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
) but does not enhance the binding of DNMT3A2 to DNA, and DNMT3L alone does not bind to DNA5
. 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
. 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
. 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 ( and Supplementary Fig. S1
). Southern blotting () 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.
Generation of epitope-tagged Dnmt3L locus and DNMT3L protein interaction screen in ES cells
We conducted anti-FLAG immunoprecipitation on lysates derived from Dnmt3LTag/Tag
ES cells. Mass spectrometry of polypeptides specific to eluates derived from Dnmt3LTag
samples () identified DNMT3A2 and DNMT3B, which have been reported to interact with Dnmt3L in vitro4
. 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
). We also found that DNMT3L was associated with the four core histones ().
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 (). 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 (, 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 (). 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 (). 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 (). These data confirm that the interactions shown in also occur in vivo.
Interaction of DNMT3L with the N terminus of histone H3 is abolished by methylation of H3 lysine 4
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 ( and Supplementary Table T1
). Although the classical methyltransferase fold that is characteristic of S
-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 ().
Structure of DNMT3L and mode of recognition of histone H3 unmethylated at lysine 4
A peptide corresponding to the first 24 amino acids of histone H3 was soaked into the DNMT3L crystal shown in . 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 (). On the basis of the binding data () 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 (). 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 ( 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
. 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
, 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.