Complex DNA sequence readout mechanisms of the DNMT3B DNA methyltransferase

Abstract

DNA methyltransferases interact with their CpG target sites in the context of variable flanking sequences. We investigated DNA methylation by the human DNMT3B catalytic domain using substrate pools containing CpX target sites in randomized flanking context and identified combined effects of CpG recognition and flanking sequence interaction together with complex contact networks involved in balancing the interaction with different flanking sites. DNA methylation rates were more affected by flanking sequences at non-CpG than at CpG sites. We show that T775 has an essential dynamic role in the catalytic mechanism of DNMT3B. Moreover, we identify six amino acid residues in the DNA-binding interface of DNMT3B (N652, N656, N658, K777, N779, and R823), which are involved in the equalization of methylation rates of CpG sites in favored and disfavored sequence contexts by forming compensatory interactions to the flanking residues including a CpG specific contact to an A at the +1 flanking site. Non-CpG flanking preferences of DNMT3B are highly correlated with non-CpG methylation patterns in human cells. Comparison of the flanking sequence preferences of human and mouse DNMT3B revealed subtle differences suggesting a co-evolution of flanking sequence preferences and cellular DNMT targets.

Flanking sequences influence the activity of TET1 and TET2 methylcytosine dioxygenases and affect genomic 5hmC patterns

“…Interestingly, the flanking sequence-dependent fluctuations of oxidation rates were higher on mCpA and mCpC sites than on CpG sites, suggesting that on the less active substrate, the quality of the fit of the flanking sequence to the enzyme’s preferences becomes more important. A similar observation had been made previously with DNMT3B 29 . Moreover, we observe that the CpG recognition is modulated by the flanking base pairs, as indicated by the strong preference of TET1 and TET2 for mCpA oxidation with a T at the +1 site.…”

“…In the last 2 years novel experimental approaches have shown pronounced effects of flanking sequences on the CpG recognition of DNMT3A, DNMT3B, and DNMT1 [27][28][29] , leading to differences in the substrate preferences of both DNMT3 paralogs 27 or DNMT3A and its somatic cancer mutant R882H 26 . Mechanistically, flanking sequences can affect indirect DNA shape readout [26][27][28] , but also direct DNA contacts and alternative contact networks between the enzymes and different flanking sequences were found 27,29 . In DNMT1, striking differences in the mechanism of DNA rearrangement after base flipping were observed in different flanking contexts that were directly coupled to enzyme activity 28 .…”

“…Initial biochemical studies detected no sequence selectivity for the DNA sequence besides their preference for the CpG dinucleotide 20 . However, novel experimental approaches recently revealed an unexpected level of flanking sequence effects on the activity, CpG recognition, and specificity of different mammalian DNA methyltransferases that is affecting cellular DNA methylation patterns [26][27][28][29][30] . Hence, further studies are needed to describe the potential effect of flanking sequences on the activity of TET enzymes as well, in particular as DNMTs and TETs employ a related base flipping mechanism that includes large conformational changes of the DNA 31 .…”

DNA Methyltransferase 1 (DNMT1) Shapes Neuronal Activity of Human iPSC-Derived Glutamatergic Cortical Neurons

“…Different DNMTs and isoforms have been reported to display different binding specificities. By structural, functional, and cellular characterization of DNMT3A and DNMT3B, a multilayered substrate-recognition mechanism was identified that accounts for their divergent genomic methylation activities [ 34 , 35 , 36 ]. Thus, the temporary change in the availability of particular DNMTs potentially contributes to the dynamic modulation of DNA methylation changes of specific genes, e.g., being associated with synaptic plasticity.…”

The interplay between DNA and histone methylation: molecular mechanisms and disease implications

“…Its function, however, can be partially compensated by catalytically inactive isoforms of DNMT3B such as DNMT3B3 (Weisenberger et al, 2004;Duymich et al, 2016;Zeng et al, 2020). DNMT3A and DNMT3B have distinct mechanisms of DNA substrate engagement and show differential preference for the flanking sequence of target CpG (Handa & Jeltsch, 2005;Dukatz et al, 2020;Gao et al, 2020;Mallona et al, 2021), and we point readers to the excellent reviews of the molecular and structural basis for the catalysis of de novo methylation by DNMT3 (Jurkowska & Jeltsch, 2016;Ren et al, 2018). Furthermore, DNMT3A/B as well as DNMT3L can interact with histone tails through shared regulatory domains.…”