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• W2027578670 abstract "Histone lysine methylation is a dynamic process that plays an important role in regulating chromatin structure and gene expression. Recent studies have identified Jhd2, a JmjC domain-containing protein, as an H3K4-specific demethylase in budding yeast. However, important questions regarding the regulation and functions of Jhd2 remain unanswered. In this study, we show that Jhd2 has intrinsic activity to remove all three states of H3K4 methylation in vivo and can dynamically associate with chromatin to modulate H3K4 methylation levels on both active and repressed genes and at the telomeric regions. We found that the plant homeodomain (PHD) finger of Jhd2 is important for its chromatin association in vivo. However, this association is not dependent on H3K4 methylation and the H3 N-terminal tail, suggesting the presence of an alternative mechanism by which Jhd2 binds nucleosomes. We also provide evidence that the JmjN domain and its interaction with the JmjC catalytic domain are important for Jhd2 function and that Not4 (an E3 ligase) monitors the structural integrity of this interdomain interaction to maintain the overall protein levels of Jhd2. We show that the S451R mutation in human SMCX (a homolog of Jhd2), which has been linked to mental retardation, and the homologous T359R mutation in Jhd2 affect the protein stability of both of these proteins. Therefore, our findings provide a mechanistic explanation for the observed defects in patients harboring this SMCX mutant and suggest the presence of a conserved pathway involving Not4 that modulates the protein stability of both yeast Jhd2 and human SMCX. Histone lysine methylation is a dynamic process that plays an important role in regulating chromatin structure and gene expression. Recent studies have identified Jhd2, a JmjC domain-containing protein, as an H3K4-specific demethylase in budding yeast. However, important questions regarding the regulation and functions of Jhd2 remain unanswered. In this study, we show that Jhd2 has intrinsic activity to remove all three states of H3K4 methylation in vivo and can dynamically associate with chromatin to modulate H3K4 methylation levels on both active and repressed genes and at the telomeric regions. We found that the plant homeodomain (PHD) finger of Jhd2 is important for its chromatin association in vivo. However, this association is not dependent on H3K4 methylation and the H3 N-terminal tail, suggesting the presence of an alternative mechanism by which Jhd2 binds nucleosomes. We also provide evidence that the JmjN domain and its interaction with the JmjC catalytic domain are important for Jhd2 function and that Not4 (an E3 ligase) monitors the structural integrity of this interdomain interaction to maintain the overall protein levels of Jhd2. We show that the S451R mutation in human SMCX (a homolog of Jhd2), which has been linked to mental retardation, and the homologous T359R mutation in Jhd2 affect the protein stability of both of these proteins. Therefore, our findings provide a mechanistic explanation for the observed defects in patients harboring this SMCX mutant and suggest the presence of a conserved pathway involving Not4 that modulates the protein stability of both yeast Jhd2 and human SMCX. Covalent modifications of histones play an important role in genome maintenance and gene regulation. In particular, methylation on the side chains of lysine and arginine in histones H3 and H4 is important in regulating chromatin structure and gene transcription (1Berger S.L. Nature. 2007; 447: 407-412Crossref PubMed Scopus (2167) Google Scholar, 2Kouzarides T. Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (8183) Google Scholar). Lysine methylation is unique among the known histone modifications, as three modification states, monomethylation (me1), dimethylation (me2), and trimethylation (me3), can be generated, adding yet another level of variation to this modification “mark.” In general, methylation at H3K4, H3K36, and H3K79 has been linked to gene activation, whereas H3K9, H3K27, and H4K20 methylation is associated with repressed genes (1Berger S.L. Nature. 2007; 447: 407-412Crossref PubMed Scopus (2167) Google Scholar, 2Kouzarides T. Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (8183) Google Scholar). However, recent studies have revealed that depending on the gene context, the methylation state of a specific modified lysine can exert an opposite effect on gene expression. For example, methylation of H3K9 within the promoter or coding region can result in gene repression or activation, respectively (3Vakoc C.R. Mandat S.A. Olenchock B.A. Blobel G.A. Mol. Cell. 2005; 19: 381-391Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar). Furthermore, whereas H3K4 methylation (especially H3K4me3) is strongly associated with gene activation (1Berger S.L. Nature. 2007; 447: 407-412Crossref PubMed Scopus (2167) Google Scholar, 2Kouzarides T. Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (8183) Google Scholar, 4Eissenberg J.C. Shilatifard A. Dev. Biol. 2010; 339: 240-249Crossref PubMed Scopus (239) Google Scholar), H3K4me2 at certain chromatin loci plays a role in repression by preventing aberrant gene expression (5Berretta J. Pinskaya M. Morillon A. Genes Dev. 2008; 22: 615-626Crossref PubMed Scopus (159) Google Scholar, 6Pinskaya M. Gourvennec S. Morillon A. EMBO J. 2009; 28: 1697-1707Crossref PubMed Scopus (140) Google Scholar). Until recently, histone lysine methylation was thought to be stable and irreversible. However, this notion was dispelled by the identification of two different classes of lysine-specific histone demethylases, amine oxidases (e.g. LSD1) and JmjC domain-containing proteins. Both classes of enzymes catalyze lysine demethylation via an oxidation reaction that generates formaldehyde, but only JmjC class demethylases require ferrous ion (Fe2+) and α-ketoglutarate as cofactors (7Klose R.J. Kallin E.M. Zhang Y. Nat. Rev. Genet. 2006; 7: 715-727Crossref PubMed Scopus (970) Google Scholar, 8Shi Y. Whetstine J.R. Mol. Cell. 2007; 25: 1-14Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar). Whereas LSD1 specifically targets mono- and dimethylated H3K4 or H3K9, the JmjC class consists of several subfamilies that remove methyl groups from the modified H3K4, H3K9, H3K27, or H3K36 residue (7Klose R.J. Kallin E.M. Zhang Y. Nat. Rev. Genet. 2006; 7: 715-727Crossref PubMed Scopus (970) Google Scholar, 8Shi Y. Whetstine J.R. Mol. Cell. 2007; 25: 1-14Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar). One member of the JmjC class is the evolutionarily conserved JARID1 family of histone demethylases, which specifically target Lys4-methylated H3 and function as transcriptional repressors (9Secombe J. Eisenman R.N. Cell Cycle. 2007; 6: 1324-1328Crossref PubMed Scopus (65) Google Scholar). In addition to the JmjC catalytic domain, the JARID1 proteins also possess a conserved N-terminal motif (JmjN domain) that is strictly associated with the JmjC domain (7Klose R.J. Kallin E.M. Zhang Y. Nat. Rev. Genet. 2006; 7: 715-727Crossref PubMed Scopus (970) Google Scholar, 9Secombe J. Eisenman R.N. Cell Cycle. 2007; 6: 1324-1328Crossref PubMed Scopus (65) Google Scholar). Metazoan JARID1 demethylases also contain several conserved functional motifs, including an ARID/BRIGHT DNA-binding domain, a C5HC2 zinc finger, and two or three plant homeodomain (PHD) 2The abbreviations used are: PHDplant homeodomainChIPchromatin immunoprecipitationWTwild typeORFopen reading frameWCEwhole-cell extractMOPS4-morpholinepropanesulfonic acidGSTglutathione S-transferase. fingers (7Klose R.J. Kallin E.M. Zhang Y. Nat. Rev. Genet. 2006; 7: 715-727Crossref PubMed Scopus (970) Google Scholar, 9Secombe J. Eisenman R.N. Cell Cycle. 2007; 6: 1324-1328Crossref PubMed Scopus (65) Google Scholar). The ARID/BRIGHT DNA-binding domain is required for the demethylation activities of JARID1 proteins (10Lee M.G. Norman J. Shilatifard A. Shiekhattar R. Cell. 2007; 128: 877-887Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 11Xiang Y. Zhu Z. Han G. Ye X. Xu B. Peng Z. Ma Y. Yu Y. Lin H. Chen A.P. Chen C.D. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 19226-19231Crossref PubMed Scopus (305) Google Scholar, 12Yamane K. Tateishi K. Klose R.J. Fang J. Fabrizio L.A. Erdjument-Bromage H. Taylor-Papadimitriou J. Tempst P. Zhang Y. Mol. Cell. 2007; 25: 801-812Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 13Tu S. Teng Y.C. Yuan C. Wu Y.T. Chan M.Y. Cheng A.N. Lin P.H. Juan L.J. Tsai M.D. Nat. Struct. Mol. Biol. 2008; 15: 419-421Crossref PubMed Scopus (83) Google Scholar). However, it is not known whether the ARID/BRIGHT domain is essential for their association with chromatin. plant homeodomain chromatin immunoprecipitation wild type open reading frame whole-cell extract 4-morpholinepropanesulfonic acid glutathione S-transferase. Although mammalian cells encode four JARID1 H3K4 demethylases (JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX, and JARID1D/SMCY), only one (Jhd2) was identified in budding yeast (14Ingvarsdottir K. Edwards C. Lee M.G. Lee J.S. Schultz D.C. Shilatifard A. Shiekhattar R. Berger S.L. Mol. Cell. Biol. 2007; 27: 7856-7864Crossref PubMed Scopus (37) Google Scholar, 15Liang G. Klose R.J. Gardner K.E. Zhang Y. Nat. Struct. Mol. Biol. 2007; 14: 243-245Crossref PubMed Scopus (100) Google Scholar, 16Seward D.J. Cubberley G. Kim S. Schonewald M. Zhang L. Tripet B. Bentley D.L. Nat. Struct. Mol. Biol. 2007; 14: 240-242Crossref PubMed Scopus (152) Google Scholar, 17Tu S. Bulloch E.M. Yang L. Ren C. Huang W.C. Hsu P.H. Chen C.H. Liao C.L. Yu H.M. Lo W.S. Freitas M.A. Tsai M.D. J. Biol. Chem. 2007; 282: 14262-14271Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Human JARID1A and JARID1B have the ability to target all three states of H3K4 methylation in vivo (11Xiang Y. Zhu Z. Han G. Ye X. Xu B. Peng Z. Ma Y. Yu Y. Lin H. Chen A.P. Chen C.D. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 19226-19231Crossref PubMed Scopus (305) Google Scholar, 12Yamane K. Tateishi K. Klose R.J. Fang J. Fabrizio L.A. Erdjument-Bromage H. Taylor-Papadimitriou J. Tempst P. Zhang Y. Mol. Cell. 2007; 25: 801-812Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 18Klose R.J. Yan Q. Tothova Z. Yamane K. Erdjument-Bromage H. Tempst P. Gilliland D.G. Zhang Y. Kaelin Jr., W.G. Cell. 2007; 128: 889-900Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). The yeast Jhd2 displayed activity toward all forms of Lys4-methylated H3 in vitro (14Ingvarsdottir K. Edwards C. Lee M.G. Lee J.S. Schultz D.C. Shilatifard A. Shiekhattar R. Berger S.L. Mol. Cell. Biol. 2007; 27: 7856-7864Crossref PubMed Scopus (37) Google Scholar) but only toward H3K4me2 and H3K4me3 in vivo (15Liang G. Klose R.J. Gardner K.E. Zhang Y. Nat. Struct. Mol. Biol. 2007; 14: 243-245Crossref PubMed Scopus (100) Google Scholar, 16Seward D.J. Cubberley G. Kim S. Schonewald M. Zhang L. Tripet B. Bentley D.L. Nat. Struct. Mol. Biol. 2007; 14: 240-242Crossref PubMed Scopus (152) Google Scholar, 17Tu S. Bulloch E.M. Yang L. Ren C. Huang W.C. Hsu P.H. Chen C.H. Liao C.L. Yu H.M. Lo W.S. Freitas M.A. Tsai M.D. J. Biol. Chem. 2007; 282: 14262-14271Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Chromatin immunoprecipitation (ChIP) assays have revealed that in the absence of JHD2, the levels of H3K4 methylation were altered compared with the wild type (WT) during activation and attenuation of GAL1 transcription (14Ingvarsdottir K. Edwards C. Lee M.G. Lee J.S. Schultz D.C. Shilatifard A. Shiekhattar R. Berger S.L. Mol. Cell. Biol. 2007; 27: 7856-7864Crossref PubMed Scopus (37) Google Scholar), suggesting a role for Jhd2 in these processes. Evidence from genetic studies indicates that Jhd2 is important for maintaining normal gene silencing at telomere and silent mating-type loci (15Liang G. Klose R.J. Gardner K.E. Zhang Y. Nat. Struct. Mol. Biol. 2007; 14: 243-245Crossref PubMed Scopus (100) Google Scholar, 19Osborne E.A. Dudoit S. Rine J. Nat. Genet. 2009; 41: 800-806Crossref PubMed Scopus (54) Google Scholar). A recent study has shown that the protein stability and steady-state levels of Jhd2 are modulated by the proteasomal degradation following E3 ligase Not4-mediated polyubiquitination (20Mersman D.P. Du H.N. Fingerman I.M. South P.F. Briggs S.D. Genes Dev. 2009; 23: 951-962Crossref PubMed Scopus (82) Google Scholar). Despite these advances in our understanding of the functions of Jhd2, many fundamental questions pertaining to its substrate specificity and selectivity, domain contributions, chromatin association, and overall regulation remain unanswered. In this study, we demonstrate that Jhd2 localizes to both transcriptionally active and inactive chromatin and regulates H3K4 methylation at these loci. Upon investigating the contributions of the conserved domains in Jhd2 to its function, we found that a proper interaction between JmjN and JmjC domains is important for Jhd2 function and that Not4 controls Jhd2 protein levels by monitoring the integrity of this JmjN-JmjC interdomain interaction. Additionally, our results show that the PHD finger is important for the interaction of Jhd2 with chromatin in vivo and that this interaction is independent of H3K4 methylation. Deletion mutants lacking Jhd2, Set1, Swd1, Sdc1, or Spp1 in strain YMH171 were created using PCR products containing the disrupted gene locus and the inserted KanMX4 selection module amplified from the genomic DNA template isolated from the respective BY4742-based yeast deletion strains (Open Biosystems). Jhd2 was genomically tagged with nine copies of Myc at its C terminus in YMH171 following PCR amplification using pYM6 as the template (21Knop M. Siegers K. Pereira G. Zachariae W. Winsor B. Nasmyth K. Schiebel E. Yeast. 1999; 15: 963-972Crossref PubMed Scopus (819) Google Scholar). MSY421, a histone H3/H4 shuffle strain (22Sun Z.W. Allis C.D. Nature. 2002; 418: 104-108Crossref PubMed Scopus (838) Google Scholar), was used to mobilize the H3 N-terminal deletion mutant (H3(1–28Δ)). The FLAG-H2B and FLAG-H2B/set1Δ strains were derived from YZS276 (22Sun Z.W. Allis C.D. Nature. 2002; 418: 104-108Crossref PubMed Scopus (838) Google Scholar). Detailed genotypes of the yeast strains described in this study are listed in supplemental Table S1. All the plasmids used in this study are listed in supplemental Table S2. For overexpressing the jumonji domain-containing proteins, PCR products containing the entire open reading frame (ORF) for RPH1, GIS1, ECM5, JHD1, or JHD2 and 500 bp of DNA upstream and downstream of each ORF were mobilized into a high copy vector, YEplac112 (23Gietz R.D. Sugino A. Gene. 1988; 74: 527-534Crossref PubMed Scopus (2528) Google Scholar). C-terminal LexA epitope-tagged Jhd2 was created by PCR amplifying the ORF of JHD2 and mobilizing it between the ADH1 promoter and a fragment of the LexA DNA-binding domain in pFBL23 (24Béranger F. Aresta S. de Gunzburg J. Camonis J. Nucleic Acids Res. 1997; 25: 2035-2036Crossref PubMed Scopus (44) Google Scholar). An XhoI-KpnI fragment containing the sequence encoding nine copies of the Myc epitope (9Myc) and the CYC1 terminator was obtained following PCR amplification and mobilized into pRS314. Subsequently, a 500-bp PCR product containing the JHD2 promoter was mobilized upstream of the region coding for 9Myc as a SacI-SpeI fragment. The entire JHD2 promoter-9Myc-CYC1 terminator module was mobilized as a SacI-KpnI fragment into pRS316. The ORF of JHD2 was then PCR-amplified and mobilized between the JHD2 promoter and the 9Myc-CYC1 terminator sequence in pRS314 or pRS316 as a SpeI-XhoI fragment. Point and truncation mutants of Jhd2 were made by PCR-based site-directed mutagenesis using pRS314-JHD2-9Myc or pRS316-JHD2-9Myc as the template. For overexpression of JHD2 or its mutant derivatives, a fragment containing the WT or mutant ORF, the promoter, and the CYC1 terminator region was excised from the pRS314-based construct and mobilized into the high copy vector pRS426. To obtain purified recombinant Jhd2 PHD finger or its variants, a sequence encoding either the WT PHD finger or its mutant derivatives was amplified by PCR and mobilized into a bacterial expression vector, pBG101 (kindly provided by the Vanderbilt Structural Biology Core). The plasmids pCS3+-6Myc and pCS3+-SMCX-6Myc were kindly provided by Ralf Janknecht (25Kim T.D. Shin S. Janknecht R. Biochem. Biophys. Res. Commun. 2008; 366: 563-567Crossref PubMed Scopus (40) Google Scholar), and the pCS3+-smcx(S451R)-6Myc construct was made by PCR-based site-directed mutagenesis. All of the constructs created using PCR amplification were verified by DNA sequencing. 3DNA primers used in this study are available upon request. To determine changes in Jhd2 levels (see Fig. 2C), whole-cell extracts (WCEs) were prepared as described (26Chandrasekharan M.B. Huang F. Sun Z.W. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 16686-16691Crossref PubMed Scopus (149) Google Scholar) and analyzed by Western blotting using antibodies raised against the Myc epitope (9E10; a gift from Ethan Lee) and Pgk1 (22C5, Molecular Probes) at 1:1000 and 1:5000 dilutions, respectively. Crude nuclear extracts were used to examine the levels of H3K4 methylation following the cell fractionation procedure described previously (26Chandrasekharan M.B. Huang F. Sun Z.W. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 16686-16691Crossref PubMed Scopus (149) Google Scholar). The following antibodies were purchased from Millipore and used in Western blotting to detect H3K4 methylation (with dilutions indicated in parentheses): anti-H3K4me1 (1:1000), anti-H3K4me2 (1:10,000), and anti-H3K4me3 (1:2500). The histone H3 loading was monitored using anti-H3 antibody (1:7500; Active Motif). For mammalian WCEs, HeLa cells were transfected with pCS3+-6Myc, pCS3+-SMCX-6Myc, or pCS3+-smcx(S451R)-6Myc using the Lipofectamine method. Following a 2-day incubation at 37 °C, cells were washed prior to and after harvesting with ice-cold 1× phosphate-buffered saline (Sigma) and boiled in 1× Laemmli sample buffer (Bio-Rad) for 10 min. After centrifugation at 16,100 × g for 5 min, equal volumes of WCEs were subjected to Western blot analysis using anti-Myc (1:1000) and anti-β-actin (1:10,000; Sigma) antibodies. WT and jhd2Δ strains were grown in yeast minimal synthetic complete medium without inositol (SC−Ino medium; Bio 101, Inc.) supplemented with 200 μm inositol and 2 mm choline (INO1 repression medium) (27Schüller H.J. Schorr R. Hoffmann B. Schweizer E. Nucleic Acids Res. 1992; 20: 5955-5961Crossref PubMed Scopus (47) Google Scholar) at 30 °C overnight. The overnight cultures were reinoculated into INO1 repression medium at 2 × 106 cells/ml and grown at 30 °C to log phase. Cells (4 × 108) were subjected to formaldehyde cross-linking for ChIP assay (INO1 repressed state). The remaining cells were harvested, washed once with SC−Ino medium, inoculated at 4 × 106 cells/ml into fresh SC−Ino medium, and grown at 30 °C for 2 h to activate INO1 expression. Again, cells (4 × 108) were set aside and subjected to formaldehyde cross-linking (INO1 induced state). To assess temporal changes in histone modification or Jhd2 occupancy following INO1 repression, cells grown in inducing medium were reinoculated into INO1 repression medium at the following initial cell densities and grown at 30 °C for the various time periods (indicated in parentheses) to obtain ∼4 × 108 cells: 7 × 106 cells/ml (20 min), 6 × 106 cells/ml (1 h), 4 × 106 cells/ml (2 h), and 2 × 106 cells/ml (4 h). Cultures grown to different incubation times following INO1 re-repression were then subjected to formaldehyde cross-linking for ChIP assay (INO1 re-repression time course). Double cross-linking using dimethyl adipimidate (Sigma) and formaldehyde was done essentially as described previously (28Gardner R.G. Nelson Z.W. Gottschling D.E. Mol. Cell. Biol. 2005; 25: 6123-6139Crossref PubMed Scopus (125) Google Scholar) with minor modifications. Following incubation in 10 mm dimethyl adipimidate, cells were washed and resuspended in 1× phosphate-buffered saline containing 1% formaldehyde and incubated at room temperature for 45 min with gentle agitation. The cross-linking was stopped by the addition of 130 mm glycine and incubation for 10 min at room temperature. Cells were then washed twice with 1× phosphate-buffered saline and harvested to prepare soluble chromatin for immunoprecipitating H3, H3K4me3, Jhd2-LexA, and Jhd2–9Myc using anti-H3, anti-H3K4me3, anti-LexA (Active Motif), and anti-Myc antibodies, respectively. The data analysis of quantitative real-time PCR following ChIP was performed as described by Chandrasekharan et al. (26Chandrasekharan M.B. Huang F. Sun Z.W. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 16686-16691Crossref PubMed Scopus (149) Google Scholar) with modifications. Briefly, occupancy of H3K4me3 was calculated using the 2−ΔΔCT method (Bio-Rad real-time PCR applications guide). Chromatin obtained from the set1Δ strain was used as a control to determine nonspecific immunoprecipitation, and any value obtained for this strain was subtracted from the H3K4me3 occupancy values obtained from all other strains. Soluble chromatins from yeast strains transformed with plasmid vectors were used as negative controls (“no tag”) for ChIP of Jhd2–9Myc and Jhd2-LexA. The 2−ΔCT value obtained for the negative control was subtracted from 2−ΔCT values obtained for test samples containing epitope-tagged WT or mutant Jhd2.3 Spheroplast preparation and nuclei isolation were performed as described previously (26Chandrasekharan M.B. Huang F. Sun Z.W. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 16686-16691Crossref PubMed Scopus (149) Google Scholar). WCEs were prepared by bead-beating spheroplasts in buffer A (1% SDS, 8 m urea, 10 mm MOPS (pH 6.8), 10 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 1 μg/ml aprotinin, and 1 μg/ml leupeptin), followed by centrifugation at 16,100 × g for 20 min. Nuclear extracts were obtained by lysing isolated nuclei in buffer A, followed by sonication and centrifugation at 16,100 × g for 15 min. For chromatin fractions, isolated nuclei were resuspended in hypotonic solution (3 mm EDTA, 0.2 mm EGTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 1 μg/ml aprotinin, and 1 μg/ml leupeptin) and incubated on ice for 30 min. After centrifugation at 1700 × g for 5 min, the chromatin pellet was washed with hypotonic solution and then resuspended in buffer A. Following a brief sonication, soluble chromatin was obtained by centrifugation at 16,100 × g for 15 min. The protein concentration of the clarified lysate was measured using a Bio-Rad DC protein assay kit following the manufacturer’s instructions, and an equal amount of total protein was subjected to Western blotting using anti-Myc, anti-Pgk1, or anti-H3 antibody. Cycloheximide treatment of yeast was done as described previously (29Belle A. Tanay A. Bitincka L. Shamir R. O'Shea E.K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 13004-13009Crossref PubMed Scopus (529) Google Scholar) with minor modifications. Briefly, a 25-ml culture was grown to log phase, and cycloheximide was added to a final concentration of 35 μg/ml to inhibit the translation machinery. Cells (3.5 × 107) were collected at 0, 20, 40, and 60 min after cycloheximide treatment and boiled in 100 μl of 1× Laemmli sample buffer for 10 min. Following centrifugation at 16,100 × g for 5 min, equal volumes of lysates were subjected to Western blot analysis using anti-Myc or anti-Pgk1 antibody. A log phase yeast culture (25 ml) was treated with either dimethyl sulfoxide or 0.1 mm MG132 (a proteasome inhibitor) for 30 min. The effect of proteasomal inhibition on Jhd2 or its mutant derivatives was examined in a pdr5Δ strain to allow efficient uptake of MG132 (20Mersman D.P. Du H.N. Fingerman I.M. South P.F. Briggs S.D. Genes Dev. 2009; 23: 951-962Crossref PubMed Scopus (82) Google Scholar). WCEs were prepared in the presence of 0.1 mm MG132 and subjected to Western blotting as described above. The sequence of the PHD domain in Jhd2 was submitted to SWISS-MODEL structure prediction (ExPASy Proteomics Server). The software uses a homology-based search and predicts the structure utilizing solved structure(s) as a reference. The template used for the structure prediction is the solution structure of the PHD finger in JARID1D/SMCY (Protein Data Bank code 2E6R). Nuclei from FLAG-H2B and FLAG-H2B/set1Δ strains were isolated as described previously (26Chandrasekharan M.B. Huang F. Sun Z.W. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 16686-16691Crossref PubMed Scopus (149) Google Scholar), and the DNA content was determined by measuring the A260 of an aliquot of nuclei diluted in 1 n NaOH. Nuclei (equivalent to 660 μg of DNA) were resuspended in 200 μl of buffer B (50 mm HEPES (pH 7.6), 0.1 m KCl, 2.5 mm MgCl2, 0.25% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 5 mmN-ethylmaleimide, 1 μg/ml pepstatin A, 1 μg/ml aprotinin, and 1 μg/ml leupeptin). Micrococcal nuclease digestion was performed twice by the addition of 5 μl of 0.1 m CaCl2 and 500 units of micrococcal nuclease (Worthington) to the nuclei suspension, followed by incubation at room temperature for 10 min, and then 4.5 μl of 0.1 m EGTA (pH 8.0) was added to stop the digestion. After centrifugation at 9300 × g for 10 min, supernatants from the two digestions were combined. The nucleosome-enriched supernatant was diluted by the addition of an equal volume of buffer B containing 10% glycerol and subjected to anti-FLAG affinity chromatography (M2, Sigma). After extensive washes with buffer B with 5% glycerol, the immobilized nucleosomes were stored at 4 °C until further use in the in vitro nucleosome binding assay. DNA was isolated from an aliquot of the immobilized nucleosomes by phenol/chloroform extraction and ethanol precipitation and resolved on a 2% agarose gel to examine the size of the nucleosomes bound to anti-FLAG antibody-conjugated agarose beads. Recombinant glutathione S-transferase (GST)-tagged WT and mutant PHD fingers (amino acid 209–320) of Jhd2 were purified from Escherichia coli using glutathione-Sepharose 4B beads (GE Healthcare) following the manufacturer’s instructions. The GST-tagged PHD finger (2 μg) was incubated with 5 μl of nucleosome-containing beads in 200 μl of binding buffer (50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 1.5 mm MgCl2, 0.2% Triton X-100, 5% glycerol, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 1 μg/ml aprotinin, and 1 μg/ml leupeptin) for 2 h at 4 °C. After three washes with binding buffer, the beads were boiled in 1× Laemmli sample buffer for 10 min. Following centrifugation at 16,100 × g for 5 min, the supernatant was subjected to Western blot analysis using anti-GST (1:20,000; GE Healthcare) and anti-FLAG (M2, 1:5000) antibodies. The JmjC domain-containing histone demethylases are evolutionarily highly conserved across many genera (7Klose R.J. Kallin E.M. Zhang Y. Nat. Rev. Genet. 2006; 7: 715-727Crossref PubMed Scopus (970) Google Scholar). There are five JmjC domain-containing proteins (Rph1, Gis1, Ecm5, Jhd1, and Jhd2) in budding yeast. Consistent with previous reports (15Liang G. Klose R.J. Gardner K.E. Zhang Y. Nat. Struct. Mol. Biol. 2007; 14: 243-245Crossref PubMed Scopus (100) Google Scholar, 17Tu S. Bulloch E.M. Yang L. Ren C. Huang W.C. Hsu P.H. Chen C.H. Liao C.L. Yu H.M. Lo W.S. Freitas M.A. Tsai M.D. J. Biol. Chem. 2007; 282: 14262-14271Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), overexpression of JHD2 through a high copy, 2μ-based plasmid led to a decrease in H3K4me3 and a modest increase in H3K4me1 levels (Fig. 1A, sixth lane). Moreover, none of the JmjC domain-containing proteins exhibited any apparent demethylation of Lys79-methylated H3 under the same conditions (supplemental Fig. S1). Thus, Jhd2 is an H3K4 methylation-specific demethylase in yeast. Given the genome-wide distribution of H3K4 methylation (30Pokholok D.K. Harbison C.T. Levine S. Cole M. Hannett N.M. Lee T.I. Bell G.W. Walker K. Rolfe P.A. Herbolsheimer E. Zeitlinger J. Lewitter F. Gifford D.K. Young R.A. Cell. 2005; 122: 517-527Abstract Full Text Full Text PDF PubMed Scopus (1105) Google Scholar, 31Millar C.B. Grunstein M. Nat. Rev. Mol. Cell Biol. 2006; 7: 657-666Crossref PubMed Scopus (226) Google Scholar) and because H3K4me3 is closely associated with gene transcription (1Berger S.L. Nature. 2007; 447: 407-412Crossref PubMed Scopus (2167) Google Scholar, 2Kouzarides T. Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (8183) Google Scholar), we tested whether Jhd2 plays a role in controlling this H3 modification during transcription. Toward this end, ChIP assays were undertaken to assess the occurrence and changes, if any, in Jhd2 occupancy and in H3K4me3 levels at the highly expressed PMA1 gene. Compared with the WT, deletion of JHD2 (jhd2Δ) or overexpression of JHD2-LexA led to a 2-fold increase or decrease in H3K4me3 at the promoter or ORF regions of PMA1, respectively (Fig. 1B). This result suggests that Jhd2 might be present on constitutively and highly expressed genes to maintain their normal H3K4 methylation levels. Indeed, ChIP data showed that Jhd2-LexA was present on the promoter and ORF regions of the PMA1 gene (Fig. 1C). Interestingly, the distribution pattern of Jhd2-LexA across the PMA1 gene was similar to that seen for H3K4me3 (Fig. 1, compare B and C). Similar results were obtained from ChIP assays of HSP104 (see Fig. 8B), which is expressed at very low levels under normal conditions (32Sanchez Y. Lindquist S.L. Science. 1990; 248: 1112-1115Crossref PubMed Scopus (659) Google Scholar). Given that H3K4me3 is the substrate for Jhd2, these data suggest that Jhd2" @default.
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• W2027578670 title "The JmjN Domain of Jhd2 Is Important for Its Protein Stability, and the Plant Homeodomain (PHD) Finger Mediates Its Chromatin Association Independent of H3K4 Methylation" @default.
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