dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression

Nucleic Acids Res. 2017 Sep 29;45(17):9901-9916. doi: 10.1093/nar/gkx578.

Abstract

Distinct epigenomic profiles of histone marks have been associated with gene expression, but questions regarding the causal relationship remain. Here we investigated the activity of a broad collection of genomically targeted epigenetic regulators that could write epigenetic marks associated with a repressed chromatin state (G9A, SUV39H1, Krüppel-associated box (KRAB), DNMT3A as well as the first targetable versions of Ezh2 and Friend of GATA-1 (FOG1)). dCas9 fusions produced target gene repression over a range of 0- to 10-fold that varied by locus and cell type. dCpf1 fusions were unable to repress gene expression. The most persistent gene repression required the action of several effector domains; however, KRAB-dCas9 did not contribute to persistence in contrast to previous reports. A 'direct tethering' strategy attaching the Ezh2 methyltransferase enzyme to dCas9, as well as a 'recruitment' strategy attaching the N-terminal 45 residues of FOG1 to dCas9 to recruit the endogenous nucleosome remodeling and deacetylase complex, were both successful in targeted deposition of H3K27me3. Surprisingly, however, repression was not correlated with deposition of either H3K9me3 or H3K27me3. Our results suggest that so-called repressive histone modifications are not sufficient for gene repression. The easily programmable dCas9 toolkit allowed precise control of epigenetic information and dissection of the relationship between the epigenome and gene regulation.

MeSH terms

  • Chromatin / chemistry*
  • Chromatin / metabolism
  • DNA (Cytosine-5-)-Methyltransferases / genetics
  • DNA (Cytosine-5-)-Methyltransferases / metabolism
  • DNA Methyltransferase 3A
  • Endonucleases / genetics*
  • Endonucleases / metabolism
  • Epigenomics / methods*
  • Gene Editing
  • Gene Silencing*
  • HCT116 Cells
  • HEK293 Cells
  • Histocompatibility Antigens / genetics
  • Histocompatibility Antigens / metabolism
  • Histone-Lysine N-Methyltransferase / genetics
  • Histone-Lysine N-Methyltransferase / metabolism
  • Histones / genetics*
  • Histones / metabolism
  • Humans
  • Methylation
  • Methyltransferases / genetics
  • Methyltransferases / metabolism
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Promoter Regions, Genetic
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Receptor, ErbB-2 / genetics
  • Receptor, ErbB-2 / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism
  • Transcription Factors / genetics
  • Transcription Factors / metabolism

Substances

  • Chromatin
  • DNMT3A protein, human
  • Histocompatibility Antigens
  • Histones
  • Nuclear Proteins
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • Transcription Factors
  • ZFPM1 protein, human
  • ZNF350 protein, human
  • SUV39H1 protein, human
  • Methyltransferases
  • DNA (Cytosine-5-)-Methyltransferases
  • DNA Methyltransferase 3A
  • EHMT2 protein, human
  • Histone-Lysine N-Methyltransferase
  • ERBB2 protein, human
  • Receptor, ErbB-2
  • Endonucleases