2. Academic Validation
  3. Two DOT1 enzymes cooperatively mediate efficient ubiquitin-independent histone H3 lysine 76 tri-methylation in kinetoplastids

Two DOT1 enzymes cooperatively mediate efficient ubiquitin-independent histone H3 lysine 76 tri-methylation in kinetoplastids

  • Nat Commun. 2024 Mar 19;15(1):2467. doi: 10.1038/s41467-024-46637-6.
Victoria S Frisbie # 1,  Hideharu Hashimoto # 1,  Yixuan Xie 2 3,  Francisca N De Luna Vitorino 2,  Josue Baeza 3,  Tam Nguyen 1,  Zhangerjiao Yuan 1,  Janna Kiselar 4,  Benjamin A Garcia 2 3,  Erik W Debler 5
Affiliations

Affiliations

  • 1 Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA.
  • 2 Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, USA.
  • 3 Epigenetics Institute, Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.
  • 4 Case Center for Proteomics and Bioinformatics, Department of Nutrition, Case Western Reserve University, School of Medicine, Cleveland, OH, USA.
  • 5 Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA. [email protected].
  • # Contributed equally.
PMID: 38503750 DOI: 10.1038/s41467-024-46637-6
Abstract

In higher eukaryotes, a single DOT1 histone H3 lysine 79 (H3K79) methyltransferase processively produces H3K79me2/me3 through histone H2B mono-ubiquitin interaction, while the kinetoplastid Trypanosoma brucei di-methyltransferase DOT1A and tri-methyltransferase DOT1B efficiently methylate the homologous H3K76 without H2B mono-ubiquitination. Based on structural and biochemical analyses of DOT1A, we identify key residues in the methyltransferase motifs VI and X for efficient ubiquitin-independent H3K76 methylation in kinetoplastids. Substitution of a basic to an acidic residue within motif VI (Gx6K) is essential to stabilize the DOT1A enzyme-substrate complex, while substitution of the motif X sequence VYGE by CAKS renders a rigid active-site loop flexible, implying a distinct mechanism of substrate recognition. We further reveal distinct methylation kinetics and substrate preferences of DOT1A (H3K76me0) and DOT1B (DOT1A products H3K76me1/me2) in vitro, determined by a Ser and Ala residue within motif IV, respectively, enabling DOT1A and DOT1B to mediate efficient H3K76 tri-methylation non-processively but cooperatively, and suggesting why kinetoplastids have evolved two DOT1 Enzymes.

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