DOI: 10.1073/pnas.93.23.12920. Complementation of methylation deficiency in embryonic stem cells by a DNA methyltransferase minigene

Complementation of methylation deficiency in embryonic stem cells by a DNA methyltransferase minigene

10.1073/pnas.93.23.12920

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

Previous attempts to express functional DNA cytosine methyltransferase (EC 2.1.1.37 ) in cells transfected with the available Dnmt cDNAs have met with little or no success. We show that the published Dnmt sequence encodes an amino terminal-truncated protein that is tolerated only at very low levels when stably expressed in embryonic stem cells. Normal expression levels were, however, obtained with constructs containing a continuation of an ORF with a coding capacity of up to 171 amino acids upstream of the previously defined start site. The protein encoded by these constructs comigrated in SDS/PAGE with the endogenous enzyme and restored methylation activity in transfected cells. This was shown by functional rescue of Dnmt mutant embryonic stem cells that contain highly demethylated genomic DNA and fail to differentiate normally. When transfected with the minigene construct, the genomic DNA became remethylated and the cells regained the capacity to form teratomas that displayed a wide variety of differentiated cell types. Our results define an amino-terminal domain of the mammalian MTase that is crucial for stable expression and function in vivo .

Bibliography

Tucker, K. L., Talbot, D., Lee, M. A., Leonhardt, H., & Jaenisch, R. (1996). Complementation of methylation deficiency in embryonic stem cells by a DNAâmethyltransferaseâminigene. Proceedings of the National Academy of Sciences, 93(23), 12920â12925.

Authors 5

Kerry Lee Tucker (first)
Dale Talbot (additional)
Min Ae Lee (additional)
Heinrich Leonhardt (additional)
Rudolf Jaenisch (additional)

References 35 Referenced 73

A Yeivin, A Razin DNA Methylation: Molecular Biology and Biological Significance, eds J P Jost, H P Saluz (Birkhauser, Basel), pp. 533–568 (1993). / DNA Methylation: Molecular Biology and Biological Significance by Yeivin A (1993)
P A Jones, W M Rideout, J C Shen, C H Spruck, Y C Tsai BioEssays 82, 9–12 (1992). / BioEssays by Jones P A (1992)
10.1126/science.270.5242.1610
10.1016/0022-2836(88)90122-2
10.1038/295620a0
10.1007/978-3-0348-9118-9_5
10.1016/0092-8674(92)90611-F
10.1242/dev.122.10.3195
10.1038/366362a0
10.1093/hmg/3.suppl_1.1487
10.1016/0092-8674(95)90329-1
10.1016/0378-1119(91)90618-L
10.1006/bbrc.1994.2562
10.1073/pnas.90.19.8891
10.1016/S0021-9258(18)42526-4
10.1101/gad.10.8.1008
10.1016/0378-1119(91)90161-4
10.1093/nar/18.4.937
10.1073/pnas.90.21.9832
10.1093/nar/18.17.5322
10.1016/0092-8674(92)90508-A
10.1038/227680a0
10.1101/gad.8.4.414
T Maniatis, E Fritsch, J Sambrook Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, 2nd Ed., Plainview, NY, 1989). / Molecular Cloning: A Laboratory Manual by Maniatis T (1989)
10.1111/j.1432-1033.1995.tb20698.x
10.1016/0092-8674(81)90305-6
10.1093/nar/19.20.5755
10.1002/aja.1002000403
10.1128/mcb.11.6.3070-3074.1991
10.1016/0092-8674(92)90561-P
10.1093/nar/20.9.2287
10.1038/321209a0
10.1128/MCB.16.8.4555
10.1073/pnas.82.9.2674
Yoder J. A. Yen R.-W. C. Vertino P. M. Bestor T. H. & Baylin S. B. (1996) J. Biol. Chem . in press.

Dates

Type	When
Created	23 years, 1 month ago (July 26, 2002, 10:40 a.m.)
Deposited	3 years, 4 months ago (April 13, 2022, 3:21 p.m.)
Indexed	1 month, 4 weeks ago (July 2, 2025, 3:07 p.m.)
Issued	28 years, 9 months ago (Nov. 12, 1996)
Published	28 years, 9 months ago (Nov. 12, 1996)
Published Online	28 years, 9 months ago (Nov. 12, 1996)
Published Print	28 years, 9 months ago (Nov. 12, 1996)

Funders 0

None

BibTeX

@article{Tucker_1996, title={Complementation of methylation deficiency in embryonic stem cells by a DNA methyltransferase minigene}, volume={93}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.93.23.12920}, DOI={10.1073/pnas.93.23.12920}, number={23}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Tucker, Kerry Lee and Talbot, Dale and Lee, Min Ae and Leonhardt, Heinrich and Jaenisch, Rudolf}, year={1996}, month=nov, pages={12920–12925} }

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