Structural basis for DNMT3A-mediated de novo DNA methylation

Abstract

DNA methylation by de novo DNA methyltransferases 3A (DNMT3A) and 3B (DNMT3B) is essential for genome regulation and development1, 2. Dysregulation of this process is implicated in various diseases, notably cancer. However, the mechanisms underlying DNMT3 substrate recognition and enzymatic specificity remain elusive. Here we report a 2.65-Å crystal structure of the DNMT3A-DNMT3L-DNA complex where two DNMT3A monomers simultaneously attack two CpG dinucleotides, with the target sites separated by fourteen base pairs within the same DNA duplex. The DNMT3A–DNA interaction involves a target recognition domain (TRD), a catalytic loop and DNMT3A homodimeric interface. A TRD residue Arg836 makes crucial contacts with CpG, ensuring DNMT3A enzymatic preference towards CpG sites in cells. Hematological cancer-associated somatic mutations of the substrate-binding residues decrease DNMT3A activity, induce CpG hypomethylation, and promote transformation of hematopoietic cells. Together, our study reveals the mechanistic basis for DNMT3A-mediated DNA methylation and establishes its etiologic link to human disease.

Comprehensive structure-function characterization of DNMT3B and DNMT3A reveals distinctivede novoDNA methylation mechanisms

“…Note that DNMT3L does not make any contact with DNA ( Fig. 3b), as previously observed for the DNMT3A-DNMT3L-DNA complex 33 and it did not show strong effects on the intrinsic flanking sequence preferences of DNMT3A or DNMT3B ( Supplementary Fig. 9).…”

“…Using liquid chromatography-mass spectrometry (LC-MS)-based quantification, we detected a global increase in cytosine methylation in TKO cells after rescue with DNMT3B ( Supplementary Fig. 4b), an effect similar to what was observed with DNMT3A rescue 33 . Next, we profiled the genome-wide methylation introduced by either DNMT3A or DNMT3B in cells by enhanced reduced representation bisulfite sequencing (eRRBS, two replicates per group; Supplementary Table 4) and generated datasets with desired high conversion rates ( Supplementary Fig.…”

“…Our previous study on the DNMT3A-DNA complex revealed that a hydrogen bond formed between residues R882 and S837 of DNMT3A provides an intramolecular link between the RD interface and the TRD loop upon DNA binding ( Fig. 6a) 33 . Interestingly, this interaction is abolished in DNMT3B, because R823 adopts a different conformation and DNA-binding mode than its DNMT3A counterpart R882 ( Fig.…”

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

“…It is a polar interface that mediates a reversible oligomerization of DNMT3A dimers (formed via the FF-interface) into a mixture of dimers, tetramers and higher aggregates depending on the buffer conditions and enzyme concentrations 17–19 . The RD interface also forms the DNA binding site of the DNMT3A complex 15,17 indicating that a tetramer is the smallest complex, which can be catalytically active.

Figure 1Position of the R882H mutation in the structure of the DNMT3A/DNMT3L C-terminal domain heterotetramer with bound DNA (green) 16 . The DNMT3L C-terminal domains are colored dark and light red, the DNMT3A C-terminal domains are dark and light blue.

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The genetic and molecular pathogenesis of myelodysplastic syndromes

“…3,29,30 DNMT3A is an essential enzyme which makes contact with CpG, subsequently catalyzing the methylation required for genomic regulation which is unsuitably committed toward myelodysplasia when DNMT3A is mutated in such a manner that inhibits appropriate DNMT3A-CpG contact and thus fosters CpG hypomethylation. 31 Mutations in DNMT3A are detected in up to 8% of cases of de novo MDS with the missense R882 methyltransferase domain mutation being the most commonly identified. 32,33 DNA sequencing of DNMT3A-mutated MDS patient marrow samples in a study by…”