Identification and functional characterization of cytoplasmic determinants of plasmid DNA nuclear import.
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.
Munkonge
Felix M
FM
Department of Gene Therapy, National Heart & Lung Institute, Faculty of Medicine, Imperial College London, London SW3 6LR, United Kingdom. f.munkonge@imperial.ac.uk
Amin
Vaksha
V
Hyde
Stephen C
SC
Green
Anne-Marie
AM
Pringle
Ian A
IA
Gill
Deborah R
DR
Smith
Joel W S
JW
Hooley
Robert P
RP
Xenariou
Stefania
S
Ward
Malcolm A
MA
Leeds
Nicola
N
Leung
Kit-Yi
KY
Chan
Mario
M
Hillery
Elizabeth
E
Geddes
Duncan M
DM
Griesenbach
Uta
U
Postel
Edith H
EH
Dean
David A
DA
Dunn
Michael J
MJ
Alton
Eric W F W
EW
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
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