title: Identification and functional characterization of cytoplasmic determinants of plasmid DNA nuclear import.
abstract: Import of exogenous plasmid DNA (pDNA) into mammalian cell nuclei represents a key intracellular obstacle to efficient non-viral gene delivery. This includes access of the pDNA to the nuclei of non-dividing cells where the presence of an intact nuclear membrane is limiting for gene transfer. Here we identify, isolate, and characterize, cytoplasmic determinants of pDNA nuclear import into digitonin-permeabilized HeLa cells. Depletion of putative DNA-binding proteins, on the basis of their ability to bind immobilized pDNA, abolished pDNA nuclear import supporting the critical role of cytoplasmic factors in this process. Elution of pDNA-bound proteins, followed by two-dimensional sodium dodecyl polyacrylamide gel electrophoresis identified several candidate DNA shuttle proteins. We show that two of these, NM23-H2, a ubiquitous c-Myc transcription-activating nucleoside diphosphate kinase, and the core histone H2B can both reconstitute pDNA nuclear import. Further, we demonstrate a significant increase in gene transfer in non-dividing HeLa cells transiently transfected with pDNA containing binding sequences from two of the DNA shuttle proteins, NM23-H2 and the homeobox transcription factor Chx10. These data support the hypothesis that exogenous pDNA binds to cytoplasmic shuttle proteins and is then translocated to the nucleus using the minimal import machinery. Importantly, increasing the binding of pDNA to shuttle proteins by re-engineering reporter plasmids with shuttle binding sequences enhances gene transfer. Increasing the potential for exogenously added pDNA to bind intracellular transport cofactors may enhance the potency of non-viral gene transfer.
authors.affiliation: Department of Gene Therapy, National Heart & Lung Institute, Faculty of Medicine, Imperial College London, London SW3 6LR, United Kingdom. f.munkonge@imperial.ac.uk
authors.forename: Felix M
authors.initials: FM
authors.lastname: Munkonge
authors.forename: Vaksha
authors.initials: V
authors.lastname: Amin
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Hyde
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Green
authors.forename: Ian A
authors.initials: IA
authors.lastname: Pringle
authors.forename: Deborah R
authors.initials: DR
authors.lastname: Gill
authors.forename: Joel W S
authors.initials: JW
authors.lastname: Smith
authors.forename: Robert P
authors.initials: RP
authors.lastname: Hooley
authors.forename: Stefania
authors.initials: S
authors.lastname: Xenariou
authors.forename: Malcolm A
authors.initials: MA
authors.lastname: Ward
authors.forename: Nicola
authors.initials: N
authors.lastname: Leeds
authors.forename: Kit-Yi
authors.initials: KY
authors.lastname: Leung
authors.forename: Mario
authors.initials: M
authors.lastname: Chan
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Hillery
authors.forename: Duncan M
authors.initials: DM
authors.lastname: Geddes
authors.forename: Uta
authors.initials: U
authors.lastname: Griesenbach
authors.forename: Edith H
authors.initials: EH
authors.lastname: Postel
authors.forename: David A
authors.initials: DA
authors.lastname: Dean
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Dunn
authors.forename: Eric W F W
authors.initials: EW
authors.lastname: Alton
mesh_terms: D021581:Active Transport, Cell Nucleus; D002457:Cell Extracts / Q000737:chemistry; D002463:Cell Membrane Permeability / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004072:Digitonin / Q000737:chemistry / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D054778:NM23 Nucleoside Diphosphate Kinases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D002457:Cell Extracts; D018398:Homeodomain Proteins; D054778:NM23 Nucleoside Diphosphate Kinases; D011506:Proteins; D014157:Transcription Factors; C490289:VSX2 protein, human; D004247:DNA; C517940:NME2 protein, human; D004072:Digitonin