Extended Data Fig. 4: H3P16oh regulates H3K4me3 and displays better affinity to KDM5APHD3.
From: Histone H3 proline 16 hydroxylation regulates mammalian gene expression
a, Immunoblots for lysates from indicated cells with DMOG or hypoxia(0.5% O2) treatment for 12 h. b, Immunoblots for 293 T cell lysates transfected with indicated plasmids followed with DMOG or hypoxia (1% O2) treatment for 12 h. c, Immunoprecipitation and immunoblots of lysates from MDA-MB-231 cells transduced with EGLN2 shCtrl or sh325 and sh327. d, Biotin-pull down assay between biotin beads or indicated biotinylated peptide with GST-KDM5APHD1. e, Titration curves and fitting curves of indicated H3 peptides titrated into His-KDM5APHD3. f, Left panel, NMR 1H-15N heteronuclear single quantum correlation (HSQC) spectrum of apo 15N-labeled PHD3 domain. Right panel, overlay of NMR 1H-15N HSQC spectra of 0.125 mM 15N-labeled KDM5APHD3 in the presence of 1.80 mM H3K4me3 (cyan) and 1.80 mM H3K4me3 P16oh (red). g, MS identification of peptides: HCD mass spectrum of 16 Da H3K4me3 P16oh peptide (Bottom panel) and H3K4me3 peptide (top panel). h, Enhanced binding of H3K4me3 P16oh peptides with KDM5APHD3 compared to H3K4me3 peptides. The MS of [M + 4H]5+ and [M + 4H]6+ ions were shown. Left, peptide mixture used as input for KDM5APHD3-binding experiment; right, KDM5APHD3-bound products. i, NMR 1H-15N heteronuclear multiple quantum coherence (HMQC) spectra overlays between apo KDM5APHD3 (black) and 0.1 mM KDM5APHD3 in the presence of 0.45 mM H3K4me3 (cyan) and H3K4me3 P16oh (red) peptides, pH=6. j, Zoomed-in region of the two peptide-bound KDM5APHD3 that highlights residues K53 and K54 with the most significant chemical shift differences between the two states.
