Biochem. J. (2014) 461, 323–334 (Printed in Great Britain) doi:10.1042/BJ20140374 Characterization of the histone methyltransferase PRDM9 using biochemical, biophysical and chemical biology techniques Xiaoying KOH-STENTA*, Joma JOY*, Anders POULSEN*, Rong LI*, Yvonne TAN*, Yoonjung SHIM†, Jung-Hyun MIN†, Liling WU‡, Anna NGO*, Jianhe PENG*, Wei Guang SEETOH*, Jing CAO*, John Liang Kuan WEE*, Perlyn Zekui KWEK*, Alvin HUNG*, Umayal LAKSHMANAN*, Horst FLOTOW*, Ernesto GUCCIONE‡ and Jeffrey HILL*1 *Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore †Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, U.S.A. ‡Institute for Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore 138669, Singapore Table S1 Validation of assay performance and robustness in a high-throughput format A library of 1600 bioactive compounds was used to evaluate performance and robustness of the methyltransferase assay in a high-throughput format. RLU (relative luminescence units) data reported are based on the average + − S.D. for 32 control wells. Plate ID DMSO controls (RLU)* Positive inhibition controls (RLU)† Signal-to-noise ratio Z ’ factor Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 335 097 + − 25 869 318 569 + − 19 257 327 784 + − 19 320 329 879 + − 15 520 326 747 + − 16 712 34 803 + − 2866 30 585 + − 3142 30 871 + − 3453 33 862 + − 25 576 29 781 + − 3357 9.6 10.4 10.6 9.7 11.0 0.713 0.767 0.770 0.583 0.797 Biochemical Journal *Reaction mixtures consisted of 180 nM PRDM9, 2.5 μM H3 peptide 1–21 and 4 μM SAM in buffer containing DMSO. † Reactions were inhibited by 1 mM suramin. www.biochemj.org SUPPLEMENTARY ONLINE DATA 1 To whom correspondence should be addressed (email [email protected]). c The Authors Journal compilation c 2014 Biochemical Society X. Koh-Stenta and others Figure S1 Analysis of purified recombinant enzyme and histone octamer Purified wild-type and mutant recombinant PRDM9 protein were analysed for accurate molecular mass and purity by (A) SDS/PAGE and (B) ESI–TOF MS. Similarly, recombinant histone octamer was purified under native conditions and analysed by (C) SDS/PAGE and (D) ESI–TOF MS to confirm the molecular mass of each of the four histone core units. Molecular masses are indicated in Da. c The Authors Journal compilation c 2014 Biochemical Society Characterization of the histone methyltransferase PRDM9 Figure S2 Biochemical characterization of PRDM9-C321P activity (A) Comparison of biochemical assay signals for wild-type and mutant PRDM9 enzymes at 3 μM H3 peptide 1–21 and 8 μM SAM. (B) Dose–response for mutant PRDM9 was extended to cover higher enzyme concentrations. Mutant PRDM9 activity was characterized as shown in (C) H3 peptide titration and (D) SAM titration experiments, performed at 5 μM PRDM9-C321P. RLU, relative luminescence unit(s). c The Authors Journal compilation c 2014 Biochemical Society X. Koh-Stenta and others Figure S3 Melting curves for (A) wild-type and (B) mutant PRDM9 show differential responses to SAM binding in a DSF assay c The Authors Journal compilation c 2014 Biochemical Society Characterization of the histone methyltransferase PRDM9 Figure S4 Recombinant histone octamer substrate modification was characterised by ESI–TOF MS for methylation of each core histone unit Samples were incubated with reaction times of (A) 1 h, (B) 2 h and (C) 3 h. c The Authors Journal compilation c 2014 Biochemical Society X. Koh-Stenta and others Figure S5 Maximum observed methylation events were characterized by ESI–TOF MS for individual recombinant whole histones (A) H2A, (B) H2B, (C) H3 and (D) H4 c The Authors Journal compilation c 2014 Biochemical Society Characterization of the histone methyltransferase PRDM9 Figure S6 MALDI–TOF MS and MALDI–TOF/TOF MS analysis of mono-, di- and tri-methylation of H3 peptide 1–21 (A) MALDI–TOF MS spectrum shows methylation states of the peptide. (B) MALDI–TOF/TOF MS of the monomethylated peptide (precursor ion of m /z 2268.4) identifies the methylation site as H3K4. (C and D) Similar MALDI–TOF/TOF MS analyses show the dimethylated (precursor ion of m /z 2282.4) and trimethylated H3K4 forms of the peptide. The quaternary amine at the trimethylated H3K4 carries a positive charge, and hence increases the b ions remarkably. c The Authors Journal compilation c 2014 Biochemical Society X. Koh-Stenta and others Figure S7 MALDI–TOF/TOF MS analysis of hexa-methylated H3 peptide 1–21 (precursor ion of m /z 2338.5) The insert shows an enlargement of m /z 480–m /z 1210, in which the b and y ions provide evidence of H3K4 trimethylation, H3K9 dimethylation and H3K18 monomethylation. c The Authors Journal compilation c 2014 Biochemical Society Characterization of the histone methyltransferase PRDM9 Figure S8 Methylation of H4 peptide 1–36 analysed by ESI ETD MS/MS (A) ETD fragmentation spectrum of the unmodified peptide with precursor ion of m /z 478.81 (8 + ). (B) ETD fragmentation spectrum of the monomethylated peptide with precursor ion of m /z 480.55 (8 + ). The methylation site was identified as H4K20. c The Authors Journal compilation c 2014 Biochemical Society X. Koh-Stenta and others Figure S9 Chemical structures of (A) SAM, (B) SAH, (C) sinefungin and (D) suramin Received 24 March 2014/24 April 2014; accepted 2 May 2014 Published as BJ Immediate Publication 2 May 2014, doi:10.1042/BJ20140374 c The Authors Journal compilation c 2014 Biochemical Society