Comprehensive structure-function characterization of DNMT3B and DNMT3A reveals distinctive de novo DNA methylation mechanisms

Abstract

Mammalian DNA methylation patterns are established by two de novo DNA methyltransferases, DNMT3A and DNMT3B, which exhibit both redundant and distinctive methylation activities. However, the related molecular basis remains undetermined. Through comprehensive structural, enzymology and cellular characterization of DNMT3A and DNMT3B, we here report a multi-layered substrate-recognition mechanism underpinning their divergent genomic methylation activities. A hydrogen bond in the catalytic loop of DNMT3B causes a lower CpG specificity than DNMT3A, while the interplay of target recognition domain and homodimeric interface fine-tunes the distinct target selection between the two enzymes, with Lysine 777 of DNMT3B acting as a unique sensor of the +1 flanking base. The divergent substrate preference between DNMT3A and DNMT3B provides an explanation for site-specific epigenomic alterations seen in ICF syndrome with DNMT3B mutations. Together, this study reveals distinctive substrate-readout mechanisms of the two DNMT3 enzymes, implicative of their differential roles during development and pathogenesis.

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

“…Strikingly, however, the 1KO methylation profile is strongly correlated with the flanking sequence preferences of DNMT3A and DNMT3B determined in our previous work (Fig. 3b) 25 . The very low residual methylation levels in TKO cells containing a triple knock-out of DNMT1, DNMT3A and DNMT3B (reduced by 99%) did not correlate with any of the DNMT preference profiles.…”

“…Therefore, we focused on further analyzing the effect of the ±2 bp (N2 = NNCGNN) flanking positions on the activity of DNMT1 and determined the average methylation for all 256 N2 flanks. In our previous work, two sets of methylation reactions with random flank substrates were carried out with the catalytic domains of DNMT3A and DNMT3B using the same substrate library and experimental approach 25 , which were used for comparison here. Correlation analysis of the DNMT1 methylation profiles with the previously obtained data for DNMT3A and DNMT3B showed that the repetitions of experiments with the same enzyme were always highly correlated (Fig.…”

“…To determine the statistical significance of this correlation, the analysis was repeated with randomly shuffled random flank data. DNMT3A and DNMT3B data were taken from our previous study for comparison 25 . c The methylation levels of NNCGNN sites of both repeats of the DNMT1 methylation reaction displayed as heatmap to show the strong correlation of both data sets (see also Supplementary Fig.…”

“…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 .…”

Complex DNA sequence readout mechanisms of the DNMT3B DNA methyltransferase

“…The structure of the heterotetrameric complex comprising the catalytic domain of DNMT3B and the C-terminal domain of DNMT3L has been solved with four different DNA substrates ( Supplementary Table S1 ) ( 13 , 14 ). The interaction between DNMT3B and the DNA involves three regions, the so-called TRD loop (residues 772–791, colored red in Figure 1A ), catalytic loop (residues 648–672, colored green in Figure 1A ), and the RD homodimeric interface region (residues 822–828, colored yellow in Figure 1A ).…”