Specific DNMT3C flanking sequence preferences facilitate methylation of young murine retrotransposons.

The DNA methyltransferase DNMT3C appeared as a duplication of the DNMT3B gene in muroids and is required for silencing of young retrotransposons in the male germline. Using specialized assay systems, we investigate the flanking sequence preferences of DNMT3C and observe characteristic preferences for cytosine at the -2 and -1 flank that are unique among DNMT3 enzymes. We identify two amino acids in the catalytic domain of DNMT3C (C543 and V547) that are responsible for the DNMT3C-specific flanking sequence preferences and evolutionary conserved in muroids. Reanalysis of published data shows that DNMT3C flanking preferences are consistent with genome-wide methylation patterns in mouse ES cells only expressing DNMT3C. Strikingly, we show that CpG sites with the preferred flanking sequences of DNMT3C are enriched in murine retrotransposons that were previously identified as DNMT3C targets. Finally, we demonstrate experimentally that DNMT3C has elevated methylation activity on substrates derived from these biological targets. Our data show that DNMT3C flanking sequence preferences match the sequences of young murine retrotransposons which facilitates their methylation. By this, our data provide mechanistic insights into the molecular co-evolution of repeat elements and (epi)genetic defense systems dedicated to maintain genomic stability in mammals

Chromatin-dependent allosteric regulation of DNMT3A activity by MeCP2

Despite their central importance in mammalian development, the mechanisms that regulate the DNA methylation machinery and thereby the generation of genomic methylation patterns are still poorly understood. Here, we identify the 5mC-binding protein MeCP2 as a direct and strong interactor of DNA methyltransferase 3( DNMT3) proteins. We mapped the interaction interface to the transcriptional repression domain of MeCP2 and the ADD domain of DNMT3A and find that binding of MeCP2 strongly inhibits the activity of DNMT3A in vitro. This effect was reinforced by cellular studies where a global reduction of DNA methylation levels was observed after overexpression of MeCP2 in human cells. By engineering conformationally locked DNMT3A variants as novel tools to study the allosteric regulation of this enzyme, we show that MeCP2 stabilizes the closed, autoinhibitory conformation of DNMT3A. Interestingly, the interaction with MeCP2 and its resulting inhibition were relieved by the binding of K4 unmodified histone H3 N-terminal tail to the DNMT3A-ADD domain. Taken together, our data indicate that the localization and activity of DNMT3A are under the combined control of MeCP2 and H3 tailmodifications where, depending on the modification status of the H3 tail at the binding sites, MeCP2 can act as either a repressor or activator of DNA methylation

The DNMT3A R882H mutant displays altered flanking sequence preferences

The DNMT3A R882H mutation is frequently observed in acute myeloid leukemia (AML). It is located in the subunit and DNA binding interface of DNMT3A and has been reported to cause a reduction in activity and dominant negative effects. We investigated the mechanistic consequences of the R882H mutation on DNMT3A showing a roughly 40% reduction in overall DNA methylation activity. Biochemical assays demonstrated that R882H does not change DNA binding affinity, protein stability or subnuclear distribution of DNMT3A. Strikingly, DNA methylation experiments revealed pronounced changes in the flanking sequence preference of the DNMT3A-R882H mutant. Based on these results, different DNA substrates with selected flanking sequences were designed to be favored or disfavored by R882H. Kinetic analyses showed that the R882H favored substrate was methylated by R882H with 45% increased rate when compared with wildtype DNMT3A, while methylation of the disfavored substrate was reduced 7-fold. Our data expand the model of the potential carcinogenic effect of the R882H mutation by showing CpG site specific activity changes. This result suggests that R882 is involved in the indirect readout of flanking sequence preferences of DNMT3A and it may explain the particular enrichment of theR882Hmutation in cancer patients by revealing mutation specific effects

Comprehensive structure-function characterization of DNMT3B and DNMT3A reveals distinctive de novo DNA methylation mechanisms

Mammalian DNA methylation patterns are established by two de novo DNA methyltransferases DNMT3A and DNMT3B, which exhibit both redundant and distinctive methylation activities. However, the related molecular basis remains undetermined. Through comprehensive structural, enzymology and cellular characterization of DNMT3A and DNMT3B, we here report a multi-layered substraterecognition mechanism underpinning their divergent genomic methylation activities. A hydrogen bond in the catalytic loop of DNMT3B causes a lower CpG specificity than DNMT3A, while the interplay of target recognition domain and homodimeric interface fine-tunes the distinct target selection between the two enzymes, with Lysine 777 of DNMT3B acting as a unique sensor of the +1 flanking base. The divergent substrate preference between DNMT3A and DNMT3B provides an explanation for site-specific epigenomic alterations seen in ICF syndrome with DNMT3B mutations. Together, this study reveals crucial and distinctive substrate-readout mechanisms of the two DNMT3 enzymes, implicative of their differential roles during development and pathogenesis

Mechanistic study on the DNA methyltransferase DNMT3A

The phenotypical and functional diversity of mammalian cell types can be attributed to a large extent to epigenetic signals that determine and stabilize gene expression profiles. One of the most important types of epigenetic signals is DNA methylation. This modification is set early in development by the de novo DNA methyltransferases DNMT3A and DNMT3B, and is found predominantly at the C5 position of cytosine bases in a CpG dinucleotide context. The accurate setting of DNA methylation patterns is critical for normal development and is determined by the precise recruitment and control of DNMT activity on chromatin. In this work, four main directions of research were undertaken, with the ultimate goal of shedding novel mechanistic insights into the mechanism of DNMT3A, its regulation by chromatin signals and interaction partners, as well as the dysregulation of this enzyme in cancer. Furthermore, the potential of DNMT3A to generate 3-methylcytosine as a side reaction was explored. The DNA methyltransferase DNMT3A has been shown to multimerize on DNA and to form large multimeric protein/DNA fibers. However, it has also been postulated that this enzyme can methylate DNA in a processive manner, a property incompatible with fiber formation. By using a dedicated set of biochemical experiments, I was able to show that the DNA methylation rate of DNMT3A increases more than linearly with increasing enzyme concentration on a long DNA substrate, but not on a short 30-mer oligonucleotide, which cannot accommodate DNMT3A polymers. Methylation experiments over a range of enzyme concentrations and with substrates containing one or two CpG sites did not provide evidence for a processive mechanism. The addition of a catalytically inactive DNMT3A mutant was found to increase the DNA methylation rate by DNMT3A on the long substrate but not on the short one. Together, these data clearly indicate that DNMT3A binds to DNA in a cooperative reaction and the formation of protein/DNA fibers increases the DNA methylation rate. These results contribute mechanistic insights into the mode by which DNA methylation patterns are established during development. The second project dealt with characterizing the effects of the R882H exchange on DNMT3A. The R882H mutation is found in the DNA binding interface of DNMT3A and is frequently observed in acute myeloid leukemia (AML). By establishing a double-tag affinity purification system, I was able to show that the mutation only leads to a minor reduction in overall DNA methylation activity in mixed R882H/wildtype DNMT3A complexes. However, a pronounced change in flanking sequence preference of the DNMT3A-R882H mutant was found. Accordingly, a substrate designed to contain the target CpG site flanked by sequences preferred by R882H was better methylated by the variant than by the wildtype enzyme. Together, these data strongly argue against a dominant-negative effect of the R882H mutation and rather propose a site-specific gain-of-activity effect. These findings are in agreement with a recently determined structure of DNMT3A in complex with DNA and they might explain the high prevalence of this specific point mutation in AML. The third project was built on previous data from the lab, documenting a strong and direct interaction between the ADD domain of DNMT3A and the TRD domain of the 5mC reading protein MECP2. These experiments revealed that through its binding, MECP2 allosterically stabilizes the autoinhibitory conformation of DNMT3A, resulting in a strong inhibition of enzymatic activity in vitro. The interaction between these two proteins and its associated inhibition could be disrupted by unmodified histone H3. In my work, I further validated the interaction between the ADD and the TRD domains by size exclusion chromatography. Also, by generating cell lines with stable over-expression of MECP2, I could show that MECP2 inhibits DNMT3A activity in cells. Together, the data from this study offer unprecedented insights into the regulation of DNMT3A by the combined action of chromatin modifications and interaction partners. Accordingly, depending on the modification status of the H3 tail at the target site, MECP2 can act as either a repressor or activator of DNA methylation. The last project dealt with the coevolution between DNA methylation and DNA repair systems, a very exciting topic that was addressed in close collaboration with the laboratory of Dr. Peter Sarkies (MRC London). By performing in vitro methylation experiments with the catalytic domain of DNMT3A, I could show that, in addition to 5mC, DNMT3A can also introduce 3mC, a modification which represents an alkylation damage of DNA. This study provides a new evolutionary perspective on the loss of DNA methylation that is observed in many species

Cooperative DNA Binding and Protein/DNA Fiber Formation Increases the Activity of the Dnmt3a DNA Methyltransferase

The Dnmt3a DNA methyltransferase has been shown to bind cooperatively to DNA and to form large multimeric protein/DNA fibers.However, it has also been reported to methylate DNA in a processive manner, a property that is incompatible with protein/DNAfiber formation. We show here that the DNA methylation rate of Dnmt3a increases more than linearly with increasing enzymeconcentration on a long DNA substrate, but not on a short 30-mer oligonucleotide substrate. We also show that addition ofa catalytically inactive Dnmt3a mutant, which carries an amino acid exchange in the catalytic center, increases the DNA methylationrate by wild type Dnmt3a on the long substrate but not on the short one. In agreement with this finding, preincubation experimentsindicate that stable protein/DNA fibers are formed on the long, but not on the short substrate. In addition, methylation experimentswith substrates containing one or two CpG sites did not provide evidence for a processive mechanism over a wide range of enzymeconcentrations. These data clearly indicate that Dnmt3a binds to DNA in a cooperative reaction and that the formation of stableprotein/DNA fibers increases the DNA methylation rate. Fiber formation occurs at low μm concentrations of Dnmt3a, which are in the range of Dnmt3a concentrations in the nucleus of embryonic stem cells. Understandingthe mechanism of Dnmt3a is of vital importance because Dnmt3a is a hotspot of somatic cancer mutations one of which has beenimplicated in changing Dnmt3a processivity

Mechanistic insights into Cytosine-N3 Methylation by DNA Methyltransferase DNMT3A

Recently, it has been discovered that different DNA-(cytosine C5)-methyltransferases including DNMT3A generate low levels of 3mC [Rosic et al. (2018), Nat. Genet., 50, 452-459]. This reaction resulted in the co-evolution of DNMTs and ALKB2 DNA repair enzymes, but its mechanism remained elusive. Here, we investigated the catalytic mechanism of DNMT3A for cytosine N3 methylation. We generated several DNMT3A variants with mutated catalytic residues and measured their activities in 5mC and 3mC generation by liquid chromatography linked to tandem mass spectrometry. Our data suggest that the methylation of N3 instead of C5 is caused by an inverted binding of the flipped cytosine target base into the active-site pocket of the DNA methyltransferase, which is partially compatible with the arrangement of catalytic amino acid residues. Given that all DNA-(cytosine C5)-methyltransferases have a common catalytic mechanism, it is likely that other enzymes of this class generate 3mC following the same mechanism

Purification and Characterization of Recombinant Expressed Apple Allergen Mal d 1

Mal d 1 is the primary apple allergen in northern Europe. To explain the differences in the allergenicity of apple varieties, it is essential to study its properties and interaction with other phytochemicals, which might modulate the allergenic potential. Therefore, an optimized production route followed by an unsophisticated purification step for Mal d 1 and respective mutants is desired to produce sufficient amounts. We describe a procedure for the transformation of the plasmid in competent E. coli cells, protein expression and rapid one-step purification. r-Mal d 1 with and without a polyhistidine-tag are purified by immobilized metal ion affinity chromatography (IMAC) and fast-protein liquid chromatography (FPLC) using a high-resolution anion-exchange column, respectively. Purity is estimated by SDS-PAGE using an image-processing program (Fiji). For both mutants an appropriate yield of r-Mal d 1 with purity higher than 85% is achieved. The allergen is characterized after tryptic in gel digestion by peptide analyses using HPLC-MS/MS. Secondary structure elements are calculated based on CD-spectroscopy and the negligible impact of the polyhistidine-tag on the folding is confirmed. The formation of dimers is proved by mass spectrometry and reduction by DTT prior to SDS-PAGE. Furthermore, the impact of the freeze and thawing process, freeze drying and storage on dimer formation is investigated

The DNMT3A R882H mutation does not cause dominant negative effects in purified mixed DNMT3A/R882H complexes

The DNA methyltransferase DNMT3A R882H mutation is observed in 25% of all AML patients. DNMT3A is active as tetramer and the R882H mutation is located in one of the subunit/subunit interfaces. Previous work has reported that formation of mixed wildtype/R882H complexes leads to a strong loss of catalytic activity observed in in vitro DNA methylation assays (Russler-Germain et al., 2014, Cancer Cell 25:442–454). To investigate this effect further, we have prepared mixed wildtype/R882H DNMT3A complexes by incubation of individually purified subunits of the DNMT3A catalytic domain and full-length DNMT3A2. In addition, we have used a double affinity tag approach and specifically purified mixed catalytic domain complexes formed after co-expression of R882H and wildtype subunits in E. coli cells. Afterwards, we determined the catalytic activity of the mixed complexes and compared it to that of purified complexes only consisting of one subunit type. In both settings, the expected catalytic activities of mixed R882H/wildtype complexes were observed demonstrating an absence of a dominant negative effect of the R882H mutation in purified DNMT3A enzymes. This result suggests that heterocomplex formation of DNMT3A and R882H is unlikely to cause dominant negative effects in human cells as well. The limitations of this conclusion and its implications are discussed