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• W2079597786 abstract "Post-translational histone methylation is a dynamic and reversible process that is involved in the spatio-temporal regulation of gene transcription and contributes to various cellular phenotypes. Methylation of histone H3 at lysine 9 (H3K9), which is generally a transcriptional repression mark, is demethylated by H3K9-specific demethylases, leading to transcriptional activation. However, how multiple demethylases with the same substrate specificity differ in their chromatin targeting mechanisms has not been well understood. Unlike other H3K9-specific demethylases, it has been reported that JMJD1A likely forms a homodimer, but a detailed mode of dimerization and the possible link between structure and enzymatic activity have remained unresolved. Here, we report the structure-function relationship of JMJD1A in detail. First, JMJD1A forms a homodimer through its catalytic domains, bringing the two active sites close together. Second, increasing the concentration of JMJD1A facilitates efficient production of unmethylated product from dimethyl-H3K9 and decreases the release of the monomethylated intermediate. Finally, substituting one of the two active sites with an inactive mutant results in a significant reduction of the demethylation rate without changing the affinity to the intermediate. Given this evidence, we propose a substrate channeling model for the efficient conversion of dimethylated H3K9 into the unmethylated state. Our study provides valuable information that will help in understanding the redundancy of H3K9-specific demethylases and the complementary activity of their unique structures and enzymatic properties for appropriate control of chromatin modification patterns. Post-translational histone methylation is a dynamic and reversible process that is involved in the spatio-temporal regulation of gene transcription and contributes to various cellular phenotypes. Methylation of histone H3 at lysine 9 (H3K9), which is generally a transcriptional repression mark, is demethylated by H3K9-specific demethylases, leading to transcriptional activation. However, how multiple demethylases with the same substrate specificity differ in their chromatin targeting mechanisms has not been well understood. Unlike other H3K9-specific demethylases, it has been reported that JMJD1A likely forms a homodimer, but a detailed mode of dimerization and the possible link between structure and enzymatic activity have remained unresolved. Here, we report the structure-function relationship of JMJD1A in detail. First, JMJD1A forms a homodimer through its catalytic domains, bringing the two active sites close together. Second, increasing the concentration of JMJD1A facilitates efficient production of unmethylated product from dimethyl-H3K9 and decreases the release of the monomethylated intermediate. Finally, substituting one of the two active sites with an inactive mutant results in a significant reduction of the demethylation rate without changing the affinity to the intermediate. Given this evidence, we propose a substrate channeling model for the efficient conversion of dimethylated H3K9 into the unmethylated state. Our study provides valuable information that will help in understanding the redundancy of H3K9-specific demethylases and the complementary activity of their unique structures and enzymatic properties for appropriate control of chromatin modification patterns. Post-translational histone modification is an important component of the epigenome, and is involved in the regulation of gene transcription in the chromatin context. Histone lysine methylation is known to contribute to human diseases, such as cancer, neuropsychiatric disorders, inflammation, and metabolic disorders (1Arrowsmith C.H. Bountra C. Fish P.V. Lee K. Schapira M. Epigenetic protein families. A new frontier for drug discovery.Nat. Rev. Drug. Discov. 2012; 11: 384-400Crossref PubMed Scopus (1060) Google Scholar). The methylation state of lysine residues can be mono, di, or tri. These different states provide information as part of the “histone code,” in conjunction with other modifications such as phosphorylation and acetylation, for fine-tuning gene expression in response to specific signals. Methylation is reversibly regulated by S-adenosylmethionine-dependent methyltransferases, the flavin-dependent demethylases, and the Jumonji family of iron and 2-oxoglutarate-dependent demethylases (1Arrowsmith C.H. Bountra C. Fish P.V. Lee K. Schapira M. Epigenetic protein families. A new frontier for drug discovery.Nat. Rev. Drug. Discov. 2012; 11: 384-400Crossref PubMed Scopus (1060) Google Scholar). Jumonji C (JmjC) 2The abbreviations used are: JmjCJumonji CJMJD1AJumonji domain containing 1AMutmutantZFzinc fingerSFStrep-FLAGSPRsurface plasmon resonanceBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. domain-containing demethylases show differential specificities toward various histone lysine residues and also demonstrate methyl group number specificity, suggesting a fine tuning role for demethylases. Jumonji C Jumonji domain containing 1A mutant zinc finger Strep-FLAG surface plasmon resonance 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. Methylation of Lys-9 in histone H3 (H3K9) is generally involved in transcriptional repression and heterochromatin formation (2Krishnan S. Horowitz S. Trievel R.C. Structure and function of histone H3 lysine 9 methyltransferases and demethylases.ChemBioChem. 2011; 12: 254-263Crossref PubMed Scopus (69) Google Scholar). High-resolution mapping of histone modification patterns has shown that di- and trimethylations of Lys-9 (H3K9me2 and H3K9me3) are enriched in the transcriptional start sites of silenced genes, whereas H3K9me1 is present in the promoter regions of active genes (3Barski A. Cuddapah S. Cui K. Roh T.Y. Schones D.E. Wang Z. Wei G. Chepelev I. Zhao K. High-resolution profiling of histone methylations in the human genome.Cell. 2007; 129: 823-837Abstract Full Text Full Text PDF PubMed Scopus (5055) Google Scholar). These findings suggest that the methylation state of H3K9 would be an important mark denoting the transcriptional status of a given gene. Within the last decade, various H3K9-specific modifying enzymes and recognition proteins, including JMJD1A/KDM3A/JHDM2A, have been isolated (2Krishnan S. Horowitz S. Trievel R.C. Structure and function of histone H3 lysine 9 methyltransferases and demethylases.ChemBioChem. 2011; 12: 254-263Crossref PubMed Scopus (69) Google Scholar, 4Collins R.E. Northrop J.P. Horton J.R. Lee D.Y. Zhang X. Stallcup M.R. Cheng X. The ankyrin repeats of G9a and GLP histone methyltransferases are mono- and dimethyllysine binding modules.Nat. Struct. Mol. Biol. 2008; 15: 245-250Crossref PubMed Scopus (214) Google Scholar, 5Nair S.S. Nair B.C. Cortez V. Chakravarty D. Metzger E. Schüle R. Brann D.W. Tekmal R.R. Vadlamudi R.K. PELP1 is a reader of histone H3 methylation that facilitates oestrogen receptor-α target gene activation by regulating lysine demethylase 1 specificity.EMBO Rep. 2010; 11: 438-444Crossref PubMed Scopus (83) Google Scholar, 6Rothbart S.B. Krajewski K. Nady N. Tempel W. Xue S. Badeaux A.I. Barsyte-Lovejoy D. Martinez J.Y. Bedford M.T. Fuchs S.M. Arrowsmith C.H. Strahl B.D. Association of UHRF1 with methylated H3K9 directs the maintenance of DNA methylation.Nat. Struct. Mol. Biol. 2012; 19: 1155-1160Crossref PubMed Scopus (264) Google Scholar). In particular, despite the similar substrate specificity of these demethylating enzymes, the differences in their kinetic properties and chromatin targeting mechanisms have remained unresolved. JMJD1A is capable of demethylating H3K9me1 and H3K9me2 specifically, but not H3K9me3 (7Yamane K. Toumazou C. Tsukada Y. Erdjument-Bromage H. Tempst P. Wong J. Zhang Y. JHDM2A, a JmjC-containing H3K9 demethylase, facilitates transcription activation by androgen receptor.Cell. 2006; 125: 483-495Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar). Unlike other H3K9me2/me1-specific demethylases, such as PHF8 and KIAA1718 (8Horton J.R. Upadhyay A.K. Qi H.H. Zhang X. Shi Y. Cheng X. Enzymatic and structural insights for substrate specificity of a family of jumonji histone lysine demethylases.Nat. Struct. Mol. Biol. 2010; 17: 38-43Crossref PubMed Scopus (297) Google Scholar), JMJD1A likely forms a homodimer (7Yamane K. Toumazou C. Tsukada Y. Erdjument-Bromage H. Tempst P. Wong J. Zhang Y. JHDM2A, a JmjC-containing H3K9 demethylase, facilitates transcription activation by androgen receptor.Cell. 2006; 125: 483-495Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar), but a detailed model of dimerization and the possible link between its structure and enzymatic activity are unknown. There are accumulating in vivo evidence on the importance of JMJD1A, such as its involvement in spermatogenesis (9Okada Y. Scott G. Ray M.K. Mishina Y. Zhang Y. Histone demethylase JHDM2A is critical for Tnp1 and Prm1 transcription and spermatogenesis.Nature. 2007; 450: 119-123Crossref PubMed Scopus (301) Google Scholar, 10Liu Z. Zhou S. Liao L. Chen X. Meistrich M. Xu J. Jmjd1a demethylase-regulated histone modification is essential for cAMP-response element modulator-regulated gene expression and spermatogenesis.J. Biol. Chem. 2010; 285: 2758-2770Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), and metabolic disorders (11Tateishi K. Okada Y. Kallin E.M. Zhang Y. Role of Jhdm2a in regulating metabolic gene expression and obesity resistance.Nature. 2009; 458: 757-761Crossref PubMed Scopus (360) Google Scholar, 12Inagaki T. Tachibana M. Magoori K. Kudo H. Tanaka T. Okamura M. Naito M. Kodama T. Shinkai Y. Sakai J. Obesity and metabolic syndrome in histone demethylase JHDM2a-deficient mice.Genes Cells. 2009; 14: 991-1001Crossref PubMed Scopus (149) Google Scholar). JMJD1A is up-regulated under hypoxic conditions (13Beyer S. Kristensen M.M. Jensen K.S. Johansen J.V. Staller P. The histone demethylases JMJD1A and JMJD2B are transcriptional targets of hypoxia-inducible factor HIF.J. Biol. Chem. 2008; 283: 36542-36552Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 14Wellmann S. Bettkober M. Zelmer A. Seeger K. Faigle M. Eltzschig H.K. Bührer C. Hypoxia up-regulates the histone demethylase JMJD1A via HIF-1.Biochem. Biophys. Res. Commun. 2008; 372: 892-897Crossref PubMed Scopus (159) Google Scholar, 15Pollard P.J. Loenarz C. Mole D.R. McDonough M.A. Gleadle J.M. Schofield C.J. Ratcliffe P.J. Regulation of Jumonji-domain-containing histone demethylases by hypoxia-inducible factor (HIF)-1α.Biochem. J. 2008; 416: 387-394Crossref PubMed Scopus (247) Google Scholar, 16Sar A. Ponjevic D. Nguyen M. Box A.H. Demetrick D.J. Identification and characterization of demethylase JMJD1A as a gene up-regulated in the human cellular response to hypoxia.Cell Tissue Res. 2009; 337: 223-234Crossref PubMed Scopus (33) Google Scholar, 17Mimura I. Nangaku M. Kanki Y. Tsutsumi S. Inoue T. Kohro T. Yamamoto S. Fujita T. Shimamura T. Suehiro J. Taguchi A. Kobayashi M. Tanimura K. Inagaki T. Tanaka T. Hamakubo T. Sakai J. Aburatani H. Kodama T. Wada Y. Dynamic change of chromatin conformation in response to hypoxia enhances the expression of GLUT3 (SLC2A3) by cooperative interaction of hypoxia-inducible factor 1 and KDM3A.Mol. Cell Biol. 2012; 32: 3018-3032Crossref PubMed Scopus (173) Google Scholar, 18Krieg A.J. Rankin E.B. Chan D. Razorenova O. Fernandez S. Giaccia A.J. Regulation of the histone demethylase JMJD1A by hypoxia-inducible factor 1α enhances hypoxic gene expression and tumor growth.Mol. Cell Biol. 2010; 30: 344-353Crossref PubMed Scopus (277) Google Scholar), and may be a good therapeutic target against various types of cancer (18Krieg A.J. Rankin E.B. Chan D. Razorenova O. Fernandez S. Giaccia A.J. Regulation of the histone demethylase JMJD1A by hypoxia-inducible factor 1α enhances hypoxic gene expression and tumor growth.Mol. Cell Biol. 2010; 30: 344-353Crossref PubMed Scopus (277) Google Scholar, 19Uemura M. Yamamoto H. Takemasa I. Mimori K. Hemmi H. Mizushima T. Ikeda M. Sekimoto M. Matsuura N. Doki Y. Mori M. Jumonji domain containing 1A is a novel prognostic marker for colorectal cancer. In vivo identification from hypoxic tumor cells.Clin. Cancer Res. 2010; 16: 4636-4646Crossref PubMed Scopus (81) Google Scholar, 20Cho H.S. Toyokawa G. Daigo Y. Hayami S. Masuda K. Ikawa N. Yamane Y. Maejima K. Tsunoda T. Field H.I. Kelly J.D. Neal D.E. Ponder B.A. Maehara Y. Nakamura Y. Hamamoto R. The JmjC domain-containing histone demethylase KDM3A is a positive regulator of the G1/S transition in cancer cells via transcriptional regulation of the HOXA1 gene.Int. J. Cancer. 2012; 131: E179-E189Crossref PubMed Scopus (80) Google Scholar, 21Yamada D. Kobayashi S. Yamamoto H. Tomimaru Y. Noda T. Uemura M. Wada H. Marubashi S. Eguchi H. Tanemura M. Doki Y. Mori M. Nagano H. Role of the hypoxia-related gene, JMJD1A, in hepatocellular carcinoma. Clinical impact on recurrence after hepatic resection.Ann. Surg. Oncol. 2012; 19: S355-S364Crossref PubMed Scopus (64) Google Scholar, 22Osawa T. Tsuchida R. Muramatsu M. Shimamura T. Wang F. Suehiro J. Kanki Y. Wada Y. Yuasa Y. Aburatani H. Miyano S. Minami T. Kodama T. Shibuya M. Inhibition of histone demethylase JMJD1A improves anti-angiogenic therapy and reduces tumor associated macrophages.Cancer Res. 2013; 73: 3019-3028Crossref PubMed Scopus (74) Google Scholar). However, there is little information thus far about the molecular basis of its enzymatic activity. Enzymological studies can provide valuable information in elucidating the biological role of JMJD1A, and understanding the catalytic mechanism of JMJD1A compared with other H3K9-specific demethylases. In this study, we investigated the structure-activity relationship of JMJD1A in detail and demonstrated how it functions as a homodimer in the sequential removal of H3K9me2 methylation. Using quantitative data during demethylating processes from formaldehyde dehydrogenase-coupled assays, surface plasmon resonance (SPR) binding assays, and MALDI-TOF mass spectrometry analyses, we investigated H3K9 demethylation by JMJD1A homodimer. Based on its kinetic properties, we propose a substrate channeling mechanism by which the two active sites cooperatively demethylate two methyl groups. A plasmid encoding human JMJD1A(1–1321) was kindly provided by Dr. Mimura (17Mimura I. Nangaku M. Kanki Y. Tsutsumi S. Inoue T. Kohro T. Yamamoto S. Fujita T. Shimamura T. Suehiro J. Taguchi A. Kobayashi M. Tanimura K. Inagaki T. Tanaka T. Hamakubo T. Sakai J. Aburatani H. Kodama T. Wada Y. Dynamic change of chromatin conformation in response to hypoxia enhances the expression of GLUT3 (SLC2A3) by cooperative interaction of hypoxia-inducible factor 1 and KDM3A.Mol. Cell Biol. 2012; 32: 3018-3032Crossref PubMed Scopus (173) Google Scholar). Strep-FLAG (SF) tag (23Gloeckner C.J. Boldt K. Schumacher A. Roepman R. Ueffing M. A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes.Proteomics. 2007; 7: 4228-4234Crossref PubMed Scopus (177) Google Scholar) and Myc-HA tag were generated by annealing complementary DNA oligos and subcloned into pcDNA3.1(+) vector. Then these tags were inserted into pcDNA3-JMJD1A to generate pcDNA3-SF-JMJD1A and pcDNA3-Myc-HA-JMJD1A. JMJD1A was also inserted into the pcDNA4-Myc-His vector to generate pcDNA4-JMJD1A-Myc-His. pcDNA3-SF-JMJD1A (H1120Y), pcDNA3-Myc-HA-JMJD1A (H1120Y), and deletion mutants (623–717, 1058–1281, 718–889, 890–1057, Δ623–717, and Δ623–1321) were generated by PCR. The Δ890–1321 deletion mutant was generated by blunting two EcoRI sites and self-ligation. All the constructs generated through PCR were verified by sequencing. 293T cells were maintained in DMEM supplemented with 10% FBS and antibiotics at 37 °C. The plasmids were transfected using Lipofectamine 2000 (Invitrogen) following standard procedures. For Blue Native-PAGE, Native-PAGETM Novex BisTris gels were purchased from Invitrogen, and the sample preparation and electrophoresis were performed according to the manufacturer's instructions. For SDS-PAGE, samples were boiled for 5 min at 95 °C in SDS-PAGE sample buffer containing 125 mm Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.01% bromphenol blue. Samples were loaded onto 7% gels. For immunoblotting analysis, the gel was electroblotted to a PVDF membrane, which was blocked in 2% skim milk. Primary antibodies used were anti-FLAG M2 (Sigma), anti-HA (Roche), and anti-Myc (Santa Cruz). Secondary detection was carried out using anti-mouse (GE Healthcare) and anti-rat (Santa Cruz) antibodies. Band intensity and relative molecular weights were determined using LAS3000 ImageGauge software (Fuji-film, Japan). Forty-eight hours after being transfected with plasmids, 293T cells were lysed in lysis buffer containing 20 mm Tris-HCl, pH 8.0, 1% Nonidet P-40, 150 mm NaCl, and a protease inhibitor mixture (Roche Applied Science). The insoluble material was removed by centrifugation at 15,000 × g for 25 min, and the soluble fraction was incubated with an anti-FLAG M2 antibody (Sigma) or an anti-Myc antibody (Santa Cruz) for 1 h. The immune complexes were pulled down with protein A-Sepharose (GE Healthcare) and washed three times with lysis buffer. Samples were eluted with SDS-PAGE sample buffer and analyzed by immunoblotting. Coupled fluorescent demethylase assays were performed in a reaction buffer containing 50 mm HEPES-KOH, pH 8.0, 50 μm Fe(NH4)2(SO4)2, 1 mm 2-oxoglutarate, 2 mm ascorbate, 500 μm NAD+, 0.025 units of formaldehyde dehydrogenase, 0.01% Tween 20, 100 nm JMJD1A, and variable concentrations of K9-methylated H3 peptide (residues 1–15). Production of NADH was monitored (excitation 355 nm/emission 460 nm) and readings were taken over 20 cycles with a cycle time of 3 min. An NADH standard curve was used to convert fluorescence to a concentration of formed product. The initial 9 min were used to calculate initial velocities, which were graphed against substrate concentration. Michaelis-Menten parameters were determined by non-linear least squares fitting using GraphPad Prism 6. All SPR experiments were carried out using a Biacore T200 system using a Series S Sensorchip SA (GE Healthcare). All sensorgrams were recorded at 10 °C using a flow rate of 30 μl/min. FLAG tag affinity purified SF-JMJD1A and SF-JMJD1A (H1120Y) were captured (∼3000 response units) on parallel channels of the chip. The dissociation constant (KD) value of the peptide-JMJD1A interaction was estimated by 5 injections for 1 min of K9-modified H3 peptides at 0.64, 1.6, 4, 10, and 25 μm in buffer containing 50 mm HEPES-KOH, 50 mm NaCl, 100 μm NiCl2, and 1 mm 2-oxoglutarate, pH 7.8. All data were corrected for nonspecific binding and buffer shifts by double subtracting binding responses collected from a blank reference cell and a buffer injection over an SF-JMJD1A or SF-JMJD1A (H1120Y) captured flow cell. For purification of JMJD1A homodimer and heterodimer by combination of wild-type and H1120Y mutant, SF-tagged and Myc-HA-tagged plasmids were cotransfected into 293T cells. Three days after transfection, cells were washed with phosphate-buffered saline (PBS) and lysed with lysis buffer (50 mm HEPES-KOH, pH 7.9, 3 mm MgCl2, 100 mm NaCl, 1 mm EGTA, and 0.5% Nonidet P-40) containing phosphatase inhibitor mixture (Sigma) and protease inhibitor mixture (Roche Applied Science). The cell lysates were incubated with M2 α-FLAG-agarose (Sigma) for 2 h at 4 °C. The beads were washed with lysis buffer three times and eluted with 200 μg/ml of FLAG peptide. The eluted fraction was then subjected to immunoprecipitation using anti-Myc antibody. The immune complexes were pulled down with protein A-Sepharose and washed three times with 50 mm HEPES-KOH, pH 7.9, 100 mm NaCl. To measure the amount of immobilized dimer, aliquots were eluted with SDS sample buffer and analyzed by SDS-PAGE with bovine serum albumin (BSA) as a standard (Fig. 4A). We quantified the amount of JMJD1A proteins with a standard curve by plotting the optical density obtained by ImageJ analysis for each BSA standard versus its amount in nanograms (Fig. 4A). In vitro demethylase assays were performed using varying amounts of the immunoprecipitated dimer in a reaction buffer containing 50 mm HEPES-KOH, pH 8.0, 50 μm Fe(NH4)2(SO4)2, 1 mm 2-oxoglutarate, 2 mm ascorbate, and 1 μm K9-methylated H3 peptide (residues 1–15) in a final volume of 20 μl. After gentle rotation at 37 °C, we collected 5-μl supernatants at the desired time points and mixed them with 5 μl of 2.5% (v/v) trifluoroacetic acid (TFA) to stop the reaction. The solution was desalted through a C-tip300 (AMR Inc.), mixed with α-cyano-4-hydroxycinnamic acid, and then analyzed with a MALDI-TOF AXIMA performance (Shimadzu). Based on fractionation of HeLa cell nuclear extracts by gel-filtration, JMJD1A likely forms a homodimer (7Yamane K. Toumazou C. Tsukada Y. Erdjument-Bromage H. Tempst P. Wong J. Zhang Y. JHDM2A, a JmjC-containing H3K9 demethylase, facilitates transcription activation by androgen receptor.Cell. 2006; 125: 483-495Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar). To test whether JMJD1A forms a homodimer, we affinity purified Strep-FLAG-tagged (SF) JMJD1A using streptactin-Sepharose, and performed Blue Native-PAGE. This technique can be used to determine the native protein masses and oligomeric states and to identify physiological protein-protein interactions (24Wittig I. Braun H.P. Schägger H. Blue native PAGE.Nat. Protoc. 2006; 1: 418-428Crossref PubMed Scopus (1261) Google Scholar). Blue Native-PAGE analysis revealed a 300-kDa band (Fig. 1A, left), which we also observed by immunoblotting with anti-FLAG antibody (Fig. 1A, right). Given that the monomer size of JMJD1A is about 150 kDa, this result indicated homodimerization of JMJD1A. Furthermore, we co-expressed SF-tagged and Myc-His-tagged JMJD1A in 293T cells to validate the interaction of these two proteins. SF-JMJD1A was co-immunoprecipitated with anti-Myc antibody after affinity purification by streptactin-Sepharose (Fig. 1B). These data strongly suggested that JMJD1A forms a homodimer. To gain more insight into the dimer interface of JMJD1A, we prepared various deletion mutants as shown in Fig. 1E, and investigated dimer formation with immunoprecipitation followed by immunoblot analysis. To date, mutational studies indicate that a JmjC domain and a zinc finger (ZF) in JMJD1A are required for its enzymatic activity (7Yamane K. Toumazou C. Tsukada Y. Erdjument-Bromage H. Tempst P. Wong J. Zhang Y. JHDM2A, a JmjC-containing H3K9 demethylase, facilitates transcription activation by androgen receptor.Cell. 2006; 125: 483-495Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar, 25Brauchle M. Yao Z. Arora R. Thigale S. Clay I. Inverardi B. Fletcher J. Taslimi P. Acker M.G. Gerrits B. Voshol J. Bauer A. Schübeler D. Bouwmeester T. Ruffner H. Protein complex interactor analysis and differential activity of KDM3 subfamily members towards H3K9 methylation.PLoS One. 2013; 8: e60549Crossref PubMed Scopus (53) Google Scholar). H1120 is also involved in demethylation, which is necessary for binding of Fe(II) (7Yamane K. Toumazou C. Tsukada Y. Erdjument-Bromage H. Tempst P. Wong J. Zhang Y. JHDM2A, a JmjC-containing H3K9 demethylase, facilitates transcription activation by androgen receptor.Cell. 2006; 125: 483-495Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar). Deletion of the ZF or JmjC domain caused the reduction of dimerization efficiency (Fig. 1C). Conversely, when co-expressing each domain with full-length JMJD1A, both the ZF and JmjC domains interacted with full-length JMJD1A (Fig. 1D). Using mutant proteins, we finally narrowed down the interaction domain to amino acids 623–717 and 890–1321 (Fig. 1E). Furthermore, we examined whether the mode of interaction between ZF and JmjC occurs in trans, cis, or a complexed mode using deletion mutants (Fig. 1F). The results indicated that each domain in each monomer interacted with both the ZF and JmjC domains in another monomer (Fig. 1F). Thus, the binding mode of homodimerization is complexed (Fig. 1G, right), bringing the two active sites close together. To examine the relationship between homodimerization and demethylase activity, we performed a demethylation assay using K9-methylated H3 peptide substrates and analyzed the demethylation products by formaldehyde dehydrogenase counterscreen and mass spectrometry. Using a fluorescent assay that follows the formation of the demethylation byproduct formaldehyde, demethylation of di- and monomethylated H3K9 peptides was analyzed (Fig. 2A). The catalytic efficiency kcat/Km of intermediate me1 is slightly higher than substrate me2, but almost the same (Table 1). Next we used mass spectrometry to measure the extent of demethylation of the dimethylated substrate as a function of time (Fig. 2B). Surprisingly, throughout the reaction the level of the intermediate substrate H3K9me1 remained low, at most 22%. These data are most consistent with a processive mechanism. However, monomethylated intermediate is also released above the enzyme concentration. Therefore, the processivity is not obligate. SPR analysis suggested that all peptides of H3K9me2, H3K9me1, and H3K9me0 showed similar dissociation constant (KD) against JMJD1A (Fig. 3, Table 2). The KD for each peptide from JMJD1A is less than the Km, indicating that the Km reflects more than just the true affinity of the enzyme for each peptide. Taken together, these results suggested partially processive whereas having a distributive mechanism for multiple demethylations.TABLE 1Kinetic analysis of JMJD1A-mediated demethylation of peptidesSubstrateKmkcatkcat/Kmμmμm min−1min−1μm−1H3K9me195.2 ± 10.516.9 ± 0.710.178 ± 0.021H3K9me2106.1 ± 11.516.5 ± 0.710.155 ± 0.018 Open table in a new tab FIGURE 3Single-cycle kinetics data reflecting the interaction of SA-captured JMJD1A with histone peptides. Six panels of wild-type (left) and H1120Y mutant (right) show experimental sensorgrams. Each run reflects five injections of analyte (H3K9me2, me1, me0) at 0.64, 1.6, 4, 10, and 25 μm. Analyte injections lasted for 60 s each and were separated by 60-s dissociation phases.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 2Effect of methylation on H3K9 peptides in binding to wild-type and H1120Y mutant JMJD1AProteinKDH3K9me0H3K9me1H3K9me2μmWT3.32 ± 0.293.59 ± 0.023.56 ± 0.13Mut3.49 ± 0.053.55 ± 0.043.48 ± 0.13 Open table in a new tab To address whether the proximal two active sites of the JMJD1A homodimer are important for its enzymatic activity, we performed a demethylase assay using a heterodimer between the wild-type and an enzymatic activity deficient mutant (H1120Y). We performed a two-step purification procedure with FLAG M2-agarose and immunoprecipitation by anti-Myc antibody to obtain immobilized FLAG-tagged/Myc-tagged WT/WT and WT/Mut dimers (Fig. 4A). To rule out the possibility that the low activity of the WT/Mut heterodimer was caused by monomerization, we performed immunoblot analysis after the demethylase assay. As shown in Fig. 4B, monomer units were not observed in the supernatant of the reaction mixture. This indicated that the homodimer of JMJD1A is stable. Time-dependent demethylation was successfully observed (Fig. 4C). Fig. 4D shows that the demethylation efficiency of the H3K9me2 peptide depends on the amount of immobilized JMJD1A homodimer, as in Fig. 2B. Furthermore, rapid accumulation of the non-methylated product relative to the formation of the monomethylated intermediate was observed, even in the presence of an excess of the dimethylated substrate, as in Fig. 2B. Time-dependent demethylation was observed using WT/WT and WT/Mut dimers (Fig. 5A). Two pseudo first-order rate constants for two demethylation steps were obtained through this analysis, k1, k2, and k1′, respectively (Table 3). Notably, the relative abundance of me0 was significantly decreased in WT/Mut compared with WT/WT (Fig. 5, A and C, Table 3). So we speculated that homodimerization of JMJD1A is important for efficient two-step demethylation. Based on our hypothesis, we performed a demethylase assay using the H3K9me1 peptide as a substrate to observe the one-step reaction. As expected, the WT/Mut heterodimer showed almost the same activity as the WT/WT homodimer (Fig. 5, B and D).TABLE 3Demethylation activity of JMJD1A homodimer and heterodimerPseudo first-order rate constantsRelative abundance after 30 minme2aSubstrate.me2aSubstrate.me1aSubstrate.me2aSubstrate.me2aSubstrate.me1aSubstrate.k1, min−1k2, min−1k1′, min−1me1, %me0, %me0, %WT/WT0.1500.0890.36919.140.663.8WT/Mut0.0760.0290.27816.74.553.1a Substrate. Open table in a new tab It has been shown that there exist several H3K9-specific demethylases (2Krishnan S. Horowitz S. Trievel R.C. Structure and function of histone H3 lysine 9 methyltransferases and demethylases.ChemBioChem. 2011; 12: 254-263Crossref PubMed Scopus (69) Google Scholar, 7Yamane K. Toumazou C. Tsukada Y. Erdjument-Bromage H. Tempst P. Wong J. Zhang Y. JHDM2A, a JmjC-containing H3K9 demethylase, facilitates transcription activation by androgen receptor.Cell. 2006; 125: 483-495Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar, 8Horton J.R. Upadhyay A.K. Qi H.H. Zhang X. Shi Y. Cheng X. Enzymatic and structural insights for substrate specificity of a family of jumonji histone lysine demethylases.Nat. Struct. Mol. Biol" @default.
• W2079597786 created "2016-06-24" @default.
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• W2079597786 title "Control of Histone H3 Lysine 9 (H3K9) Methylation State via Cooperative Two-step Demethylation by Jumonji Domain Containing 1A (JMJD1A) Homodimer" @default.
• W2079597786 cites W1510561425 @default.
• W2079597786 cites W1963909260 @default.
• W2079597786 cites W1975511655 @default.
• W2079597786 cites W1980435226 @default.
• W2079597786 cites W1989646912 @default.
• W2079597786 cites W1992538495 @default.
• W2079597786 cites W1999560052 @default.
• W2079597786 cites W2001401534 @default.
• W2079597786 cites W2014023175 @default.
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• W2079597786 cites W2029865263 @default.
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• W2079597786 cites W2035526755 @default.
• W2079597786 cites W2040402810 @default.
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• W2079597786 cites W2049365130 @default.
• W2079597786 cites W2053146271 @default.
• W2079597786 cites W2053351022 @default.
• W2079597786 cites W2060740961 @default.
• W2079597786 cites W2060903701 @default.
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• W2079597786 cites W2063430478 @default.
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• W2079597786 cites W2074310370 @default.
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• W2079597786 cites W2104047605 @default.
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