes (13.Lai A. Lee J.M. Yang W.M. DeCaprio J.A. Kaelin Jr., W.G. Seto E. Branton P.E. Mol. Cell. Biol. 1999; 19: 6632-6641Crossref PubMed Scopus (142) Google Scholar, 14.Lai A. Kennedy B.K. Barbie D.A. Bertos N.R. Yang X.J. Theberge M.C. Tsai S.C. Seto E. Zhang Y. Kuzmichev A. Lane W.S. Reinberg D. Harlow E. Branton P.E. Mol. Cell. Biol. 2001; 21: 2918-2932Crossref PubMed Scopus (165) Google Scholar). The JMJD2 family of proteins has recently been identified in silico (15.Katoh M. Int. J. Oncol. 2004; 24: 1623-1628PubMed Google Scholar). Here we describe the molecular characterization of JMJD2A, a nuclear and cytoplasmic member of this family that participates as a pRb-HDAC complex component. JMJD2A contains two LAP/PHD (leukemia-associated protein/plant homeodomain) zinc fingers, one JmjN, one JmjC (containing an internal RBBP2-like sequence), and two Tudor domains, and promotes histone deacetylation and gene repression in vivo, through its association with HDACs and pRb. Multiple truncated fragments of JMJD2A bind pRb, suggesting that the pRb A/B pocket is not the only interaction site. JMJD2A also binds the viral protein Tax in HTLV-1-infected cells. The property of JMJD2A to both associate with pRb/HDACs and Tax and to mediate transcriptional repression suggests an important role for this protein in cell proliferation and transformation, and provides new insights into the molecular mechanisms of pRb function. Library Screening and Plasmid Construction—A human fetal liver λgt11 cDNA library (Clontech number HL1064b) was screened under low stringency conditions, using as a probe a DNA fragment corresponding to the DNA-binding domain of human Sp1, as described previously (16.Ladias J.A.A. J. Biol. Chem. 1994; 269: 5944-5951Abstract Full Text PDF PubMed Google Scholar). The full-length KIAA0677 cDNA was a gift from T. Nagase (Kazusa DNA Research Institute). The region of JMJD2A (aa 661–1064) originally identified in the Sp1 screen was cloned directly into pFGAL94XR (17.Green V.J. Kokkotou E. Ladias J.A.A. J. Biol. Chem. 1998; 273: 29950-29957Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), for use in repression assay and chromatin immunoprecipitation experiments. The dihydrofolate reductase (DHFR) promoter-Luc construct containing three E2F-binding sites, pSG5-HA-pRb, pSG5-HA-pRbΔ22, and GAL4-pRb (18.Sellers W.R. Novitch B.G. Miyake S. Heith A. Otterson G.A. Kaye F.J. Lassar A.B. Kaelin Jr., W.G. Genes Dev. 1998; 12: 95-106Crossref PubMed Scopus (288) Google Scholar), were gifts from W. Kaelin (Dana-Farber Cancer Institute). A full-length FLAG-tagged cDNA for human HDAC3 was obtained from E. Seto (Moffitt Cancer Center) (19.Yang W.M. Yao Y.L. Sun J.M. Davie J.R. Seto E. J. Biol. Chem. 1997; 272: 28001-28007Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). A full-length fusion of JMJD2A to EYFP was generated by PCR of the full-length JMJD2A cDNA with primers incorporating appropriate restriction enzyme sites: EYFP-N1 JMJD2A FWD (HindIII), 5′-CGAAGCTTATGGCTTCTGAGTCTGAAACTCTGAATCCC-3′; EYFP-N1 JMJD2A REV (SalI), 5′-GGATGTCGACTTCTCCATGATGGCCCGGTATAGTGC-3′. The PCR products were first cloned into pCR II (Invitrogen), from which JMJD2A was isolated using HindIII/SalI and cloned into similarly digested EYFP-N1 (Clontech). The construct was sequenced to verify that the fusion was in-frame. Bioinformatic and Phylogenetic Analysis—The accession numbers for the protein sequences used for alignments are as follows: hJMJD2A (NP_055478), hJMJD2B (NP_055830), hJMJD2C/hGASC1 (NP_055876), hJMJD2D (XP_061849), hJMJD2E (AP002383.3), hJMJD2F (AP001264.4), mJMJD2A (AAH28866), mJMJD2B (AAH07145), mJMJD2C/mGASC1 (NP_659036), mJMJD2D (XP_143503), rJMJD2A (XP_233441), rJMJD2D (XP_235826), cJMJD2A (XP_422410), cJMJD2B (XP_425914), cJMJD2C (XP_424815), xtJMJD2A (C_scaffold_25000012), fJMJD2A (FRUP00000161146), fJMJD2C (FRUP00000151439), tnJMJD2.1 (CAF92874), tnJMJD2.2 (CAF91827), tnJMJD2.3 (CAG01941), zJMJD2A (AAH47193), dJMJD2s (AAF59172 and AAF58413), agJMJDs (XP_309467 and XP_308975), amJMJD2 (XP_395616), crJMJD2 (C_50086) and ceJMJD2D (CAB54451), where h stands for Homo sapiens, m for the mouse Mus musculus, r for the rat Rattus norvegicus, c for the chicken Gallus gallus, xt for the frog Xenopus tropicalis, f for the pufferfish Fugu rubripes, tn for the pufferfish Tetraodon nigroviridis, z for the zebrafish Danio rerio, d for Drosophila melanogaster, ag for the mosquito Anopheles gambiae, am for the honeybee Apis mellifera, cr for Clamydomonas reinhardtii, and ce for Caenorhabditis elegans. Protein sequences were obtained from the following public data bases: NCBI (www.ncbi.nlm.nih.gov), the UCSC Genome Browser (genome.ucsc.edu), the FUGU Rubripes genome data base version 3.0 (genome.jgi-psf.org/fugu), Xenopus tropicalis genome data base assembly version 2.0 (genome.jgi-psf.org/xenopus), and the Chlamydomonas genome assembly version 2.0 (genome.jgi-psf.org/chlamy). Protein sequence alignments were generated using ClustalW (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html), and the conserved residues obtained from this analysis were highlighted using BOXSHADE (www.ch.embnet.org/software/Box_form.html). Phylogenetic analysis was carried out using the MEGA 3 software package (www.megasoftware.net) (20.Kumar S. Tamura K. Nei M. Brief Bioinform. 2004; 5: 150-163Crossref PubMed Scopus (10656) Google Scholar). Structurally conserved elements were identified using SMART (smart.embl.de) (21.Letunic I. Copley R.R. Schmidt S. Ciccarelli F.D. Doerks T. Schultz J. Ponting C.P. Bork P. Nucleic Acids Res. 2004; 32: D142-D144Crossref PubMed Google Scholar). Zinc Binding Assay—GST fusion proteins were expressed in Escherichia coli BL21 cells and were purified on glutathione-Sepharose 4B (Amersham Biosciences). Protein samples were analyzed by SDS-PAGE, transferred onto a nitrocellulose filter, denatured with 6 m guanidine hydrochloride for 30 min, followed by three washes in metal binding buffer A (100 mm Tris, 50 mm NaCl, 5 mm dithiothreitol, pH 7.5) for 1 h with three buffer changes. Subsequently, the blot was probed with 50 μCi of 65ZnCl2 (PerkinElmer Life Sciences) in 20 ml of buffer A for 1 h, washed three times with buffer A, rinsed with deionized H2O, and autoradiographed for 16 h at -80 °C with an intensifying screen. After autoradiography, the filter was stained with 0.1% Amido Black (Sigma) in 45% methanol, 10% acetic acid for protein visualization. Cell Cultures and Treatment—Hep3B, ACHN, HeLa, U2OS, Saos-2, and 293 cell lines were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 2 mm l-glutamine, and 100 units/100 μg/ml of penicillin/streptomycin. HT1376 bladder carcinoma cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 10 mm Hepes, 2 mm l-glutamine, and 100 units/100 μg/ml of penicillin/streptomycin. Saos-2 cells were supplemented with l-glutamine (BioWhittaker). T-cell clones were stimulated with the polyclonal activator phytohemagglutinin (1 μg/ml) and irradiated feeders for continuous growth. All cells were plated on sterile Petri dishes or 96-well plates and incubated at 37 °C with 6% CO2 and 96% humidity. Human myeloid leukemia THP-1 cells, Epstein-Barr virus lymphoblasts, HUT102, and C81 cells were grown in complete medium (RPMI media supplemented with 10% fetal calf serum, 10 mm Hepes, 2 mm l-glutamine, and 100 units/100 μg/ml of penicillin/streptomycin). Complete medium plus interleukin-2-containing T-Stim (Collaborative Biomedical Products) was used for T-cell clone cultures. Trichostatin A was purchased from Upstate Biochemicals, dissolved in Me2SO, and cells were treated as previously described (22.Gray S.G. Ekström T.J. Biochem. Biophys. Res. Commun. 1998; 245: 423-427Crossref PubMed Scopus (46) Google Scholar). Transient Transfections—THP-1 cells were transfected by electroporation as reported previously (23.Dangond F. Hafler D.A. Tong J.K. Randall J. Kojima R. Utku N. Gullans S.R. Biochem. Biophys. Res. Commun. 1998; 242: 648-652Crossref PubMed Scopus (105) Google Scholar), and other cells were transfected with various amounts of effector plasmids, 1.7 μg of luciferase reporter and 1 μg of CMV-β-galactosidase using the calcium phosphate coprecipitation method, as described previously (17.Green V.J. Kokkotou E. Ladias J.A.A. J. Biol. Chem. 1998; 273: 29950-29957Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). 293 cells used for the mammalian two-hybrid assay were transfected by lipofection with the Lipofectamine Plus kit (Invitrogen). Plasmid pcDNA3 was used as filler DNA to balance the amount of transfected DNA in all experiments. Cells were harvested 48 h post-transfection, and luciferase and β-galactosidase activities were measured in the lysates, as described (17.Green V.J. Kokkotou E. Ladias J.A.A. J. Biol. Chem. 1998; 273: 29950-29957Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Transfections performed in triplicate are presented as a mean ± S.E. For fluorescent protein analysis HEK-293T cells were transfected using FuGENE (Roche) according to the manufacturer's instructions. Plasmid pBluescript II SK(-) (Stratagene) was used as filler DNA. EYFP fluorescence was monitored 48 h post-transfection. For E2F promoter construct luciferase assay, 293 cells were trypsinized and plated in 24-well plates at a final concentration of 5 × 105 cells per well in 2 ml of medium, 24 h prior to transfection. Using the FuGENE 6 (Roche) transfection reagent, cells were transfected with the following various concentrations of effector plasmids, 1.0 μg of JPTR1, 0.5 μg of pRb, 0.7 μg of dihydrofolate reductase luciferase reporter, and 0.1 μg of pCMV-β-Gal. Cells were harvested 48 h post-transfection, and levels of luciferase and β-galactosidase were measured using the Dual Light System (Applied Biosystem), a chemiluminescent reporter gene assay system for the combined detection of luciferase and β-galactosidase. Antibody Generation, Immunocytochemistry, and Western Blot Analyses—Rabbit anti-JMJD2A and -JMJD2B were raised using the following synthetic peptides: JMJD2A, TEKEVKQEKKRQ; and JMJD2B, STFSKLKMEIK. Immunocytochemistry with these antibodies was then performed on rat brain tissues (medulla and pons). Adult Sprague-Dawley rats were transcardially perfused with 4% para-formaldehyde/phosphate-buffered saline, and the brain dissected out. Fifty-μm thick frozen sections were taken from brains for subsequent immunocytochemical analysis. The procedure for immunocytochemistry has been described elsewhere (24.Koibuchi N. Gibbs R.B. Suzuki M. Pfaff D.W. Endocrinology. 1991; 129: 3208-3216Crossref PubMed Scopus (27) Google Scholar), and immunoreactivity was visualized with 3,3′-diaminobenzidine. For Western blot analysis, cells were harvested, washed, and lysed in extraction buffer (50 mm Tris, 10% glycerol, 150 mm NaCl, pH 8.0) by rocking at 4 °C for 30 min. Following centrifugation, the whole cell extracts were removed and stored at -80 °C prior to use. Proteins were resolved by electrophoresis using precast a 4–20% gradient Tris glycine gel (NOVEX, San Diego, CA), and transferred to a nitrocellulose membrane filter (Millipore, Bedford, MA). The membranes were blocked with 5% nonfat milk and probed with rabbit polyclonal antibodies to JMJD2A at a dilution of 1:1000 for 1 h, then with horseradish peroxidase-conjugated anti-rabbit antibodies (Affinity Bioreagents, Golden, CO) at a dilution of 1:10,000. α-FLAG and α-HA antibodies were also used. The bands were visualized by using the enhanced chemiluminescence system (PerkinElmer Life Sciences) and exposing to x-ray film (Eastman Kodak). RNA Isolation and RT-PCR from Multiple Cell Lines—Total RNA was isolated from 5 × 106 cells using the TRIzol method (Invitrogen). RNA was resuspended in diethyl pyrocarbonate (Sigma)-treated water, and its concentration and purity were determined using OD 260/280 (Carey 64 UV Spectrophotometer). RNA (1 μg) was used in a cDNA synthesis reaction using 200 units of Moloney murine leukemia virus-reverse transcriptase (Invitrogen) with Retroscript Kit reagents (Ambion, Austin, TX) for RT-PCR analysis. Following the manufacturer's protocol, RNA was incubated with 5 μl of oligo(dT) primer (Ambion) at 70 °C for 10 min. RNA was transferred to ice, the Superscript II mixture was added, and cDNA synthesis was allowed to occur at 42 °C for 1 h in the presence of ∼20 units of RNasin (Promega). Three μl of cDNA were used in 50 μl of PCR using recombinant Taq polymerase (Invitrogen) at 0.25 units/sample. One μl of 10 mm dNTP mixture (Invitrogen) per sample was used for each reaction. PCR was performed for JMJD2A, HDAC3, and β-actin. Specific primers were as follows: JMJD2A, forward, 5′-AATCGGATCCATGGCTTCTGAGTCTGAAACTC-3′, reverse, 5′-AGCTGGGCCCGGATCCTCTTGAGTCACCTTGTCAAA-3′; HDAC3, forward, 5′-CGCCGGCACCATGGCCAAGA-3′, reverse, 5′-GCTGGGTTGCTCCTTGCAGA-3′; β-actin, 5′-AACCCCAAGGCCAACCGCGAGAAGATGACC-3′, reverse, 5′-GGTGATGACCTGGCCGTCAGGAGCTCGTA-3′. The JMJD2A primers yield a 790-base product, which spans nucleotides (1–790), and includes the JmjN and most of the JmjC domains. The HDAC3 primers yield a PCR product of 427 bases. The β-actin primers yield a PCR product of 406 bases. PCR conditions were as follows: for JMJD2A, denaturing was performed at 94 °C for 1 min, annealing at 62 °C for 1 min, and extension at 72 °C for 2 min; for HDAC3, denaturing was done at 94 °C for 1 min, annealing at 68 °C for 1 min, and extension at 72 °C for 2 min; for β-actin, denaturing was done at 94 °C for 1 min, annealing at 68 °C for 1 min, and extension at 72 °C for 2 min. Following 35 PCR cycles, all samples were allowed to extend at 72 °C for 10 min. All PCR were performed in a model PTC-100 programmable thermal controller (MJ Research, Inc., Watertown, MA). Twenty μl of each of the PCR products were electrophoresed in 1% agarose (Invitrogen) gels. The gels were then stained with ethidium bromide, placed on a UV transilluminator, and photographed. Northern Blot Analysis—Human multiple tissue and tumor cell line Northern blots (Clontech numbers 7760-1 and 7757-1, respectively) were probed with a radiolabeled DNA fragment corresponding to JMJD2A residues 661–1064, at 42 °C for 16 h. The blots were washed twice with 1× SSC, 0.5% SDS at room temperature for 15 min, followed by 0.5× SSC, 0.5% SDS at 65 °C for 15 min. After autoradiography, the probe was removed, and hybridization was repeated using a β-actin probe (Clontech). RNase Protection Assay (RPA)—T7 and Sp6 RNA polymerases (Invitrogen) were used to make antisense probes from the following templates according to the protocol provided in the RPA II kit (Ambion). When incorporating radioactivity into the probe, radioactive [32P]UTP with a specific activity of 800 Ci/mmol was used. Cold UTP was added such that the final UTP specific activity was 80 Ci/mmol for the β-actin probe and 400 Ci/mmol for the others. The β-actin probe used in this study is commercially available (Ambion). The probes used to measure JMJD2A and JMJD2B were prepared as follows. To measure JMJD2A expression, a 300-base pair fragment was amplified from full-length JMJD2A cDNA (nucleotides 1375–1675) using the amplification conditions as described above and the following primers: JMJD2A RPA forward, 5′-CCACTGAAGTCAAATTTGAAGAGC-3′; JMJD2A RPA reverse, 5′-GCTCTCCCACAGTGACCCTGCC-3′. The PCR amplification product was isolated from agarose using a Qiaex II Gel extraction kit (Qiagen) and cloned into pCRII using TOPO TA cloning (Invitrogen). The resulting plasmid could be linearized using EcoRV, and an appropriate antisense probe generated using Sp6 RNA polymerase of which 300 bases hybridize specifically to JMJD2A RNA. This region spans amino acids 459–558, between the JmJC and PHD domains of JMJD2A. JMJD2B expression was measured using a 351-bp EcoRI/XbaI fragment of JMJD2B cDNA cloned into the same sites of pGem3zf(+) (Promega). This region spans nucleotides 3582–3933 of the full-length JMJD2B mRNA and lies within the 3′-untranslated region. Following linearization with EcoRI, Sp6 RNA polymerase was used to transcribe probe. Both plasmid templates described were sequence verified to confirm the veracity of the cloned insert. Quantification of the RPA results was obtained using a BAS-100 phosphorimager (Fuji). The levels of β-actin mRNA were utilized as the internal control in each case. The values for each experimental gene examined were normalized to the internal control for each sample. Mammalian Two-hybrid Analysis—-Protein-protein interactions in vivo were detected using the mammalian two-hybrid system (Stratagene number 211344) in 293 cells. In this assay, a gene encoding a protein of interest (protein X) was fused to the DNA-binding domain of the yeast protein GAL4, whereas another gene suspected of interacting with protein X (protein Y) was fused to the transcriptional activation domain of the mouse protein NF-κB. If protein X and protein Y interact, they create a functional transcription activator by bringing the activation domain into close proximity with the DNA-binding domain; this can be detected by expression from a luciferase reporter gene under control of a promoter with GAL4-binding sites. Various JMJD2A cDNA fragments were cloned into the pCMV-AD vector (Stratagene), to produce JMJD2A fused to the NF-κB activation domain (AD). These constructs (0.1 μg) were used in calcium phosphate-based cotransfection experiments with the reporter plasmid pFR-Luc (1.7 μg, Stratagene), which contains a synthetic promoter with five GAL4-binding sites upstream of the luciferase gene, along with control activation domain constructs pAD-SV40T and pAD-TRAF, and binding domain constructs pBD-Rb and control pBD-p53 (0.1 μg each; all from Stratagene). Luciferase activity was then measured in cell extracts. The full-length cDNA and fragments of JPTR1 used in the mammalian two-hybrid experiments described within were derived by PCR, and the primer sets incorporated a BamHI restriction site for cloning purposes and were as follows: full-length (aa 1–1064), forward 5′-AATCGGATCCATGGCTTCTGAGTCTGAAACTC-3′, reverse, 5′-AATCGATCCCTACTCCATGATGGCCCGTTATAG-3′. Fragments Thr-660 (aa 1–660 containing the JmjN and JmjC domains), and Met-867 (aa 1–867 containing the JmjN, JmjC, and PHD domains) used the same forward primer from the full-length oligo pair but had the following reverse primers: 5′-AATCGGATCCGGTCTCATTGAATTCCTTCTCA-3′ (Thr-660) and 5′-AATCGGATCCCATCACACCGGCAGCCTGGGCGCAG-3′ (Met-867). Fragments Met-661 (aa 661–1064 containing the PHD and Tudor domains) and Met-868 (aa 868–1064 containing only the Tudor domains) use the reverse primer from the full-length oligo pair but have the following forward primers: 5′-AATCGGATCCATGGCCCAACAGGCCCCTCA-3′ (Met-661) and 5′-AATCGGATCCATGCAGCCTGACGACTGGCCT-3′ (Met-868). The amino acid prior to the numbers indicate at which each fragment starts and the numbers indicate the position of this amino acid in relation to the full-length protein. The conditions used to amplify these fragments were as follows: ∼100 ng of template (full-length JMJD2A cDNA plasmid) was combined with 0.5 μm of each primer in the presence of 1.5 mm MgCl2, 0.2 mm dNTPs, and 1 unit of Taq DNA polymerase with the supplied buffer (Invitrogen) in a total volume of 50 μl. Cycling conditions were 95 °C for 5 min followed by 30 cycles of 1 min at 95 °C, 1 min at 55 °C, 2 min at 72 °C with a final extension at 72 °C for 10 min. PCR products were extracted once with phenol-chloroform, precipitated, and then subsequently digested with BamHI. Following digestion the fragments were run into 1% agarose, and the bands were isolated and purified using the Qiaquick gel extraction kit (Qiagen). Following purification the fragments were subsequently ligated into pCMV-AD (Stratagene) for the mammalian two-hybrid assay (used against Rb-GAL4 from W. Kaelin), and transformed into E. coli. Immunoprecipitation—Thirty-six hours post-transfection, HeLa cells were washed with phosphate-buffered saline and harvested in lysis buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 10% glycerol, 0.5% Triton X-100) containing protease inhibitors. The cells were lysed for 20 min at 4 °C, followed by centrifugation at 14,000 × g for 10 min. Proteins were immunoprecipitated from the supernatant by overnight incubation at 4 °C with anti-human pRb G3-245 (BD Pharmingen), anti-His (Invitrogen), anti-HA (Santa Cruz Biotechnology), anti-mSin3 (Santa Cruz Biotechnology), anti-HDAC1 (Affinity Bioreagents), anti-HDAC3 (Affinity Bioreagents), or M2 anti-FLAG antibodies. Immunoprecipitated proteins were recovered on protein A-agarose (Upstate Biotechnology), washed three times for 5 min at room temperature with 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA, and 0.1% Nonidet P-40, followed by SDS-PAGE analysis of the proteins. For enzyme activity assays, the beads were washed three times with lysis buffer. Chromatin Immunoprecipitation Assay—HeLa cells were transfected with 5 μg of UAS-TK-Luc (25.Heinzel T. Lavinsky R.M. Mullen T.M. Soderstrom M. Laherty C.D. Torchia J. Yang W.M. Brard G. Ngo S.D. Davie J.R. Seto E. Eisenman R.N. Rose D.W. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 43-48Crossref PubMed Scopus (1086) Google Scholar), and 15 μg of the GAL4-VP16-JMJD2A (aa 661–1064) or the parent expression-only GAL4-VP16 control, using the calcium-phosphate method. After 36 h, histones were cross-linked to DNA by adding formaldehyde directly to culture medium to a final concentration of 1%, and incubated for 10 min at 37 °C with mild shaking. Cells were harvested and suspended in lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris-HCl, pH 8.1) with protease inhibitors. After sonication and dilution of lysates, half of the lysate amount was saved for a no-antibody control, whereas the remaining was incubated overnight at 4 °C with an anti-acetyl-histone H3 antibody (Upstate Biotechnology number 06-599). Immunoprecipitated complexes were collected using salmon sperm DNA/protein-agarose. Precipitants were washed, and the immune complex was eluted by adding 250 μl of elution buffer (1% SDS, 0.1 m NaHCO3). Subsequently, 20 μl of 5 m NaCl was added to reverse the formaldehyde cross-linking at 65 °C for 4 h. Following incubation with proteinase K, 0.5 m EDTA, and 1 m Tris-HCl, DNA was recovered by phenol/chloroform and ethanol precipitated. Pellets were suspended in water