Figure 3. H3K4me3 - A redox sensitive histone modification involved in stress gene expression and resistance.

(a) Heat shock gene transcripts in sorted subpopulations before and after heat shock. n = 3 independent sorting experiments; two-way ANOVA with Tukey correction. (b) Venn diagram of upregulated genes in L2^ox and ash-2 RNAi worms ¹⁰ (complete list in Supplementary Table 1). (c) Quantification of global H3K4me3 levels in L2^ox and L2^red worms. n = 7 independent sorting experiments; unpaired t-test, two-sided. (d) Quantification of H3K4me3 levels in HeLa cells before and after H₂O₂ treatment. n = 3 independent experiments; one-way ANOVA with Dunnett correction. (e) In vitro histone methyltransferase assays of core HMC members; ^ox: pre-treated with 1 mM (+) or 2 mM (++) H₂O₂ for 30 min prior to activity assay. Thiol-reducing agent dithiothreitol (DTT) was added after the H₂O₂ treatment. n = 3 independent experiments; one-way ANOVA with Sidak correction. (f) Reverse thiol trapping of oxidized and reduced MLL1_SET. A 500-Da mass increase per oxidized thiol can be detected on non-reducing SDS-PAGE. (g) Heat shock survival of C. elegans N2 wild-type, wdr-5, set-2, rbr-2 and set2/rbr-2 mutants after 48 hours (n = 3 independent experiments; one-way ANOVA with Dunnett correction) or N2jrIs2[Prpl-17::Grx1-roGFP2] worms treated with ash-2 or control RNAi after 24 hours (n = 5 independent experiments; unpaired t-test, two-sided. E.V. empty vector). (h) Heat shock survival of ASH2L-siRNA treated HeLa cells. n = 5 independent experiments; two-way ANOVA with Tukey correction. (i) Transcript levels of heat shock genes after 30 min heat stress treatment of ASH2L siRNA treated HeLa cells. N.T., non-targeting. n = 3 independent experiments; unpaired t-test, two-sided. Data in (a), (c), (d), (e) and (g-i) represent mean ± SEM. For blot and gel source images, see Supplementary Fig. 1–3.