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Mammals use DNA methylation for the heritable silencing of retrotransposons and imprinted genes and for the inactivation of the X chromosome in females. The establishment of patterns of DNA methylation during gametogenesis depends in part on DNMT3L, an enzymatically inactive regulatory factor that is related in sequence to the DNA methyltransferases DNMT3A and DNMT3B1,2. The main proteins that interact in vivo with the product of an epitope-tagged allele of the endogenous Dnmt3L gene were identified by mass spectrometry as DNMT3A2, DNMT3B and the four core histones. Peptide interaction assays showed that DNMT3L specifically interacts with the extreme amino terminus of histone H3; this interaction was strongly inhibited by methylation at lysine 4 of histone H3 but was insensitive to modifications at other positions. Crystallographic studies of human DNMT3L showed that the protein has a carboxy-terminal methyltransferase-like domain and an N-terminal cysteine-rich domain. Cocrystallization of DNMT3L with the tail of histone H3 revealed that the tail bound to the cysteine-rich domain of DNMT3L, and substitution of key residues in the binding site eliminated the H3 tail-DNMT3L interaction. These data indicate that DNMT3L recognizes histone H3 tails that are unmethylated at lysine 4 and induces de novo DNA methylation by recruitment or activation of DNMT3A2.
Null alleles of Dnmt3L are lethal when transmitted through the maternal germ line as a result of a failure to establish maternal genomic imprints during oogenesis, and mutant males show reactivation of retrotransposons and extreme meiotic defects in spermatocytes. The loss of DNA methylation in DNMT3L-deficient male germ cells also causes hyperacetylation of histones and a loss of methylation from lysine 9 of histone H3 (ref. 3). DNMT3L stimulates de novo methylation by DNMT3A2 (refs 4,5) but does not enhance the binding of DNMT3A2 to DNA, and DNMT3L alone does not bind to DNA5,6. However, the nature of the cues that regulate DNMT3L are unknown.
Embryonic stem (ES) cells express both DNMT3L and DNMT3A2, a germ-cell-specific isoform of DNMT3A that is also required for genomic imprinting4,7. ES cells show much higher rates of de novo methylation than do differentiated somatic cells, and methylation imprints are gained and lost at high rates in ES cells8,9. ES cells are therefore an appropriate cell type in which to carry out biochemical identification of proteins that interact with DNMT3L. We introduced a tandem His6-FLAG tag into the N terminus of the endogenous Dnmt3L gene by homologous recombination in ES cells to generate the Dnmt3LTag allele (Fig. 1a and Supplementary Fig. S1). Southern blotting (Fig. 1a) and DNA sequencing confirmed that the tag was targeted correctly, and expression of the tagged DNMT3L protein was confirmed on immunoblots probed with an anti-FLAG antibody (Supplementary Fig. S1). Mice that were homozygous for the tagged allele were of normal phenotype and both sexes were fertile, which indicated that the tag did not interfere with the function of DNMT3L.
We conducted anti-FLAG immunoprecipitation on lysates derived from Dnmt3LTag/Tag ES cells. Mass spectrometry of polypeptides specific to eluates derived from Dnmt3LTag samples (Fig. 1b) identified DNMT3A2 and DNMT3B, which have been reported to interact with Dnmt3L in vitro4,5,10. DNMT3A2 has been reported to be required for de novo DNA methylation in germ cells, and germ-cell-specific conditional alleles of Dnmt3A2 phenocopy null alleles of Dnmt3L (ref. 7). We also found that DNMT3L was associated with the four core histones (Fig. 1b).
Peptide interaction assays with biotinylated tails of the four core histones showed that recombinant human DNMT3L bound only to the N-terminal tail of histone H3 (Fig. 2a). Peptide interaction assays using different regions of the N-terminal histone H3 tail revealed that DNMT3L binding required the first seven N-terminal amino acids of H3 (Fig. 2a, right). The binding of DNMT3L to histone H3 was abolished by mono-, di- or trimethylation of lysine 4 on histone H3, but was insensitive to modifications at other positions (Fig. 2b). These data indicate that the interaction between DNMT3L and the N-terminal tail of histone H3 depends on the methylation state of H3 lysine 4. Fluorescence polarization data showed that the dissociation constant (Kd) for the interaction between the unmodified H3 N-terminal tail and full-length DNMT3L was 2.1 μM; a single methyl group increased the Kd to 38.5 μM, and no interaction could be detected when the pep tide was di- or trimethylated at lysine 4 (Fig. 2d). Isolation of mononucleosomes by treatment of nuclei with micrococcal nuclease before anti-FLAG immunoprecipitation showed that DNMT3L is associated with nucleosomes that are depleted for H3 methylated at lysine 4 (Fig. 2e). These data confirm that the interactions shown in Fig. 2a, b also occur in vivo.
The results of crystallographic studies have suggested a mechanism for the histone-DNMT3L interaction. The structure of full-length human DNMT3L was determined to a resolution of 3.29 Å. The three endogenous zinc ions within the cysteine-rich region established X-ray phasing (Fig. 3a and Supplementary Table T1). Although the classical methyltransferase fold that is characteristic of S-adenosyl l-methionine-dependent methyltransferases was present11, the DNA recognition domain12 was absent and a cysteine-rich region organized around the three zinc ions was located opposite to the region where the catalytic centre is located in active DNA (cytosine-5) methyltransferases (Fig. 3a).
A peptide corresponding to the first 24 amino acids of histone H3 was soaked into the DNMT3L crystal shown in Fig. 3a. Crystallographic analysis of the complex showed electron density for approximately seven amino acids bound to the zinc-binding domain; the remainder of the peptide was unstructured (Fig. 3b). On the basis of the binding data (Fig. 2) and the structural comparison with the PHD-like domain of BHC80, a component of the LSD1 histone H3 lysine 4 demethylation complex13, we deduced that the structured portion of the peptide is the first seven amino acids of H3. BHC80 selectively binds unmethylated H3 lysine 4 peptides, and this binding is mediated by a PHD-like domain that has strong structural similarities to the cysteine-rich domain of DNMT3L (Supplementary Fig. S2 and ref. 14). Mutations that introduce a bulky tryptophan side chain into the H3 binding site of DNMT3L (I107W) or that disrupt the interaction of an aspartic acid side chain with the amino group of H3 lysine 4 (D90A) eliminate the interaction of the H3 tail with DNMT3L (Fig. 3c). The basis for methylation-sensitive binding of H3 to DNMT3L is steric occlusion of the interaction between aspartic acid 90 in DNMT3L and lysine 4 of histone H3 (Fig. 3b, c and data not shown).
These data indicate that Dnmt3L responds to states of histone modification to regulate de novo DNA methylation. Under this model, DNMT3L triggers de novo DNA methylation by activation or recruitment of DNMT3A2 upon contact with nucleosomes that contain unmethylated H3 lysine 4, while other sequences are masked from DNMT3L by the inhibitory effects of nucleosomes that contain histone H3 methylated at lysine 4.
Methylation of lysine 9 on histone H3 is required for DNA methylation in vegetative cells of the ascomycete Neurospora crassa, and for methylation of some non-CpG sequences in Arabidopsis thaliana15,16. Mouse ES cells that lack the H3 lysine 9 methyltransferases Suv39h1 and Suv39h2 show slight demethylation of satellite DNA17. In each of these cases, the signal for DNA methylation is the presence of methylation at H3 lysine 9 rather than the absence of methylation at H3 lysine 4. Methylation of lysine 4 on histone H3 has recently been suggested to protect gene promoters from de novo DNA methylation in mammalian somatic cells18,19, and this suggestion is fully compatible with the findings presented here.
DNMT3L is required for the de novo methylation of imprinting control regions in female germ cells and for the de novo methylation of dispersed repeated sequences in male germ cells. The data presented here point towards a novel mechanism whereby DNMT3L could convert patterns of histone H3 lysine 4 methylation, which are not known to be transmitted by mitotic inheritance, into patterns of DNA methylation that mediate the heritable transcriptional silencing of the affected sequences.
A synthetic oligonucleotide coding for His6 followed by the FLAG epitope was introduced into the Dnmt3L gene by homologous recombination in ES cells. Once we had confirmed correct targeting, we excised the neomycin resistance cassette by transient exposure to Cre, before injecting the cells into blastocysts to generate chimaeric mice. Dnmt3LTag/Tag ES cells were derived from blastocysts from homozygous breedings.
We immunoprecipitated clarified lysates from wild-type and Dnmt3LTag/Tag ES cells using EZview Red Anti-FLAG M2 Affinity Gel (Sigma) and eluted them with FLAG peptide (Sigma) according to the manufacturer’s protocol.
The interaction of DNMT3L with biotinylated histone tails was assayed essentially as described20.
Protein bands were excised and digested with trypsin and then batch fractionated on a RP micro-tip, and the peptide mixtures were analysed by matrix assisted laser resorption/ionization reflectron time-of-flight (MALDI-reTOF) mass spectrometry (UltraFlex TOF/TOF; Bruker Daltonics) as described21, 22. We took selected experimental masses (m/z) to search a non-redundant protein database (‘NR’; ~3.7 × 106 entries; National Center for Biotechnology Information; Bethesda, Maryland), using the PeptideSearch algorithm (M. Mann, Southern Denmark University, Odense, Denmark). Mass spectrometric sequencing of selected peptides was done by MALDI-TOF/TOF (MS/MS) analysis on the same prepared samples, using the UltraFlex instrument in ‘LIFT’ mode. We used fragment ion spectra to search the NR database using the MASCOT MS/MS Ion Search program (Matrix Science Ltd.).
We solved the structure of full-length human DNMT3L by three-wavelength Zn anomalous diffraction data (Supplementary Table T1).
We thank C.S. Lin for advice and assistance, D. Bourc’his, M. Damelin, C. Schaefer, X. Zhang and R. E. Collins for helpful discussions, J. R. Horton for collection of X-ray diffraction data, A. Ruthenberg for recombinant WDR5 protein, and K. Anderson, D. Bourc’his and C. Schaefer for criticism of the manuscript. This work was supported by grants from the National Institutes of Health to C. D. A., X. C., P. T. and T. H. B. and by a fellowship from the European Molecular Biology Organisation to S.K.T.O.
We synthesized the right and left homologous arms of the Dnmt3L targeting construct by cloning the indicated PCR products into pBluescript SK (Stratagene). We designed forward and reverse primers using the available mouse genome sequence data. The synthetic oligonucleotide (5′-GG CAC CAT CAC CAC CAT CAC GAC TAC AAA GAC GAT GAC GAT AAA TCC C-3′) encoding a His6 tag followed by FLAG epitope was introduced into a SmaI site within exon 2, which contains the ATG start codon. An additional loxP site in inverse orientation was introduced into a ClaI site between exons 1 and 2 using the synthetic oligonucleotide 5′-ATA ACT TCG TAT AAT GTA TGC TAT ACG AAG TTA TAT CG-3′. The floxed neomycin resistance cassette was introduced from plasmid pEasyFlox (a gift from T. Ludwig) into NotI and XbaI sites within the pBluescript SK multiple cloning site between exons 3 and 4. Following gene targeting and G418 selection of 129Sv/Ev ES cell, we identified clones that had been correctly targeted by homologous recombination at the endogenous Dnmt3L locus by southern blotting with a 516-bp PCR-amplified probe, generated with 5′-AGA ATT CGC GGG CCC TAT GGA GAT ATA CAA GT-3′ forward and 5′- ATG GAT CCG AAG TTC AGG AAG GTG TGT GTG T-3′ reverse primers. The neomycin cassette was removed by Cre-mediated excision after transfection of the Cre-expressing plasmid pCAGGS-Cre (a gift from V. E. Papaioannou). Cre-mediated site-specific recombination was verified by southern blotting and sequencing of PCR products that spanned LoxP sites. Targeted clones that had deleted the neomycin cassette were injected into blastocysts and chimaeric mice were identified by southern blotting. Both male and female animals that are homozygous for the Dnmt3LTag allele are viable and fertile. Dnmt3LTag/Tag ES cells were derived from blastocysts from homozygous breedings.
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.