Methylation-directed glycosylation of chromatin factors represses retrotransposon promoters

Proc Natl Acad Sci U S A. 2020 Jun 23;117(25):14292-14298. doi: 10.1073/pnas.1912074117. Epub 2020 Jun 10.


The mechanisms by which methylated mammalian promoters are transcriptionally silenced even in the presence of all of the factors required for their expression have long been a major unresolved issue in the field of epigenetics. Repression requires the assembly of a methylation-dependent silencing complex that contains the TRIM28 protein (also known as KAP1 and TIF1β), a scaffolding protein without intrinsic repressive or DNA-binding properties. The identity of the key effector within this complex that represses transcription is unknown. We developed a methylation-sensitized interaction screen which revealed that TRIM28 was complexed with O-linked β-N-acetylglucosamine transferase (OGT) only in cells that had normal genomic methylation patterns. OGT is the only glycosyltransferase that modifies cytoplasmic and nuclear protein by transfer of N-acetylglucosamine (O-GlcNAc) to serine and threonine hydroxyls. Whole-genome analysis showed that O-glycosylated proteins and TRIM28 were specifically bound to promoters of active retrotransposons and to imprinting control regions, the two major regulatory sequences controlled by DNA methylation. Furthermore, genome-wide loss of DNA methylation caused a loss of O-GlcNAc from multiple transcriptional repressor proteins associated with TRIM28. A newly developed Cas9-based editing method for targeted removal of O-GlcNAc was directed against retrotransposon promoters. Local chromatin de-GlcNAcylation specifically reactivated the expression of the targeted retrotransposon family without loss of DNA methylation. These data revealed that O-linked glycosylation of chromatin factors is essential for the transcriptional repression of methylated retrotransposons.

Keywords: DNA methylation; gene silencing; protein O-glycosylation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / metabolism
  • Animals
  • Chromatin / metabolism*
  • DNA (Cytosine-5-)-Methyltransferase 1 / genetics
  • DNA (Cytosine-5-)-Methyltransferase 1 / metabolism
  • DNA Methylation
  • Epigenesis, Genetic
  • Gene Silencing
  • Glycosylation
  • Humans
  • Methylation
  • N-Acetylglucosaminyltransferases
  • Nuclear Proteins / metabolism
  • Promoter Regions, Genetic*
  • Protein Processing, Post-Translational
  • Proteomics
  • Repressor Proteins / metabolism
  • Retroelements / physiology*
  • Transcription Factors / metabolism
  • Tripartite Motif-Containing Protein 28 / metabolism*


  • Chromatin
  • Nuclear Proteins
  • Repressor Proteins
  • Retroelements
  • Transcription Factors
  • DNA (Cytosine-5-)-Methyltransferase 1
  • DNMT1 protein, human
  • Tripartite Motif-Containing Protein 28
  • N-Acetylglucosaminyltransferases
  • UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase
  • Acetylglucosamine