RESUMO
DNA methylation is an epigenetic mechanism that introduces a methyl group at the C5 position of cytosine. This reaction is catalyzed by DNA methyltransferases (DNMTs) and is essential for the regulation of gene transcription. The DNMT1 and DNMT3A or -3B family proteins are known targets for the inhibition of DNA hypermethylation in cancer cells. A selective non-nucleoside DNMT3A inhibitor was developed that mimics S-adenosyl-l-methionine and deoxycytidine; however, the mechanism of selectivity is unclear because the inhibitor-protein complex structure determination is absent. Therefore, we performed docking and molecular dynamics simulations to predict the structure of the complex formed by the association between DNMT3A and the selective inhibitor. Our simulations, binding free energy decomposition analysis, structural isoform comparison, and residue scanning showed that Arg688 of DNMT3A is involved in the interaction with this inhibitor, as evidenced by its significant contribution to the binding free energy. The presence of Asn1192 at the corresponding residues in DNMT1 results in a loss of affinity for the inhibitor, suggesting that the interactions mediated by Arg688 in DNMT3A are essential for selectivity. Our findings can be applied in the design of DNMT-selective inhibitors and methylation-specific drug optimization procedures.
Assuntos
DNA (Citosina-5-)-Metiltransferases , DNA Metiltransferase 3A , Inibidores Enzimáticos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Metilação de DNA , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/química , Sítios de LigaçãoRESUMO
The DNMT3A DNA methyltransferase and MECP2 methylation reader are highly expressed in neurons. Both proteins interact via their DNMT3A-ADD and MECP2-TRD domains, and the MECP2 interaction regulates the activity and subnuclear localization of DNMT3A. Here, we mapped the interface of both domains using peptide SPOT array binding, protein pull-down, equilibrium peptide binding assays, and structural analyses. The region D529-D531 on the surface of the ADD domain was identified as interaction point with the TRD domain. This includes important residues of the histone H3 N-terminal tail binding site to the ADD domain, explaining why TRD and H3 binding to the ADD domain is competitive. On the TRD domain, residues 214-228 containing K219 and K223 were found to be essential for the ADD interaction. This part represents a folded patch within the otherwise largely disordered TRD domain. A crystal structure analysis of ADD revealed that the identified H3/TDR lysine binding pocket is occupied by an arginine residue from a crystallographic neighbor in the ADD apoprotein structure. Finally, we show that mutations in the interface of ADD and TRD domains disrupt the cellular interaction of both proteins in NIH3T3 cells. In summary, our data show that the H3 peptide binding cleft of the ADD domain also mediates the interaction with the MECP2-TRD domain suggesting that this binding site may have a broader role also in the interaction of DNMT3A with other proteins leading to complex regulation options by competitive and PTM specific binding.
Assuntos
DNA (Citosina-5-)-Metiltransferases , DNA Metiltransferase 3A , Proteína 2 de Ligação a Metil-CpG , Sítios de Ligação , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Proteína 2 de Ligação a Metil-CpG/química , Proteína 2 de Ligação a Metil-CpG/metabolismo , Células NIH 3T3 , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Histonas/química , Histonas/metabolismo , HumanosRESUMO
Cytosine methylation at the C5-position-generating 5-methylcytosine (5mC)-is a DNA modification found in many eukaryotic organisms, including fungi, plants, invertebrates, and vertebrates, albeit its levels vary greatly in different organisms. In mammals, cytosine methylation occurs predominantly in the context of CpG dinucleotides, with the majority (60-80%) of CpG sites in their genomes being methylated. DNA methylation plays crucial roles in the regulation of chromatin structure and gene expression and is essential for mammalian development. Aberrant changes in DNA methylation and genetic alterations in enzymes and regulators involved in DNA methylation are associated with various human diseases, including cancer and developmental disorders. In mammals, DNA methylation is mediated by two families of DNA methyltransferases (Dnmts), namely Dnmt1 and Dnmt3 proteins. Over the last three decades, genetic manipulations of these enzymes, as well as their regulators, in mice have greatly contributed to our understanding of the biological functions of DNA methylation in mammals. In this chapter, we discuss genetic studies on mammalian Dnmts, focusing on their roles in embryogenesis, cellular differentiation, genomic imprinting, and human diseases.
Assuntos
DNA (Citosina-5-)-Metiltransferases , Metilação de DNA , Humanos , Camundongos , Animais , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferase 1 , Metilases de Modificação do DNA/genética , 5-Metilcitosina , Mamíferos/genética , Mamíferos/metabolismo , DNA/metabolismoRESUMO
DNMT1 maintains the parental DNA methylation pattern on newly replicated hemimethylated DNA. The failure of this maintenance process causes aberrant DNA methylation that affects transcription and contributes to the development and progression of cancers such as acute myeloid leukemia. Here, we structurally characterized a set of newly discovered DNMT1-selective, reversible, non-nucleoside inhibitors that bear a core 3,5-dicyanopyridine moiety, as exemplified by GSK3735967, to better understand their mechanism of inhibition. All of the dicyanopydridine-containing inhibitors examined intercalate into the hemimethylated DNA between two CpG base pairs through the DNA minor groove, resulting in conformational movement of the DNMT1 active-site loop. In addition, GSK3735967 introduces two new binding sites, where it interacts with and stabilizes the displaced DNMT1 active-site loop and it occupies an open aromatic cage in which trimethylated histone H4 lysine 20 is expected to bind. Our work represents a substantial step in generating potent, selective, and non-nucleoside inhibitors of DNMT1.
Assuntos
DNA (Citosina-5-)-Metiltransferases , Metilação de DNA , Sítios de Ligação , Domínio Catalítico , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismoRESUMO
Dnmt2, a member of the DNA methyltransferase superfamily, catalyzes the formation of 5-methylcytosine at position 38 in the anticodon loop of tRNAs. Dnmt2 regulates many cellular biological processes, especially the production of tRNA-derived fragments and intergenerational transmission of paternal metabolic disorders to offspring. Moreover, Dnmt2 is closely related to human cancers. The tRNA substrates of mammalian Dnmt2s are mainly detected using bisulfite sequencing; however, we lack supporting biochemical data concerning their substrate specificity or recognition mechanism. Here, we deciphered the tRNA substrates of human DNMT2 (hDNMT2) as tRNAAsp(GUC), tRNAGly(GCC) and tRNAVal(AAC). Intriguingly, for tRNAAsp(GUC) and tRNAGly(GCC), G34 is the discriminator element; whereas for tRNAVal(AAC), the inosine modification at position 34 (I34), which is formed by the ADAT2/3 complex, is the prerequisite for hDNMT2 recognition. We showed that the C32U33(G/I)34N35 (C/U)36A37C38 motif in the anticodon loop, U11:A24 in the D stem, and the correct size of the variable loop are required for Dnmt2 recognition of substrate tRNAs. Furthermore, mammalian Dnmt2s possess a conserved tRNA recognition mechanism.
Assuntos
5-Metilcitosina/metabolismo , Anticódon/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , RNA de Transferência/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Anticódon/genética , Sequência de Bases , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/genética , Células HEK293 , Células HeLa , Humanos , Inosina/metabolismo , Camundongos , Modelos Moleculares , Células NIH 3T3 , Conformação de Ácido Nucleico , Ligação Proteica , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência de Ácido Aspártico/química , RNA de Transferência de Ácido Aspártico/genética , RNA de Transferência de Ácido Aspártico/metabolismo , RNA de Transferência de Glicina/química , RNA de Transferência de Glicina/genética , RNA de Transferência de Glicina/metabolismo , RNA de Transferência de Valina/química , RNA de Transferência de Valina/genética , RNA de Transferência de Valina/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Especificidade por SubstratoRESUMO
Precise deposition of CpG methylation is critical for mammalian development and tissue homeostasis and is often dysregulated in human diseases. The localization of de novo DNA methyltransferase DNMT3A is facilitated by its PWWP domain recognizing histone H3 lysine 36 (H3K36) methylation1,2 and is normally depleted at CpG islands (CGIs)3. However, methylation of CGIs regulated by Polycomb repressive complexes (PRCs) has also been observed4-8. Here, we report that DNMT3A PWWP domain mutations identified in paragangliomas9 and microcephalic dwarfism10 promote aberrant localization of DNMT3A to CGIs in a PRC1-dependent manner. DNMT3A PWWP mutants accumulate at regions containing PRC1-mediated formation of monoubiquitylated histone H2A lysine 119 (H2AK119ub), irrespective of the amounts of PRC2-catalyzed formation of trimethylated histone H3 lysine 27 (H3K27me3). DNMT3A interacts with H2AK119ub-modified nucleosomes through a putative amino-terminal ubiquitin-dependent recruitment region, providing an alternative form of DNMT3A genomic targeting that is augmented by the loss of PWWP reader function. Ablation of PRC1 abrogates localization of DNMT3A PWWP mutants to CGIs and prevents aberrant DNA hypermethylation. Our study implies that a balance between DNMT3A recruitment by distinct reader domains guides de novo CpG methylation and may underlie the abnormal DNA methylation landscapes observed in select human cancer subtypes and developmental disorders.
Assuntos
Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Proteínas do Grupo Polycomb/metabolismo , Animais , Catálise , Linhagem Celular , DNA (Citosina-5-)-Metiltransferases/química , DNA Metiltransferase 3A , Predisposição Genética para Doença , Genoma Humano , Histonas/metabolismo , Humanos , Lisina/metabolismo , Camundongos , Mutação/genética , Nucleossomos/metabolismo , Domínios Proteicos , UbiquitinaçãoRESUMO
5-Methylcytosine (5mC), the central epigenetic mark of mammalian DNA, plays fundamental roles in chromatin regulation. 5mC is written onto genomes by DNA methyltransferases (DNMT), and perturbation of this process is an early event in carcinogenesis. However, studying 5mC functions is limited by the inability to control individual DNMTs with spatiotemporal resolution in vivo. We report light-control of DNMT catalysis by genetically encoding a photocaged cysteine as a catalytic residue. This enables translation of inactive DNMTs, their rapid activation by light-decaging, and subsequent monitoring of de novo DNA methylation. We provide insights into how cancer-related DNMT mutations alter de novo methylation in vivo, and demonstrate local and tuneable cytosine methylation by light-controlled DNMTs fused to a programmable transcription activator-like effector domain targeting pericentromeric satellite-3 DNA. We further study early events of transcriptome alterations upon DNMT-catalyzed cytosine methylation. Our study sets a basis to dissect the order and kinetics of diverse chromatin-associated events triggered by normal and aberrant DNA methylation.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos da radiação , Luz , 5-Metilcitosina/metabolismo , Biocatálise , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/genética , Células HEK293 , Humanos , Mutação , Transcriptoma/efeitos da radiaçãoRESUMO
Despite widespread interest for understanding how modified bases have evolved their contemporary functions, limited experimental evidence exists for measuring how close an organism is to accidentally creating a new, modified base within the framework of its existing genome. Here, we describe the biochemical and structural basis for how a single-point mutation in E. coli's naturally occurring cytosine methyltransferase can surprisingly endow a neomorphic ability to create the unnatural DNA base, 5-carboxymethylcytosine (5cxmC), in vivo. Mass spectrometry, bacterial genetics, and structure-guided biochemistry reveal this base to be exclusively derived from the natural but sparse secondary metabolite carboxy-S-adenosyl-L-methionine (CxSAM). Our discovery of a new, unnatural DNA modification reveals insights into the substrate selectivity of DNA methyltransferase enzymes, offers a promising new biotechnological tool for the characterization of the mammalian epigenome, and provides an unexpected model for how neomorphic bases could arise in nature from repurposed host metabolites.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/genética , DNA Bacteriano/química , Mutação PuntualRESUMO
CpG methylation by de novo DNA methyltransferases (DNMTs) 3A and 3B is essential for mammalian development and differentiation and is frequently dysregulated in cancer1. These two DNMTs preferentially bind to nucleosomes, yet cannot methylate the DNA wrapped around the nucleosome core2, and they favour the methylation of linker DNA at positioned nucleosomes3,4. Here we present the cryo-electron microscopy structure of a ternary complex of catalytically competent DNMT3A2, the catalytically inactive accessory subunit DNMT3B3 and a nucleosome core particle flanked by linker DNA. The catalytic-like domain of the accessory DNMT3B3 binds to the acidic patch of the nucleosome core, which orients the binding of DNMT3A2 to the linker DNA. The steric constraints of this arrangement suggest that nucleosomal DNA must be moved relative to the nucleosome core for de novo methylation to occur.
Assuntos
Microscopia Crioeletrônica , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , Nucleossomos/metabolismo , Animais , Biocatálise , Montagem e Desmontagem da Cromatina , DNA/química , DNA/metabolismo , Metilação de DNA , DNA Metiltransferase 3A , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Nucleossomos/química , Ligação Proteica , Domínios Proteicos , Xenopus/genética , DNA Metiltransferase 3BRESUMO
DNA methyltransferase DNMT3A is essential for establishment of mammalian DNA methylation during development. The R882H DNMT3A is a hotspot mutation in acute myeloid leukemia (AML) causing aberrant DNA methylation. However, how this mutation affects the structure and function of DNMT3A remains unclear. Here we report structural characterization of wild-type and R882H-mutated DNMT3A in complex with DNA substrates with different sequence contexts. A loop from the target recognition domain (TRD loop) recognizes the CpG dinucleotides in a +1 flanking site-dependent manner. The R882H mutation reduces the DNA binding at the homodimeric interface, as well as the molecular link between the homodimeric interface and TRD loop, leading to enhanced dynamics of TRD loop. Consistently, in vitro methylation analyses indicate that the R882H mutation compromises the enzymatic activity, CpG specificity and flanking sequence preference of DNMT3A. Together, this study uncovers multiple defects of DNMT3A caused by the R882H mutation in AML.
Assuntos
DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Mutação/genética , Sequência de Bases , DNA/genética , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
Large bodies of studies have shown that the CRISPR/Cas9-based library screening is a very powerful tool for the identification of gene functions. However, most of these studies have focused on protein-coding genes, and, furthermore, very few studies have used gene reporters for screening. In the present study, we generated DNA methyltransferase 3B (DNMT3B) reporter and screened a CRISPR/Cas9 synergistic activation mediator (SAM) library against a focused group of lncRNAs. With this screening approach, we identified Rhabdomyosarcoma 2-Associated Transcript (RMST) as a positive regulator for DNMT3B. This was confirmed by activation of the endogenous RMST by SAM or ectopic expression of RMST. Moreover, RMST knockout (KO) suppresses DNMT3, while rescue with RMST in the KO cells restores the DNMT3 level. Finally, RMST KO suppresses global DNA methylation, leading to the upregulation of methylation-regulated genes. Mechanistically, RMST promotes the interaction between the RNA-binding protein HuR and DNMT3B 3' UTR, increasing the DNMT3B stability. Together, these results not only provide the feasibility of a reporter system for CRISPR library screening but also demonstrate the previously uncharacterized factor RMST as an important player in the modulation of DNA methylation.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , RNA Longo não Codificante/metabolismo , Regulação para Cima/genética , Regiões 3' não Traduzidas , Sistemas CRISPR-Cas , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Proteína Semelhante a ELAV 1/química , Estabilidade Enzimática/genética , Técnicas de Inativação de Genes , Genes Reporter , Células HCT116 , Células HEK293 , Humanos , Células MCF-7 , RNA Guia de Cinetoplastídeos/genética , RNA Longo não Codificante/genética , Transfecção , DNA Metiltransferase 3BRESUMO
In mammals, DNA methylation is necessary for the maintenance of genomic stability, gene expression regulation, and other processes. During malignant diseases progression, changes in both DNA methylation patterns and DNA methyltransferase (MTase) genes are observed. Human de novo MTase DNMT3A is most frequently mutated in acute myeloid leukemia (AML) with a striking prevalence of R882H mutation, which has been extensively studied. Here, we investigate the functional role of the missense mutations (S714C, R635W, R736H, R771L, P777R, and F752V) found in the catalytic domain of DNMT3A in AML patients. These were accordingly mutated in the murine Dnmt3a catalytic domain (S124C, R45W, R146H, R181L, P187R, and F162V) and in addition, one-site CpG-containing DNA substrates were used as a model system. The 3-15-fold decrease (S124C and P187R) or complete loss (F162V, R45W, and R146H) of Dnmt3a-CD methylation activity was observed. Remarkably, Pro 187 and Arg 146 are not located at or near the Dnmt3a functional motives. Regulatory protein Dnmt3L did not enhance the methylation activity of R45W, R146H, P187R, and F162V mutants. The key steps of the Dnmt3a-mediated methylation mechanism, including DNA binding and transient covalent intermediate formation, were examined. There was a complete loss of DNA-binding affinity for R45W located in the AdoMet binding region and for R146H. Dnmt3a mutants studied in vitro suggest functional impairment of DNMT3A during pathogenesis.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Leucemia Mieloide Aguda/enzimologia , Mutação de Sentido Incorreto , Sequência de Aminoácidos , Domínio Catalítico , DNA (Citosina-5-)-Metiltransferases/química , Metilação de DNA , DNA Metiltransferase 3A , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , S-Adenosilmetionina/metabolismo , Alinhamento de SequênciaRESUMO
DNMT3A (DNA methyltransferase 3A) is a de novo DNA methyltransferase responsible for establishing CpG methylation patterns within the genome. DNMT3A activity is essential for normal development, and its dysfunction has been linked to developmental disorders and cancer. DNMT3A is frequently mutated in myeloid malignancies with the majority of mutations occurring at Arg-882, where R882H mutations are most frequent. The R882H mutation causes a reduction in DNA methyltransferase activity and hypomethylation at differentially-methylated regions within the genome, ultimately preventing hematopoietic stem cell differentiation and leading to leukemogenesis. Although the means by which the R882H DNMT3A mutation reduces enzymatic activity has been the subject of several studies, the precise mechanism by which this occurs has been elusive. Herein, we demonstrate that in the context of the full-length DNMT3A protein, the R882H mutation stabilizes the formation of large oligomeric DNMT3A species to reduce the overall DNA methyltransferase activity of the mutant protein as well as the WT-R882H complex in a dominant-negative manner. This shift in the DNMT3A oligomeric equilibrium and the resulting reduced enzymatic activity can be partially rescued in the presence of oligomer-disrupting DNMT3L, as well as DNMT3A point mutations along the oligomer-forming interface of the catalytic domain. In addition to modulating the oligomeric state of DNMT3A, the R882H mutation also leads to a DNA-binding defect, which may further reduce enzymatic activity. These findings provide a mechanistic explanation for the observed loss of DNMT3A activity associated with the R882H hot spot mutation in cancer.
Assuntos
DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , Mutação , Multimerização Proteica , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Humanos , Modelos Moleculares , Estrutura Quaternária de ProteínaRESUMO
Somatic DNMT3A mutations at R882 are frequently observed in AML patients including the very abundant R882H, but also R882C, R882P and R882S. Using deep enzymology, we show here that DNMT3A-R882H has more than 70-fold altered flanking sequence preferences when compared with wildtype DNMT3A. The R882H flanking sequence preferences mainly differ on the 3' side of the CpG site, where they resemble DNMT3B, while 5' flanking sequence preferences resemble wildtype DNMT3A, indicating that R882H behaves like a DNMT3A/DNMT3B chimera. Investigation of the activity and flanking sequence preferences of other mutations of R882 revealed that they cause similar effects. Bioinformatic analyses of genomic methylation patterns focusing on flanking sequence effects after expression of wildtype DNMT3A and R882H in human cells revealed that genomic methylation patterns reflect the details of the altered flanking sequence preferences of R882H. Concordantly, R882H specific hypermethylation in AML patients was strongly correlated with the R882H flanking sequence preferences. R882H specific DNA hypermethylation events in AML patients were accompanied by R882H specific mis-regulation of several genes with strong cancer connection, which are potential downstream targets of R882H. In conclusion, our data provide novel and detailed mechanistic understanding of the pathogenic mechanism of the DNMT3A R882H somatic cancer mutation.
Assuntos
Região 5'-Flanqueadora/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Arginina/genética , Sítios de Ligação/genética , Domínio Catalítico , Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferases/química , DNA Metiltransferase 3A , Células HCT116 , Histidina/genética , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Especificidade por Substrato/genéticaRESUMO
DNA methylations are carried out by DNA methyltransferases (DNMTs) that are key enzymes during gene expression. Many chemicals, including pesticides, have shown modulation of epigenetic functions by inhibiting DNMTs. In this work, human DNMTs were evaluated as a potential target for pesticides through virtual screening of 1038 pesticides on DNMT1 (3SWR) and DNMT3A (2QRV). Molecular docking calculations for DNMTs-pesticide complexes were performed using AutoDock Vina. Binding-affinity values and contact patterns were employed as selection criteria of pesticides as virtual hits for DNMTs. The best three DNMT-pesticides complexes selected according to their high absolute affinity values (kcal/mol), for both DNMT1 and DNMT3A, were flocoumafen (-12.5; -9.9), brodifacoum (-12.4; -8.4) and difenacoum (-12.1; -8.7). These chemicals belong to second-generation rodenticides. The most frequent predicted interacting residues for DNMT1-pesticide complexes were Trp1170A, Phe1145A, Asn1578A, Arg1574A and Pro1225A; whereas for DNMT3A those were Arg271B, Lys740A, and Glu303B. These results suggest that rodenticides used for pest control are potential DNMT ligands and therefore, may modulate DNA methylations. This finding has important environmental and clinical implications, as epigenetic pathways are critical in many biochemical processes leading to diseases.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/química , DNA (Citosina-5-)-Metiltransferases/química , Inibidores Enzimáticos/química , Metiltransferases/metabolismo , Praguicidas/química , 4-Hidroxicumarinas/química , Simulação por Computador , Metilação de DNA , DNA Metiltransferase 3A , Bases de Dados de Compostos Químicos , Humanos , Simulação de Acoplamento Molecular , Relação Quantitativa Estrutura-Atividade , Reprodutibilidade dos TestesRESUMO
The PWWP domain of DNMT3 DNA methyltransferases binds to histone H3 tails containing methylated K36, and this activity is important for heterochromatic targeting. Here, we show that the PWWP domain of mouse DNMT3A binds to H3K36me2 and H3K36me3 with a slight preference for H3K36me2. PWWP domains have also been reported to bind to DNA, and the close proximity of H3K36 and nucleosomal DNA suggests a combined binding to H3K36me2/3 and DNA. We show here that the DNMT3A PWWP domain binds to DNA with a weak preference for AT-rich sequences and that the designed charge reversal R362E mutation disrupts DNA binding. The K295E mutation, as well as K295I recently identified in paraganglioma, a rare neuroendocrine neoplasm, disrupts both DNA and H3K36me2/3 binding, which is in agreement with the proximity of K295 to residues involved in K36me2/3 methyllysine binding. Nucleosome pulldown experiments show that DNA binding and H3K36me2/3 binding are important for the interaction of the DNMT3A PWWP domain with nucleosomes. Localization studies of transiently transfected fluorescently-tagged wild-type and PWWP-mutated full-length DNMT3A indicate that both interactions contribute to the subnuclear localization of DNMT3A in mouse cells. In summary, our data demonstrate that the combined binding of the DNMT3A PWWP domain to the H3 tail containing K36me2/3 and to the nucleosomal or linker DNA is important for its chromatin interaction and subnuclear targeting of DNMT3A in living cells.
Assuntos
Cromatina/genética , Cromatina/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA/metabolismo , Histonas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Animais , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , DNA Metiltransferase 3A , Espaço Intracelular/metabolismo , Camundongos , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Transporte ProteicoRESUMO
A myriad of protein partners modulate the activity of the human DNA methyltransferase 3A (DNMT3A), whose interactions with these other proteins are frequently altered during oncogenesis. We show here that the tumor suppressor p53 decreases DNMT3A activity by forming a heterotetramer complex with DNMT3A. Mutational and modeling experiments suggested that p53 interacts with the same region in DNMT3A as does the structurally characterized DNMT3L. We observed that the p53-mediated repression of DNMT3A activity is blocked by amino acid substitutions within this interface, but surprisingly, also by a distal DNMT3A residue, R882H. DNMT3A R882H occurs frequently in various cancers, including acute myeloid leukemia, and our results suggest that the effects of R882H and other DNMT3A mutations may go beyond changes in DNMT3A methylation activity. To further understand the dynamics of how protein-protein interactions modulate DNMT3A activity, we determined that p53 has a greater affinity for DNMT3A than for DNMT3L and that p53 readily displaces DNMT3L from the DNMT3A:DNMT3L heterotetramer. Interestingly, this occurred even when the preformed DNMT3A:DNMT3L complex was actively methylating DNA. The frequently identified p53 substitutions (R248W and R273H), whereas able to regulate DNMT3A function when forming the DNMT3A:p53 heterotetramer, no longer displaced DNMT3L from the DNMT3A:DNMT3L heterotetramer. The results of our work highlight the complex interplay between DNMT3A, p53, and DNMT3L and how these interactions are further modulated by clinically derived mutations in each of the interacting partners.
Assuntos
DNA (Citosina-5-)-Metiltransferases/química , Mutação de Sentido Incorreto , Proteína Supressora de Tumor p53/química , Regulação Alostérica , Substituição de Aminoácidos , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA Metiltransferase 3A , DNA de Neoplasias/química , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Multimerização Proteica , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
DNA methyltransferases (DNMTs), responsible for the regulation of DNA methylation, have been regarded as promising drug targets for cancer therapy. However, high structural conservation of the catalytic domains of DNMTs poses a big challenge to design selective inhibitors for a specific DNMT isoform. In this study, molecular dynamics (MD) simulations, end-point free energy calculations and umbrella sampling (US) simulations were performed to reveal the molecular basis of the binding selectivity of three representative DNMT inhibitors towards DNMT1 and DNMT3A, including SFG (DNMT1 and DNMT3A dual inhibitors), DC-05 (DNMT1 selective inhibitor) and GSKex1 (DNMT3A selective inhibitor). The binding selectivity of the studied inhibitors reported in previous experiments is reproduced by the MD simulation and binding free energy prediction. The simulation results also suggest that the driving force to determine the binding selectivity of the studied inhibitors stems from the difference in the protein-inhibitor van der Waals interactions. Meanwhile, the per-residue free energy decomposition reveals that the contributions from several non-conserved residues in the binding pocket of DNMT1/DNMT3A, especially Val1580/Trp893, Asn1578/Arg891 and Met1169/Val665, are the key factors responsible for the binding selectivity of DNMT inhibitors. In addition, the binding preference of the studied inhibitors was further validated by the potentials of mean force predicted by the US simulations. This study will provide valuable information for the rational design of novel selective inhibitors targeting DNMT1 and DNMT3A.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Inibidores Enzimáticos/química , Simulação de Dinâmica Molecular , Sítios de Ligação , Domínio Catalítico , DNA (Citosina-5-)-Metiltransferase 1/química , DNA (Citosina-5-)-Metiltransferases/química , Metilação de DNA , DNA Metiltransferase 3A , Ligação Proteica , Conformação Proteica , TermodinâmicaRESUMO
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.
Assuntos
DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/genética , Mutação , Domínio Catalítico , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA Metiltransferase 3A , Humanos , Modelos Moleculares , Multimerização ProteicaRESUMO
Dnmt2 methylates cytosine at position 38 of tRNAAsp in a variety of eukaryotic organisms. A correlation between the presence of the hypermodified nucleoside queuosine (Q) at position 34 of tRNAAsp and the Dnmt2 dependent C38 methylation was recently found in vivo for S. pombe and D. discoideum. We demonstrate a direct effect of the Q-modification on the methyltransferase catalytic efficiency in vitro, as Vmax/K0.5 of purified S. pombe Dnmt2 shows an increase for in vitro transcribed tRNAAsp containing Q34 to 6.27 ∗ 10-3 s-1 µM-1 compared to 1.51 ∗ 10-3 s-1 µM-1 for the unmodified substrate. Q34tRNAAsp exhibits an only slightly increased affinity for Dnmt2 in comparison to unmodified G34tRNA. In order to get insight into the structural basis for the Q-dependency, the crystal structure of S. pombe Dnmt2 was determined at 1.7 Å resolution. It closely resembles the known structures of human and E. histolytica Dnmt2, and contains the entire active site loop. The interaction with tRNA was analyzed by means of mass-spectrometry using UV cross-linked Dnmt2-tRNA complex. These cross-link data and computational docking of Dnmt2 and tRNAAsp reveal Q34 positioned adjacent to the S-adenosylmethionine occupying the active site, suggesting that the observed increase of Dnmt2 catalytic efficiency by queuine originates from optimal positioning of the substrate molecules and residues relevant for methyl transfer.