Net155v001mc

HMT assay was performed according to a previously described method with minor modifications^{29 (link)}. Briefly, FLAG IP eluates of FLAG-tagged MLL3/4 PHD and SET fusion proteins were mixed with 0.5 μg of substrates (wild type or mutated recombinant nucleosomes) and 2.5 μCi of [³H]-labeled S-adenosyl-L-methionine ([³H]-SAM, PerkinElmer, NET155V001MC). All reactions were performed in HMT buffer with a final volume of 20 μL. After incubation at 30 °C for 16 h, the reactions were terminated by adding 2xSDS sample buffer and were subjected to SDS-PAGE. The signals for HMT activity were detected by autoradiography. The input of the MLL3/4 proteins and nucleosomes were detected by mouse monoclonal anti-FLAG M2 (Sigma-Aldrich, F3165, 1:1000) and rabbit polyclonal anti-H3 antibody (Abcam, ab1791, 1:1000), respectively. All the uncropped scans of these western blot were shown in Supplementary Figs. 10–12.

Approximately 1 μg of recombinant GST-SET8 (in 50% glycerol) and 2 μg of purified tubulin (MP Biomedicals, 08771151), recombinant MBP-α-tubulin, or recombinant MBP-β-tubulin were incubated with 6 μm radioactively labeled [³H]AdoMet (PerkinElmer Life Sciences, NET155V001MC) in 5 mm Tris (pH 8.0) and 0.5 mm DTT at room temperature overnight. As indicated, histone H4 (New England Biolabs, M2504S), recombinant His-LSF protein, or FQI1 inhibitor was added to the reaction. Samples were separated by electrophoresis through a 10% N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine gel (Invitrogen), and the gel was stained with Coomassie Brilliant Blue (shown in grayscale in the figures) and incubated with EN3HANCE (PerkinElmer Life Sciences) solution. The gel was dried and exposed to autoradiography film for 1 week. For the peptide assays, the specific peptides of α-tubulin were synthesized from AnaSpec Inc. Sequences are listed in Table S1. 2 μg of each peptide and 2 μg of purified WT or mutant SET8 or SETD2 (aa 1392–2564, Active Motif) were incubated with radioactively labeled [³H]AdoMet at room temperature overnight. Samples were spotted onto P81 filters (Whatman, 3698325), and the filters were washed three times with 0.3 m ammonium bicarbonate. The level of incorporated [³H]CH₃ was determined using liquid scintillation counting.

[³H]S-Adenosylmethionine ([³H]AdoMet), containing the ³H radiolabel on the labile methyl group, was obtained from PerkinElmer Life Sciences (catalog number NET155V001MC, Specific activity range 12–18 Ci/mmol). S-adenosylmethionine (AdoMet) was obtained from AK Scientific (Union City, CA). Biotinylated peptide substrates were obtained from Peptide2.0 (Chantilly, VA) or Tufts University Peptide Synthesis Core Facility (Boston, MA). All other chemicals were obtained from Sigma or LifeTech and were reagent grade or higher. Peptide sequences were as follows: H3(1–25), ARTKQTARKSTGGKAPRKQLATKAA-GK-biotin; H3K4A, ARTAQTARKSTGGKAPRKQLATKAA-GK-biotin; H3K4me1, ART(Kme1)QTARKSTGGKAPRKQLATKAA-GK-biotin; H3K4me2, ART(Kme2)QTARKSTGGKAPRKQLATKAA-GK-biotin; H3(21–44), ATKAARKSAPATGGVKKPHRYRPG-GK-biotin; H3K36me1, ATKAARKSAPATGGV(Kme1)KPHRYRPG-GK-biotin; H3K36me2, ATKAARKSAPATGGV(Kme2)KPHRYRPG-GK-biotin.