title: Stable X chromosome inactivation involves the PRC1 Polycomb complex and requires histone MACROH2A1 and the CULLIN3/SPOP ubiquitin E3 ligase.
abstract: X inactivation involves the stable silencing of one of the two X chromosomes in XX female mammals. Initiation of this process occurs during early development and involves Xist (X-inactive-specific transcript) RNA coating and the recruitment of Polycomb repressive complex (PRC) 2 and PRC1 proteins. This recruitment results in an inactive state that is initially labile but is further locked in by epigenetic marks such as DNA methylation, histone hypoacetylation, and MACROH2A deposition. Here, we report that the E3 ubiquitin ligase consisting of SPOP and CULLIN3 is able to ubiquitinate the Polycomb group protein BMI1 and the variant histone MACROH2A. We find that in addition to MACROH2A, PRC1 is recruited to the inactivated X chromosome in somatic cells in a highly dynamic, cell cycle-regulated manner. Importantly, RNAi-mediated knock-down of CULLIN3 or SPOP results in loss of MACROH2A1 from the inactivated X chromosome (Xi), leading to reactivation of the Xi in the presence of inhibitors of DNA methylation and histone deacetylation. Likewise, Xi reactivation is also seen on MacroH2A1 RNAi under these conditions. Hence, we propose that the PRC1 complex is involved in the maintenance of X chromosome inactivation in somatic cells. We further demonstrate that MACROH2A1 deposition is regulated by the CULLIN3/SPOP ligase complex and is actively involved in stable X inactivation, likely through the formation of an additional layer of epigenetic silencing.
authors.affiliation: Division of Molecular Genetics, The Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands.
authors.forename: Inmaculada
authors.initials: I
authors.lastname: Hernández-Muñoz
authors.forename: Anders H
authors.initials: AH
authors.lastname: Lund
authors.forename: Petra
authors.initials: P
authors.lastname: van der Stoop
authors.forename: Erwin
authors.initials: E
authors.lastname: Boutsma
authors.forename: Inhua
authors.initials: I
authors.lastname: Muijrers
authors.forename: Els
authors.initials: E
authors.lastname: Verhoeven
authors.forename: Dmitri A
authors.initials: DA
authors.lastname: Nusinow
authors.forename: Barbara
authors.initials: B
authors.lastname: Panning
authors.forename: York
authors.initials: Y
authors.lastname: Marahrens
authors.forename: Maarten
authors.initials: M
authors.lastname: van Lohuizen
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D004303:Dosage Compensation, Genetic; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006657:Histones / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D017404:In Situ Hybridization, Fluorescence; D046912:Multiprotein Complexes / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D063150:Polycomb Repressive Complex 1; D063146:Polycomb-Group Proteins; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D034622:RNA Interference; D012097:Repressor Proteins / Q000378:metabolism*; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
chemical_list: C069077:BMI1 protein, human; D004268:DNA-Binding Proteins; D006657:Histones; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D063146:Polycomb-Group Proteins; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; C120756:macroH2A histone; D049452:Green Fluorescent Proteins; D063150:Polycomb Repressive Complex 1; C489841:RNF2 protein, human; D044767:Ubiquitin-Protein Ligases