title: Identification of RNF168 as a PML nuclear body regulator.
abstract: Promyelocytic leukemia (PML) protein forms the basis of PML nuclear bodies (PML NBs), which control many important processes. We have screened an shRNA library targeting ubiquitin pathway proteins for effects on PML NBs, and identified RNF8 and RNF168 DNA-damage response proteins as negative regulators of PML NBs. Additional studies confirmed that depletion of either RNF8 or RNF168 increased the levels of PML NBs and proteins, whereas overexpression induced loss of PML NBs. RNF168 partially localized to PML NBs through its UMI/MIU1 ubiquitin-interacting region and associated with NBs formed by any PML isoform. The association of RNF168 with PML NBs resulted in increased ubiquitylation and SUMO2 modification of PML. In addition, RNF168 was found to associate with proteins modified by SUMO2 and/or SUMO3 in a manner dependent on its ubiquitin-binding sequences, suggesting that hybrid SUMO-ubiquitin chains can be bound. In vitro assays confirmed that RNF168, preferentially, binds hybrid SUMO2-K63 ubiquitin chains compared with K63-ubiquitin chains or individual SUMO2. Our study identified previously unrecognized roles for RNF8 and RNF168 in the regulation of PML, and a so far unknown preference of RNF168 for hybrid SUMO-ubiquitin chains.
authors.affiliation: Department of Molecular Genetics, University of Toronto, 1 Kings College Circle, Toronto, Ontario, Canada M5S 1A8.
authors.forename: Kathy
authors.initials: K
authors.lastname: Shire
authors.affiliation: Department of Molecular Genetics, University of Toronto, 1 Kings College Circle, Toronto, Ontario, Canada M5S 1A8.
authors.forename: Andrew I
authors.initials: AI
authors.lastname: Wong
authors.affiliation: Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Tatham
authors.affiliation: Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee.
authors.forename: Oliver F
authors.initials: OF
authors.lastname: Anderson
authors.affiliation: Department of Molecular Genetics, University of Toronto, 1 Kings College Circle, Toronto, Ontario, Canada M5S 1A8.
authors.forename: David
authors.initials: D
authors.lastname: Ripsman
authors.affiliation: Department of Molecular Genetics, University of Toronto, 1 Kings College Circle, Toronto, Ontario, Canada M5S 1A8.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Gulstene
authors.affiliation: Department of Molecular Genetics, University of Toronto, 1 Kings College Circle, Toronto, Ontario, Canada M5S 1A8.
authors.forename: Jason
authors.initials: J
authors.lastname: Moffat
authors.affiliation: Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee.
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Hay
authors.affiliation: Department of Molecular Genetics, University of Toronto, 1 Kings College Circle, Toronto, Ontario, Canada M5S 1A8 lori.frappier@utoronto.ca.
authors.forename: Lori
authors.initials: L
authors.lastname: Frappier
mesh_terms: D045744:Cell Line, Tumor; D006801:Humans; D045586:Intranuclear Inclusion Bodies / Q000378:metabolism*; D007951:Leukemia, Myeloid / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D000071156:Promyelocytic Leukemia Protein; D011485:Protein Binding / Q000502:physiology; D020033:Protein Isoforms / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology
chemical_list: D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D020033:Protein Isoforms; D025841:Small Ubiquitin-Related Modifier Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; C070576:PML protein, human; C535210:RNF168 protein, human; D044767:Ubiquitin-Protein Ligases
keywords: PML nuclear bodies; RNF168; RNF8; SUMO