Complementation of methylation deficiency in embryonic stem cells by a DNA methyltransferase minigene.
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.
Whitehead Institute for Biomedical Research, Cambridge, MA, USA.
K L
KL
Tucker
D
D
Talbot
M A
MA
Lee
H
H
Leonhardt
R
R
Jaenisch
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D002460:Cell Line; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D017931:DNA Primers; D005091:Exons; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D051379:Mice; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus; D016366:Open Reading Frames; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013234:Stem Cells; D014162:Transfection
D000906:Antibodies; D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004248:DNA (Cytosine-5-)-Methyltransferases