FIH hydroxylates G9a and GLP at asparagine residues. FIH hydroxylates G9a and GLP at asparagine residues. A, Interaction between endogenous FIH and G9a/GLP proteins. Proteins in HEK293 cell lysates were precipitated by anti-FIH antibody and the precipitates were immunoblotted using the indicated antibodies (n = 3). B, Interaction between ectopic FIH and G9a/GLP. HEK293 cells, which had been cotransfected with HA-FIH and Flag-G9a/GLP-HA plasmids, were subjected to immunoprecipitation and immunoblotting (n = 3). C, HEK293 cells were incubated under normoxia or hypoxia for 24 hours. Total lysates (T) were fractionated to cytosolic (C) and nuclear (N) components, which were immunoprecipitated and immunoblotted (n = 3). D, Schematic diagram of the F/S-tagged fragments of G9a (top). HA-FIH and one of three G9a fragments were coexpressed in HEK293 cells, and the cell lysates were subjected to coimmunoprecipitation with anti-FLAG (bottom left). HA-FIH and F/S-G9a fragments were purified from HEK293T cells using HA and FLAG affinity beads, and incubated together in test tubes. F/S-G9a fragments were pulled down using streptavidin beads and immunoblotted (bottom right; n = 3). E, Recombinant GST-G9a_ARD was reacted with recombinant GST-FIH and cofactors in a hydroxylation buffer. GST-G9a_ARD was electrophoresed on SDS-PAGE and digested in gel. LC/MS-MS spectra for identifying the unmodified G9a-N779 in sample [a] and the hydroxylated G9a-N779 in sample [b]. F,In vitro hydroxylation assay. Recombinant GST-G9a_ARD (left) or GST-GLP_ARD (right) peptides were reacted with recombinant GST-FIH in a hydroxylation buffer. 14CO2 molecules generated from the enzymatic reaction were captured and quantified using a scintillation counter (mean + SD, n = 3; *, P < 0.05; n.s., not significant). G, Putative FIH target motifs in the ankyrin repeat domains of G9a and GLP. The conserved amino acids among FIH-targeted proteins are highlighted. Jengmin Kang et al. Cancer Res 2018;78:1184-1199 ©2018 by American Association for Cancer Research