Tsukada et al. 3
Histone Demethylation by A Family of JmjC Domain-containing Proteins
Yu-ichi Tsukada, Jia Fang, Hediye Erdjument-Bromage,
Maria E. Warren, Christoph H. Borchers, Paul Tempst, and Yi Zhang
Supplementary Methods
Purification of the H3-K36 demethylase activity from HeLa cells
Separation of HeLa S3 nuclear proteins into nuclear extract and nuclear pellet, followed by solubilization of nuclear pellet proteins, fractionation on DEAE52, and P11 columns were performed as described 1. The P11 fraction, which eluted with BC300 [40 mM HEPES-KOH (pH 7.9), 0.2 mM EDTA, 1 mM DTT, 0.2 mM PMSF, and 10% glycerol, 300 mM KCl] was dialyzed into buffer D [40 mM HEPES-KOH (pH 7.9), 0.2 mM EDTA, 1 mM DTT, 0.2 mM PMSF, and 10% glycerol] containing 20 mM ammonium sulfate (BD20) and loaded to a 45 ml DE5PW column (TosoHaas). The bound proteins were eluted with a 12 column volume (cv) liner gradient from BD50 to BD500. The fractions containing the demethylase activity, which eluted between 140-185 mM ammonium sulfate, were combined and adjusted to 700 mM ammonium sulfate before they were loaded onto a 22 ml Phenyl Sepharose column (Pharmacia). The bound proteins were eluted with 8 cv linear gradient from BD700 to BD0. The active fractions, which eluted from the column between 450-360 mM ammonium sulfate, were pooled and concentrated to 0.5 ml before loading onto a 24 ml Superose 6 gel filtration column (Pharmacia). The Superose 6 column was eluted with BC400 (buffer C with 400 mM KCl). The active fractions, which eluted between 240-320 kDa, were then combined and adjusted to 200 mM KCl with BC50 before being loaded onto a 0.1 ml mini-MonoQ column (Pharmacia). The bound proteins were eluted with 20 cv linear gradient from BC200 to BC500. The active fractions eluted from the column between 315-345 mM KCl. The proteins in the active fractions were combined and resolved in a 6.5-15% gradient SDS-PAGE. After Coomassie staining, candidate polypeptides were excised for protein identification.
Purification of HMTs and Flag-JHDM1, Flag-Epe1
GST and CBP fusion HMTs were expressed in E. coli and purified on glutathione-immobilized agarose beads (Sigma), or calmodulin affinity resin (Stratagene) using standard protocols. Purification of the EZH2 complex was carried out as described 2. Generation of baculovirus expressing Flag-JHDM1A, Flag-Epe1 and purification of Flag-JHDM1A and Flag-Epe1 from infected Sf9 cells were performed as described 2. For purification of wild-type and mutant Flag-JHDM1A from COS-7 cells, plasmids were transfected into the cells with FuGENE 6 following the manufacture’s instruction. Two days after transfection, cells were washed with phosphate-buffered saline (PBS) and lysed with lysis buffer (20 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 100 mM NaCl, 1 mM Na3VO4, 10 mM NaF, 20 mM b-Glycerophosphate, 1 mM EGTA, and 0.5% NP-40) containing protease inhibitor cocktail (Roche) and 1 mM phenylmetyl sulfonate fluoride. The cell lysates were incubated with M2 a-Flag agarose (Sigma) for 3 h at 40C. After spinning down, the beads were washed with lysis buffer once and with BC50 without EDTA twice. The immunoprecipitated proteins were used for demethylase assay and Western blotting.
Protein identification and mass spectrometry
For protein identification, the candidate polypeptides were digested with trypsin and the proteins were identified as previously described 3. For peptide substrate analysis, an aliquot (1ml) of the demethylation reaction mixture was diluted 100-fold with 0.1 % formic acid, and loaded onto a 2-µl bed volume of Poros 50 R2 (PerSeptive Biosystems, Framingham, MA, USA) reversed-phase beads packed into an Eppendorf gel-loading tip. The peptides were eluted with 5 µl of 30% acetonitrile/0.1% formic acid. A fraction (0.5 ml) of this peptide pool was analyzed by matrix-assisted laser-desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS), using a BRUKER UltraFlex TOF/TOF instrument (Bruker Daltonics; Bremen, Germany), as described 4. For detection of formaldehyde and succinate, the reaction mixture was diluted 1:10 with aqueous 0.1% triflouro acetic acid and directly analyzed by nano-electrospray mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS) using an Applied Biosystems QSTAR quadrupole time-of-flight instrument. A 5-minute acquisition time and Proxeon (Odense, Denmark) nanospray needles were used, under the conditions previously described 5. For optimum sensitivity, only the masses of protonated formaldehyde and succinate were selected by the quadrupole and analyzed by the time-of-flight analyzer (selected ion monitoring). The fragmentation analysis of succinate by ESI-MS/MS was performed as previously described 5. LC-MS/MS on succinate was performed with an UPLC (Acquity, Waters) connected to a TSQ Quantum triple quadrupole (Thermo-Finnigan, San Jose, Ca) using a AcquityTMUPLC BEH C18 column (1 ´ 50 mm). Succinate was eluted with a linear gradient of 100% water/0.1% acetic acid to 50% acetonitrile/water/0.1% acetic acid in 2 min at flow rate of 200 µl/min. Succinate was detected in ESI negative ion SRM mode by monitoring the transition of m/z 117 to m/z 73. Five ml fractions of the reaction mixture were injected.
Immunostaining
For immunostaining, 293T cells were plated on glass coverslips and cultured for 1 day. After washing with PBS, cells were fixed in 4% paraformaldehyde for 10 min. The cells were then washed once with cold PBS and permeabilized for 5 min with cold PBS containing 0.2% Triton X-100. Permeabilized cells were then washed three times with blocking buffer (1% bovine serum albumin in PBS) and blocked for 30 min before being incubated with primary antibodies for 1 h in a humidified chamber. After three consecutive 5-min washes with PBS, cells were incubated with secondary antibodies for 1 hour before being washed with PBS and stained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) in PBS. Cells were washed again twice with PBS and then mounted in fluorescent mounting medium (Dako) before viewing under a fluorescence microscope.
Constructs and antibodies
Plasmids encoding GST-SET7, GST-hDOT1L (1-416), GST-PRMT1 and components of the EZH2 complex have been previously described 1, 2, 6, 7. Plasmids encoding GST-G9a (621-1000), CBP-Set2-Flag (S. pombe), and GST-Suv4-20h1 were kindly provided by Drs. Shinkai, Strahl, and Jenuwein, respectively. A plasmid encoding Flag-JHDM1A was constructed by PCR amplification from an EST clone (IMAGE 5534384), and was inserted into NotI and XbaI sites of an N-terminal Flag-tagged pcDNA3 vector or an N-terminal Flag-tagged pFASTBAC vector. The pcDNA3-Flag-JHDM1A (H212A), the deletions of the JmjC (148-316 aa), zf-CXXC (563-609 aa), PHD (619-676 aa), FBOX (893-933 aa), and LRRs (1000-1118 aa) were generated by two-step PCR. Plasmids encoding GST-scJHDM1 (S. cerevisiae) were constructed by PCR amplification of S. cerevisiae genomic DNA. The GST-scJHDM1(H305A) mutant was generated by two-step PCR. Epe1 was amplified by PCR from S. pombe genomic DNA and cloned into NotI and SphI sites of N-terminal Flag-tagged pFASTBAC vector for baculovirus production. All the constructs generated through PCR were verified by sequencing.
Sources of the antibodies used are as follows: H3 monomethyl-K4 (Abcam 8895), H3 trimethyl-K4 (Abcam 8580), H3 monomethyl-K9 (Abcam 9045), H3 dimethyl-K9 (Upstate 05-768), H3 monomethyl-K27 (Upstate 07-448), H3 dimethy-K27 (Upstate 07-452), H3 trimethyl-K27 H3 monomethyl-K79 (Abcam 2886), H3 trimethyl-K79 (Abcam 2621), H3 monomethyl-K36 (Abcam 9048), H3 trimethyl-K36 (Abcam 9050), H3 dimethyl R17 (Abcam 8284), H4 monomethyl-K20 (Upstate 07-440), H4 trimethyl-K20 (Upstate 07-463), and H4 dimethyl-R3 (Abcam 5823). The antibody against H3 dimethyl-K36 was generated in rabbits by injection of a synthetic peptide (STGGVKKPHRY-C), in which K36 (underlined) was dimethylated. Antibodies against H3 trimethyl-K9, H3 trimethyl-K27, H3 dimethyl-K79, H3 dimethyl-K4 and H4 dimethyl-K20 have been previously described 8-10. Antibodies against H3 and H4 were kindly provided by Dr. Verreault. The antibody against Flag and secondary antibodies for immunofluorescence were purchased from Sigma and Jackson ImmunoResearch Laboratories, respectively.