title: Targeting of EZH2 to a defined genomic site is sufficient for recruitment of Dnmt3a but not de novo DNA methylation.
abstract: Polycomb-mediated gene silencing and DNA methylation underlie many epigenetic processes important in normal development as well as in cancer. An interaction between EZH2 of the Polycomb repressive complex 2 (PRC2), which trimethylates lysine 27 on Histone 3 (H3K27me3), and all three DNA methyltransferases (DNMTs) has been reported, implicating a role for PRC2 in directing DNA methylation. Interestingly, however, the majority of H3K27me3 marked genes lack DNA methylation in ES cells, indicating that EZH2 recruitment may not be sufficient to promote DNA methylation. Here, we employed a Gal4DBD/gal4UAS-based system to directly test if EZH2 binding at a defined genomic site is sufficient to promote de novo DNA methylation in a murine erythroleukaemia cell line. Targeting of a Gal4DBD-EZH2 fusion to an intergenic transgene bearing a gal4 binding-site array promoted localized recruitment of Suz12 and Bmi1, subunits of PRC2 and PRC1, respectively, and deposition of H3K27me3. Further analysis of the H3K27me3-marked site revealed the persistence of H3K4me2, a mark inversely correlated with DNA methylation. Strikingly, while Dnmt3a was also recruited in an EZH2-dependent manner, de novo DNA methylation of the transgene was not observed. Thus, while targeting of EZH2 to a specific genomic site is sufficient for recruitment of Dnmt3a, additional events are required for de novo DNA methylation.
authors.affiliation: Department of Medical Genetics, Life Sciences Institute, The University of British Columbia, Vancouver, BC, CA.
authors.forename: Margaret
authors.initials: M
authors.lastname: Rush
authors.forename: Ruth
authors.initials: R
authors.lastname: Appanah
authors.forename: Sandra
authors.initials: S
authors.lastname: Lee
authors.forename: Lucia L
authors.initials: LL
authors.lastname: Lam
authors.forename: Preeti
authors.initials: P
authors.lastname: Goyal
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Lorincz
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D000071221:Enhancer of Zeste Homolog 2 Protein; D044127:Epigenesis, Genetic; D016678:Genome; D011495:Histone-Lysine N-Methyltransferase / Q000032:analysis / Q000378:metabolism / Q000502:physiology*; D006657:Histones / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D051379:Mice; D063151:Polycomb Repressive Complex 2; D063146:Polycomb-Group Proteins; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism
chemical_list: C000715535:Dnmt3a protein, mouse; D006657:Histones; D063146:Polycomb-Group Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D000071221:Enhancer of Zeste Homolog 2 Protein; C498410:Ezh2 protein, mouse; D011495:Histone-Lysine N-Methyltransferase; D063151:Polycomb Repressive Complex 2; D008239:Lysine