Tomasz P. Jurkowski

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.

DNA methylation analysis of chromosome 21 gene promoters at single base pair and single allele resolution.

Differential DNA methylation is an essential epigenetic signal for gene regulation, development, and disease processes. We mapped DNA methylation patterns of 190 gene promoter regions on chromosome 21 using bisulfite conversion and subclone sequencing in five human cell types. A total of 28,626 subclones were sequenced at high accuracy using (long-read) Sanger sequencing resulting in the measurement of the DNA methylation state of 580427 CpG sites. Our results show that average DNA methylation levels are distributed bimodally with enrichment of highly methylated and unmethylated sequences, both for amplicons and individual subclones, which represent single alleles from individual cells. Within CpG-rich sequences, DNA methylation was found to be anti-correlated with CpG dinucleotide density and GC content, and methylated CpGs are more likely to be flanked by AT-rich sequences. We observed over-representation of CpG sites in distances of 9, 18, and 27 bps in highly methylated amplicons. However, DNA sequence alone is not sufficient to predict an amplicons DNA methylation status, since 43% of all amplicons are differentially methylated between the cell types studied here. DNA methylation in promoter regions is strongly correlated with the absence of gene expression and low levels of activating epigenetic marks like H3K4 methylation and H3K9 and K14 acetylation. Utilizing the single base pair and single allele resolution of our data, we found that i) amplicons from different parts of a CpG island frequently differ in their DNA methylation level, ii) methylation levels of individual cells in one tissue are very similar, and iii) methylation patterns follow a relaxed site-specific distribution. Furthermore, iv) we identified three cases of allele-specific DNA methylation on chromosome 21. Our data shed new light on the nature of methylation patterns in human cells, the sequence dependence of DNA methylation, and its function as epigenetic signal in gene regulation. Further, we illustrate genotype–epigenotype interactions by showing novel examples of allele-specific methylation.

Human DNMT2 methylates tRNAAsp molecules using a DNA methyltransferase-like catalytic mechanism

Although their amino acid sequences and structure closely resemble DNA methyltransferases, Dnmt2 proteins were recently shown by Goll and colleagues to function as RNA methyltransferases transferring a methyl group to the C5 position of C38 in tRNA(Asp). We observe that human DNMT2 methylates tRNA isolated from Dnmt2 knock-out Drosophila melanogaster and Dictyostelium discoideum. RNA extracted from wild type D. melanogaster was methylated to a lower degree, but in the case of Dictyostelium, there was no difference in the methylation of RNA isolated from wild-type and Dnmt2 knock-out strains. Methylation of in vitro transcribed tRNA(Asp) confirms it to be a target of DNMT2. Using site directed mutagenesis, we show here that the enzyme has a DNA methyltransferase-like mechanism, because similar residues from motifs IV, VI, and VIII are involved in catalysis as identified in DNA methyltransferases. In addition, exchange of C292, which is located in a CFT motif conserved among Dnmt2 proteins, strongly reduced the catalytic activity of DNMT2. Dnmt2 represents the first example of an RNA methyltransferase using a DNA methyltransferase type of mechanism.

Targeted Methylation and Gene Silencing of VEGF-A in Human Cells by Using a Designed Dnmt3a–Dnmt3L Single-Chain Fusion Protein with Increased DNA Methylation Activity

The C-terminal domain of the Dnmt3a de novo DNA methyltransferase (Dnmt3a-C) forms a complex with the C-terminal domain of Dnmt3L, which stimulates its catalytic activity. We generated and characterized single-chain (sc) fusion proteins of both these domains with linker lengths between 16 and 30 amino acid residues. The purified sc proteins showed about 10-fold higher DNA methylation activities than Dnmt3a-C in vitro and were more active in bacterial cells as well. After fusing the Dnmt3a-3L sc enzyme to an artificial zinc-finger protein targeting the vascular endothelial cell growth factor A (VEGF-A) promoter, we demonstrate successful targeting of DNA methylation to the VEGF-A promoter in human cells and observed that almost complete methylation of 12 CpG sites in the gene promoter could be achieved. Targeted methylation by the Dnmt3a-3L sc enzymes was about twofold higher than that of Dnmt3a-C, indicating that Dnmt3a-3L sc variants are more efficient as catalytic modules in chimeric DNA methyltransfeases than Dnmt3a-C. Targeted methylation of the VEGF-A promoter with the Dnmt3a-3L sc variant led to a strong silencing of VEGF-A expression, indicating that the artificial DNA methylation of an endogenous promoter is a powerful strategy to achieve silencing of the corresponding gene in human cells.

On the evolutionary origin of eukaryotic DNA methyltransferases and Dnmt2.

The Dnmt2 enzymes show strong amino acid sequence similarity with eukaryotic and prokaryotic DNA-(cytosine C5)-methyltransferases. Yet, Dnmt2 enzymes from several species were shown to methylate tRNA-Asp and had been proposed that eukaryotic DNA methyltransferases evolved from a Dnmt2-like tRNA methyltransferase ancestor [Goll et al., 2006, Science, 311, 395-8]. It was the aim of this study to investigate if this hypothesis could be supported by evidence from sequence alignments. We present phylogenetic analyses based on sequence alignments of the methyltransferase catalytic domains of more than 2300 eukaryotic and prokaryotic DNA-(cytosine C5)-methyltransferases and analyzed the distribution of DNA methyltransferases in eukaryotic species. The Dnmt2 homologues were reliably identified by an additional conserved CFT motif next to motif IX. All DNA methyltransferases and Dnmt2 enzymes were clearly separated from other RNA-(cytosine-C5)-methyltransferases. Our sequence alignments and phylogenetic analyses indicate that the last universal eukaryotic ancestor contained at least one member of the Dnmt1, Dnmt2 and Dnmt3 families of enzymes and additional RNA methyltransferases. The similarity of Dnmt2 enzymes with DNA methyltransferases and absence of similarity with RNA methyltransferases combined with their strong RNA methylation activity suggest that the ancestor of Dnmt2 was a DNA methyltransferase and an early Dnmt2 enzyme changed its substrate preference to tRNA. There is no phylogenetic evidence that Dnmt2 was the precursor of eukaryotic Dnmts. Most likely, the eukaryotic Dnmt1 and Dnmt3 families of DNA methyltransferases had an independent origin in the prokaryotic DNA methyltransferase sequence space.

Molecular signatures of plastic phenotypes in two eusocial insect species with simple societies

Significance In eusocial insect societies, such as ants and some bees and wasps, phenotypes are highly plastic, generating alternative phenotypes (queens and workers) from the same genome. The greatest plasticity is found in simple insect societies, in which individuals can switch between phenotypes as adults. The genomic, transcriptional, and epigenetic underpinnings of such plasticity are largely unknown. In contrast to the complex societies of the honeybee, we find that simple insect societies lack distinct transcriptional differentiation between phenotypes and coherently patterned DNA methylomes. Instead, alternative phenotypes are largely defined by subtle transcriptional network organization. These traits may facilitate genomic plasticity. These insights and resources will stimulate new approaches and hypotheses that will help to unravel the genomic processes that create phenotypic plasticity. Phenotypic plasticity is important in adaptation and shapes the evolution of organisms. However, we understand little about what aspects of the genome are important in facilitating plasticity. Eusocial insect societies produce plastic phenotypes from the same genome, as reproductives (queens) and nonreproductives (workers). The greatest plasticity is found in the simple eusocial insect societies in which individuals retain the ability to switch between reproductive and nonreproductive phenotypes as adults. We lack comprehensive data on the molecular basis of plastic phenotypes. Here, we sequenced genomes, microRNAs (miRNAs), and multiple transcriptomes and methylomes from individual brains in a wasp (Polistes canadensis) and an ant (Dinoponera quadriceps) that live in simple eusocial societies. In both species, we found few differences between phenotypes at the transcriptional level, with little functional specialization, and no evidence that phenotype-specific gene expression is driven by DNA methylation or miRNAs. Instead, phenotypic differentiation was defined more subtly by nonrandom transcriptional network organization, with roles in these networks for both conserved and taxon-restricted genes. The general lack of highly methylated regions or methylome patterning in both species may be an important mechanism for achieving plasticity among phenotypes during adulthood. These findings define previously unidentified hypotheses on the genomic processes that facilitate plasticity and suggest that the molecular hallmarks of social behavior are likely to differ with the level of social complexity.

Bisulfite sequencing Data Presentation and Compilation (BDPC) web server—a useful tool for DNA methylation analysis

During bisulfite genomic sequencing projects large amount of data are generated. The Bisulfite sequencing Data Presentation and Compilation (BDPC) web interface (http://biochem.jacobs-university.de/BDPC/) automatically analyzes bisulfite datasets prepared using the BiQ Analyzer. BDPC provides the following output: (i) MS-Excel compatible files compiling for each PCR product (a) the average methylation level, the number of clones analyzed and the percentage of CG sites analyzed (which is an indicator of data quality), (b) the methylation level observed at each CG site and (c) the methylation level of each clone. (ii) A methylation overview table compiling the methylation of all amplicons in all tissues. (iii) Publication grade figures in PNG format showing the methylation pattern for each PCR product embedded in an HMTL file summarizing the methylation data, the DNA sequence and some basic statistics. (iv) A summary file compiling the methylation pattern of different tissues, which is linked to the individual HTML result files, and can be directly used for presentation of the data in the internet. (v) A condensed file, containing all primary data in simplified format for further downstream data analysis and (vi) a custom track file for display of the results in the UCSC genome browser.

Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase.

Abstract DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a–Dnmt3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters. We show that targeting of these loci with single gRNAs leads to efficient and widespread methylation of the promoters. Multiplexing of several guide RNAs does not increase the efficiency of methylation. Peaks of targeted methylation were observed around 25 bp upstream and 40 bp downstream of the PAM site, while 20–30 bp of the binding site itself are protected against methylation. Potent methylation is dependent on the multimerization of Dnmt3a/Dnmt3L complexes on the DNA. Furthermore, the introduced methylation causes transcriptional repression of the targeted genes. These new programmable epigenetic editors allow unprecedented control of the DNA methylation status in cells and will lead to further advances in the understanding of epigenetic signaling.

Synthetic epigenetics—towards intelligent control of epigenetic states and cell identity

Epigenetics is currently one of the hottest topics in basic and biomedical research. However, to date, most of the studies have been descriptive in nature, designed to investigate static distribution of various epigenetic modifications in cells. Even though tremendous amount of information has been collected, we are still far from the complete understanding of epigenetic processes, their dynamics or even their direct effects on local chromatin and we still do not comprehend whether these epigenetic states are the cause or the consequence of the transcriptional profile of the cell. In this review, we try to define the concept of synthetic epigenetics and outline the available genome targeting technologies, which are used for locus-specific editing of epigenetic signals. We report early success stories and the lessons we have learned from them, and provide a guide for their application. Finally, we discuss existing limitations of the available technologies and indicate possible areas for further development.

Pmt1, a Dnmt2 homolog in Schizosaccharomyces pombe, mediates tRNA methylation in response to nutrient signaling

The fission yeast Schizosaccharomyces pombe carries a cytosine 5-methyltransferase homolog of the Dnmt2 family (termed pombe methyltransferase 1, Pmt1), but contains no detectable DNA methylation. Here, we found that Pmt1, like other Dnmt2 homologs, has in vitro methylation activity on cytosine 38 of tRNAAsp and, to a lesser extent, of tRNAGlu, despite the fact that it contains a non-consensus residue in catalytic motif IV as compared with its homologs. In vivo tRNA methylation also required Pmt1. Unexpectedly, however, its in vivo activity showed a strong dependence on the nutritional status of the cell because Pmt1-dependent tRNA methylation was induced in cells grown in the presence of peptone or with glutamate as a nitrogen source. Furthermore, this induction required the serine/threonine kinase Sck2, but not the kinases Sck1, Pka1 or Tor1 and was independent of glucose signaling. Taken together, this work reveals a novel connection between nutrient signaling and tRNA methylation that thus may link tRNA methylation to processes downstream of nutrient signaling like ribosome biogenesis and translation initiation.