Chromatin binding and N-terminal domains of DNMT3B1 confer specificity for developmentally regulated CpG island methylation

CpG methylation in mammalian genomes is established by the closely related de novo DNA methyltransferases DNMT3A and DNMT3B. While both enzymes contribute to genome-wide CpG methylation, DNMT3B has a unique role in developmentally regulated CpG island (CGI) methylation on the inactive X chromosome and at other sites in the genome. The mechanistic basis for this specificity is poorly understood. Here we have developed an in vitro embryonic stem cell model system to dissect critical determinants of DNMT3B specificity. Our model faithfully recapitulates developmentally regulated CGI methylation and additionally provides novel insights into CpG methylation at cis -regulatory elements. Using genetic complementation, we show that DNMT3B specificity is attributable solely to the major catalytic isoform DNMT3B1. Domain swap experiments demonstrate a role for the PWWP–ADD chromatin binding domains, while deletion analysis reveals the importance of the unstructured N-terminal domain, including a requirement for a specific subregion in CGI methylation on Xi. Together, these findings advance our mechanistic understanding of the unique roles of DNMT3 enzymes in establishing CpG methylation in development.