Nuclear Scaffold/Matrix Attached Region Modules Linked to a Transcription Unit Are Sufficient for Replication and Maintenance of a Mammalian Episome

The activation of mammalian origins of replication depends so far on ill understood epigenetic events, such as binding of transcription factors, chromatin structure, and nuclear localization. Understanding these mechanisms is not only a scientific challenge but also represents a prerequisite for the...

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Published in:Proceedings of the National Academy of Sciences - PNAS 2004-08, Vol.101 (31), p.11322-11327
Main Authors: Andreas C. W. Jenke, Stehle, Isa M., Herrmann, Frank, Eisenberger, Tobias, Baiker, Armin, Bode, Jürgen, Fackelmayer, Frank O., Lipps, Hans J., Groudine, Mark T.
Format: Article
Language:English
Subjects:
Animals
Biological Sciences
Cell lines
Cellular biology
CHO Cells
Cricetinae
Crosslinking
Deoxyribonucleic acid
DNA
DNA Replication - physiology
Escherichia coli
Genetic vectors
Genetics
HeLa cells
Mammals
Mitosis - physiology
Nuclear matrix
Nuclear Matrix - genetics
Nuclear Matrix-Associated Proteins - physiology
Plasmids
Plasmids - genetics
Polymerase chain reaction
Replication origin
Transcription, Genetic - physiology
Transgenes - genetics
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Summary:The activation of mammalian origins of replication depends so far on ill understood epigenetic events, such as binding of transcription factors, chromatin structure, and nuclear localization. Understanding these mechanisms is not only a scientific challenge but also represents a prerequisite for the rational design of nonviral episomal vectors for mammalian cells. In this paper, we demonstrate that a tetramer of a 155-bp minimal nuclear scaffold/matrix attached region DNA module linked to an upstream transcription unit is sufficient for replication and mitotic stability of a mammalian episome in the absence of selection. Fluorescence in situ hybridization analyses, crosslinking with cis-diammineplatinum(II)-dichloride and chromatin immunoprecipitation demonstrate that this vector associates with the nuclear matrix or scaffold in vivo by means of specific interaction of the nuclear scaffold/matrix attached region with the nuclear matrix protein SAF-A. Results presented in this paper define the minimal requirements of an episomal vector for mammalian cells on the molecular level.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0401355101