The PWWP Domain of Dnmt3a and Dnmt3b Is Required for Directing DNA Methylation to the Major Satellite Repeats at Pericentric Heterochromatin

Abstract

Dnmt3a and Dnmt3b are responsible for the establishment of DNA methylation patterns during development. These proteins contain, in addition to a C-terminal catalytic domain, a unique N-terminal regulatory region that harbors conserved domains, including a PWWP domain. The PWWP domain, characterized by the presence of a highly conserved proline-tryptophan-tryptophan-proline motif, is a module of 100 to 150 amino acids found in many chromatin-associated proteins. However, the function of the PWWP domain remains largely unknown. In this study, we provide evidence that the PWWP domains of Dnmt3a and Dnmt3b are involved in functional specialization of these enzymes. We show that both endogenous and green fluorescent protein-tagged Dnmt3a and Dnmt3b are particularly concentrated in pericentric heterochromatin. Mutagenesis analysis indicates that their PWWP domains are required for their association with pericentric heterochromatin. Disruption of the PWWP domain abolishes the ability of Dnmt3a and Dnmt3b to methylate the major satellite repeats at pericentric heterochromatin. Furthermore, we demonstrate that the Dnmt3a PWWP domain has little DNA-binding ability, in contrast to the Dnmt3b PWWP domain, which binds DNA nonspecifically. Collectively, our results suggest that the PWWP domains of Dnmt3a and Dnmt3b are essential for targeting these enzymes to pericentric heterochromatin, probably via a mechanism other than protein-DNA interactions.

Histone tails regulate DNA methylation by allosterically activating de novo methyltransferase

“…The PHD domain of Dnmt3a is able to bind histone tails in vitro [14,15], but is dispensable for the enzyme association with chromatin in vivo [19,20]. These observations suggest that recruitment of Dnmt3a to chromatin could be independent of the PHD domain-mediated binding to the H3 N-terminus.…”

“…As previous studies indicate that the Dnmt3a PHD domain is not required for its chromatin association [19,20], we reasoned that the binding of H3 peptide via the PHD domain might regulate the Dnmt3a function through allosteric enzymatic activation rather than chromatin recruitment. As the allosteric effect of H3 binding might be mediated by interaction between the PHD and catalytic domains, we sought to characterize crucial residues at the interaction interfaces, whose substitutions would block allosteric activation of enzymatic activity but have no effect on the H3 binding and the basal catalytic activities.…”

“…DKO (Dnmt3a and Dnmt3b double knockout [39]) stable ES cells ectopically expressing Dnmt3a were established essentially as described [20]. TKO (Dnmt31, Dnmt3a, Dnmt3b TKO [24]) stable ES cells lines ectopically expressing Dnmt3a were established by infection with lentivirus carrying a Dnmt3a expression cassette linked to the EGFP gene.…”

Diverse Domains of (Cytosine-5)-DNA Methyltransferases: Structural and Functional Characterization

“…Deletion of the PWWP domain does not influence the Dnmt3b methylation efficiency in vitro [117]. On the contrary, the PWWP domain of Dnmt3a is almost unable to bind DNA [121]. The PWWP domains of Dnmt3a and Dnmt3b are necessary for targeting these MTases to pericentromeric heterochromatin [121][122][123].…”

“…The PWWP domain of Dnmt3b has a positively charged surface with an approximate area of 45 × 32 Å 2 and can bind DNA nonspecifically [117,121]. The PWWP domain binds 30 bp duplexes with unmethylated, mono-, and dimethylated 5′-CG-3′/3′-GC-5′ sites.…”