Extended Data Fig. 4: KDM3A catalysed hydroxylation of N^ε-acetyl-lysine 9 or demethylation of N^ε-dimethyl-lysine 9 on histone H3 peptides in the absence of an assay component, or in the presence of the inhibitor IOX1.

KDM3A_CD reactions of H3(1-21)K9ac (10 µM) or H3(1-21)K9me₂ (10 µM) under standard conditions, but with the absence of one component. a,g, standard conditions, b,h, in the absence of l-ascorbate, c,i, in the absence of Fe(II), d,j, in the absence of 2OG, e,k, in the absence of TCEP, and f,l in the absence of KDM3A_CD. Representative MALDI-TOF MS spectra are shown for each peptide (n: 3 independent assays). m KDM3A_CD catalysed demethylation of H3(1-21)K9me2 in the absence of one assay component. The bar graph shows the relative abundance of substrate and demethylated products (mean ± SD, n = 3 independent assays). Conditions: l-ascorbate (500 µM), Fe(II) (50 µM), 2OG (100 µM), and TCEP (500 µM) were reacted for (60 min, 37 °C). Black t: 0 min, red t: 60 min. n, KDM3A_CD (0.5 µM) was pre-incubated (15 min) with DMSO or IOX1 (varied concentrations) before addition of H3(1-35)K9ac (5 µM), l-ascorbate (500 µM), Fe(II) (50 µM), 2OG (100 µM) and TCEP (500 µM). The reaction was quenched after 30 min (RT), then analysed by LC-MS. Hydroxylation activity is plotted relative to DMSO (%). Mean, n = 2 (IOX1) or 6 (DMSO) independent assays.

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