Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation

Abstract

Genetic imprinting, found in flowering plants and placental mammals, uses DNA methylation to yield gene expression that is dependent on the parent of origin 1 . DNA methyltransferase 3a (Dnmt3a) and its regulatory factor, DNA methyltransferase 3-like protein (Dnmt3L), are both required for the de novo DNA methylation of imprinted genes in mammalian germ cells. Dnmt3L interacts specifically with unmethylated lysine 4 of histone H3 through its amino-terminal PHD (plant homeodomain)-like domain 2 . Here we show, with the use of crystallography, that the carboxyterminal domain of human Dnmt3L interacts with the catalytic domain of Dnmt3a, demonstrating that Dnmt3L has dual functions of binding the unmethylated histone tail and activating DNA methyltransferase. The complexed C-terminal domains of Dnmt3a and Dnmt3L showed further dimerization through Dnmt3a-Dnmt3a interaction, forming a tetrameric complex with two active sites. Substitution of key non-catalytic residues at the Dnmt3a-Dnmt3L interface or the Dnmt3a-Dnmt3a interface eliminated enzymatic activity. Molecular modelling of a DNA-Dnmt3a dimer indicated that the two active sites are separated by about one DNA helical turn. The C-terminal domain of Dnmt3a oligomerizes on DNA to form a nucleoprotein filament. A periodicity in the activity of Dnmt3a on long DNA revealed a correlation of methylated CpG sites at distances of eight to ten base pairs, indicating that oligomerization leads Dnmt3a to methylate DNA in a periodic pattern. A similar periodicity is observed for the frequency of CpG sites in the differentially methylated regions of 12 maternally imprinted mouse genes. These results suggest a basis for the recognition and methylation of differentially methylated regions in imprinted genes, involving the detection of both nucleosome modification and CpG spacing.In both flowering plants and placental mammals, DNA methylation has a central role in imprinting, but in neither case is it clear how imprinted genes are targeted for methylation. Imprinted genes in mammals are often associated with differentially methylated regions (DMRs) 3 , which show DNA methylation patterns that depend on the parent of origin. How the imprinting machinery recognizes DMRs is unknown. The Dnmt3 family includes three members: two de novo CpG methyltransferases, namely Dnmt3a and Dnmt3b (ref. 4), and anCorrespondence and requests for materials should be addressed to A.J. [E-mail: a.jeltsch@jacobs-university.de] or X.C. [E-mail: xcheng@emory.edu]. * These authors contributed equally to this work.Author Information The X-ray structure of Dnmt3a-Dnmt3L C-terminal tetramer complex is deposited in the Protein Data Bank under ID code 2QRV. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. We ...

Detection of High-Frequency and Novel DNMT3A Mutations in Acute Myeloid Leukemia by High-Resolution Melting Curve Analysis

“…3,4 The R882 residue resides in the dimerization domain of DNMT3A and is important for enzymatic activity. 25 Mutation of R882 decreases enzyme activity by 50%, leading to suppressed DNA methylation and aberrant gene expression. 2 Seven mutations detected in our study (p.C306X, p.C330X, p.I407S, p.I407T, p.C583Y, p.N710S, and p.W893R) have not been reported previously, thus adding to the growing list of DNMT3A mutations in AML.…”

“…24 The PDB structure files used for modeling studies were from structures reported previously for DNMT3A (PDB codes 2QRV, 3A1A, and 3LLR). 25 …”

Genomic imprinting and its relevance to congenital disease, infertility, molar pregnancy and induced pluripotent stem cell

“…Factors and features identified so far as specificity determinants for imprinting in oocytes include: (1) transcription traversing ICRs (Figure 1), 26 (2) unmethylated lysine-4 of histone H3 (H3K4), 27 (3) 10-bp CpG spacing, 28 (4) a histone H3K4 demethylase Kdm1b (Figure 1) 29 and (5) a Krüppel-associated box (KRAB) zinc finger protein Zfp57 (Figure 1). 30 Among these, the first three seem common to all oocyte-methylated ICRs and could be prerequisites for imprint establishment.…”

“…Transcription through ICRs may make the chromatin more accessible by the Dnmt3a-Dnmt3L complex; 26 unmethylated, but not methylated, H3K4 is the high-affinity binding target of Dnmt3L; 27 two CpG sites located 10-bp apart fit very well with the catalytic centers of the heterotetrameric Dnmt3a-Dnmt3L complex. 28 However, these features can be found elsewhere in the genome and are not specific to ICRs. By contrast, Kdm1b acts only on a subset of ICRs (for example, ICRs at Mest, Grb10, Plagl1 and Impact loci), 29 although demethylation of H3K4 by this enzyme is consistent with the preference of Dnmt3L for unmethylated H3K4.…”

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“…Dnmt3a can also function as a DNA demethylase [54]. Dnmt3L is a catalytically inactive homologue of Dnmt3a and Dnmt3b that functions as an essential regulatory cofactor for tetrameric complex assembly [55].…”