Structural insight into hierarchical DNMT3A autoinhibition and its dysregulation in disease

Domain: chemistry
Published: 2026-02-18
Editorial outcome: minor_revision

Abstract
DNA methyltransferase DNMT3A-mediated DNA methylation is important for genomic imprinting and transcriptional regulation. However, how the regulatory domains of DNMT3A cooperate with its methyltransferase domain and histone marks to orchestrate genomic methylation remains unclear. Here we report the cryo-EM structure of DNMT3A2 with regulatory factor DNMT3L, revealing an intricate domain interaction underlying multilayered autoinhibition. The PWWP domain interacts with the ADD and methyltransferase domains to block the target recognition domain and the H3K36me2-binding pocket, thereby coupling the H3K36me2 binding with DNMT3A activation, adding a layer of allosteric regulation distinct from the previously characterized ADD-H3K4me0 regulation. Molecular dynamics simulations of the DNMT3A-DNMT3L complex further reveal that relief of DNMT3A autoinhibition involves disengagement of the CpG-recognition loop of the target recognition domain from autoinhibitory interaction, leading to enhanced accessibility of the target recognition domain loop for DNA binding and DNMT3A activation. Importantly, our combined structural, biochemical and genomic methylation analysis demonstrates that disrupting the PWWP-ADD interaction by disease-associated DNMT3A mutations leads to impaired DNMT3A autoinhibition and substrate specificity, providing a potential explanation to aberrant DNA methylation in disease.

Introduction
DNA methylation is an important epigenetic mechanism essential for gene regulation and genome stability1–3. In mammals, DNA methylation mainly occurs within the symmetric CpG dinucleotides, established by de novo DNA methyltransferases DNMT3A and DNMT3B during gametogenesis and early embryogenesis4,5. Subsequently, the DNA methylation landscape continues to evolve throughout development, through a coordinated action between DNMT3A, DNMT3B and maintenance DNA methyltransferase DNMT16–8. Proper regulation of the DNA methylation activities of DNA methyltransferases across various regions of the genome is critical for transcriptional programming and other cellular processes9.

To date, two isoforms of DNMT3A have been identified: DNMT3A1 and DNMT3A2, differing in a DNMT3A1-unique N-terminal ubiquitin-dependent recruitment region (UDR) that recognizes histone H2A lysine-119 mono-ubiquitylation (H2AK119ub1)10–14. On the other hand, both isoforms contain a methyltransferase (MTase) domain preceded by a Pro-Trp-Trp-Pro (PWWP) domain and an ATRX-DNMT3-DNMT3L (ADD) domain4,15, which serve as specific readers for di-methylated histone H3 lysine-36 (H3K36me2) and unmethylated H3 lysine-4 (H3K4me0) respectively11,16–21. It has been shown that the H3K4me0 interaction relieves the ADD-mediated DNMT3A autoinhibition, thereby stimulating DNMT3A-mediated DNA methylation in the corresponding genomic regions22,23. Likewise, recent studies showed that the presence of a H3K36me2 peptide led to enhanced DNA methylation activity of DNMT3A in vitro24, although the underlying mechanism is unclear.

DNMT3A-mediated DNA methylation is further regulated by DNMT3-like (DNMT3L) protein during gametogenesis and early embryogenesis25–28. DNMT3L harbors an N-terminal ADD domain that reads the H3K4me0 mark29 and a C-terminal methyltransferase-like (ML) domain that associates with the DNMT3A MTase domain to stimulate the DNA methylation activity of the latter26,30. Previous studies on the DNMT3A MTase and DNMT3L ML fragment (DNMT3AMTase-DNMT3LML) and the DNMT3A ADD-MTase and DNMT3L ML fragment (DNMT3AADD-MTase-DNMT3LML) revealed a 3A-3L-3L-3A linear assembly, mediated by a hydrophilic interface between the DNMT3A subunits (a.k.a. RD interface) and a hydrophobic interface (a.k.a. FF interface) between the DNMT3A and DNMT3L subunits22,31. Furthermore, the substrate recognition of DNMT3A-DNMT3L complex is mainly mediated by three structural elements: the catalytic loop (residues 707–731), a loop from the target recognition domain (TRD loop: residues 831–847), and a helical element (residues 881–887) at the RD interface32,33. In addition, a crystal structure of the DNMT3AADD-MTase-DNMT3LML complex revealed an autoinhibitory conformation in which the direct interaction between the ADD and MTase domains of DNMT3A leads to occlusion of its catalytic site and H3K4me0-binding site22, lending an explanation to the coupling between the H3K4me0 binding and the enzymatic activation of DNMT3A23. However, how DNMT3A transits from such an autoinhibitory state to an active state remains elusive.

Mutations of DNMT3A have been associated with a wide spectrum of human diseases and developmental disorders34–38. For instance, mutation at the site of R882 within the MTase domain represents a hotspot mutation in acute myeloid leukemia (AML) and clonal haematopoiesis35. Likewise, mutations within the ADD domain (e.g. E545G) have been associated with AML, Tatton-Brown-Rahman syndrome and myelodysplastic syndrome (MDS)38–40. In addition, mutations within the PWWP domain (e.g. W330R and D333N) have been linked to microcephalic dwarfism34. Mechanistically, the R882 mutations promote high-order DNMT3A multimerization, causing a dominant-negative effect in its DNA methylation activity41–44. The PWWP mutations were shown to lead to gain-of-function DNA methylation in polycomb targets11,34,36, in part attributed to the loss of the PWWP-H3K36me2 interaction and the competing chromatin-targeting mechanism underpinned by the interaction between DNMT3A1 UDR and histone H2AK119ub1-marked nucleosome12–14. Due to the lack of structure-function characterization of a DNMT3A fragment harboring the PWWP, ADD and MTase domains, a comprehensive understanding of how the mutations within the regulatory domains of DNMT3A contribute to disease progression remains lacking.

To elucidate the functional regulation of DNMT3A, we solved the cryo-EM structure of DNMT3A2 bound to full-length DNMT3L, revealing a multilayered autoinhibition. The DNMT3A PWWP domain dynamically interacts with the ADD and MTase domains to strengthen the previously characterized autoinhibition by the ADD domain. Furthermore, the PWWP domain-mediated autoinhibition leads to occlusion of the H3K36me2-binding pocket and the CpG-engaging TRD loop, thereby coupling the H3K36me2 binding with DNMT3A activation. Through molecular dynamics (MD) simulation of DNMT3A-DNMT3L under apo and histone-bound states, we observed that following relief of the PWWP domain-mediated autoinhibition, the ADD-H3K4me0 interaction disengages the TRD loop from autoinhibitory interaction, releasing the latter for potential DNA contact. On the other hand, the positioning of the ADD domain remains largely intact, suggesting that engagement with both histone and DNA might be required for dislodging the ADD domain toward full activation of DNMT3A. Importantly, our combined structural, biochemical and genomic methylation analysis demonstrates that disrupting the disease-associated PWWP-ADD interface mutations W330R, D333N and E545G leads to impaired DNMT3A autoinhibition and target specificity, which may contribute to the aberrant DNA methylation patterns in disease associated with these mutations. Together, this study uncovers a hierarchical autoinhibitory regulation of DNMT3A, sheds light onto the mechanism of DNMT3A activation and links the PWWP domain-mediated autoinhibition to disease.

Results
Cryo-EM structure of the DNMT3A2-DNMT3L complex
To elucidate the functional interplay between the individual domains of DNMT3A and DNMT3L, we focused on a fragment of DNMT3A2 (residues 281–912) in complex with full-length DNMT3L for structure determination (Fig. 1a). The structure of the DNMT3A2-DNMT3L complex was solved at 3.66-Å overall resolution (Fig. 1b–d and Supplementary Figs. 1, 2 and Supplementary Table 1). We were able to trace the PWWP, ADD and MTase domains for one of the DNMT3A subunits (3A-1), the ADD and MTase domains for the other DNMT3A subunit (3A-2), and the ADD and ML domains for both DNMT3L subunits (Fig. 1f). Each of the DNMT3A subunits harbors a S-adenosyl-homocysteine (SAH) molecule, the by-product of cofactor S-adenosyl-methionine (SAM) (Fig. 1c). The DNMT3A2-DNMT3L complex assumes a heterotetrameric assembly resembling that of the DNMT3AMTase-DNMT3LML complex31 (Fig. 1b, c), with a root-mean-square deviation (RMSD) of 0.93 Å over 845 aligned Cα atoms (Supplementary Fig. 3a).

As expected, the two DNMT3A subunits of the DNMT3A2-DNMT3L complex are highly similar, with an RMSD of 0.70 Å over 386 aligned Cα atoms (Fig. 1d and Supplementary Fig. 2b). Nevertheless, they differ in the conformational state of the PWWP domain: the PWWP domain in the 3A-1 subunit packs against the ADD and MTase domains into a compact fold, whereas the corresponding region lacks defined density in the 3A-2 subunit (Fig. 1d). In line with this difference, the two DNMT3A subunits show a subtle structural variation in the PWWP-interacting site of the ADD domain, in which residues E469-L475 run in parallel with PWWP residues S319-E323 in the 3A-1 subunit but adopts a helical conformation in the PWWP-disordered 3A-2 subunit (Supplementary Fig. 3b). On the other hand, both DNMT3L subunits show an elongated fold where the ADD domain stacks against the ML domain at an interface distant from the DNMT3A-DNMT3L interface (Fig. 1f). In fact, structural alignment of both DNMT3L subunits with the previously reported DNMT3L molecule bound to a histone H3K4me0 peptide (PDB 2PVC) gave RMSD of 0.96 Å and 0.84 Å over 333 and 307 aligned Cα atoms, respectively, indicative of high structural similarity. Notably, the H3K4me0-binding site of the DNMT3L ADD domain in the DNMT3A2-DNMT3L complex is solvent accessible, poised for potential histone binding (Fig. 1f).

It is worth noting that detailed 3D classification of the DNMT3A2-DNMT3L complex also reveals a population of DNMT3A2-DNMT3L particles lacking the density of the PWWP domain in both DNMT3A subunits (Supplementary Fig. 1), highlighting the dynamic nature of the interaction between the PWWP domain and the ADD-MTase domains of DNMT3A.

The PWWP domain joins the ADD domain to underpin an autoinhibitory conformation of DNMT3A
Next, we aligned the DNMT3A2-DNMT3L complex with the previously reported DNMT3AMTase-DNMT3LML-DNA complex (PDB 5YX2). The DNA-binding site of DNMT3A MTase domain in the DNMT3A2-DNMT3L complex is blocked by the ADD domain (Fig. 2a, b), as observed previously22. In addition, the DNA binding is blocked by the PWWP domain of the 3A-1 subunit, but not the 3A-2 subunit, indicative of a role for the dynamic PWWP-ADD-MTase interaction in strengthening the autoinhibitory state of DNMT3A (Fig. 2a, b).

Detailed structural analysis of the DNMT3A2-DNMT3L complex reveals that within the 3A-1 subunit, the PWWP domain interacts with the ADD domain as well as the MTase domain (Fig. 2c). At the PWWP-ADD interface, residue R544 of the ADD domain inserts its side chain guanidinium group into the aromatic cage of the PWWP domain, lined by residues F303, W306, W330 and D333 (Fig. 2c). Furthermore, residues E473, E477, Y536 and E545 of the ADD domain interact with residues R301, W306, D333, C340, and P363 of the PWWP domain through hydrogen-bonding and/or van der Waals contacts (Fig. 2c). Together, the PWWP-ADD association leads to a buried surface area of ~900 Å2. At the PWWP-MTase interface, residues K397 and E400 of the PWWP domain are within a distance for van der Waals and/or electrostatic contacts with residues R836-S837 from the TRD loop of the MTase domain (Fig. 2c), a structural element known for CpG-specific DNA recognition32,33. As a result of these interactions, the PWWP domain is positioned in front of the substrate-binding site of DNMT3A, blocking the latter from potential DNA binding (Fig. 2a, b).

Disrupting the PWWP-ADD interaction boosts DNMT3A-mediated DNA methylation
To test the structural observation, we performed isothermal titration calorimetry (ITC) binding assays for the DNMT3AADD-MTase-DNMT3LML complex and the isolated PWWP domain of DNMT3A. Wild-type (WT) PWWP domain binds to DNMT3AADD-MTase-DNMT3LML with a dissociation constant (Kd) of 86 μM, supporting the structural observation that the PWWP domain directly interacts with the ADD-MTase fragment of DNMT3A. In contrast, no appreciable interaction was observed between the DNMT3AADD-MTase-DNMT3LML complex and the W330A-mutated PWWP or between the R544A-mutated DNMT3AADD-MTase-DNMT3LML and WT PWWP (Fig. 2d). We further performed two-dimensional 1H-15N Heteronuclear Single-Quantum Correlation (HSQC) NMR experiment for the DNMT3A ADD domain in the absence or presence of the PWWP domain. Spectral comparison of the ADD domain among various conditions reveals that the addition of the PWWP domain leads to notable chemical shift perturbation of several NMR peaks (Fig. 2e), in line with the observed PWWP-ADD interaction. In contrast, replacement of WT PWWP with the W330A-mutated PWWP domain led to a shift of these peaks toward the positions corresponding to the ADD domain alone, indicative of impaired PWWP-ADD interaction (Fig. 2e). These observations lend strong support to the observed PWWP-ADD interaction of DNMT3A.

To examine how the PWWP-ADD interaction affects the DNA binding of DNMT3A2-DNMT3L, we performed the fluorescence polarization (FP) experiment to measure the DNA-binding affinity of WT or mutant DNMT3A2-DNMT3L for a 24-mer DNA duplex. Consistent with a previous observation22, WT DNMT3A2-DNMT3L binds to the DNA with Kd of 8 μM. In contrast, introducing the R544A or W330A mutation led to >10-fold increase in binding affinity, with Kd of 0.6 μM and 0.8 μM, respectively (Fig. 2f). This observation suggests that disrupting the PWWP-ADD interaction substantially relieves DNMT3A autoinhibition. Consistently, our in vitro DNA methylation assay for WT and mutant DNMT3A2-DNMT3L over 36-mer (GTC)12/(GAC)12 DNA duplex showed that the R544A and W330A mutations led to a 3.0- and 2.4-fold higher methylation efficiency, respectively. Together, these data support the notion that the intramolecular interaction of the PWWP domain with the ADD and MTase domains gives rise to one layer of autoinhibition for DNMT3A2-DNMT3L.

Sequence analysis of DNMT3A and DNMT3B indicates that the residues involved in the PWWP interaction with the ADD-MTase domains of DNMT3A are largely preserved in DNMT3B (Supplementary Fig. 4), suggesting that the PWWP-mediated autoinhibition likely represents a common regulatory mechanism for the DNMT3A-DNMT3L and DNMT3B-DNMT3L complexes. In support of this notion, a recent study showed that the presence of H3K36me3 peptide leads to enzymatic activation of DNMT3B-DNMT3L45.

The ADD domain engages the TRD loop for autoinhibition
Structural analysis of the DNMT3A2-DNMT3L complex reveals that the interaction between the ADD and MTase domains is mainly mediated by a zinc-coordinating loop of the ADD domain (ADD loop: E523-V560) and the TRD loop (Fig. 3a). Notably, the side chain of TRD-loop residue R836 engages in electrostatic contact with the tri-aspartate motif (D529-D530-D531) of the ADD loop, the side chain of TRD-loop residue R831 engages in cation-π interaction with the aromatic ring of ADD-loop residue Y528, and TRD-loop residues F827, D845, Q846 and H847 are within a distance for van der Waals contact with ADD-loop residues D531, N552 and N553 (Fig. 3a). In addition, the ADD-MTase interaction is supported by van der Waals and hydrophobic contacts involving residues V716, M779, H789 and Y793 from the MTase domain and residues Y526, Y528, D529 and F609 from the ADD domain (Fig. 3a). Collectively, these interactions lead to buried surface area of ~1199 Å2 between the MTase and ADD domains. Consistent with the structural observation, our ITC binding assay shows that while the ADD domain binds to the MTase domain with a Kd of 11 μM, consistent with the previous observation22, introducing the D529A/D531A and R836A mutations reduced the binding by ~3 and ~8 fold, respectively (Fig. 3b).

It is worth noting that the ADD-MTase interface of the DNMT3A2-DNMT3L complex is distinct from what was previously observed for the crystal structure of DNMT3AADD-MTase-DNMT3LML complex (PDB 4U7P): unlike in the DNMT3A2-DNMT3L complex the ADD-loop approaches the TRD loop for electrostatic contact, in the crystal structure the ADD loop moves away by ~13 Å to engage in electrostatic contact with motif-VIII residues R790 and R792 (Supplementary Fig. 5a–c), reflecting the conformational dynamics of this domain. In addition, analysis of the crystal structure of DNMT3AADD-MTase-DNMT3LML complex reveals that the ADD domain is involved in crystal packing (Supplementary Fig. 5d). Whether such an effect contributes to the observed structural difference awaits further investigation.

Sequence analysis of DNMT3A and DNMT3B indicates that the residues involved in the ADD-MTase interaction of DNMT3A are largely preserved in DNMT3B (Supplementary Fig. 4). In fact, structural overlay of the DNMT3A2-DNMT3L complex with the previously reported DNMT3B homotetramer (PDB 8EIH)46 reveals high structural similarity between their central subunits, with an RMSD of 1.46 Å over 406 aligned Cα atoms. Notably, the ADD domains of DNMT3A and DNMT3B are positioned similarly next to the TRD loop (Supplementary Fig. 5e), suggesting that, as with the PWWP interaction, the ADD-MTase interaction is likely evolutionarily conserved between DNMT3A and DNMT3B.

Functional interplay between the PWWP-H3K36me2 and ADD-H3K4me0 readouts
Structural overlay of the DNMT3A2-DNMT3L complex with the previously reported DNMT3A ADD domain bound to a H3K4me0 peptide (PDB 4QBQ)19 as well as the DNMT3B PWWP domain bound to a H3K36me3 peptide (PDB 5CIU)47 mapped the H3K4me0 and H3K36me2-binding sites of the DNMT3A2-DNMT3L complex onto the ADD-MTase interface and the PWWP-ADD interface, respectively (Fig. 4a–c). At the ADD-MTase interface, the H3-binding site is largely accessible for the H3 binding, except that ADD residues D529 and D531 engage in electrostatic contact with TRD-loop residue R836, which competes against their potential salt-bridge interactions with H3K4me0 (Fig. 4b). At the PWWP-ADD interface, the H3K36me2 pocket, formed by PWWP residues F303, W306, W330 and D333, is blocked from potential H3K36me2 binding by ADD residue R544 (Fig. 4c). These observations link the H3K4me0 and H3K36me2/3 binding to the relief of the PWWP-mediated autoinhibition and ADD-mediated autoinhibition of DNMT3A, respectively.

To further elaborate how the PWWP- and ADD-mediated autoinhibition crosstalk with histone bindings, we performed in vitro DNA methylation analysis of DNMT3A2-DNMT3L, WT or mutant, on nucleosome substrates either unmodified or modified with H3 lysine-4 tri-methylation analogue (H3Kc4me3, denoted as H3K4me3 herein) or H3 lysine-36 di-methylation analogue (H3Kc36me2, denoted as H3K36me2 herein)48. On the H3K4me3 nucleosome substrates, the D529A/D531A-, R544A- and W330A-mutated DNMT3A2-DNMT3L show 6.5, 3.3 and 3.2-fold higher DNA methylation activity than WT DNMT3A2-DNMT3L (Fig. 4d), supporting the notion that these residues play a role in DNMT3A autoinhibition. In line with previous observations22,24,49,50, replacement of the H3K4me3 nucleosome with unmodified (H3K4me0) nucleosome led to 7-fold higher methylation efficiency for WT DNMT3A2-DNMT3L; the presence of H3K4me0/K36me2 dual mark further enhanced the DNA methylation efficiency of DNMT3A by 1.7-fold (Fig. 4d). On the other hand, introducing the D529A/D531A mutation at the ADD-MTase interface reduced the H3K4me0-mediated DNMT3A activation to 1.4-fold, while failing to affect the H3K36me2-mediated DNMT3A stimulation (Fig. 4d). Introducing the R544A or W330A mutation at the PWWP-ADD interface reduced the H3K36me2-mediated stimulation to 1.2-fold (for R544A) or even abolished the H3K36me2-mediated stimulation (for W330A); yet, the H3K4me0-mediated stimulation remains strong for both mutants (6.1-fold for R544A and 5.3-fold for W330A) (Fig. 4d). Finally, introducing the D529A/D531A/W330A triple mutation led to reduced (for H3K4me0) or completely lost (for H3K36me2) stimulation by either histone mark (Fig. 4d). Together, these observations support the notion that the respective interplay between DNMT3A PWWP and ADD domains with H3K4me0 and H3K36me2 marks lead to distinct yet additive functional regulation of DNMT3A.

It is worth noting that introducing the R836A mutation, while reducing the ADD-MTase association (Fig. 3b), led to decrease of DNA methylation activity on all three substrates (Supplementary Fig. 6), consistent with the fact that residue R836 plays a key role in CpG interaction33.

MD simulation analysis of DNMT3A activation
The TRD loop has previously been shown to mediate the specific recognition of CpG sites by DNMT3A and DNMT3B, with residue R836 of DNMT3A recognizing the CpG guanine (G1) of the substrate (Fig. 5a)32,33,51. In this context, structural overlay of the DNMT3A2-DNMT3L complex with the DNMT3AMTase-DNMT3LML-CpG DNA complex (PDB 5YX2) reveals strong steric clashes between the CpG DNA and the ADD and PWWP domains (Fig. 5a), suggesting that the PWWP-ADD-MTase interaction blocks TRD-residue R836 from potential contact with the CpG DNA.

Next, we sought to elucidate the activation mechanism of DNMT3A via MD simulation. Toward this, we generated a structural model of the apo-form DNMT3AADD-MTase-DNMT3L (apo-DNMT3AADD-MTase-DNMT3L) to mimic the functional state of DNMT3A2-DNMT3L in which the PWWP domain-mediated autoinhibition is relieved. In addition, a structural model of the DNMT3AADD-MTase-H3K4me0-DNMT3L complex was generated based on the structures of DNMT3A2-DNMT3L and DNMT3A ADD-H3K4me0 (PDB 4QBQ). The structural models were analyzed after 200-ns MD simulation (Fig. 5b, c). We found that the DNMT3A ADD domain remains positioned similarly between the DNMT3A2-DNMT3L complex, apo-DNMT3AADD-MTase-DNMT3L and the DNMT3AADD-MTase-H3-DNMT3L complex (Fig. 5b). However, the TRD loop undergoes a notable movement away from the ADD domain, resulting in increased solvent exposure (Fig. 5b). Consistently, our root-mean-square fluctuation (RMSF) analysis of the three structural models reveals that the RMSF of the TRD loop is the highest in the DNMT3AADD-MTase-H3-DNMT3L complex but the lowest in the DNMT3A2-DNMT3L complex under the PWWP domain-mediated autoinhibition (Fig. 5c). These observations suggest that relieving the PWWP domain-mediated autoinhibition and gaining the ADD-H3K4me0 interaction would lead to disengagement of TRD residue R836 from the autoinhibitory interaction, allowing the TRD loop to turn away from the ADD domain for potential DNA contact. On the other hand, the observation that the ADD domain remains positioned in place after the H3K4me0 binding implies that the interaction with both histone and DNA is likely required for the full activation of DNMT3A.

Functional analysis of the autoinhibition-disrupting disease mutations
The disease mutations of DNMT3A spread across all the functional domains of DNMT3A35,37,39,52, including those at the PWWP-ADD interface, such as AML-associated ADD-mutation E545G39 and microcephalic dwarfism-associated PWWP-mutation W330R and D333N34 (Fig. 6a). Our 1H,15N-HSQC NMR spectroscopic analysis of the ADD domain in the presence or absence of the PWWP domain reveals that introducing the W330R, D333N and E545G mutations all led to much reduced chemical shift perturbations of the NMR signals of the ADD domain, indicative of impaired PWWP-ADD interaction (Fig. 6b, c). To assess the impact of these mutations on DNMT3A activity, we performed in vitro DNA methylation assays of WT or mutant DNMT3A2-DNMT3L on DNA or nucleosome substrates. Compared with WT DNMT3A2-DNMT3L, the W330R, D333N and E545G mutations all led to increased DNA methylation activity on (GAC)12/(GTC)12 DNA duplex, reinforcing the notion that these mutations lead to impaired DNMT3A autoinhibition (Fig. 6d). On the other hand, these disease mutants exhibit WT-distinct substrate specificity on the nucleosome substrates: Compared with WT DNMT3A2-DNMT3L, the E545G mutant shows reduced DNA methylation activity on H3K4me0 and H3K36me2 nucleosomes but increased activity on the H3K4me3 nucleosome substrates, while W330R and D333N mutants both show increased activity on all three nucleosome substrates (Fig. 6e). The caveat of these assays is that they were performed in the presence of excessive amount of substrates, in contrast to the in vivo condition where the enzyme-substrate association also constitutes a crucial reaction step. Nevertheless, these nucleosome context-dependent activity alterations of DNMT3A by the PWWP-ADD interface mutations provide an explanation for the aberrant DNA methylation patterns associated with these mutations in disease.

The DNMT3A W330R mutation has previously been shown to shift the DNMT3A1-mediated DNA methylation to CpG islands (CGIs), in part attributed to abolished PWWP-H3K36me2 binding and the interaction between the DNMT3A1 UDR and the H2AK119ub1-modified nucleosome10–14. In light of the observation above that the W330R mutations also lead to disruption of the PWWP-mediated autoinhibition, we asked whether this mutation causes an increase of DNA methylation in the genomic regions beyond the CGIs. To address this, we analyzed the Reduced Representation Bisulfite Sequencing (RRBS) methylome, focusing on non-CGI regions, in mouse embryonic stem cells (mESCs) with Dnmt1/Dnmt3A/Dnmt3B triple knockout (TKO) or Dnmt1/Dnmt3A/Dnmt3B/Dnmt3L quadruple knockout (QKO) transduced with WT or W330R DNMT3A113. In line with the previous observation11,13,34, in the H3K36me2-positive regions, the TKO cells with DNMT3A1 W330R show a lower level of DNA methylation than those with WT DNMT3A1. In contrast, in the H3K36me2-poor regions the W330R cells show a significantly higher level of DNA methylation than WT cells (Fig. 6f). Consistently, in contrast to the activity of WT DNMT3A1 that is positively correlated with the H3K36me2 level, the activity of W330R is largely independent of H3K36me2 and greater than that of WT DNMT3A1 at the low-H3K36me2 regions (Fig. 6g). A similar, although modest, trend of DNA methylation patterns was observed for the QKO cells with WT or W330R DNMT3A1 (Supplementary Fig. 7). These observations suggest that in addition to the impact by the UDR-H2AK119ub1 interaction, disruption of the PWWP domain-mediated DNMT3A autoinhibition by the W330R mutation leads to promiscuous DNA methylation throughout the genome.

Methods
Protein expression and purification
The DNA encoding human DNMT3A PWWP-ADD-MTase fragment (residue 281–912) and DNMT3L (residue 1–386) were separately inserted into an in-house bacterial expression vector in which the inserted gene is preceded by a hexa-histidine (His6)-MBP tag and a TEV cleavage site. BL21(DE3)RIL cells harboring the expression plasmids grew at 37 °C and were induced by addition of 0.13 mM (for DNMT3A) or 0.2 mM (for DNMT3L) isopropyl β-D-1-thiogalactopyranoside (IPTG) with 50 μM ZnCl2 when the cell density reached A600 of 1.0. The cells continued to grow at 16 °C overnight. The DNMT3A and DNMT3L cells were collected and co-lysed in a buffer containing 50 mM Tris-HCl (pH 8.0), 1 M NaCl, 25 mM Imidazole, 10% glycerol, 10 μg/mL DNase I and 1 mM PMSF. Subsequently, the fusion proteins were purified through a nickel column, followed by removal of His6-MBP tag by TEV cleavage, HiTrap Heparin HP (Cytiva) ion-exchange chromatography and size-exclusion chromatography on a 16/600 Superdex 200pg column (Cytiva) in a buffer containing 20 mM Tris (pH 8.0), 250 mM NaCl, 5% Glycerol, and 5 mM DTT. The mutants were generated by site-directed mutagenesis and purified in the same way as described above. The primers used in this study were summarized in Supplementary Table 2

The DNA encoding DNMT3AMTase (residue 628–912) and DNMT3AADD-MTase (residue 469–912), WT or mutants, and DNMT3LML (residue 178–386) were inserted in tandem into a modified pRSFDuet-1 vector (Novagen), preceded by a His6-Sumo tag and a ULP1 cleavage site. Expression and purification of the DNMT3AMTase–DNMT3LML and DNMT3AADD-MTase–DNMT3LML complex followed the previously described protocol33, involving nickel chromatography, ion-exchange chromatography on a HiTrap Heparin HP column and size-exclusion chromatography on a 16/600 Superdex 200pg column (Cytiva), pre-equilibrated with a buffer containing 20 mM Tris (pH 8.0), 250 mM NaCl, and 5 mM DTT. For analysis of domain interactions, DNMT3A PWWP (residues 281–427), ADD (residues 476–614) and mutants were each cloned into the modified pRSFDuet-1 vector, and expressed and purified following the previously described protocol46. In essence, the DNMT3A PWWP domain was purified sequentially through nickel chromatography, ion-exchange chromatography on a HiTrap Heparin HP column and size-exclusion chromatography on a 16/600 Superdex 75pg column (Cytiva), pre-equilibrated with a buffer containing 20 mM Tris (pH 8.0), 300 mM NaCl and 5 mM DTT. The DNMT3A ADD domain was purified sequentially through nickel chromatography, ion-exchange chromatography on a HiTrap Q HP column (Cytiva) and size-exclusion chromatography on a 16/600 Superdex 75pg column (Cytiva), pre-equilibrated with a buffer containing 20 mM Tris (pH 8.0), 300 mM NaCl and 5 mM DTT. For the NMR experiments, 15N-labeled DNMT3A ADD domain was produced in M9 minimal media supplemented with 15NH4Cl as sole nitrogen resource and purified as described above. All the purified protein samples were concentrated and stored in -80 °C freezer.

Cryo-EM sample preparation and data acquisition
For cryo-EM sample preparation, concentrated DNMT3A2-DNMT3L complex was subjected to size-exclusion chromatography on a Superdex 200 increase 10/300 column (Cytiva) pre-equilibrated with a buffer containing 20 mM Tris–HCl (pH 8.0), 100 mM NaCl, 5% glycerol and 5 mM DTT. The peak fraction, without treatment by chemical crosslinker, was used for subsequent handling. An aliquot of 2.5 μL of DNMT3A2-DNMT3L sample at a concentration of ~1.23 mg/ml was applied to a Quantifoil holey carbon grid (Au, R1.2/1.3, 300 mesh). The grids were glow-discharged for 1 min with H2/O2 through Gatan Plasma clean System (SOLARUS) before use. The grids were blotted and plunge-frozen in liquid ethane cooled by liquid nitrogen with a Vitrobot IV (Thermo Fisher) at 8 °C under 100% humidity. The frozen grids were stored in liquid nitrogen before use. High-resolution cryo-EM data were collected on a Titan Krios electron microscope operating at 300 keV, equipped with a Falcon IV at Pacific Northwest Center for Cryo-EM (PNCC). Movies were recorded at a nominal magnification of ×130,000 with a pixel of 0.7675 Å with the defocus range of −0.8 to −2.3 μm. Each micrograph was recorded with 29 frames with a total dose rate of around 50 e−/Å2.

Cryo-EM data processing and model building
The cryo-EM data were processed using cryoSPARC (v4.4.1)54. The movies were motion-corrected and dose-weighted using the patch-based motion correction module. The contrast transfer function (CTF) of the resulting images was then subjected to patch-based estimation. Automatic particle picking was performed using the TOPAZ method55. In total 862,300 particles were extracted from 4756 images, with a down-scaled pixel size of 3.21 Å. After two rounds of clean-up by 2D classifications, those classes with identifiable secondary or tertiary structures, comprised of 527,682 particles, were selected. Initial models were then generated by 3D ab initio reconstruction, followed by one round of heterogeneous refinement (K=4) applying C1 symmetry. The class with well-defined density for the PWWP domain was re-extracted with a pixel size of 1.203 Å and subjected to another round of heterogeneous refinement (K=3). Finally, 88,258 particles were selected for non-uniform and CTF refinement, resulting in a map with a resolution of 3.64 Å. To improve the density of the PWWP domain, the refined map was used to perform template-based particle picking and 2,634,423 particles were extracted with down-scaled pixel size of 3.21 Å. The extracted particles were evenly divided into six subgroups, while 88,258 before mentioned high-quality particles were treated as “seed” particles and a round of seed-facilitated 3D classification56 was performed. After removing duplicate particles, 876,165 high-quality particles were selected, re-extracted with a pixel size of 1.203 Å and subjected to two rounds of heterogeneous refinement (K=3 and K=4). Then 209,307 “good” particles were selected. Subsequently, a 3D focused classification with a mask applied to the density of the ADD domain and PWWP was conducted (K=4), and 65,578 particles with identified PWWP features were selected for the final non-uniform and CTF refinement. The final map was sharpened with uniform B factor of −116.28 Å2 for further model building and deepEMhancer57 for visualization. The resolution for the DNMT3A-3L tetramer was 3.66 Å as given by the Fourier shell correlation criteria (FSC0.143).

For model building, the reported crystal structures of the DNMT3AADD-MTase (PDB 4U7P), PWWP (PDB 3LLR) and DNMT3L (PDB 2PV0) were manually fit into the sharpened map in Chimera (v1.17.3)58. The initial structural model was then subjected to iterative model building using Coot (v0.9.8)59 and real-space refinement using Phenix (v1.21.2_5419)60. For the structure, a model-map Fourier shell correlation was calculated using the criterion of 0.5.

Isothermal Titration Calorimetry (ITC) binding assay
WT DNMT3AMTase-DNMT3LML, DNMT3AADD-MTase-DNMT3LML, ADD, PWWP and mutants were dialyzed at 4 °C overnight against a buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 1 mM β-mercaptoethanol. The final concentrations, determined based on ultraviolet absorption at 280 nm, were 30–50 μM for DNMT3A-DNMT3L tetramer and mutants and 0.3–0.5 mM for the PWWP and ADD domains, WT or mutant.

A MicroCal iTC200 system (GE Healthcare) was used to conduct ITC measurements. A total of 15–17 injections with a spacing of 180 s and a reference power of 5 μcal/s were performed after the temperature was equilibrated to 4 °C. The ITC curves were processed with software Origin (MicroCal) using one-site fitting model.

Preparation of chemically modified nucleosome
Histones (H2A, H2B, WT or K4C/C110S or K36C/C110S-mutated H3, and H4) from Xenopus laevis were expressed and purified under denatured condition as previously described12. Briefly, WT and mutant histones were expressed in Escherichia coli BL21(DE3)RIL at 37 °C. The cells were induced by addition of 0.4 mM IPTG when the cell density reached A600 of 1.0 and continued to grow 1 h. The cells were harvested and lysed in a buffer containing 50 mM Tris-HCl (pH 8.0), 0.2 M NaCl and 1% Triton X-100. Histone inclusion bodies were solubilized and denatured in 20 mM Tris-HCl (pH 7.5), 7 M guanidine hydrochloride and 10 mM DTT. Subsequently, denatured histones were purified through sequential ion-exchange chromatography on Q-XL and SP-HP columns (Cytiva) using the buffer containing 20 mM Tris-HCl (pH 7.5), 7 M urea and 2 mM β-mercaptoethanol with a KCl gradient of 0 to 1 M. The purified histone proteins were thoroughly dialyzed against distilled water containing 2 mM β-mercaptoethanol, lyophilized and stored at −80 °C. The K4C and K36C-mutated H3 proteins were chemically alkylated to Kc4me3 (denoted as H3K4me3 herein) and Kc36me2 (denoted as H3K36me2 herein) respectively as previously described48. In essence, lyophilized histones (5–10 mg) were each dissolved in ~1 mL of alkylation buffer containing 1 M Hepes (pH 7.8), 4 M guanidine hydrochloride, 10 mM D/L-methionine, and then mixed with 20 μL of 1 M DTT and incubated at 37 °C for 1 h. Alkylation of H3K36C was carried at room temperature, with the reaction mixture subject to sequential additional of 50 μL of 1 M (2-chloroethyl)-dimethylammonium chloride (Sigma-Aldrich), 10 μL of 1 M DTT, and 50 μL of 1 M (2-chloroethyl)-dimethylammonium chloride. Alkylation of H3K4C was performed at 50 °C, with the reaction mixture subject to sequential addition of 100 mg (2-bromoethyl)trimethylammonium bromide and 10 μL of 1 M DTT. All alkylation reactions were quenched by addition of β-mercaptoethanol (50 μL, 14.2 M), followed by dialysis against distilled water containing 2 mM β-mercaptoethanol. The histone octamer was reconstituted by mixing four histones with equal molar and dialysis in refolding buffer (20 mM Tris-HCl pH 7.5, 2 M NaCl, 1 mM EDTA and 5 mM 2-mercaptoethanol) and further purified by size-exclusion chromatography on a HiLoad 16/600 Superdex 200pg column.

The Widom 601 DNA flanked by a 35-bp DNA sequence (5’-GTCGTCGTCGTCGTCGTCGTCGTCGTCGTCGTCGTATCGAGAATCCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACATCCGAT-3’; Widom 601 DNA sequence is underlined) was PCR amplified and purified as previously described12. To assemble the nucleosome, purified histone octamer was mixed with the 601 DNA in a 1:1 molar ratio, followed by stepwise dialysis against a buffer containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM DTT and 0.25–2 M KCl. Finally, the assembled NCP was dialyzed against a buffer containing 10 mM Tris-HCl (pH 8.0) and 1 mM DTT overnight. The assembled NCP was analyzed in 5% Tris-borate-EDTA (TBE) native gel in 0.2× TBE buffer (89 mM TBE, pH 8.3). The DNA band was visualized using SYBR Gold staining and scanned using a ChemiDoc imager (Bio-Rad).

In vitro enzymatic assay
For comparison of the DNA methylation activity between WT and mutant DNMT3A-DNMT3L on nucleosomes, a 20-μL reaction mixture contained 0.1 μM DNMT3A-DNMT3L, 0.4 μM assembled nucleosome, 0.55 μM S-adenosyl-L-[methyl-3H]methionine (specific activity 87.9 Ci/mmol, PerkinElmer), 0.65 μM non-radioactive AdoMet in 50 mM Tris–HCl (pH 8.0), 75 mM NaCl, 0.05% β-mercaptoethanol, 0.02% NP-40, 10% glycerol and 200 μg/mL BSA was incubated at 37 °C for 30 min and quenched by addition of 5 μL of 10 mM non-radioactive AdoMet. For detection, 8 μL of the reaction mixture was spotted on Amersham Hybond-N+ paper (Cytiva) and air dried. The paper was then washed with 0.2 M cold ammonium bicarbonate (pH 8.2) (twice), Milli Q water and ethanol. Subsequently, the paper was air dried and transferred to scintillation vials filled with 3 mL ScintiVerse cocktail (Thermo fisher). The radioactivity of tritium was measured with a Beckman LS6500 counter. For the comparison of activity on naked DNA, a 36-mer (GAC)12/(GTC)12 DNA duplex was used as the substrate and the reaction condition is similar as above, except that the concentration of DNMT3A-DNMT3L and dsDNA were kept at 0.2 μM and 0.75 μM, respectively. Each methylation assay was carried out in triplicate and plotted in the GraphPad Prism (v.10).

Fluorescence polarization (FP) assay
For FP assay, 15 nM FAM-labeled 24-mer DNA duplex (upper strand: /5FAM-T/CGGACAGGATGTATATATCTGAC; lower strand: GTCAGATATATACATCCTGTCCGA) was mixed with various concentrations (0.01, 0.02, 0.04, 0.08, 0.15, 0.31, 0.62, 1.25, 2.5, and 5 μM) of DNMT3A2-DNMT3L for 30 min at 4 °C in a buffer containing 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5% glycerol, and 5 mM DTT. The FP measurements were performed on a ClarioStar plus plate reader (BMG LABTECH) at 25 °C. The DNA bound fractions were calculated as (mP − baseline mP)/(maximum mP − baseline mP), where mP represents the FP value. Each reaction was carried out in triplicate and the bound fractions were fit against protein concentrations using the specific binding model with Hill slope in GraphPad Prism (v.10).

NMR spectroscopy
Uniformly 15N-labeled WT or mutant DNMT3A ADD, ADDE545G and PWWP domains were dialyzed overnight against buffer containing 10 mM Tris–HCl (pH 7.2), 100 mM NaCl, and 1 mM DTT and quantified based on ultraviolet absorption at 280 nm. The NMR spectroscopy was performed at 20 °C on a 700 MHz spectrometer (Bruker Avance) equipped with a cryogenic probe in a buffer containing 10 mM Tris–HCl (pH 7.2), 50 mM NaCl, 1 mM dithiothreitol (DTT), and 10% (v/v) D2O. 1H,15N–HSQC NMR spectra were acquired for 0.2 mM 15N-labeled DNMT3A ADD in absence or presence of equimolar amounts of PWWP, WT or mutant. Similarly, the 1H,15N–HSQC NMR spectra of 0.2 mM 15N-labeled DNMT3A ADD E545G were collected in the absence or presence of equimolar WT DNMT3A PWWP. The NMR spectra were processed using TopSpin (Bruker BioSpin) and analyzed with Mnova NMR (Mestrelab Research).

Molecular dynamics (MD) simulation
The structural models of the DNMT3A2-DNMT3L, DNMT3AADD-MTase-DNMT3L and DNMT3AADD-MTase-H3-DNMT3L complexes were used for molecular dynamic (MD) simulation of the DNMT3APWWP-ADD-MTase, DNMT3AADD-MTase and DNMT3AADD-MTase-H3 states. In essence, the structural model of DNMT3AADD-MTase-DNMT3L was generated by removal of the PWWP domain in the DNMT3A2-DNMT3L complex. The structural model of DNMT3AADD-MTase-H3-DNMT3L was obtained based on the structural overlay between the DNMT3A2-DNMT3L complex and the DNMT3A ADD-H3K4me0 complex (PDB 4QBQ). MD simulations were performed using the AMBER20 package61. The partial charges of S-adenosylhomocysteine (SAH) were calculated using AM1-BCC. MD simulations were initiated using the ff14SB force field for protein and GAFF2 for ligand. ZAFF force field was used to stabilize the zinc finger62. Covalent bonds were added to the 4 cysteine nearest to each zinc, for a total of 48 zinc-cysteine covalent bonds. All systems were solved in water boxes 12 Å from the protein edge using TIP3P explicit solvent model at temperature of 298 K with NPT ensemble. Na+ ions were added to neutralize the system. A 12-Å cutoff was used for short-range non-bonded interactions and the long-range electrostatic interactions were computed by the particle mesh Ewald method (PME). The water molecules were minimized for 10,000 steps, followed by minimization of the entire system for 20,000 steps (Supplementary Table 3). The solvated system was equilibrated under constant pressure and temperature (NPT ensemble) at 50 K for 200 ps, from 75 K to 275 K with 25 K increments and 100 ps each, and finally at 298 K for 400 ps. Production runs were also performed in the NPT ensemble at 298 K using a Langevin Thermostat with 2-fs time-steps. We performed 200-ns MD saving every 1-ps interval, then resaved the trajectories every 1 ns, resulting in 200 frames for analysis. To understand the differences in dynamics, simulations were visualized using Visual Molecular Dynamics (VMD) software63. The RMSF of each alpha carbon was calculated based on alignment of DNMT3A residues directly in VMD. The RMSF values were averaged with standard deviation across three replicas and plotted versus residue position. The coordinates resulting from the MD simulations were summarized in Supplementary Data 1.

CUT&RUN and reduced representation bisulfite sequencing (EM-seq) data analysis
H3K36me2 CUT&RUN sequencing data from QKO (Dnmt1/Dnmt3A/Dnmt3B/Dnmt3L KO) mESC (GSE247019) was analyzed using the multiBigwigSummary function from DeepTools (v3.5.1)64 to quantify H3K36me2 CUT&RUN signal in 10-kb genome-wide bins. H3K36me2 domains were called using SEACR (v1.3)65. Bismark coverage files of TKO (Dnmt1/Dnmt3A/Dnmt3B KO) and QKO mESC EM-seq data (DNA Methylation sequencing) (GSE247019) were analyzed using methylkit (v1.26)66. All CpGs with at least 2 reads were retained, and the DNA methylation status of 10-kb bins with at least 5 CpGs were retained for analysis. To focus on the non-CGI genomic regions, 10-kb bins overlapping CGIs were left out of the downstream analysis.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript reports the cryoEM structure of the hetero-tetrameric DNMT3A-DNMT3L complex. In this structure, one of\nthe DNMT3A subunits displays a well resolved PWWP domain assembled together with the ADD and MTase domains. This\narrangement reveals that not only is the DNA-binding loop in the MTase domain blocked by ADD and PWWP domains, but\nthe H3-binding pockets within both the ADD and PWWP domains are also occluded. Based on these observations, the\nauthors propose a hierarchical autoinhibition mechanism that may underlie the differential DNMT3A activity observed on\nvariously modified nucleosomes. Overall, this study provides the first structural visualization of the PWWP, ADD and MTase\ndomains of DNMT3A assembled in an auto-inhibitory conformation. These findings offer mechanistic insight into how\nDNMT3A adopts its inactive state and explains why disease-linked mutations at the PWWP-ADD interface lead to aberrant\nDNA methylation. A few specific concerns and suggestions are outlined below.\n1. Several previous studies have shown that the PWWP domain is highly flexible in both DNMT3A and DNMT3B. However,\nthis manuscript unexpectedly resolves the structure of the PWWP domain assembled with the ADD and MTase domains in\nan auto-inhibitory conformation. Although this finding is surprising, the ITC experiment presented in Figure 2d indeed\nsupports interactions between the PWWP domain and the PWWP-truncated DNMT3A-DNMT3L complex. Could the authors\nelaborate on the possible reasons why the PWWP domain appears less flexible in their study?\n2. In the active state, the ADD domain in DNMT3A undergoes a positional shift relative to the MTase domain as\ndemonstrated in previous structural studies. Similarly, the PWWP domain is also expected to change its position in the\nactive state, as suggested by the model shown in Figure 7. To further support this model, do the authors have any evidence\nindicating that the PWWP domain interacts differently with the PWWP-truncated DNMT3A-DNMT3L complex in the\npresence of the nucleosome substrates?", "reviewer_2": "This is an exciting manuscript centered on DNMT3A/L structures that reveal a new autoregulatory mechanism, whose\nphenomenology has been described, but whose molecular details remained mysterious. The key breakthrough here is the\nnew structural observation of a PWWP domain of DNMT3A packing against the enzyme active site and occluding histone\nrecognition pockets, that is convincingly argued to be a novel autoinhibited state of the complex. The experiments are highly\nrigorous and combine the strengths of NMR and cryoEM, as well as convincing accompanying biochemistry to define the\nnovel mode of PWWP interaction with the ADD and methyltransferase domains that serves to sharpen the previously noted\nnucleosome substrate specify, that was thought to be purely driven by favorable binding enthalpy as opposed to a dynamic\ninterplay of the PWWP domain occluding the active site and histone binding pockets.\nThis goes beyond merely defining the structural and biochemical basis of H3K36me regulation of DNMT3a/L, the reanalysis\nof prior W330R mutant methylation patterns is consistent with the proposed model, but also can be interpreted as reduced\naffinity for H3K36me3 by ablation of the aromatic cage that binds H3K36me3, a mutant that separates this function from\nPWWP autoinhibition would be needed to make a more robust conclusion from these data, but given the strength of the in\nvitro data, I am convinced that their newly discovered mechanism is operant in cells. Several noted disease associated\nmutations can be reinterpreted in light of the toggling in between the autoinhibited state and the open state, and these are\ntested in vitro as well.\nAs this is an extremely important enzyme in cancer, development and epigenetic inheritance, this new regulatory\nmechanism will be of interest to a broad readership of Nature Communications, and likely extends to the related DNMT3B/L\ncomplex.\nWhile the gross features are similar to prior structures, one of the two 3A protomers in the tetramer has the PWWP domain\nmodellable, and it is oriented to block the DNA methyltransferase active site, the ADD domain that binds unmethylated H3\ntail, and the PWWP’s binding pocket that has been previously shown to bind H3K36me2.\nEvidence that the newly discovered autoinhibition interface is real and important is quite strong—mutations, ITC and NMR\nall support the interpretation that the PWWP-ADD autoinhibited interface observed in the new structure represents state in\ncells that is likely populated. Moreover, the inhibition of histone binding and methyltransferase activity being relieved by\nmutants that disrupt the PWWP-ADD and PWWP-MTase interfaces strengthens this interpretation. It is speculated based on\nTRD loop position and superposition with CpG DNA bound forms of the complex this PWWP packing inhibition mode would\nalso occlude this interface, but it is not explicitly tested beyond and MD simulation (and would be challenging to actually\ntest). Importantly, the authors also show that this autoinhibition mode also sharpens the nucleosomal substrate preferences\npreviously noted in the literature for H3K4me0 and H3K36me2 for the ADD domain and PWWP-domain, respectively.\nMajor point—a mutant that disrupts the autoinhibited state that has clean separation of function from other individual module\nfunctions, tested in living cells would improve the case that this mechanism is relevant in cells, but I do not think is essential\nas the accumulated in vitro data is quite compelling on its own and consistent with prior cell and organism based studies.\nApart from a few typos, the paper is well written and figures well made to convey the key structural details, the rigor of the\ndata is high and key points are made by multiple lines of unambiguous evidence.\nMinor points:\nMethylysine analogs are used in this work rather to mimic the normal counterparts. Given noted deviation of MLAs from the\nnative modifications observed in other contexts, I think this should be more prominently noted in the main text rather than\nappearing solely in the methods to avoid confusion.\nThe Kd reported for the ADD-PWWP interface needs to be appropriately truncated for significant digits (8+/-2 uM). Similar\nconsiderations apply to Fig 3b Kds.\nA few typos to fix: line 56: “H3K36m2” , line 254 “bindings”", "reviewer_3": "In this manuscript the authors present a thorough and relevant study that nicely integrates structural information with\nbiophysical, biochemical and molecular modeling studies to offer mechanistic insight into orchestration of the\nDNMT3A2/DNMT3L association with histone mark recognition, DNA substrate binding, and enzymatic methylation activity.\nThis not only provides key mechanistic insight into the regulation of DNMT3A activity in normal cell function but importantly\noffers mechanistic perspective for how site-specific disease associated protein mutations disrupt key auto-inhibiting\ninteractions to support observed hypermethylated DNA disease phenotypes. The inclusion of mutational studies in\northogonal biophysical and biochemical assays provides compelling support for the structural studies and the core\nconclusions presented. Overall, this is a comprehensive, timely and important investigation that advances our understanding\nof the molecular mechanisms by which epigenetic alterations and aberrant DNA methylation accumulation may occur to\npromote disease conditions. The findings are highly valuable for advancing the field. Nevertheless, there are a few points\nthat should be addressed prior to publication that would enhance clarity of presentation, strengthening the major findings.\n1) In the Abstract, most of the acronyms are defined, but not PWWP and ADD. It is recommended to define these to be\nconsistent with defining all the other acronyms.\n2) Fig. 2: In Fig. 2d, as well as 3b, there does not seem to be error bars included on the presented ITC data, which should be\nadded in.\nRegarding the presented 1H/15N HSQC in Fig. 2e, it is difficult to differentiate the magenta from red peaks, making it difficult\nto readily evaluate the author’s conclusions. It is also stated in the text that addition of WT PWWP results in “…substantial\nchemical shift perturbation of several…” ADD NMR resonances, though there seems to be very few ADD resonances that\nexperience a chemical shift perturbation and the majority of the ones that do have small chemical shift perturbations. In\naddition, it is claimed that addition of the PWWP W330A variant shifts the ADD resonances “…back to..” the non-interacting\nform. The presented spectra support the conclusion that the ADD resonances move back toward the unbound ADD form, but\ndo not revert back fully. It is thus recommended to attenuate the claims in these two statements. Having said that, it is again\ndifficult to globally assess these claims given the lack of contrast between the red and magenta spectra.\n3) The Fig. 4 legend denotes f, rather than d. It is stated in regard to the DNA methylation assay data that neither variant\nR544A or W330A affected “…H3K4me0-mediated stimulation appreciably…”. It is presumed this statement is being made\nrelative to WT, in which it appears that the difference between H3K4me3 and H3K4me0 substrates in activating DNMT3A is\ngreater for both variants than the 7-fold difference observed for the WT."}, "author_response": "General Response\nWe thank all reviewers for their collective efforts in reviewing our manuscript, their positive\nview of our work, and their constructive comments for improving the manuscript. As\noutlined below in the point-by-point response (marked in blue), we have now\nsystematically addressed all the raised critiques and have incorporated them in the\nrevised manuscript (marked in red).\nPoint-by-point Response\nResponse to Reviewer 1\nThis manuscript reports the cryoEM structure of the hetero-tetrameric DNMT3A-DNMT3L\ncomplex. In this structure, one of the DNMT3A subunits displays a well resolved PWWP\ndomain assembled together with the ADD and MTase domains. This arrangement reveals\nthat not only is the DNA-binding loop in the MTase domain blocked by ADD and PWWP\ndomains, but the H3-binding pockets within both the ADD and PWWP domains are also\noccluded. Based on these observations, the authors propose a hierarchical autoinhibition\nmechanism that may underlie the differential DNMT3A activity observed on variously\nmodified nucleosomes. Overall, this study provides the first structural visualization of the\nPWWP, ADD and MTase domains of DNMT3A assembled in an auto-inhibitory\nconformation. These findings offer mechanistic insight into how DNMT3A adopts its\ninactive state and explains why disease-linked mutations at the PWWP-ADD interface\nlead to aberrant DNA methylation. A few specific concerns and suggestions are outlined\nbelow.\nWe thank the Reviewer for his/her positive assessment of this work and have addressed\nhis/her concerns below.\n1. Several previous studies have shown that the PWWP domain is highly flexible in both\nDNMT3A and DNMT3B. However, this manuscript unexpectedly resolves the structure of\nthe PWWP domain assembled with the ADD and MTase domains in an auto-inhibitory\nconformation. Although this finding is surprising, the ITC experiment presented in Figure\n2d indeed supports interactions between the PWWP domain and the PWWP-truncated\nDNMT3A-DNMT3L complex. Could the authors elaborate on the possible reasons why\nthe PWWP domain appears less flexible in their study?\nThe Reviewer’s point is well taken. Indeed, compared with the stable ADD-MTase\ninteraction, the interaction of the PWWP domain with the ADD and MTase domains is\nrather dynamic: within the DNMT3A homodimer, the PWWP domain is only traceable for\none DNMT3A subunit, but not the other; in contrast, the ADD domains in both DNMT3A\nsubunits are well defined. To define the conformational state of the PWWP domain, we\nresorted to a “seed”-facilitated 3D classification approach, combined with focused\nclassification with a mask applied to the density of the ADD domain and PWWP. These\ndata processing approaches permitted us to obtain the defined density for the PWWP\ndomain. We have described our data processing pipeline in detail in the Method section\nand supplementary materials.\n2. In the active state, the ADD domain in DNMT3A undergoes a positional shift relative to\nthe MTase domain as demonstrated in previous structural studies. Similarly, the PWWP\ndomain is also expected to change its position in the active state, as suggested by the\nmodel shown in Figure 7. To further support this model, do the authors have any evidence\nindicating that the PWWP domain interacts differently with the PWWP-truncated\nDNMT3A-DNMT3L complex in the presence of the nucleosome substrates?\nOur structural analysis indicates that the PWWP-ADD interaction leads to occlusion of\nthe H3K36me2-binding site in the PWWP domain. Therefore, in the active state, the\ninteraction of the PWWP domain with the nucleosome substrate would conceivably\ncompete against the ADD-PWWP interaction, leading to repositioning of the PWWP\ndomain. Our unpublished data supports this notion. Following the Reviewer’s comments,\nwe have clarified this point in the discussion section (Line 372-375) of the revised\nmanuscript.\nResponse to Reviewer 2\nReviewer #2 (Remarks to the Author):\nThis is an exciting manuscript centered on DNMT3A/L structures that reveal a new\nautoregulatory mechanism, whose phenomenology has been described, but whose\nmolecular details remained mysterious. The key breakthrough here is the new structural\nobservation of a PWWP domain of DNMT3A packing against the enzyme active site and\noccluding histone recognition pockets, that is convincingly argued to be a novel\nautoinhibited state of the complex. The experiments are highly rigorous and combine the\nstrengths of NMR and cryoEM, as well as convincing accompanying biochemistry to\ndefine the novel mode of PWWP interaction with the ADD and methyltransferase domains\nthat serves to sharpen the previously noted nucleosome substrate specify, that was\nthought to be purely driven by favorable binding enthalpy as opposed to a dynamic\ninterplay of the PWWP domain occluding the active site and histone binding pockets.\nThis goes beyond merely defining the structural and biochemical basis of H3K36me\nregulation of DNMT3a/L, the reanalysis of prior W330R mutant methylation patterns is\nconsistent with the proposed model, but also can be interpreted as reduced affinity for\nH3K36me3 by ablation of the aromatic cage that binds H3K36me3, a mutant that\nseparates this function from PWWP autoinhibition would be needed to make a more\nrobust conclusion from these data, but given the strength of the in vitro data, I am\nconvinced that their newly discovered mechanism is operant in cells. Several noted\ndisease associated mutations can be reinterpreted in light of the toggling in between the\nautoinhibited state and the open state, and these are tested in vitro as well.\nAs this is an extremely important enzyme in cancer, development and epigenetic\ninheritance, this new regulatory mechanism will be of interest to a broad readership of\nNature Communications, and likely extends to the related DNMT3B/L complex.\nWhile the gross features are similar to prior structures, one of the two 3A protomers in the\ntetramer has the PWWP domain modellable, and it is oriented to block the DNA\nmethyltransferase active site, the ADD domain that binds unmethylated H3 tail, and the\nPWWP’s binding pocket that has been previously shown to bind H3K36me2.\nEvidence that the newly discovered autoinhibition interface is real and important is quite\nstrong—mutations, ITC and NMR all support the interpretation that the PWWP-ADD\nautoinhibited interface observed in the new structure represents state in cells that is likely\npopulated. Moreover, the inhibition of histone binding and methyltransferase activity being\nrelieved by mutants that disrupt the PWWP-ADD and PWWP-MTase interfaces\nstrengthens this interpretation. It is speculated based on TRD loop position and\nsuperposition with CpG DNA bound forms of the complex this PWWP packing inhibition\nmode would also occlude this interface, but it is not explicitly tested beyond and MD\nsimulation (and would be challenging to actually test). Importantly, the authors also show\nthat this autoinhibition mode also sharpens the nucleosomal substrate preferences\npreviously noted in the literature for H3K4me0 and H3K36me2 for the ADD domain and\nPWWP-domain, respectively.\nThe Reviewer has summarized this study very well. We thank the Reviewer for his/her\npositive view of this work.\nMajor point—a mutant that disrupts the autoinhibited state that has clean separation of\nfunction from other individual module functions, tested in living cells would improve the\ncase that this mechanism is relevant in cells, but I do not think is essential as the\naccumulated in vitro data is quite compelling on its own and consistent with prior cell and\norganism based studies.\nWe agree with the Reviewer that inclusion of a mutant that not only disrupts the\nautoinhibitory state but also has clear separation from other individual module functions\nin cells would further clarify the importance of this autoinhibitory mechanism in vivo.\nHowever, the lack of a detailed molecular understanding of the interaction between\nDNMT3A and its in vivo substrate, nucleosomes, makes a clear-cut design of such\nmutation rather challenging. To alleviate the Reviewer’s concern, we have clarified this\npoint by including the statement “Future investigation of such DNMT3A autoinhibition, e.g.\nvia a mutation that disrupts autoinhibition but not any other regulations of DNMT3A, may\nfurther clarify this link.” (Line 403-405) in the discussion in the revised manuscript.\nApart from a few typos, the paper is well written and figures well made to convey the key\nstructural details, the rigor of the data is high and key points are made by multiple lines\nof unambiguous evidence.\nWe thank the reviewer again for his/her assessment of this paper.\nMinor points:\nMethylysine analogs are used in this work rather to mimic the normal counterparts. Given\nnoted deviation of MLAs from the native modifications observed in other contexts, I think\nthis should be more prominently noted in the main text rather than appearing solely in the\nmethods to avoid confusion.\nFollowing the Reviewer’s suggestion, we have clarified this in the result section (Line 259-\n261).\nThe Kd reported for the ADD-PWWP interface needs to be appropriately truncated for\nsignificant digits (8+/-2 uM). Similar considerations apply to Fig 3b Kds.\nWe have rounded up the Kd values in Fig 2 & 3 accordingly.\nA few typos to fix: line 56: “H3K36m2” , line 254 “bindings”\nThese typos have been fixed.\nResponse to Reviewer 3\nIn this manuscript the authors present a thorough and relevant study that nicely integrates\nstructural information with biophysical, biochemical and molecular modeling studies to\noffer mechanistic insight into orchestration of the DNMT3A2/DNMT3L association with\nhistone mark recognition, DNA substrate binding, and enzymatic methylation activity. This\nnot only provides key mechanistic insight into the regulation of DNMT3A activity in normal\ncell function but importantly offers mechanistic perspective for how site-specific disease\nassociated protein mutations disrupt key auto-inhibiting interactions to support observed\nhypermethylated DNA disease phenotypes. The inclusion of mutational studies in\northogonal biophysical and biochemical assays provides compelling support for the\nstructural studies and the core conclusions presented. Overall, this is a comprehensive,\ntimely and important investigation that advances our understanding of the molecular\nmechanisms by which epigenetic alterations and aberrant DNA methylation accumulation\nmay occur to promote disease conditions. The findings are highly valuable for advancing\nthe field. Nevertheless, there are a few points that should be addressed prior to\npublication that would enhance clarity of presentation, strengthening the major findings.\nWe thank the Reviewer for his/her positive assessment of this work.\n1) In the Abstract, most of the acronyms are defined, but not PWWP and ADD. It is\nrecommended to define these to be consistent with defining all the other acronyms.\nWe thank the reviewer for the suggestion. We have defined the PWWP and ADD domains\nin the abstract in the revised manuscript.\n2) Fig. 2: In Fig. 2d, as well as 3b, there does not seem to be error bars included on the\npresented ITC data, which should be added in.\nWe wish to clarify that the K values and error estimates in Fig. 2d & 3b were derived from\nd\ntwo independent ITC experiments. For clarity, only one representative ITC curve was\nshown in these figures. Therefore, there are no error bars associated with the presented\ndata. Following the reviewer’s comment, we have clarified this in the figure legends in the\nrevised manuscript.\nRegarding the presented 1H/15N HSQC in Fig. 2e, it is difficult to differentiate the\nmagenta from red peaks, making it difficult to readily evaluate the author’s conclusions. It\nis also stated in the text that addition of WT PWWP results in “…substantial chemical shift\nperturbation of several…” ADD NMR resonances, though there seems to be very few\nADD resonances that experience a chemical shift perturbation and the majority of the\nones that do have small chemical shift perturbations. In addition, it is claimed that addition\nof the PWWP W330A variant shifts the ADD resonances “…back to..” the non-interacting\nform. The presented spectra support the conclusion that the ADD resonances move back\ntoward the unbound ADD form, but do not revert back fully. It is thus recommended to\nattenuate the claims in these two statements. Having said that, it is again difficult to\nglobally assess these claims given the lack of contrast between the red and magenta\nspectra.\nWe thank the reviewer for the suggestions. In the revised manuscript, we have toned\ndown the statements for the NMR results, such as “the addition of the PWWP domain\nleads to notable chemical shift perturbation” (Line 183) and “replacement of WT PWWP\nwith the W330A-mutated PWWP domain led to a shift of these peaks toward the positions”\n(Line 186) of the non-interacting form. In addition, we have changed the color for NMR\nsignals associated with the PWWP W330A mutant from magenta to blue.\n3) The Fig. 4 legend denotes f, rather than d. It is stated in regard to the DNA methylation\nassay data that neither variant R544A or W330A affected “…H3K4me0-mediated\nstimulation appreciably…”. It is presumed this statement is being made relative to WT,\nin which it appears that the difference between H3K4me3 and H3K4me0 substrates in\nactivating DNMT3A is greater for both variants than the 7-fold difference observed for the\nWT.\nWe apologize for the confusion. In the revised manuscript, we have clarified the\ndescription by stating that “the H3K4me0-mediated stimulation remains strong for both\nmutants (6.1-fold for R544A and 5.3-fold for W330A)” (Line 274-275)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "This is a well-written manuscript describing the cryoEM structure of the hetero-tetrameric DNMT3A-DNMT3L complex. The\nauthors have satisfactorily addressed the questions I previously raised.", "reviewer_2": "The revisions have addressed all of my concerns, which were very minor to begin with. I fully endorse its publication in its\npresent form.", "reviewer_3": "The authors were highly response to the Reviewer's concerns and have sufficiently addressed the points raised, leading to\nan overall improved manuscript. This reviewer has no further concerns that would need to be addressed prior to publication."}, "author_response": ""}]

Decision letter