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. 2020 Jan 17;432(2):569-575.
doi: 10.1016/j.jmb.2019.11.004. Epub 2019 Nov 11.

Dissect the DNMT3A- and DNMT3B-mediated DNA Co-methylation through a Covalent Complex Approach

Linfeng Gao  1 , Hiwot Anteneh  2 , Jikui Song  3
Affiliations

Dissect the DNMT3A- and DNMT3B-mediated DNA Co-methylation through a Covalent Complex Approach

Linfeng Gao et al. J Mol Biol. .

Abstract

DNA methylation plays a critical role in regulating gene expression, genomic stability, and cell fate commitment. Mammalian DNA methylation, which mostly occurs in the context of CpG dinucleotide, is installed by two denovo DNA methyltransferases, DNMT3A and DNMT3B. Oligomerization of DNMT3A and DNMT3B permits both enzymes to comethylate two CpG sites located on the same DNA substrates. However, how DNMT3A- and DNMT3B-mediated co-methylation contributes to the DNA methylation patterns remain unclear. Here we generated covalent enzyme-substrate complexes of DNMT3A and DNMT3B, and performed bisulfite sequencing-based single-turnover methylation analysis on both complexes. Our results showed that both DNMT3A- and DNMT3B-mediated co-methylation preferentially gives rise to a methylation spacing of 14 base pairs, consistent with the previous structural observation for DNMT3A in complex with regulatory protein DNMT3L and CpG DNA. This study provides a novel method for mechanistic investigation of DNMT3A- and DNMT3B-mediated DNA co-methylation.

Keywords: Covalent complex; CpG spacing; DNA methylation; DNMT3A; DNMT3B.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.. Co-methylation model of DNMT3A- and DNMT3B
(A) Schematic model of co-methylation vs sequential methylation. (B) Structural overlay of the DNMT3ADNMT3L tetramer bound to two short DNA duplexes containing single CpG site (PDB 6F57) and the DNMT3A-DNMT3L tetramer bound to one long DNA substrate containing two CpG sites (PDB 5YX2). Both structures suggest co-methylation spacing of 14-bp.
Figure 2.
Figure 2.. Schematic view of single-turnover methylation assay for measurement of co-methylation spacing
(A) Schematics for generation of the covalent complex of DNMT3A and DNMT3B with Zebularine-containing DNA. (B) Workflow for the single-turnover methylation assay. The Zebularine and unmodified cytosine are denoted as “Z” and “C”, respectively. The target strand contains a 5′ overhang for the purpose of strand-specific PCR amplification. (C) The relative abundance of indicated even-numbered CpG co-methylation spacing mediated by the DNMT3A-DNMT3L complex. The average and standard deviation were derived from analysis of three independent batches of sequencing data (See Table S2). (D) The relative abundance of indicated even-numbered CpG co-methylation spacing mediated by the DNMT3B-DNMT3L complex. The average and standard deviation were derived from analysis of three independent batches of sequencing data (See Table S3). (E) The relative abundance of indicated odd-numbered CpG co-methylation spacing mediated by the DNMT3A-DNMT3L complex. The average and standard deviation were derived from analysis of two independent batches of sequencing data (See Table S4).
Figure 3.
Figure 3.. Model for DNMT3A- and DNMT3B-mediated DNA co-methylation
(A) The 14-bp co-methylation spacing arises from the two concurrent methylation events of DNMT3A and DNMT3B. The CpG sites are denoted by letters ‘C’ and ‘G’. The DNMT3A or DNMT3B monomers are represented by oval spheres. The flipped ‘C’ represents the insertion of target cytosine into the active site of each monomer, corresponding to a productive methylation state. (B) DNMT3A/3B may catalyze the methylation of one CpG site first, and then transit into the catalytic state for methylating the next CpG site. At each catalytic stage, only one monomer undergoes productive methylation, while the other monomer adopts a non-productive state, which does not involve base flipping of target cytosine. During the transition of the methylation events, the DNMT3A/3B dimer may remain associated with the DNA substrate.
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