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The DNMT3A R882H mutation does not cause dominant negative effects in purified mixed DNMT3A/R882H complexes.

ABSTRACT: The DNA methyltransferase DNMT3A R882H mutation is observed in 25% of all AML patients. DNMT3A is active as tetramer and the R882H mutation is located in one of the subunit/subunit interfaces. Previous work has reported that formation of mixed wildtype/R882H complexes leads to a strong loss of catalytic activity observed in in vitro DNA methylation assays (Russler-Germain et al., 2014, Cancer Cell 25:442-454). To investigate this effect further, we have prepared mixed wildtype/R882H DNMT3A complexes by incubation of individually purified subunits of the DNMT3A catalytic domain and full-length DNMT3A2. In addition, we have used a double affinity tag approach and specifically purified mixed catalytic domain complexes formed after co-expression of R882H and wildtype subunits in E. coli cells. Afterwards, we determined the catalytic activity of the mixed complexes and compared it to that of purified complexes only consisting of one subunit type. In both settings, the expected catalytic activities of mixed R882H/wildtype complexes were observed demonstrating an absence of a dominant negative effect of the R882H mutation in purified DNMT3A enzymes. This result suggests that heterocomplex formation of DNMT3A and R882H is unlikely to cause dominant negative effects in human cells as well. The limitations of this conclusion and its implications are discussed.

SUBMITTER: Emperle M

PROVIDER: S-EPMC6125428 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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The DNMT3A R882H mutation does not cause dominant negative effects in purified mixed DNMT3A/R882H complexes.

Emperle Max M Dukatz Michael M Kunert Stefan S Holzer Katharina K Rajavelu Arumugam A Jurkowska Renata Z RZ Jeltsch Albert A

Scientific reports 20180905 1

The DNA methyltransferase DNMT3A R882H mutation is observed in 25% of all AML patients. DNMT3A is active as tetramer and the R882H mutation is located in one of the subunit/subunit interfaces. Previous work has reported that formation of mixed wildtype/R882H complexes leads to a strong loss of catalytic activity observed in in vitro DNA methylation assays (Russler-Germain et al., 2014, Cancer Cell 25:442-454). To investigate this effect further, we have prepared mixed wildtype/R882H DNMT3A compl ...[more]

PMID: 30185810

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Project description:Mutation of DNA methyltransferase 3A at arginine 882 (DNMT3AR882mut) is prevalent in hematologic cancers and disorders. Recently, DNMT3AR882mut has been shown to have hypomorphic, dominant-negative, and/or gain-of-function effects on DNA methylation under different biological contexts. However, the causal role for such a multifaceted effect of DNMT3AR882mut in leukemogenesis remains undetermined. Here, we report TF-1 leukemia cells as a robust system useful for modeling the DNMT3AR882mut-dependent transformation and for dissecting the cause-effect relationship between multifaceted activities of DNMT3AR882mut and leukemic transformation. Ectopic expression of DNMT3AR882mut and not wild-type DNMT3A promoted TF-1 cell transformation characterized by cytokine-independent growth, and induces CpG hypomethylation predominantly at enhancers. This effect was dose dependent, acted synergistically with the isocitrate dehydrogenase 1 (IDH1) mutation, and resembled what was seen in human leukemia patients carrying DNMT3AR882mut. The transformation- and hypomethylation-inducing capacities of DNMT3AR882mut relied on a motif involved in heterodimerization, whereas its various chromatin-binding domains were dispensable. Mutation of the heterodimerization motif that interferes with DNMT3AR882mut binding to endogenous wild-type DNMT proteins partially reversed the CpG hypomethylation phenotype caused by DNMT3AR882mut, thus supporting a dominant-negative mechanism in cells. In mice, bromodomain inhibition repressed gene-activation events downstream of DNMT3AR882mut-induced CpG hypomethylation, thereby suppressing leukemogenesis mediated by DNMT3AR882mut. Collectively, this study reports a model system useful for studying DNMT3AR882mut, shows a requirement of the dominant-negative effect by DNMT3AR882mut for leukemogenesis, and describes an attractive strategy for the treatment of leukemias carrying DNMT3AR882mut. SIGNIFICANCE: These findings highlight a model system to study the functional impact of a hotspot mutation of DNMT3A at R882 in leukemia.

Dataset Information

The DNMT3A R882H mutation does not cause dominant negative effects in purified mixed DNMT3A/R882H complexes.

Publications

The DNMT3A R882H mutation does not cause dominant negative effects in purified mixed DNMT3A/R882H complexes.

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