Histone Demethylation by a Family of Jmjc Domain-Containing Proteins

Tsukada et al. 1

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 Figure Legends

Figure S1. Schematic illustration of the demethylation assay.

Figure S2. Schematic representation of the steps used in purifying the demethylase activity from HeLa cells. Numbers represent the salt concentrations (mM) at which the histone demethylase activity elutes from the column.

Figure S3. Comparison of the JHDM1 family of proteins. a. Diagrammatic representation of the JHDM1 family proteins in different organisms. Two highly related proteins were identified in human and mouse, but only one homolog was found in each of the other organisms. The various functional domains present in this family of proteins are shown based on analysis using the SMART program. b. Alignment of the JmjC domain of the JHDM1 family members with that of FIH1 (Q9NWT6) using the PAPIA system ( The accession numbers for the JHDM1A proteins are: NP_036440 (human), XP_355123 (mouse). JHDM1 proteins are AAH82636 (Xenopus), NP_649864 (Drosophila), AAN65291 (C. elegans), CAA21872 (S. pombe), and NP_010971 (S. cerevisiae). The JHDM1B proteins are NP_115979 (human) and NP_001003953 (mouse). The numbers represent the amino acid position in each protein. The amino acids in FIH1 that are involved in Fe(II) and -KG binding are indicated by “*” and “#”, respectively. The conserved tyrosine that is critical for Epe1 function is indicated by a “$”. Conservation of four out of seven sequences is highlighted with black background.

Figure S4. A Coomassie stained protein gel containing recombinant Flag-JHDM1A purified from Sf9 cells. Quantification was achieved by comparison with known amount of BSA.

Figure S5. Analysis of substrate specificity of recombinant Flag-JHDM1A purified from Sf9 cells. Equal amounts of nucleosome substrates were subjected to histone demethylase assays in the presence or absence of Flag-JHDM1A. Changes in histone methylation levels were analyzed by site-specific methyl-lysine or methyl-arginine antibodies. Western blotting with antibodies against histone H3 or H4 was used to demonstrate equal loading.

Figure S6. Flag-JHDM1A demethylates an H3 dimethyl-K36 peptide (STGGV2mKKPHRY-C) in a concentration and time dependent manner. a. The percentage of di-, mono-, and un-methyl peptides at different times (0, 5, 15, 60, 180 min), with different enzyme concentrations (enzyme:substrate= 1:80, 1:40, or 1:20) are indicated in the table. Samples taken at different times were subject to mass spectrometry analysis to determine the methylation states. b. Graphic representation of the total percentage of released methyl groups in relation to the reaction time. The graph was generated using the data presented in panel a.

Figure S7. Over-expression of Flag-JHDM1A in 293T cells does not change the levels of monomethyl-K36, trimethyl-K36, or dimethyl-K4 forms of histone H3.

Figure S8. Generation of succinate is stimulated by the presence of methylated substrate peptide. Succinate generated in the different reactions were separated by liquid chromatography and quantified by mass spectrometry. The relative amount of succinate in the different reaction is presented. The reaction conditions and detection of succinate were described in Supplementary Methods. The presence (+) or absence (-) of enzyme or substrate is indicated.

Figure S9. Epe1 lacks histone demethylase activity. a, Western blotting of recombinant Flag-Epe1 and Flag-JHDM1A purified from Sf9 cells. About 0.2 g of Flag-Epe1 and Flag-JHDM1A, quantified by western blotting, were used for histone demethylase assay presented in panel b. b, Recombinant Flag-Epe1 shown in panel a was used to demethylate various methylated histone substrates in vitro. The histone methyltransferases and their sites of methylation are indicated on top of the panel. The demethylase assays were performed with equal inputs, either by counts or by histone amounts, with similar results. Recombinant Flag-JHDM1A was used as a positive control for the assay.