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De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells
1Cardiovascular Research Center, Massachusetts General Hospital-East, Charlestown 02129, USA.
Abstract:
It has been a controversial issue as to how many DNA cytosine methyltransferase mammalian cells have and whether de novo methylation and maintenance methylation activities are encoded by a single gene or two different genes. To address these questions, we have generated a null mutation of the only known mammalian DNA methyltransferase gene through homologous recombination in mouse embryonic stem cells and found that the development of the homozygous embryos is arrested prior to the 8-somite stage. Surprisingly, the null mutant embryonic stem cells are viable and contain low but stable levels of methyl cytosine and methyltransferase activity, suggesting the existence of a second DNA methyltransferase in mammalian cells. Further studies indicate that de novo methylation activity is not impaired by the mutation as integrated provirus DNA in MoMuLV-infected homozygous embryonic stem cells become methylated at a similar rate as in wild-type cells. Differentiation of mutant cells results in further reduction of methyl cytosine levels, consistent with the de novo methylation activity being down regulated in differentiated cells. These results provide the first evidence that an independently encoded DNA methyltransferase is present in mammalian cells which is capable of de novo methylating cellular and viral DNA in vivo.
Insights
Mammalian cells possess a second DNA methyltransferase, distinct from the known gene, capable of de novo methylation. This discovery clarifies the mechanisms of DNA methylation in mammals.
Area of Science:
- Epigenetics
- Molecular Biology
- Genetics
Background:
- The number and function of DNA cytosine methyltransferase genes in mammalian cells have been debated.
- It was unclear if de novo and maintenance methylation were controlled by one or two genes.
Purpose of the Study:
- To investigate the existence and function of DNA methyltransferases in mammalian cells.
- To determine if a single gene encodes both de novo and maintenance methylation activities.
Main Methods:
- Generation of a null mutation in the known mammalian DNA methyltransferase gene using homologous recombination in mouse embryonic stem cells.
- Analysis of homozygous null mutant embryos and embryonic stem cells for DNA methylation levels and activity.
- Assessment of de novo methylation activity using MoMuLV-infected cells.
Main Results:
- Homozygous null mutant embryos showed developmental arrest before the 8-somite stage.
- Mutant embryonic stem cells remained viable with detectable methyl cytosine and methyltransferase activity.
- De novo methylation of integrated provirus DNA was not impaired in mutant cells, indicating a second DNA methyltransferase.
Conclusions:
- Mammalian cells contain an independently encoded DNA methyltransferase responsible for de novo methylation.
- This second DNA methyltransferase can methylate cellular and viral DNA in vivo.
- The findings resolve the controversy regarding the number and function of DNA methyltransferase genes in mammals.