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1.
Nat Commun ; 11(1): 3355, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620778

RESUMO

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 substrate-recognition 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 distinctive substrate-readout mechanisms of the two DNMT3 enzymes, implicative of their differential roles during development and pathogenesis.


Assuntos
Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Animais , Domínio Catalítico , Linhagem Celular , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/ultraestrutura , Células-Tronco Embrionárias , Ensaios Enzimáticos , Epigênese Genética , Face/anormalidades , Humanos , Camundongos , Mutação , Doenças da Imunodeficiência Primária/genética , Relação Estrutura-Atividade , Especificidade por Substrato/genética , Difração de Raios X
2.
Nat Commun ; 11(1): 3671, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32699299

RESUMO

Epigenetic reprogramming is a cancer hallmark, but how it unfolds during early neoplastic events and its role in carcinogenesis and cancer progression is not fully understood. Here we show that resetting from primed to naïve human pluripotency results in acquisition of a DNA methylation landscape mirroring the cancer DNA methylome, with gradual hypermethylation of bivalent developmental genes. We identify a dichotomy between bivalent genes that do and do not become hypermethylated, which is also mirrored in cancer. We find that loss of H3K4me3 at bivalent regions is associated with gain of methylation. Additionally, we observe that promoter CpG island hypermethylation is not restricted solely to emerging naïve cells, suggesting that it is a feature of a heterogeneous intermediate population during resetting. These results indicate that transition to naïve pluripotency and oncogenic transformation share common epigenetic trajectories, which implicates reprogramming and the pluripotency network as a central hub in cancer formation.


Assuntos
Transformação Celular Neoplásica/genética , Reprogramação Celular , Metilação de DNA , Epigênese Genética , Neoplasias/genética , Animais , Linhagem Celular , Técnicas de Cocultura , Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Fibroblastos , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Histonas/genética , Histonas/metabolismo , Células-Tronco Embrionárias Humanas , Humanos , Camundongos , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/metabolismo
3.
Nat Commun ; 11(1): 3199, 2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32581223

RESUMO

De novo establishment of DNA methylation is accomplished by DNMT3A and DNMT3B. Here, we analyze de novo DNA methylation in mouse embryonic fibroblasts (2i-MEFs) derived from DNA-hypomethylated 2i/L ES cells with genetic ablation of Dnmt3a or Dnmt3b. We identify 355 and 333 uniquely unmethylated genes in Dnmt3a and Dnmt3b knockout (KO) 2i-MEFs, respectively. We find that Dnmt3a is exclusively required for de novo methylation at both TSS regions and gene bodies of Polycomb group (PcG) target developmental genes, while Dnmt3b has a dominant role on the X chromosome. Consistent with this, tissue-specific DNA methylation at PcG target genes is substantially reduced in Dnmt3a KO embryos. Finally, we find that human patients with DNMT3 mutations exhibit reduced DNA methylation at regions that are hypomethylated in Dnmt3 KO 2i-MEFs. In conclusion, here we report a set of unique de novo DNA methylation target sites for both DNMT3 enzymes during mammalian development that overlap with hypomethylated sites in human patients.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Animais , Diferenciação Celular/genética , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/genética , Repressão Epigenética/genética , Feminino , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Mutação , Especificidade de Órgãos , Proteínas do Grupo Polycomb , Sítio de Iniciação de Transcrição
4.
Nat Commun ; 11(1): 3153, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561758

RESUMO

Mouse embryos acquire global DNA methylation of their genome during implantation. However the exact roles of DNA methyltransferases (DNMTs) in embryos have not been studied comprehensively. Here we systematically analyze the consequences of genetic inactivation of Dnmt1, Dnmt3a and Dnmt3b on the methylome and transcriptome of mouse embryos. We find a strict division of function between DNMT1, responsible for maintenance methylation, and DNMT3A/B, solely responsible for methylation acquisition in development. By analyzing severely hypomethylated embryos, we uncover multiple functions of DNA methylation that is used as a mechanism of repression for a panel of genes including not only imprinted and germline genes, but also lineage-committed genes and 2-cell genes. DNA methylation also suppresses multiple retrotransposons and illegitimate transcripts from cryptic promoters in transposons and gene bodies. Our work provides a thorough analysis of the roles of DNA methyltransferases and the importance of DNA methylation for transcriptome integrity in mammalian embryos.


Assuntos
DNA (Citosina-5-)-Metiltransferases , Metilação de DNA , Desenvolvimento Embrionário/genética , Animais , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Metilação de DNA/fisiologia , Embrião de Mamíferos/metabolismo , Epigenômica , Regulação da Expressão Gênica , Genoma , Camundongos , Transcriptoma
5.
Nat Commun ; 11(1): 3140, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561780

RESUMO

MeCP2 plays a multifaceted role in gene expression regulation and chromatin organization. Interaction between MeCP2 and methylated DNA in the regulation of gene expression is well established. However, the widespread distribution of MeCP2 suggests it has additional interactions with chromatin. Here we demonstrate, by both biochemical and genomic analyses, that MeCP2 directly interacts with nucleosomes and its genomic distribution correlates with that of H3K27me3. In particular, the methyl-CpG-binding domain of MeCP2 shows preferential interactions with H3K27me3. We further observe that the impact of MeCP2 on transcriptional changes correlates with histone post-translational modification patterns. Our findings indicate that MeCP2 interacts with genomic loci via binding to DNA as well as histones, and that interaction between MeCP2 and histone proteins plays a key role in gene expression regulation.


Assuntos
Regulação da Expressão Gênica/fisiologia , Histonas/metabolismo , Proteína 2 de Ligação a Metil-CpG/metabolismo , Transcrição Genética/fisiologia , Animais , Sequenciamento de Cromatina por Imunoprecipitação , DNA/genética , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/fisiologia , Técnicas de Inativação de Genes , Loci Gênicos , Células HCT116 , Células HEK293 , Histonas/genética , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Knockout , Nucleossomos/genética , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Sítio de Iniciação de Transcrição/fisiologia
6.
PLoS One ; 15(6): e0235217, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32574225

RESUMO

In sheep, polyunsaturated fatty acid (PUFA) supplementations in late gestation increases the growth of offspring; however, there is a lack of evidence on the effect of PUFA supplementation during early gestation. Thus, the objective of this study was to evaluate the effect of dietary supplementation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in early gestation pregnant ewes on fatty acid concentration of fetal liver (FL) and fetal central nervous system (FCNS), and relative abundance of the mRNA for genes associated with transport and metabolism of fatty acids in FL and placenta. A total of 12 ewes, block for stage of gestation were fed a diet containing 1.6% (dry matter basis) monounsaturated fatty acids (MUFA) or EPA+DHA during the first 45 days of gestation. A cesarean section was conducted on day 45 of gestation to collect placenta (caruncle and cotyledon), FL, and FCNS. Relative abundance of mRNA in FL and FCNS and fatty acid concentration were analyzed using a 2x2 factorial arrangement of treatments considering fatty acid supplementation and tissue as the main factors. Concentrations of C18:1 isomers increase (P < 0.05) in FL and FCNS with MUFA supplementation; the FL and FCNS had a greater concentration of C20:3(n-6), C20:3(n-3), C22:1, C22:5 and C22:6 (P < 0.05) with EPA+DHA supplementation. In FL, the relative abundance of LPL mRNA was greater (P = 0.02) as a result of MUFA supplementation. In placenta, there was a FA x tissue interaction for relative abundance of DNMT3b and FFAR-4 mRNA (P < 0.05). Fetus from MUFA-supplemented dams had a greater relative abundance of FABP-4 mRNA (P < 0.05). Results indicate supplementation with EPA+DHA during early gestation increases the total EPA and DHA in FL. For the placenta, EPA+DHA supplementation led to an increase in the relative abundance of lipid mRNA for transport genes.


Assuntos
Sistema Nervoso Central/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/análogos & derivados , Ácidos Graxos/análise , Feto/efeitos dos fármacos , Placenta/efeitos dos fármacos , RNA Mensageiro/metabolismo , Animais , DNA (Citosina-5-)-Metiltransferases/genética , Suplementos Nutricionais , Ácido Eicosapentaenoico/farmacologia , Proteínas de Ligação a Ácido Graxo/genética , Ácidos Graxos/química , Feminino , Feto/metabolismo , Idade Gestacional , Lipase Lipoproteica/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Placenta/metabolismo , Gravidez , Ovinos
7.
Nat Genet ; 52(8): 819-827, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32514123

RESUMO

Mammalian cells stably maintain high levels of DNA methylation despite expressing both positive (DNMT3A/B) and negative (TET1-3) regulators. Here, we analyzed the independent and combined effects of these regulators on the DNA methylation landscape using a panel of knockout human embryonic stem cell (ESC) lines. The greatest impact on global methylation levels was observed in DNMT3-deficient cells, including reproducible focal demethylation at thousands of normally methylated loci. Demethylation depends on TET expression and occurs only when both DNMT3s are absent. Dynamic loci are enriched for hydroxymethylcytosine and overlap with subsets of putative somatic enhancers that are methylated in ESCs and can be activated upon differentiation. We observe similar dynamics in mouse ESCs that were less frequent in epiblast stem cells (EpiSCs) and scarce in somatic tissues, suggesting a conserved pluripotency-linked mechanism. Taken together, our data reveal tightly regulated competition between DNMT3s and TETs at thousands of somatic regulatory sequences within pluripotent cells.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Elementos Facilitadores Genéticos/genética , Oxigenases de Função Mista/genética , Células-Tronco Pluripotentes/fisiologia , Proteínas Proto-Oncogênicas/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/fisiologia , Epigênese Genética/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Camadas Germinativas/fisiologia , Humanos , Camundongos , Camundongos Knockout
8.
Nat Commun ; 11(1): 2996, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32533060

RESUMO

Multiple myeloma (MM) is a plasma-cell neoplasm that is treated with high-dose chemotherapy, autologous stem cell transplant (ASCT) and long-term immunomodulatory drug (IMiD) maintenance. The presence of somatic mutations in the peripheral blood is termed clonal hematopoiesis of indeterminate potential (CHIP) and is associated with adverse outcomes. Targeted sequencing of the stem cell product from 629 MM patients treated by ASCT at the Dana-Farber Cancer Institute (2003-2011) detects CHIP in 136/629 patients (21.6%). The most commonly mutated genes are DNMT3A, TET2, TP53, ASXL1 and PPM1D. Twenty-one from fifty-six patients (3.3%) receiving first-line IMiD maintenance develop a therapy-related myeloid neoplasm (TMN). However, regardless of CHIP status, the use of IMiD maintenance associates with improved PFS and OS. In those not receiving IMiD maintenance, CHIP is associated with decreased overall survival (OS) (HR:1.34, p = 0.02) and progression free survival (PFS) (HR:1.45, p < 0.001) due to an increase in MM progression.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Hematopoese/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Mieloma Múltiplo/terapia , Mutação , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/genética , Intervalo Livre de Progressão , Proteínas Proto-Oncogênicas/genética , Transplante Autólogo , Proteína Supressora de Tumor p53/genética , Adulto Jovem
9.
Nat Commun ; 11(1): 2834, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503981

RESUMO

Recruitment of DNA repair proteins to DNA damage sites is a critical step for DNA repair. Post-translational modifications of proteins at DNA damage sites serve as DNA damage codes to recruit specific DNA repair factors. Here, we show that mRNA is locally modified by m5C at sites of DNA damage. The RNA methyltransferase TRDMT1 is recruited to DNA damage sites to promote m5C induction. Loss of TRDMT1 compromises homologous recombination (HR) and increases cellular sensitivity to DNA double-strand breaks (DSBs). In the absence of TRDMT1, RAD51 and RAD52 fail to localize to sites of reactive oxygen species (ROS)-induced DNA damage. In vitro, RAD52 displays an increased affinity for DNA:RNA hybrids containing m5C-modified RNA. Loss of TRDMT1 in cancer cells confers sensitivity to PARP inhibitors in vitro and in vivo. These results reveal an unexpected TRDMT1-m5C axis that promotes HR, suggesting that post-transcriptional modifications of RNA can also serve as DNA damage codes to regulate DNA repair.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/metabolismo , Animais , Linhagem Celular Tumoral , Citosina/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Resistencia a Medicamentos Antineoplásicos/genética , Técnicas de Silenciamento de Genes , Humanos , Metilação , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Nat Commun ; 11(1): 2680, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32471981

RESUMO

DNA methylation is considered a stable epigenetic mark, yet methylation patterns can vary during differentiation and in diseases such as cancer. Local levels of DNA methylation result from opposing enzymatic activities, the rates of which remain largely unknown. Here we developed a theoretical and experimental framework enabling us to infer methylation and demethylation rates at 860,404 CpGs in mouse embryonic stem cells. We find that enzymatic rates can vary as much as two orders of magnitude between CpGs with identical steady-state DNA methylation. Unexpectedly, de novo and maintenance methylation activity is reduced at transcription factor binding sites, while methylation turnover is elevated in transcribed gene bodies. Furthermore, we show that TET activity contributes substantially more than passive demethylation to establishing low methylation levels at distal enhancers. Taken together, our work unveils a genome-scale map of methylation kinetics, revealing highly variable and context-specific activity for the DNA methylation machinery.


Assuntos
Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Desmetilação do DNA , Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Sítios de Ligação/genética , Linhagem Celular , Mapeamento Cromossômico , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Dioxigenases/metabolismo , Epigênese Genética/genética , Genoma/genética , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismo , Transcrição Genética/genética
11.
Mol Cell ; 78(3): 493-505.e8, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32353257

RESUMO

The promyelocytic leukemia (PML) body is a phase-separated nuclear structure physically associated with chromatin, implying its crucial roles in genome functions. However, its role in transcriptional regulation is largely unknown. We developed APEX-mediated chromatin labeling and purification (ALaP) to identify the genomic regions proximal to PML bodies. We found that PML bodies associate with active regulatory regions across the genome and with ∼300 kb of the short arm of the Y chromosome (YS300) in mouse embryonic stem cells. The PML body association with YS300 is essential for the transcriptional activity of the neighboring Y-linked clustered genes. Mechanistically, PML bodies provide specific nuclear spaces that the de novo DNA methyltransferase DNMT3A cannot access, resulting in the steady maintenance of a hypo-methylated state at Y-linked gene promoters. Our study underscores a new mechanism for gene regulation in the 3D nuclear space and provides insights into the functional properties of nuclear structures for genome function.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação da Expressão Gênica , Corpos de Inclusão Intranuclear/genética , Cromossomo Y/genética , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , RNA Helicases DEAD-box/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Células-Tronco Embrionárias/fisiologia , Endonucleases/genética , Sequenciamento de Nucleotídeos em Larga Escala , Corpos de Inclusão Intranuclear/metabolismo , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/genética , Enzimas Multifuncionais/genética , Família Multigênica , Estresse Oxidativo , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Proteínas/genética , Fatores de Transcrição/genética , Cromossomo Y/metabolismo
12.
Nat Commun ; 11(1): 2214, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32371941

RESUMO

MSH1 is a plant-specific protein. RNAi suppression of MSH1 results in phenotype variability for developmental and stress response pathways. Segregation of the RNAi transgene produces non-genetic msh1 'memory' with multi-generational inheritance. First-generation memory versus non-memory comparison, and six-generation inheritance studies, identifies gene-associated, heritable methylation repatterning. Genome-wide methylome analysis integrated with RNAseq and network-based enrichment studies identifies altered circadian clock networks, and phytohormone and stress response pathways that intersect with circadian control. A total of 373 differentially methylated loci comprising these networks are sufficient to discriminate memory from nonmemory full sibs. Methylation inhibitor 5-azacytidine diminishes the differences between memory and wild type for growth, gene expression and methylation patterning. The msh1 reprogramming is dependent on functional HISTONE DEACETYLASE 6 and methyltransferase MET1, and transition to memory requires the RNA-directed DNA methylation pathway. This system of phenotypic plasticity may serve as a potent model for defining accelerated plant adaptation during environmental change.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Metilação de DNA , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Característica Quantitativa Herdável , Interferência de RNA , Transgenes/genética , Adaptação Fisiológica/genética , DNA (Citosina-5-)-Metiltransferases/genética , Epigênese Genética , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla/métodos , Desacetilase 6 de Histona/genética , Padrões de Herança/genética , Plantas Geneticamente Modificadas , Transdução de Sinais/genética
13.
Mol Cell ; 79(1): 127-139.e4, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32437639

RESUMO

C.neoformans Dnmt5 is an unusually specific maintenance-type CpG methyltransferase (DNMT) that mediates long-term epigenome evolution. It harbors a DNMT domain and SNF2 ATPase domain. We find that the SNF2 domain couples substrate specificity to an ATPase step essential for DNA methylation. Coupling occurs independent of nucleosomes. Hemimethylated DNA preferentially stimulates ATPase activity, and mutating Dnmt5's ATP-binding pocket disproportionately reduces ATPase stimulation by hemimethylated versus unmethylated substrates. Engineered DNA substrates that stabilize a reaction intermediate by mimicking a "flipped-out" conformation of the target cytosine bypass the SNF2 domain's requirement for hemimethylation. This result implies that ATP hydrolysis by the SNF2 domain is coupled to the DNMT domain conformational changes induced by preferred substrates. These findings establish a new role for a SNF2 ATPase: controlling an adjoined enzymatic domain's substrate recognition and catalysis. We speculate that this coupling contributes to the exquisite specificity of Dnmt5 via mechanisms related to kinetic proofreading.


Assuntos
Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA Fúngico/metabolismo , Proteínas Fúngicas/metabolismo , Nucleossomos/metabolismo , Adenosina Trifosfatases/genética , Cryptococcus neoformans/genética , Cryptococcus neoformans/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA Fúngico/química , DNA Fúngico/genética , Proteínas Fúngicas/genética , Hidrólise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
Nat Commun ; 11(1): 2294, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385248

RESUMO

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-Atividade
16.
J Appl Oral Sci ; 28: e20190583, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32267380

RESUMO

BACKGROUND: Genetic and epigenetic changes have been associated with periodontitis in various genes; however, little is known about genes involved in epigenetic mechanisms and in oxidative stress. OBJECTIVE: This study aims to investigate the association of polymorphisms C677T in MTHFR (rs1801133) and -149C→T in DNMT3B (rs2424913), as well as the methylation profiles of MTHFR, miR-9-1, miR-9-3, SOD1, and CAT with periodontitis. The association between polymorphisms and DNA methylation profiles was also analyzed. METHODOLOGY: The population studied was composed of 100 nonsmokers of both sexes, divided into healthy and periodontitis groups. Genomic DNA was extracted from the epithelial buccal cells, which were collected through a mouthwash. Polymorphism analysis was performed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), while methylation-specific PCR (MSP) or combined bisulfite restriction analysis techniques were applied for methylation analysis. RESULTS: For DNMT3B, the T allele and the TT genotype were detected more frequently in the periodontitis group, as well as the methylated profile on the miR-9-1 promoter region. There was also a tendency towards promoter region methylation on the CAT sequence of individuals with periodontal disease. CONCLUSION: The polymorphism -149C→T in DNMT3B (rs2424913) and the methylated profile of the miR-9-1 promoter region are associated with periodontitis.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , MicroRNAs/genética , Periodontite/genética , Polimorfismo Genético , Adulto , Idoso , Estudos de Casos e Controles , Catalase/genética , Feminino , Estudos de Associação Genética , Genótipo , Humanos , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Superóxido Dismutase-1/genética
17.
Am J Clin Pathol ; 154(1): 48-56, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32112088

RESUMO

OBJECTIVES: To compare the clinical significance of SF3B1/DNMT3A Comutations with SF3B1 or DNMT3A mutation alone in myelodysplastic syndrome (MDS) and clonal cytopenia of undetermined significance (CCUS). METHODS: We identified and compared 31 patients with only DNMT3A mutation, 48 patients with only SF3B1 mutation, and 16 patients with only SF3B1/DNMT3A comutations. RESULTS: SF3B1/DNMT3A comutations were found to be more common in MDS, whereas DNMT3A mutation alone was more common in CCUS. The patients with SF3B1/DNMT3A comutations were less likely to have poor cytogenetics than patients with DNMT3A mutation alone. Patients with SF3B1/DNMT3A comutations showed significantly longer median survival time and better overall survival than patients with DNMT3A mutation alone. CONCLUSIONS: Patients with SF3B1/DNMT3A comutations appear to have better clinical outcomes than patients with isolated DNMT3A mutation. These findings suggest that the favorable prognosis of SF3B1 mutation in is not abrogated by the concurrent presence of a DNMT3A mutation.


Assuntos
Doenças da Medula Óssea/genética , DNA (Citosina-5-)-Metiltransferases/genética , Síndromes Mielodisplásicas/genética , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Idoso , Doenças da Medula Óssea/mortalidade , Feminino , Humanos , Masculino , Mutação , Síndromes Mielodisplásicas/mortalidade
18.
Nucleic Acids Res ; 48(8): 4081-4099, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32187373

RESUMO

Cytosine methylation is a ubiquitous modification in mammalian DNA generated and maintained by several DNA methyltransferases (DNMTs) with partially overlapping functions and genomic targets. To systematically dissect the factors specifying each DNMT's activity, we engineered combinatorial knock-in of human DNMT genes in Komagataella phaffii, a yeast species lacking endogenous DNA methylation. Time-course expression measurements captured dynamic network-level adaptation of cells to DNMT3B1-induced DNA methylation stress and showed that coordinately modulating the availability of S-adenosyl methionine (SAM), the essential metabolite for DNMT-catalyzed methylation, is an evolutionarily conserved epigenetic stress response, also implicated in several human diseases. Convolutional neural networks trained on genome-wide CpG-methylation data learned distinct sequence preferences of DNMT3 family members. A simulated annealing interpretation method resolved these preferences into individual flanking nucleotides and periodic poly(A) tracts that rotationally position highly methylated cytosines relative to phased nucleosomes. Furthermore, the nucleosome repeat length defined the spatial unit of methylation spreading. Gene methylation patterns were similar to those in mammals, and hypo- and hypermethylation were predictive of increased and decreased transcription relative to control, respectively, in the absence of mammalian readers of DNA methylation. Introducing controlled epigenetic perturbations in yeast thus enabled characterization of fundamental genomic features directing specific DNMT3 proteins.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Epigênese Genética , Saccharomycetales/genética , Engenharia Celular , Centrômero , Cromatina/química , DNA (Citosina-5-)-Metiltransferases/genética , Técnicas de Introdução de Genes , Genoma Fúngico , Humanos , Redes Neurais de Computação , S-Adenosilmetionina/metabolismo , Saccharomycetales/metabolismo , Estresse Fisiológico/genética , Telômero , Transcrição Genética
19.
Circ J ; 84(4): 616-625, 2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32115441

RESUMO

BACKGROUND: Ischemic postconditioning (IPostC) is an endogenous protective mechanism to reduce ischemia-reperfusion (I/R) injury. However, whether IPostC protects aged cardiomyocytes against I/R injury is not fully understood. Considering the protective function of microRNA 30a (miR-30a) against ischemia-induced injury in H9C2 cells, its role in the protective effects of IPostC on I/R injury of aged cardiomyocytes was investigated further.Methods and Results:To mimic I/R and IPostC in vitro, the aged cardiomyocyte model for hypoxia postconditioning (HPostC) treatment was established by 9 days of incubation with 8 mg/mL D-galactose and then followed by exposure to hypoxic environment. HPostC significantly alleviated hypoxia/reoxygenation (H/R) injury and reduced autophagy of aged cardiomyocytes, as evidenced by decreased LC3B-II expression and increased p62 by Western blot. Quantified by quantitative real-time polymerase chain reaction (qRT-PCR), miR-30a was increased in aged cardiomyocytes treated with HPostC compared with I/R injury group. Overexpression of miR-30a by LV3-rno-miR-30a mimic promoted cardioprotective effect of HPostC in aged cardiomyocytes by suppressing BECN1-mediated autophagy, all of which was abrogated by knockdown of miR-30a expression. Epigenetic analyses demonstrated that HPostC reduced DNA methyltransferase 3b-mediated DNA hypomethylation levels at miR-30a promoter, leading to upregulation of miR-30a. CONCLUSIONS: HPostC protected aged cardiomyocytes survival against H/R injury via DNMT3b-dependent activation of miR-30a. miR-30a could be a potential therapeutic target for ischemic myocardial infarction.


Assuntos
Autofagia , Senescência Celular , Metilação de DNA , Epigênese Genética , MicroRNAs/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Hipóxia Celular , Linhagem Celular , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , MicroRNAs/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Regiões Promotoras Genéticas , Ratos , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais
20.
Nat Genet ; 52(4): 378-387, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203468

RESUMO

Mutations in genes involved in DNA methylation (DNAme; for example, TET2 and DNMT3A) are frequently observed in hematological malignancies1-3 and clonal hematopoiesis4,5. Applying single-cell sequencing to murine hematopoietic stem and progenitor cells, we observed that these mutations disrupt hematopoietic differentiation, causing opposite shifts in the frequencies of erythroid versus myelomonocytic progenitors following Tet2 or Dnmt3a loss. Notably, these shifts trace back to transcriptional priming skews in uncommitted hematopoietic stem cells. To reconcile genome-wide DNAme changes with specific erythroid versus myelomonocytic skews, we provide evidence in support of differential sensitivity of transcription factors due to biases in CpG enrichment in their binding motif. Single-cell transcriptomes with targeted genotyping showed similar skews in transcriptional priming of DNMT3A-mutated human clonal hematopoiesis bone marrow progenitors. These data show that DNAme shapes the topography of hematopoietic differentiation, and support a model in which genome-wide methylation changes are transduced to differentiation skews through biases in CpG enrichment of the transcription factor binding motif.


Assuntos
Diferenciação Celular/genética , Metilação de DNA/genética , Hematopoese/genética , Animais , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Hematopoéticas/fisiologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mutação/genética , Transcrição Genética/genética , Transcriptoma/genética
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