DNMT3B PWWP mutations cause hypermethylation of heterochromatin

Abstract

The correct establishment of DNA methylation patterns is vital for mammalian development and is achieved largely by the de novo DNA methyltransferases DNMT3A and DNMT3B. Mutations in DNMT3B can cause immunodeficiency-centromeric instability-facial anomalies type 1 (ICF1) syndrome which is characterised by hypomethylated heterochromatin. However, in the genome, DNMT3B primarily localises to actively transcribing gene bodies through the interaction of its PWWP domain with the histone modification H3K36me3 and it is unclear how it is recruited to heterochromatin. Here we show that in DNMT3B knockout cells, loss of DNA methylation predominantly occurs in heterochromatic domains marked by H3K9me3. We also find that PWWP domain mutations which disrupt DNMT3B's interaction with H3K36me3 result in striking increases of DNA methylation in H3K9me3-marked heterochromatin. Gains of methylation are also observed when the PWWP domain of DNMT3B is deleted. In contrast, we find that the ICF1 syndrome-causing PWWP mutation, S270P, does not result in hypermethylation of heterochromatin and destabilises the protein. We also show that removal of the N-terminus region of DNMT3B affects its recruitment to chromatin and ability to methylate H3K9me3 marked regions. Our results suggest that DNMT3B is recruited to H3K9me3 marked heterochromatin in a PWWP-independent manner and that this recruitment is facilitated by the protein's N-terminus. More generally, we suggest that DNMT3B plays a role in DNA methylation homeostasis at heterochromatin, a process which is disrupted in ICF syndrome, cancer and aging.

Structural basis for the allosteric regulation and dynamic assembly of DNMT3B

“…3C), presumably owing to a structural perturbation effect of this mutation on DNMT3B as shown by our thermal shift analysis (Fig. S9A-C) and a recent study (56). Collectively, these data confirm the interaction between the PWWP and ADD domains.…”

“…3C), presumably owing to a structural perturbation effect of this mutation on DNMT3B as shown by our thermal shift analysis (Fig. S9A-C) and a recent study (56).…”

“…In addition, the PWWP domain, through interaction with both the ADD domain and the catalytic loop of DNMT3B, might reinforce the ADD domain-mediated autoinhibition. In these contexts, previous studies have demonstrated that the ICF mutation S270P, located near to the PWWP-ADD interface, impairs the interaction between DNMT3B and H3K36me3-marked nucleosome in vitro (44), reduces the stability of DNMT3B in cells (58), and affects the recruitment of DNMT3B to the major satellite repeats at pericentric heterochromatin (59). This study showed that this mutation causes abolished PWWP-ADD interaction and reduced thermal stability of DNMT3B.…”

Structural basis for the allosteric regulation and dynamic assembly of DNMT3B

“…In contrast, introducing the K276A mutation to the PWWP domain and the D470A/D472A double mutation to the ADD domain reduced the binding affinity by ∼3- and 2-fold, respectively, under the experimental condition (Figure 3C ). Strikingly, introducing the ICF-associated S270P mutation to the PWWP domain completely abolished the PWWP–ADD interaction (Figure 3C ), presumably owing to a structural perturbation effect of this mutation on DNMT3B as shown by our thermal shift analysis (Figure S9A–C) and a recent study ( 56 ). Collectively, these data confirm the interaction between the PWWP and ADD domains.…”

“…This inter-domain interaction leads to occlusion of the H3K4me0-binding sites of the ADD domain but not the H3K36me3-binding sites of the PWWP domain, which might impact chromatin targeting of DNMT3B. Along the line, introducing the DNMT3B K276E mutation at the PWWP–ADD interface was recently shown to increase the heterochromatin localization of DNMT3B ( 56 ), supporting a link between the PWWP–ADD interaction and the targeting specificity of DNMT3B. Furthermore, the PWWP–ADD-MTase interaction appears to help stabilize DNMT3B homotetramer, as indicated by our structural and DLS analysis, thereby influencing the dynamic equilibrium of DNMT3B assembly.…”