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Published on: September 20, 2016
A DNMT3A mutation common in AML exhibits dominant-negative effects in murine ES cells
Soo Jin Kim1, Hongbo Zhao, Swanand Hardikar
1Department of Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Smithville, TX;
Abstract:
Somatic heterozygous mutations of the DNA methyltransferase gene DNMT3A occur frequently in acute myeloid leukemia and other hematological malignancies, with the majority (∼60%) of mutations affecting a single amino acid, Arg882 (R882), in the catalytic domain. Although the mutations impair DNMT3A catalytic activity in vitro, their effects on DNA methylation in cells have not been explored. Here, we show that exogenously expressed mouse Dnmt3a proteins harboring the corresponding R878 mutations largely fail to mediate DNA methylation in murine embryonic stem (ES) cells but are capable of interacting with wild-type Dnmt3a and Dnmt3b. Coexpression of the Dnmt3a R878H (histidine) mutant protein results in inhibition of the ability of wild-type Dnmt3a and Dnmt3b to methylate DNA in murine ES cells. Furthermore, expression of Dnmt3a R878H in ES cells containing endogenous Dnmt3a or Dnmt3b induces hypomethylation. These results suggest that the DNMT3A R882 mutations, in addition to being hypomorphic, have dominant-negative effects.
Insights
Mutations in the DNA methyltransferase gene DNMT3A, common in acute myeloid leukemia, impair its function. These DNMT3A R882 mutations exhibit dominant-negative effects, inhibiting DNA methylation in cells.
Area of Science:
- Molecular Biology
- Epigenetics
- Cancer Genetics
Background:
- Somatic heterozygous mutations in DNMT3A are frequent in acute myeloid leukemia (AML) and other hematological malignancies.
- The majority of these mutations (∼60%) affect the Arg882 (R882) amino acid in the DNMT3A catalytic domain.
- While in vitro studies show impaired catalytic activity, the in-cell effects of DNMT3A mutations on DNA methylation remain largely unexplored.
Purpose of the Study:
- To investigate the cellular effects of DNMT3A R882 mutations on DNA methylation.
- To determine if DNMT3A R882 mutations have dominant-negative effects on DNA methylation in a cellular context.
Main Methods:
- Exogenous expression of mouse Dnmt3a proteins with corresponding R878 mutations in murine embryonic stem (ES) cells.
- Assessment of DNA methylation activity of mutant and wild-type Dnmt3a and Dnmt3b proteins.
- Analysis of protein interactions between mutant and wild-type Dnmt3a and Dnmt3b.
- Evaluation of DNA methylation levels in ES cells expressing mutant Dnmt3a R878H.
Main Results:
- Exogenously expressed mouse Dnmt3a R878 mutants largely failed to mediate DNA methylation in murine ES cells.
- Mutant Dnmt3a proteins were capable of interacting with wild-type Dnmt3a and Dnmt3b.
- Coexpression of Dnmt3a R878H inhibited the DNA methylation activity of wild-type Dnmt3a and Dnmt3b in ES cells.
- Expression of Dnmt3a R878H induced hypomethylation in ES cells with endogenous Dnmt3a or Dnmt3b.
Conclusions:
- DNMT3A R882 mutations are hypomorphic, meaning they have reduced but not absent function.
- These mutations exert dominant-negative effects, interfering with the function of wild-type DNMT3A and DNMT3B.
- The findings provide cellular insights into the mechanisms by which DNMT3A mutations contribute to hematological malignancies.
