Stacey L. Thomas

Proper function and switching of regulatory elements are essential for vertebrate development and are regulated by DNA methylation. We use isoform-specific knockouts of the de novo methyltransferase Dnmt3a1 and Dnmt3a2 to probe their roles during embryogenesis and postnatal development. Mice lacking Dnmt3a1 show minimal embryonic methylation loss but are smaller and die postnatally. In contrast, Dnmt3a2 ^−/− mice exhibit widespread hypomethylation at enhancers, CTCF sites and imprinted genes, which are largely repaired postnatally. These mice are viable but display sporadic abnormalities including anophthalmia, hydrocephalus, hydronephrosis and male infertility due to absence of sperm. Interestingly, the fertile Dnmt3a2 ^−/− mice produce sperm with sporadic imprinting defects. These findings suggest that the two isoform have distinct, developmentally regulated roles, with Dnmt3a2 being crucial for maintaining proper methylation of regulatory elements, especially for enhancers, CTCF sites and imprinted genes, and preventing stochastic phenotypic outcomes after birth.

De novo DNA methylation is mediated by DNA methyltransferases DNMT3A and DNMT3B, in cooperation with the catalytically inactive paralogs DNMT3L and DNMT3B3. DNMT3L is predominantly expressed in embryonic stem cells to establish methylation patterns and is silenced upon differentiation, with DNMT3B3 substituting in somatic cells. Here we present high-resolution cryo-electron microscopy structures of nucleosome-bound, full-length DNMT3A2–3L and its oligomeric assemblies in the nucleosome-free state. We identified the critical role of DNMT3L as a histone modification sensor, guiding chromatin engagement through a mechanism distinct from DNMT3B3. The structures show a 180° rotated ‘switching helix’ in DNMT3L that prevents direct interaction with the nucleosome acidic patch. Instead, nucleosome binding is mediated by the DNMT3L ADD domain, while the DNMT3A PWWP domain exhibits reduced engagement in the absence of H3K36 methylation. The oligomeric arrangement of DNMT3A2–3L in nucleosome-free states highlights its dynamic assembly and potential allosteric regulation. We further capture dynamic structural movements of DNMT3A2–3L on nucleosomes. These findings uncover a previously unknown mechanism by which DNMT3A–3L mediates de novo DNA methylation on chromatin through complex assembly, histone tail sensing, dynamic DNA search and regulated nucleosome engagement, providing insights into epigenetic regulation.

Proper function and switching of regulatory elements are essential for the development of vertebrates and is known to be controlled by DNA methylation. We used isoform-specific knockouts of the de novo methyltransferase Dnmt3a, namely Dnmt3a1 and Dnmt3a2, to probe their roles in regulatory element methylation during embryogenesis and postnatal development. Mouse embryos lacking Dnmt3a1 showed minimal loss of methylation, suggesting limited involvement in embryonic development. However, they were smaller than their littermates and died about 4 weeks after birth with considerable postnatal demethylation as previously reported. In contrast, embryos lacking Dnmt3a2 showed widespread hypomethylation particularly at enhancers, CTCF sites and imprinted genes. These methylation deficits were largely repaired after birth, presumably by Dnmt3a1. The mice lacking Dnmt3a2 were viable; however, they showed an increased prevalence of sporadic abnormalities previously observed at a low frequency in laboratory mice, including anophthalmia, hydrocephalus, hydronephrosis and male infertility. Interestingly, hypomethylation of several imprinted genes was observed in sperm which might explain the infertility phenotype. Therefore, the interaction between the two isoforms is developmentally regulated, with Dnmt3a2 playing a key role in ensuring the methylation states of enhancers, CTCF sites and imprinted genes, thereby reducing the likelihood of stochastic phenotypes emerging after birth.

De novo DNA methylation is a fundamental epigenetic process essential for early development. It is mediated by DNA methyltransferase DNMT3A and DNMT3B, in cooperation with catalytically inactive DNMT3L. Abnormal DNA methylation contributes to genomic instability and oncogenesis, yet the mechanisms governing its establishment and regulation remain unclear. Here, we present high-resolution cryo-EM structures of the nucleosome-bound full-length DNMT3A2-3L and its oligomeric assemblies in the nucleosome-free state. Strikingly, DNMT3L C-terminal “Switching Helix” displayed a distinct conformation with a 180° rotational difference compared to its DNMT3B3 counterpart in the presence of nucleosome, preventing direct interactions with the nucleosome acidic patch. Instead, DNMT3L ADD domain promotes nucleosome binding, while DNMT3A PWWP domain inhibits it, indicating multi-layer regulation. Additionally, the novel oligomeric arrangement of DNMT3A2-3L in nucleosome-free states highlights its dynamic assembly and potential allosteric regulation. Furthermore, we identified the critical role of DNMT3L as a sensor of histone modifications, guiding the complex’s chromatin interaction. These findings uncover a previously unknown mechanism by which DNMT3A-3L mediates de novo DNA methylation on chromatin through complex reorganization, histone tail sensing, dynamic DNA search, and nucleosome engagement, providing key insights into epigenetic regulation.

CpG methylation generally occurs on both DNA strands and is essential for mammalian development and differentiation. Until recently, hemimethylation, in which only one strand is methylated, was considered to be simply a transitory state generated during DNA synthesis. The discovery that a subset of CCCTC-binding factor (CTCF) binding sites is heritably hemimethylated suggests that hemimethylation might have an unknown biological function. Here we show that the binding of CTCF is profoundly altered by which DNA strand is methylated and by the specific CTCF binding motif. CpG methylation on the motif strand can inhibit CTCF binding by up to 7-fold, whereas methylation on the opposite strand can stimulate binding by up to 4-fold. Thus, hemimethylation can alter binding by up to 28-fold in a strand-specific manner. The mechanism for sensing methylation on the opposite strand requires two critical residues, V454 and S364, within CTCF zinc fingers 7 and 4. Similar to methylation, CpG hydroxymethylation on the motif strand can inhibit CTCF binding by up to 4-fold. However, hydroxymethylation on the opposite strand removes the stimulatory effect. Strand-specific methylation states may therefore provide a mechanism to explain the transient and dynamic nature of CTCF-mediated chromatin interactions.

Significance: Genomic imprinting is essential for human development and occurs in germ cells before fertilization. The noncoding RNA, nc886 , is the only known example of more than 100 such human genes which shows variable frequencies of maternal imprinting. Here, we show that the DNA methylation imprint is present in oocytes and that the probability of imprinting increases as a function of maternal age. Importantly, we demonstrate that alcohol consumption but not cigarette smoking is associated with a lower frequency of imprinting. While most studies focus on the postconceptional developmental time, our work indicates that maternal age and exposures the year prior to pregnancy may alter the epigenome and therefore the developing child.