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.
Highlights
Epigenetic regulation conveys heritable alterations in gene expression that are not caused by changes in the primary DNA sequence[1]
We initially used the catalytic domain of DNMT3AC, because it can be purified with different affinity tags in good yields and sufficient quantities for the planned biochemical work[15,17]
In vitro methylation experiments using identical concentrations of DNMT3AC wildtype and R882H showed that the R882H mutant has a residual activity of approximately 65% with a 30 mer DNA substrate (Fig. 2A,C) that has been used as standard substrate in many of our previous publications[17,18,25,27,28]
Summary
Epigenetic regulation conveys heritable alterations in gene expression that are not caused by changes in the primary DNA sequence[1]. Mutations in the DNMT3A DNA methyltransferase were reported for the first time in 2010, when it was found that a high frequency of missense mutations in DNMT3A occurs in AML11,12. Russler-Germain et al (2014) described that purified mixed enzyme preparations obtained after co-expression of wildtype and R882H in mammalian cells showed only 12% of residual activity in kinetic enzyme activity assays carried out in vitro. This observation led them to the conclusion that the R882H mutant has a dominant negative effect at the biochemical level and the formation of mixed complexes of wildtype and R882H subunits strongly reduces the activity of the mutant and wildtype DNMT3A subunits[22]
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