Structural basis for the allosteric regulation and dynamic assembly of DNMT3B

Abstract

Oligomerization of DNMT3B, a mammalian de novo DNA methyltransferase, critically regulates its chromatin targeting and DNA methylation activities. However, how the N-terminal PWWP and ADD domains interplay with the C-terminal methyltransferase (MTase) domain in regulating the dynamic assembly of DNMT3B remains unclear. Here, we report the cryo-EM structure of DNMT3B under various oligomerization states. The ADD domain of DNMT3B interacts with the MTase domain to form an autoinhibitory conformation, resembling the previously observed DNMT3A autoinhibition. Our combined structural and biochemical study further identifies a role for the PWWP domain and its associated ICF mutation in the allosteric regulation of DNMT3B tetramer, and a differential functional impact on DNMT3B by potential ADD–H3K4me0 and PWWP–H3K36me3 bindings. In addition, our comparative structural analysis reveals a coupling between DNMT3B oligomerization and folding of its substrate-binding sites. Together, this study provides mechanistic insights into the allosteric regulation and dynamic assembly of DNMT3B.

Structure-guided functional suppression of AML-associated DNMT3A hotspot mutations

“…The coupling between the RD interface formation and folding of the substrate-binding sites is thermodynamically governed by compensated enthalpy and entropy, which influences the equilibrium of DNMT3A/DNMT3B oligomerization. Along the line, our cryo-EM study of DNMT3B reveals the co-existence of homotetramer with other alternative assemblies, such as homohexamer and homotrimer, in solution 36 . This study, through structural and biochemical characterization of DNMT3A homo-oligomer, suggests that the assembly of DNMT3A may undergo a similar transition between alternative oligomerization states in solution (Fig.…”

“…1c ), with the TRD and SAM-binding pocket structurally ordered for the central subunits but disordered for the external subunits (Fig. 1d, e ) 36 . Together, these observations suggest oligomerization-coupled RD interface folding as a recurrent mechanism for the DNMT3 family of DNA methyltransferases.…”

“…First, DNMT3A MTase eluted in a much broader peak on a size-exclusion chromatography column than that of the heterotetrameric complex between DNMT3A MTase and the DNMT3L C-terminal domain (Supplementary Fig. 5a, b ), suggesting that DNMT3A, like DNMT3B 36 , may exist as a mixture of homotetramer and other alternative assembly states in solution. Consistently, our measurement of the hydrodynamic radii ( R h ) of DNMT3A and DNMT3B using the dynamic light scattering (DLS) method reveals that, under the experimental condition, WT DNMT3A shows a size distribution profile centered at R h of 22 nm, similar to that of DNMT3B (Fig.…”

“…In the absence of regulatory factor DNMT3L/DNMT3B3, DNMT3A, and DNMT3B have been shown to exist dominantly in homotetrameric forms in vitro 15 , 31 , 34 , 35 , which might interact with DNA to form filament-like macro-oligomers 31 , 33 . To illuminate the mechanism of DNMT3B homo-oligomerization, we recently solved the crystal structure of the MTase domain of DNMT3B, which assumes a helical polymer in crystals via alternating RD and FF interfaces 33 , as well as the cryo-EM structures of DNMT3B PWWP-ADD-MTase tetramer and other oligomers with ascending or descending order (e.g., trimer or hexamer) 36 . Structural analysis of these complexes reveals a coupling between the formation of the RD interface and the folding of the underlying structural elements, comprised of the TRD and S -adenosyl methionine (SAM)-binding site 36 , establishing an intricate link between DNMT3B oligomerization, RD interface conformation and substrate binding.…”

“…To illuminate the mechanism of DNMT3B homo-oligomerization, we recently solved the crystal structure of the MTase domain of DNMT3B, which assumes a helical polymer in crystals via alternating RD and FF interfaces 33 , as well as the cryo-EM structures of DNMT3B PWWP-ADD-MTase tetramer and other oligomers with ascending or descending order (e.g., trimer or hexamer) 36 . Structural analysis of these complexes reveals a coupling between the formation of the RD interface and the folding of the underlying structural elements, comprised of the TRD and S -adenosyl methionine (SAM)-binding site 36 , establishing an intricate link between DNMT3B oligomerization, RD interface conformation and substrate binding. However, due to a lack of structure for homo-oligomeric DNMT3A, the molecular basis for DNMT3A homo-oligomerization remains elusive.…”