title: Retention of the Native Epigenome in Purified Mammalian Chromatin.
abstract: A protocol is presented for the isolation of native mammalian chromatin as fibers of 25-250 nucleosomes under conditions that preserve the natural epigenetic signature. The material is composed almost exclusively of histones and DNA and conforms to the structure expected by electron microscopy. All sequences probed for were retained, indicating that the material is representative of the majority of the genome. DNA methylation marks and histone marks resembled the patterns observed in vivo. Importantly, nucleosome positions also remained largely unchanged, except on CpG islands, where nucleosomes were found to be unstable. The technical challenges of reconstituting biochemical reactions with native mammalian chromatin are discussed.
authors.affiliation: Mechanisms of Transcription Laboratory, Clare Hall Laboratories, The Francis Crick Insitute, London Research Institute, South Mimms, United Kingdom.
authors.forename: Andreas H
authors.initials: AH
authors.lastname: Ehrensberger
authors.affiliation: DNA Editing Lab, Clare Hall Laboratories, Cancer Research UK, London Research Institute, South Mimms, United Kingdom; DNA Editing in Immunity and Epigenetics, IFOM-Fondazione Instituto FIRC di Oncologia Molecolare, Milano, Italy.
authors.forename: Don-Marc
authors.initials: DM
authors.lastname: Franchini
authors.affiliation: Bioinformatics and Biostatistics Group, The Francis Crick Insitute, London Research Institute, London, United Kingdom.
authors.forename: Philip
authors.initials: P
authors.lastname: East
authors.affiliation: Protein Purification Facility, The Francis Crick Insitute, London Research Institute, London, United Kingdom.
authors.forename: Roger
authors.initials: R
authors.lastname: George
authors.affiliation: Advanced Sequencing Facility, The Francis Crick Insitute, London Research Institute, London, United Kingdom.
authors.forename: Nik
authors.initials: N
authors.lastname: Matthews
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, United Kingdom.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Maslen
authors.affiliation: Mechanisms of Transcription Laboratory, Clare Hall Laboratories, The Francis Crick Insitute, London Research Institute, South Mimms, United Kingdom.
authors.forename: Jesper Q
authors.initials: JQ
authors.lastname: Svejstrup
mesh_terms: D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D018899:CpG Islands; D019175:DNA Methylation; D006657:Histones / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D051379:Mice; D008854:Microscopy, Electron; D009707:Nucleosomes / Q000737:chemistry / Q000378:metabolism; D051381:Rats
chemical_list: D002843:Chromatin; D006657:Histones; D009707:Nucleosomes