title: Visualizing the Spatial Relationship of the Genome with the Nuclear Envelope Using Fluorescence In Situ Hybridization.
abstract: The genome has a special relationship with the nuclear envelope in cells. Much of the genome is anchored at the nuclear periphery, tethered by chromatin binding proteins such nuclear lamins and other integral membrane proteins. Even though there are global assays such as DAM-ID or ChIP to assess what parts of the genome are associated with the nuclear envelope, it is also essential to be able to visualize regions of the genome in order to reveal their individual relationships with nuclear structures in single cells. This is executed by fluorescence in situ hybridization (FISH) in 2-dimensional flattened nuclei (2D-FISH) or 3-dimensionally preserved cells (3D-FISH) in combination with indirect immunofluorescence to reveal structural proteins. This chapter explains the protocols for 2D- and 3D-FISH in combination with indirect immunofluorescence and discusses options for image capture and analysis. Due to the nuclear envelope proteins being part of the non-extractable nucleoskeleton, we also describe how to prepare DNA halos through salt extraction and how they can be used to study genome behavior and association when combined with 2D-FISH.
authors.affiliation: Division of Biosciences, College of Life and Health Sciences, Brunel University London, Uxbridge, London, UB8 3PH, UK.
authors.forename: Craig S
authors.initials: CS
authors.lastname: Clements
authors.affiliation: Division of Biosciences, College of Life and Health Sciences, Brunel University London, Uxbridge, London, UB8 3PH, UK.
authors.forename: Ural
authors.initials: U
authors.lastname: Bikkul
authors.affiliation: Division of Biosciences, College of Life and Health Sciences, Brunel University London, Uxbridge, London, UB8 3PH, UK.
authors.forename: Mai Hassan
authors.initials: MH
authors.lastname: Ahmed
authors.affiliation: Division of Biosciences, College of Life and Health Sciences, Brunel University London, Uxbridge, London, UB8 3PH, UK.
authors.forename: Helen A
authors.initials: HA
authors.lastname: Foster
authors.affiliation: Division of Biosciences, College of Life and Health Sciences, Brunel University London, Uxbridge, London, UB8 3PH, UK.
authors.forename: Lauren S
authors.initials: LS
authors.lastname: Godwin
authors.affiliation: Division of Biosciences, College of Life and Health Sciences, Brunel University London, Uxbridge, London, UB8 3PH, UK. Joanna.bridger@brunel.ac.uk.
authors.forename: Joanna M
authors.initials: JM
authors.lastname: Bridger
mesh_terms: D015415:Biomarkers; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D015342:DNA Probes; D016678:Genome; D017404:In Situ Hybridization, Fluorescence; D019394:Ki-67 Antigen / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009685:Nuclear Envelope / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D002843:Chromatin; D015342:DNA Probes; D019394:Ki-67 Antigen
keywords: 2D-FISH; 3D-FISH; Chromosome territories; Fluorescence in situ hybridization; Gene positioning; Genome organization; Nuclear envelope; Nuclear lamins