Analogue-Sensitive Inhibition of Histone Demethylases Uncovers Member-Specific Function in Ribosomal Protein Synthesis

Abstract

Lysine demethylases (KDMs) catalyze the oxidative removal of the methyl group from histones using earth-abundant iron and the metabolite 2-oxoglutarate (2OG). KDMs have emerged as master regulators of eukaryotic gene expression and are novel drug targets; small-molecule inhibitors of KDMs are in the clinical pipeline for the treatment of human cancer. Yet, mechanistic insights into the functional heterogeneity of human KDMs are limited, necessitating the development of chemical probes for precision targeting. Herein, we identify analogue-sensitive (as) mutants of the KDM4 subfamily to elucidate member-specific biological functions in a temporally defined manner. By replacing the highly conserved phenylalanine residue in the active site of KDM4 members with alanine, we develop mutants with intact catalytic activity and substrate specificity indistinguishable from those of the wild type congener. Unlike the wild type demethylases, mutants were sensitized toward cofactor-competitive N-oxalyl glycine (NOG) analogues carrying complementary steric appendage. Particularly notable is N-oxalyl leucine (NOL) which inhibited the KDM4 mutants reversibly with submicromolar efficacy. Cell-permeable NOL prodrugs inhibited as enzymes in cultured human cells to modulate lysine methylation on nucleosomal histones. Through conditional perturbation of the orthogonal enzymes, we uncover a KDM4A-specific role in ribosomal protein synthesis and map a remarkably dynamic signaling cascade involving locus-specific histone demethylation leading to fast rRNA expression, enhanced ribosome assembly, and protein synthesis. The results provide a mechanistic clue into KDM4A’s role in cancers that rely on heightened ribosomal activity to support uncontrolled cellular proliferation.