Albert Jeltsch

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

Genetic imprinting, found in flowering plants and placental mammals, uses DNA methylation to yield gene expression that is dependent on the parent of origin. 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. Here we show, with the use of crystallography, that the carboxy-terminal 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.

Beyond watson and crick: DNA methylation and molecular enzymology of DNA methyltransferases

DNA methyltransferases catalyze the transfer of a methyl group from S‐adenosyl‐L‐methionine to cytosine or adenine bases in DNA. These enzymes challenge the Watson/Crick dogma in two instances: 1) They attach inheritable information to the DNA that is not encoded in the nucleotide sequence. This so‐called epigenetic information has many important biological functions. In prokaryotes, DNA methylation is used to coordinate DNA replication and the cell cycle, to direct postreplicative mismatch repair, and to distinguish self and nonself DNA. In eukaryotes, DNA methylation contributes to the control of gene expression, the protection of the genome against selfish DNA, maintenance of genome integrity, parental imprinting, X‐chromosome inactivation in mammals, and regulation of development. 2) The enzymatic mechanism of DNA methyltransferases is unusual, because these enzymes flip their target base out of the DNA helix and, thereby, locally disrupt the B‐DNA helix. This review describes the biological functions of DNA methylation in bacteria, fungi, plants, and mammals. In addition, the structures and mechanisms of the DNA methyltransferases, which enable them to specifically recognize their DNA targets and to induce such large conformational changes of the DNA, are discussed.

Biochemistry and biology of mammalian DNA methyltransferases.

Abstract.DNA methylation is a stable but not irreversible epigenetic signal that silences gene expression. It has a variety of important functions in mammals, including control of gene expression, cellular differentiation and development, preservation of chromosomal integrity, parental imprinting and X-chromosome inactivation. In addition, it has been implicated in brain function and the development of the immune system. Somatic alterations in genomic methylation patterns contribute to the etiology of human cancers and ageing. It is tightly interwoven with the modification of histone tails and other epigenetic signals. Here we review our current understanding of the molecular enzymology of the mammalian DNA methyltransferases Dnmt1, Dnmt3a, Dnmt3b and Dnmt2 and the roles of the enzymes in the above-mentioned biological processes.

Structure and Function of Mammalian DNA Methyltransferases

DNA methylation plays an important role in epigenetic signalling, having an impact on gene regulation, chromatin structure, development and disease. Here, we review the structures and functions of the mammalian DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b, including their domain structures, catalytic mechanisms, localisation, regulation, post‐translational modifications and interaction with chromatin and other proteins, summarising data obtained in genetic, cell biology and enzymatic studies. We focus on the question of how the molecular and enzymatic properties of these enzymes are connected to the dynamics of DNA methylation patterns and to the roles the enzymes play in the processes of de novo and maintenance DNA methylation. Recent enzymatic and genome‐wide methylome data have led to a new model of genomic DNA methylation patterns based on the preservation of average levels of DNA methylation in certain regions, rather than the methylation states of individual CG sites.

THE DNMT3A PWWP domain reads histone 3 lysine 36 trimethylation and guides DNA methylation

The Dnmt3a DNA methyltransferase contains in its N-terminal part a PWWP domain that is involved in chromatin targeting. Here, we have investigated the interaction of the PWWP domain with modified histone tails using peptide arrays and show that it specifically recognizes the histone 3 lysine 36 trimethylation mark. H3K36me3 is known to be a repressive modification correlated with DNA methylation in mammals and heterochromatin in Schizosaccharomyces pombe. These results were confirmed by equilibrium peptide binding studies and pulldown experiments with native histones and purified native nucleosomes. The PWWP-H3K36me3 interaction is important for the subnuclear localization of enhanced yellow fluorescent protein-fused Dnmt3a. Furthermore, the PWWP-H3K36me3 interaction increases the activity of Dnmt3a for methylation of nucleosomal DNA as observed using native nucleosomes isolated from human cells after demethylation of the DNA with 5-aza-2′-deoxycytidine as substrate for methylation with Dnmt3a. These data suggest that the interaction of the PWWP domain with H3K36me3 is involved in targeting of Dnmt3a to chromatin carrying that mark, a model that is in agreement with several studies on the genome-wide distribution of DNA methylation and H3K36me3.

Protein lysine methyltransferase G9a acts on non-histone targets

By methylation of peptide arrays, we determined the specificity profile of the protein methyltransferase G9a. We show that it mostly recognizes an Arg-Lys sequence and that its activity is inhibited by methylation of the arginine residue. Using the specificity profile, we identified new non-histone protein targets of G9a, including CDYL1, WIZ, ACINUS and G9a (automethylation), as well as peptides derived from CSB. We demonstrate potential downstream signaling pathways for methylation of non-histone proteins.

Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail

Using peptide arrays and binding to native histone proteins, we show that the ADD domain of Dnmt3a specifically interacts with the H3 histone 1–19 tail. Binding is disrupted by di- and trimethylation of K4, phosphorylation of T3, S10 or T11 and acetylation of K4. We did not observe binding to the H4 1–19 tail. The ADD domain of Dnmt3b shows the same binding specificity, suggesting that the distinct biological functions of both enzymes are not related to their ADD domains. To establish a functional role of the ADD domain binding to unmodified H3 tails, we analyzed the DNA methylation of in vitro reconstituted chromatin with Dnmt3a2, the Dnmt3a2/Dnmt3L complex, and the catalytic domain of Dnmt3a. All Dnmt3a complexes preferentially methylated linker DNA regions. Chromatin substrates with unmodified H3 tail or with H3K9me3 modification were methylated more efficiently by full-length Dnmt3a and full-length Dnmt3a/3L complexes than chromatin trimethylated at H3K4. In contrast, the catalytic domain of Dnmt3a was not affected by the H3K4me3 modification. These results demonstrate that the binding of the ADD domain to H3 tails unmethylated at K4 leads to the preferential methylation of DNA bound to chromatin with this modification state. Our in vitro results recapitulate DNA methylation patterns observed in genome-wide DNA methylation studies.

Mechanism of stimulation of catalytic activity of Dnmt3A and Dnmt3B DNA-(cytosine-C5)-methyltransferases by Dnmt3L

Dnmt3L has been identified as a stimulator of the catalytic activity of de novo DNA methyltransferases. It is essential in the development of germ cells in mammals. We show here that Dnmt3L stimulates the catalytic activity of the Dnmt3A and Dnmt3B enzymes by directly binding to their respective catalytic domains via its own C-terminal domain. The catalytic activity of Dnmt3A and -3B was stimulated ∼15-fold, and Dnmt3L directly binds to DNA but not to S-adenosyl-l-methionine (AdoMet). Complex formation between Dnmt3A and Dnmt3L accelerates DNA binding by Dnmt3A 20-fold and lowers its Km for DNA. Interaction of Dnmt3L with Dnmt3A increases the binding of the coenzyme AdoMet to Dnmt3A, and it lowers the Km of Dnmt3A for AdoMet. On the basis of our data we propose a model in which the interaction of Dnmt3A with Dnmt3L induces a conformational change of Dnmt3A that opens the active site of the enzyme and promotes binding of DNA and the AdoMet. We demonstrate that the interaction of Dnmt3A and Dnmt3L is transient, and after DNA binding to Dnmt3A, Dnmt3L dissociates from the complex. Following dissociation of Dnmt3L, Dnmt3A adopts a closed conformation leading to slow rates of DNA release. Therefore, Dnmt3L acts as a substrate exchange factor that accelerates DNA and AdoMet binding to de novo DNA methyltransferases.

DNA of Drosophila melanogaster contains 5-methylcytosine

It is commonly accepted that the DNA of Drosophila melanogaster does not contain 5‐methylcytosine, which is essential in the development of most eukaryotes. We have developed a new, highly specific and sensitive assay to detect the presence of 5‐methylcytosine in genomic DNA. The DNA is degraded to nucleosides, 5‐methylcytosine purified by HPLC and, for detection by 1D‐ and 2D‐TLC, radiolabeled using deoxynucleoside kinase and [γ‐32P]ATP. Using this assay, we show here that 5‐methylcytosine occurs in the DNA of D.melanogaster at a level of ∼1 in 1000–2000 cytosine residues in adult flies. DNA methylation is detectable in all stages of D.melanogaster development.

New concepts in DNA methylation

The widely-cited model of maintenance of DNA methylation at CpG sites implies that DNA methylation is introduced by the Dnmt3 de novo DNA methyltransferases during early development, and methylation at hemimethylated CpG sites is specifically maintained by the Dnmt1 maintenance methyltransferase. However, substantial experimental evidence from the past decade indicates that this simple model needs to be revised. DNA methylation can be described by a dynamic stochastic model, in which DNA methylation at each site is determined by the local activity of DNA methyltransferases (Dnmts), DNA demethylases, and the DNA replication rate. Through the targeting and regulation of these enzymes, DNA methylation is controlled by the network of chromatin marks.