Fig. 3: KDM4E-catalysed hydroxylation occurs at Arg-20 on histone H2a peptide.

a LC–MS/MS analysis of H2a(1–20) after KDM4E treatment. Two potential hydroxylation sites, R17 and R20, were identified, with the latter more likely (Supplementary Fig. 9). b Peptide variant studies support Arg-20 hydroxylation; specific activities: H2a(1–20), 0.65 nmol min⁻¹ mg⁻¹ SD: 0.073; H2a(1–20) with a C-terminal carboxylic acid (indicated with an -OH), no activity; H2a(1–20)R20A, no activity; H2a(1–20)R20K*, no activity; H2a(1–20)R20Kme3*, 0.44 nmol min⁻¹ mg⁻¹ SD: 0.04; H2a(1–20)R3me2a*, 0.51 nmol min⁻¹ mg⁻¹ SD: 0.05; H2a(1–20)R3A, 0.37 nmol min⁻¹ mg⁻¹ SD: 0.002; H2a(1–21)*, 0.65 nmol min⁻¹ mg⁻¹ SD 0.02; H2a(1–25, 0.35 nmol min⁻¹ mg⁻¹ SD: 0.044; and H2a(10–29), 0.02 nmol min⁻¹ mg⁻¹ SD: 0.013. Measurements employed MALDI–TOF MS except when labelled with*, which employed LC–MS (Supplementary Fig. 10 for representative MS data). n = 3 independent assays. c KDM, RDM and hydroxylation (+16 Da) activities of KDM4E are sequence and context dependent. Histone peptides (H2a, H3, H4) and variants (wild type residue in red, replaced with either K, Kme3, R or Rme2a) were incubated with KDM4E and analysed on MALDI–TOF MS. KDM, RDM and hydroxylation (+16 Da) were detected in some but not all peptides (Supplementary Fig. 10 for representative MS data). (−) No mass changes observed. n = 3 independent assays. See Supplementary Table 6 for conditions.