Allosteric control of mammalian DNA methyltransferases - a new regulatory paradigm

In mammals, DNA methylation is introduced by the DNMT1, DNMT3A and DNMT3B methyltransferases, which are all large multi-domain proteins containing a catalytic C-terminal domain and an N-terminal part with regulatory functions. Recently, two novel regulatory principles of DNMTs were uncovered. It was shown that their catalytic activity is under allosteric control of N-terminal domains with autoinhibitory function, the RFT and CXXC domains in DNMT1 and the ADD domain in DNMT3. Moreover, targeting and activity of DNMTs were found to be regulated in a concerted manner by interactors and posttranslational modifications (PTMs). In this review, we describe the structures and domain composition of the DNMT1 and DNMT3 enzymes, their DNA binding, catalytic mechanism,multimerization and the processes controlling their stability in cells with a focus on their regulation and chromatin targeting by PTMs, interactors and chromatin modifications. We propose that the allosteric regulation of DNMTs by autoinhibitory domains acts as a general switch for the modulation of the function of DNMTs, providing numerous possibilities for interacting proteins, nucleic acids or PTMs to regulate DNMT activity and targeting. The combined regulation of DNMT targeting and catalytic activity contributes to the precise spatiotemporal control of DNMT function and genome methylation in cells

The UHRF1 protein stimulates the activity and specificity of the maintenance DNA methyltransferase DNMT1 by an allosteric mechanism

The ubiquitin-like, containing PHD and RING finger domains protein 1 (UHRF1) is essential for maintenance DNA methylationby DNA methyltransferase 1 (DNMT1). UHRF1 has been shown to recruit DNMT1 to replicated DNA by the ability of its SET andRING-associated (SRA) domain to bind to hemimethylated DNA. Here, we demonstrate that UHRF1 also increases the activity ofDNMT1 by almost 5-fold. This stimulation is mediated by a direct interaction of both proteins through the SRA domain of UHRF1and the replication focus targeting sequence domain of DNMT1, and it does not require DNA binding by the SRA domain. Disruptionof the interaction between DNMT1 and UHRF1 by replacement of key residues in the replication focus targeting sequence domainled to a strong reduction of DNMT1 stimulation. Additionally, the interaction with UHRF1 increased the specificity of DNMT1for methylation of hemimethylated CpG sites. These findings show that apart from the targeting of DNMT1 to the replicatedDNA UHRF1 increases the activity and specificity of DNMT1, thus exerting a multifaceted influence on the maintenance of DNAmethylatio

Function and disruption of DNA Methyltransferase 3a cooperative DNA binding and nucleoprotein filament formation

The catalytic domain of Dnmt3a cooperatively multimerizes on DNA forming nucleoprotein filaments. Based on modeling, we identified the interface of Dnmt3a complexes binding next to each other on the DNA and disrupted it by charge reversal of critical residues. This prevented cooperative DNA binding and multimerization of Dnmt3a on the DNA, as shown by the loss of cooperative complex formation in electrophoretic mobility shift assay, the loss of cooperativity in DNA binding in solution, the loss of a characteristic 8- to 10-bp periodicity in DNA methylation and direct imaging of protein–DNA complexes by scanning force microscopy. Non-cooperative Dnmt3a-C variants bound DNA well and retained methylation activity, indicating that cooperative DNA binding and multimerization of Dnmt3a on the DNA are not required for activity. However, one non-cooperative variant showed reduced heterochromatic localization in mammalian cells. We propose two roles of Dnmt3a cooperative DNA binding in the cell: (i) either nucleofilament formation could be required for periodic DNA methylation or (ii) favorable interactions between Dnmt3a complexes may be needed for the tight packing of Dnmt3a at heterochromatic regions. The complex interface optimized for tight packing would then promote the cooperative binding of Dnmt3a to naked DNA in vitro

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

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

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

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

A solid‐phase transfection platform for arrayed CRISPR screens [Corrigendum]

Since the publication of this study, it has come to our attention that a citation to the study by Bulkescher et al (2017) was omitted from the Introduction. The following sentence should have been included in the introduction: “A previously reported solid‐phase reverse transfection method for proteins (Bulkescher et al , 2017) was used for the delivery of RNPs for three endogenous genes suggesting the potential of solid‐phase reverse transfection for CRISPR/Cas9‐based gene editing, despite its low efficiency”. We apologise for any inconvenience this omission may have caused

A solid-phase transfection platform for arrayed CRISPR screens

Arrayed CRISPR‐based screens emerge as a powerful alternative to pooled screens making it possible to investigate a wide range of cellular phenotypes that are typically not amenable to pooled screens. Here, we describe a solid‐phase transfection platform that enables CRISPR‐based genetic screens in arrayed format with flexible readouts. We demonstrate efficient gene knockout upon delivery of guide RNAs and Cas9/guide RNA ribonucleoprotein complexes into untransformed and cancer cell lines. In addition, we provide evidence that our platform can be easily adapted to high‐throughput screens and we use this approach to study oncogene addiction in tumor cells. Finally demonstrating that the human primary cells can also be edited using this method, we pave the way for rapid testing of potential targeted therapies

Formation of nucleoprotein filaments by mammalian DNA methyltransferase Dnmt3a in complex with regulator Dnmt3L

The C-terminal domains of Dnmt3a and Dnmt3L form elongated heterotetramers (3L-3a-3a-3L). Analytical ultracentrifugation confirmed the Dnmt3a-C/3L-C complex exists as a 2:2 heterotetramer in solution. The 3a–3a interface is the DNA-binding site, while both interfaces are essential for AdoMet binding and catalytic activity. Hairpin bisulfite analysis shows correlated methylation of two CG sites in a distance of ∼8-10 bp in the opposite DNA strands, which corresponds to the geometry of the two active sites in one Dnmt3a-C/3L-C tetramer. Correlated methylation was also observed for two CG sites at similar distances in the same DNA strand, which can be attributed to the binding of two tetramers next to each other. DNA-binding experiments show that Dnmt3a-C/3L-C complexes multimerize on the DNA. Scanning force microscopy demonstrates filament formation rather than binding of single tetramers and shows that protein–DNA filament formation leads to a 1.5-fold shortening of the DNA length

EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells

Introduction: Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. Results: In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. Conclusion: In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management. 1 Introductio