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Exogenous expression of mouse Dnmt3 induces apoptosis in Xenopus early embryos
Hironobu Kimura1, Isao Suetake, Shoji Tajima
1Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan.
Abstract:
Mouse DNA (cytosine-5) methyltransferases Dnmt3a and Dnmt3b are expected to be de novo-type DNA methyltransferases. In the present study, we found that exogenously expressed mouse Dnmt3a or Dnmt3b induced abnormal cell clusters at the gastrulation stage in Xenopus embryos. The abnormal cells were judged to be apoptotic from the positive staining with the TdT dUTP nucleotide end-labeling method and the rescue by hBcl-x(L), a Bcl-2 homologue. On the other hand, neither bacterial DNA (cytosine-5) methyltransferase nor Dnmt3b3, one of the three isoforms of Dnmt3b that has no DNA methylation activity, induced apoptosis. In addition, mutant Dnmt3a and the other two Dnmt3b isoforms, Dnmt3b1 and Dnmt3b2, which have no DNA methylation activity due to a change of the cysteine residue in the catalytic center to an alanine residue, retained the ability to induce apoptosis. This indicates that the apoptosis was not induced by DNA methylation activity. The domain of Dnmt3b1 (3b2) responsible for the apoptosis is the catalytic domain in the carboxyl-terminal half.
Insights
Mouse DNA methyltransferases Dnmt3a and Dnmt3b can trigger apoptosis in Xenopus embryos during gastrulation. This cell death is independent of DNA methylation activity, suggesting a non-enzymatic role for these proteins.
Area of Science:
- Developmental Biology
- Molecular Biology
- Epigenetics
Background:
- DNA methyltransferases (DNMTs) are crucial for establishing DNA methylation patterns.
- Mouse DNA (cytosine-5) methyltransferases Dnmt3a and Dnmt3b are characterized as de novo methyltransferases.
- Their precise roles beyond DNA methylation, especially in developmental contexts, require further investigation.
Purpose of the Study:
- To investigate the function of mouse DNA methyltransferases Dnmt3a and Dnmt3b in early embryonic development.
- To determine if DNA methylation activity is essential for the observed developmental effects.
- To identify the specific domains responsible for any non-epigenetic functions.
Main Methods:
- Exogenous expression of mouse Dnmt3a and Dnmt3b in Xenopus embryos.
- Assessment of embryonic development and cell viability.
- Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to detect apoptosis.
- Expression of mutant Dnmt3a and Dnmt3b isoforms lacking DNA methylation activity.
- Rescue experiments using hBcl-x(L).
Main Results:
- Exogenous expression of Dnmt3a or Dnmt3b induced abnormal cell clusters and apoptosis at the gastrulation stage in Xenopus embryos.
- Apoptosis was confirmed by TUNEL staining and rescue with hBcl-x(L).
- Bacterial DNA methyltransferase and Dnmt3b isoforms lacking DNA methylation activity (Dnmt3b3, mutant Dnmt3a, Dnmt3b1, Dnmt3b2) did not induce apoptosis, indicating the effect is not due to DNA methylation.
- Mutant forms of Dnmt3a and Dnmt3b isoforms lacking catalytic activity retained the ability to induce apoptosis.
- The catalytic domain in the carboxyl-terminal half of Dnmt3b1 was identified as responsible for apoptosis induction.
Conclusions:
- Mouse Dnmt3a and Dnmt3b can induce apoptosis in Xenopus embryos independently of their DNA methylation activity.
- The catalytic domain of Dnmt3b possesses non-epigenetic functions that can trigger cell death during development.
- These findings suggest that Dnmt3a and Dnmt3b may have roles beyond DNA methylation in embryonic development.
