Abstract
The DNTM3A and DNMT3B de novo DNA methyltransferases (DNMTs) are responsible for setting genomic DNA methylation patterns, a key layer of epigenetic information. Here, using an in vivo episomal methylation assay and extensive bisulfite methylation sequencing, we show that human DNMT3A and DNMT3B possess significant and distinct flanking sequence preferences for target CpG sites. Selection for high or low efficiency sites is mediated by the base composition at the −2 and +2 positions flanking the CpG site for DNMT3A, and at the −1 and +1 positions for DNMT3B. This intrinsic preference reproducibly leads to the formation of specific de novo methylation patterns characterized by up to 34-fold variations in the efficiency of DNA methylation at individual sites. Furthermore, analysis of the distribution of signature methylation hotspot and coldspot motifs suggests that DNMT flanking sequence preference has contributed to shaping the composition of CpG islands in the human genome. Our results also show that the DNMT3L stimulatory factor modulates the formation of de novo methylation patterns in two ways. First, DNMT3L selectively focuses the DNA methylation machinery on properly chromatinized DNA templates. Second, DNMT3L attenuates the impact of the intrinsic DNMT flanking sequence preference by providing a much greater boost to the methylation of poorly methylated sites, thus promoting the formation of broader and more uniform methylation patterns. This study offers insights into the manner by which DNA methylation patterns are deposited and reveals a new level of interplay between members of the de novo DNMT family.
Highlights
Cytosine DNA methylation, which is primarily focused at symmetrical CpG sites in mammalian cells, represents a critical epigenetic mark broadly associated with silent genomic regions
We examine the function of the proteins responsible for laying down the initial DNA methylation patterns in the human genome
Our study clearly establishes that DNMT3A and DNMT3B do not methylate DNA at random but rather that they show strong and distinct preferences for their target sites in vivo
Summary
Cytosine DNA methylation, which is primarily focused at symmetrical CpG sites in mammalian cells, represents a critical epigenetic mark broadly associated with silent genomic regions. Once DNA methylation is established on both DNA strands at a CpG site, it is propagated with high fidelity at each cell division [7]. This stems directly from the fact that hemi-methylated CpG sites, a key intermediate generated by replicating through a fully methylated CpG sequence, are preferentially methylated back to a fully methylated state by the maintenance DNA methyltransferase DNMT1 [8,9] in association with other interacting factors such as UHRF1 [10,11]. DNA methylation profiles represent an important form of epigenetic memory
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