E3-Independent Constitutive Monoubiquitination Complements Histone Methyltransferase Activity of SETDB1

Mol Cell. 2016 Jun 16;62(6):958-966. doi: 10.1016/j.molcel.2016.04.022. Epub 2016 May 26.


Ubiquitination typically occurs through the sequential action of three enzymes catalyzing ubiquitin activation (E1), conjugation (E2), and ligation (E3) and regulates diverse eukaryotic cellular processes. Although monoubiquitination commonly confers nondegradative activities, mechanisms underlying its temporal and spatial regulation and functional plasticity still remain largely unknown. Here we demonstrate that SETDB1, a major histone H3K9 methyltransferase is monoubiquitinated at the evolutionarily conserved lysine-867 in its SET-Insertion domain. This ubiquitination is directly catalyzed by UBE2E family of E2 enzymes in an E3-independent manner while the conjugated-ubiquitin (Ub) is protected from active deubiquitination. The resulting constitutive lysine-867 monoubiquitination is essential for SETDB1's enzymatic activity and endogenous retrovirus silencing in murine embryonic stem cells. Furthermore, the canonical hydrophobic patch on the conjugated-Ub is critical for Ub protection and function. Together, our findings highlight an E3-independent mechanism for monoubiquitination and reveal mechanistic details of SETDB1's enzymatic activity and the functional significance of its SET-Insertion.

MeSH terms

  • Amino Acid Motifs
  • Animals
  • CRISPR-Cas Systems
  • Catalysis
  • DNA Methylation*
  • Embryonic Stem Cells / enzymology*
  • Endogenous Retroviruses / genetics
  • Gene Silencing
  • HEK293 Cells
  • HeLa Cells
  • Histone-Lysine N-Methyltransferase / chemistry
  • Histone-Lysine N-Methyltransferase / genetics
  • Histone-Lysine N-Methyltransferase / metabolism*
  • Histones / metabolism*
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Lysine
  • MCF-7 Cells
  • Mice
  • Protein Conformation
  • Protein Methyltransferases / chemistry
  • Protein Methyltransferases / genetics
  • Protein Methyltransferases / metabolism*
  • Structure-Activity Relationship
  • Transfection
  • Ubiquitin-Conjugating Enzymes / chemistry
  • Ubiquitin-Conjugating Enzymes / genetics
  • Ubiquitin-Conjugating Enzymes / metabolism*
  • Ubiquitination*


  • Histones
  • Protein Methyltransferases
  • Histone-Lysine N-Methyltransferase
  • SETDB1 protein, human
  • SETDB1 protein, mouse
  • Ubiquitin-Conjugating Enzymes
  • Lysine