Mutations of R882 change flanking sequence preferences of the DNA methyltransferase DNMT3A and cellular methylation patterns

Abstract

Somatic DNMT3A mutations at R882 are frequently observed in AML patients including the very abundant R882H, but also R882C, R882P and R882S. Using deep enzymology, we show here that DNMT3A-R882H has more than 70-fold altered flanking sequence preferences when compared with wildtype DNMT3A. The R882H flanking sequence preferences mainly differ on the 3′ side of the CpG site, where they resemble DNMT3B, while 5′ flanking sequence preferences resemble wildtype DNMT3A, indicating that R882H behaves like a DNMT3A/DNMT3B chimera. Investigation of the activity and flanking sequence preferences of other mutations of R882 revealed that they cause similar effects. Bioinformatic analyses of genomic methylation patterns focusing on flanking sequence effects after expression of wildtype DNMT3A and R882H in human cells revealed that genomic methylation patterns reflect the details of the altered flanking sequence preferences of R882H. Concordantly, R882H specific hypermethylation in AML patients was strongly correlated with the R882H flanking sequence preferences. R882H specific DNA hypermethylation events in AML patients were accompanied by R882H specific mis-regulation of several genes with strong cancer connection, which are potential downstream targets of R882H. In conclusion, our data provide novel and detailed mechanistic understanding of the pathogenic mechanism of the DNMT3A R882H somatic cancer mutation.

Structural basis for impairment of DNA methylation by the DNMT3A R882H mutation

“…Along the line, the enzymatic preference toward the flanking sequences of CpG target sites has previously been observed for both DNMT3A and DNMT3B [35][36][37][38] , yet the mechanism remains unclear. The enzymatic analysis in this study not only supports an earlier notion that the DNMT3A R882H mutation shifts the enzymatic preference of DNMT3A toward a purine (G or A) on the +1 flanking site 25 ( Supplementary Fig. 6d), but also indicates that the R882H mutation reduces the CpG/CpH specificity of DNMT3A, both of which presumably impact the DNA methylation landscape across the genome 25 .…”

“…The enzymatic analysis in this study not only supports an earlier notion that the DNMT3A R882H mutation shifts the enzymatic preference of DNMT3A toward a purine (G or A) on the +1 flanking site 25 ( Supplementary Fig. 6d), but also indicates that the R882H mutation reduces the CpG/CpH specificity of DNMT3A, both of which presumably impact the DNA methylation landscape across the genome 25 . Importantly, the crosstalk between the RD interface and the TRD loop provides a mechanism by which the R882H mutation affects the substrate specificity of DNMT3A.…”

“…Furthermore, analysis of the relative methylation levels of the CGX (X = G, A, T or C) motif in the DNA substrate also indicates that the R882H mutation shifts the relative methylation efficiencies of DNMT3A WT -DNMT3L toward the CG(G/A) sites ( Supplementary Fig. 6i), consistent with the observation in a recent report 25 . Together, these data lend a strong support to the structural observation that the R882H mutation results in impaired CpG recognition of DNMT3A.…”

“…Consistently, it has been reported that the R882H mutation impairs the enzymatic activity of DNMT3A 16,17,[20][21][22][23][24] , and destabilizes its tetrameric form in vitro and in cells 17,22,23 . Furthermore, recent studies have suggested that the R882H mutation may affect the relative enzymatic preference of DNMT3A toward different CpGflanking sequences: in comparison with wild-type DNMT3A, the R882H mutant shows stronger preference for the CG(G/A) motif over the CG(T/C) motif as substrate 25 , which correlates with the aberrant DNA methylation and gene expression in AML, suggesting an off-targeting effect of the DNMT3A R882H mutation that may contribute to AML pathogenesis 21 .…”

DNA sequence-dependent activity and base flipping mechanisms of DNMT1 regulate genome-wide DNA methylation

“…However, increasing evidence has indicated that the activity of DNMTs can be strongly influenced by sequences outside the core CpG site, called flanking sequence here. For instance, the DNMT3 enzymes have been reported for their pronounced flanking sequence preferences [19][20][21][22][23][24][25] . Recently, it has even been demonstrated that alteration of the flanking sequence preferences of DNMT3A provides a key mechanistic basis for cancer promoting effects of the somatic DNMT3A R882H mutation, which is frequently observed in acute myeloid leukemia (AML) 23,24 .…”