Extended Data Fig. 5: Redox sensitivity of in vivo H3K4m3e3 levels and in vitro histone methyltransferase complex activity. | Nature

Extended Data Fig. 5: Redox sensitivity of in vivo H3K4m3e3 levels and in vitro histone methyltransferase complex activity.

From: Developmental ROS individualizes organismal stress resistance and lifespan

Extended Data Fig. 5

a, Global H3K4me3 levels in the sorted L2ox and L2red worms. A representative western blot using antibodies against H3K4me3 is shown. b, c, Quantification of global H3K27ac (b) and H3K27me3 (c) levels by western blot. n = 3 independent sorting experiments. P = 0.3793 (b) and P = 0.0905 (c); unpaired two-sided t-test. Data represent mean ± s.e.m. df, Global H3K4me3 (d), ASH2L (e) and MLL1 (f) levels in HeLa cells before and after H2O2 treatment, as assessed by western blot. g, Time course of the in vitro methyltransferase reaction for core COMPASS subunits (SET domain of MLL1–WDR5–ASH2L–RBBP5). Reaction rates were derived from the first 20 min of the linear range. hj, In vitro histone methyltransferase assays of core COMPASS subunits, consisting of purified GST–WDR5 (WDR5), GST–ASH2L (ASH2L), GST–RBBP5 (RBBP5) and either GST–MLL1 SET domain or untagged MLL1 SET domain (h), GST–SET1A SET domain (i) or GST–SET1B SET domain (j). Superscript ox indicates that the protein was pre-treated with either 1 mM (+) or 2 mM (++) H2O2 for 30 min before the activity assay. DTT was added after the H2O2 treatment. n = 3 independent experiments; one-way ANOVA with Sidak correction. Data are mean ± s.e.m. k, The MLL1 SET domain was treated with either 2 mM DTT, 2 mM H2O2 or 2 mM H2O2, followed by 4 mM DTT. Catalase was used to quench the H2O2. The proteins were denatured and thiols were modified with NEM before loading onto non-reducing SDS–PAGE to prevent non-specific thiol oxidation. The proteins were visualized by silver staining. M, marker. l, MLL1 SET domain treated with either 2 mM DTT, 2 mM H2O2 or 2 mM H2O2, followed by 4 mM DTT. All reduced protein thiols were then labelled with the 500-Da thiol-reactive compound AMS, causing a 500-Da mass decrease per oxidized thiol—detectable on reducing SDS–PAGE. M, protein marker. m, Cysteine oxidation state in MLL1 SET domain after treatment with either 2 mM DTT or 2 mM H2O2 followed by NEM labelling as assessed by LC–MS/MS. The peptide containing Cys3967 could not be detected. n, Schematic representation of the redox sensitivity of the MLL1 SET domain. For blot and gel source images, see Supplementary Figs. 1 and 3. o, Sequence alignment of the SET domain. All cysteines in MLL1 are shown in bold, and the five absolutely conserved cysteines are highlighted in yellow. Cysteines shown to be involved in zinc coordination are marked with an asterisk. NCBI protein BLAST and Clustal Omega Multiple Sequence Alignment, Clustal O (1.2.4) were used.

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