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1.
Nat Commun ; 11(1): 5417, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33110091

RESUMO

De novo DNA methylation (DNAme) during mammalian spermatogenesis yields a densely methylated genome, with the exception of CpG islands (CGIs), which are hypomethylated in sperm. While the paternal genome undergoes widespread DNAme loss before the first S-phase following fertilization, recent mass spectrometry analysis revealed that the zygotic paternal genome is paradoxically also subject to a low level of de novo DNAme. However, the loci involved, and impact on transcription were not addressed. Here, we employ allele-specific analysis of whole-genome bisulphite sequencing data and show that a number of genomic regions, including several dozen CGI promoters, are de novo methylated on the paternal genome by the 2-cell stage. A subset of these promoters maintains DNAme through development to the blastocyst stage. Consistent with paternal DNAme acquisition, many of these loci are hypermethylated in androgenetic blastocysts but hypomethylated in parthenogenetic blastocysts. Paternal DNAme acquisition is lost following maternal deletion of Dnmt3a, with a subset of promoters, which are normally transcribed from the paternal allele in blastocysts, being prematurely transcribed at the 4-cell stage in maternal Dnmt3a knockout embryos. These observations uncover a role for maternal DNMT3A activity in post-fertilization epigenetic reprogramming and transcriptional silencing of the paternal genome.


Assuntos
Blastocisto/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Genoma , Herança Materna , Herança Paterna , Alelos , Animais , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Epigenômica , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos Endogâmicos DBA , Oócitos/metabolismo , Espermatozoides/metabolismo
2.
Int J Nanomedicine ; 15: 5963-5975, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32884259

RESUMO

Background: Previous studies have reported that quercetin (Q) has a potential antibacterial and anticancer activity. However, its application is limited by many important factors including high hydrophobicity and low absorption. Methodology: In the current study, we synthesized and characterized (Patent) a novel chitosan-based quercetin nanohydrogel (ChiNH/Q). Encapsulation efficiency was confirmed by UV/VIS spectrophotometer. Physicochemical characterization of ChiNH/Q was assessed by PDI, DLS, SEM, FTIR, and XRD. The toxicity of the ChiNH/Q against five strains of the pathogen and HepG2 cells was examined. Moreover, the quantification of ChiNH/Q on genomic global DNA methylation and expression of DNMTs (DNMT1/3A/3B) in HepG2 cancer cells were evaluated by ELISA and real-time PCR, respectively. Results: Under the SEM-based images, the hydrodynamic size of the ChiNH/Q was 743.6 nm. The changes in the PDI were 0.507, and zeta potential was obtained as 12.1 mV for ChiNH/Q. The FTIR peak of ChiNH/Q showed the peak at 627 cm-1 corresponded to tensile vibrational of NH2-groups related to Q, and it is the indication of Q loading in the formulation. Moreover, XRD data have detected the encapsulation of ChiNH/Q. The ChiNH/Q showed a potent antimicrobial inhibitory effect and exerted cytotoxic effects against HepG2 cancer cells with IC50 values of 100 µg/mL. Moreover, our data have shown that ChiNH/Q effectively reduced (65%) the average expression level of all the three DNMTs (p<0.05) and significantly increased (1.01%) the 5-methylated cytosine (5-mC) levels in HepG2 cells. Conclusion: Our results showed for the first time the bioavailability and potentiality of ChiNH/Q as a potent antimicrobial and anticancer agent against cancer cells. Our result provided evidence that ChiNH/Q could effectively reduce cellular DNMT expression levels and increase genomic global DNA methylation in HepG2 cancer cells. Our results suggest a potential clinical application of nanoparticles as antimicrobial and anticancer agents in combination cancer therapy.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Epigênese Genética/efeitos dos fármacos , Hidrogéis/química , Nanoestruturas/química , Quercetina/farmacologia , Anti-Infecciosos/administração & dosagem , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacocinética , Quitosana/química , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Células Hep G2 , Humanos , Hidrogéis/administração & dosagem , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Nanoestruturas/administração & dosagem , Quercetina/administração & dosagem , Quercetina/química , Quercetina/farmacocinética , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X
3.
Nature ; 586(7827): 151-155, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32968275

RESUMO

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 , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Nucleossomos/química , Ligação Proteica , Domínios Proteicos , Xenopus/genética
4.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(9): 1295-1300, 2020 Sep 30.
Artigo em Chinês | MEDLINE | ID: mdl-32990224

RESUMO

OBJECTIVE: To investigate the expression of DNMT3b in human bladder cancer tissues and its correlation with postoperative survival of patients with bladder cancer. METHODS: Thirty-eight pairs of surgically resected human bladder cancer tissues and adjacent bladder tissues were detected by immunohistochemistry for DNMT3b expression, and the correlations of DNMT3b expression level were analyzed with the patients'age, gender, pathological grade, tumor size, T stage, lymph node metastasis and TNM stages. Kaplan-Meier survival analysis was performed to assess the effect of DNMT3b expression on survival outcomes of the patients. RESULTS: High DNMT3b protein expression was detected in 63.16% of the bladder cancer tissues and in 13.16% of the adjacent tissues (P < 0.05). The expression level of DNMT3b was associated with the pathological grade (P=0.002), tumor size (P < 0.001), T stage (P < 0.001), lymphatic metastasis (P=0.039) and TNM stage (P < 0.001), but not with gender or age of the patients. Multivariate logistic regression analysis showed that the protein expression level of DNMT3b was correlated with tumor size (P=0.008) and TNM grades of the tumor (P=0.042). Kaplan-Meier analysis showed that the patients with a high DNMT3b expression had a significantly shorter overall survival than those with a low DNMT3b expression (P=0.021). CONCLUSIONS: DNMT3b overexpression in bladder cancer is closely related to such clinicopathological factors as pathological grade, tumor size, T stage, lymphatic metastasis, and TNM stage and a shorter overall survival of the patients, suggesting the potential value of DNMT3b as a prognostic marker and a new therapeutic target for bladder cancer.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Neoplasias da Bexiga Urinária , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Neoplasias da Bexiga Urinária/patologia
5.
Nat Commun ; 11(1): 4118, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807789

RESUMO

Epigenetic information is transmitted from mother to daughter cells through mitosis. Here, to identify factors that might play a role in conveying epigenetic memory through cell division, we report on the isolation of unfixed, native chromosomes from metaphase-arrested cells using flow cytometry and perform LC-MS/MS to identify chromosome-bound proteins. A quantitative proteomic comparison between metaphase-arrested cell lysates and chromosome-sorted samples reveals a cohort of proteins that were significantly enriched on mitotic ESC chromosomes. These include pluripotency-associated transcription factors, repressive chromatin-modifiers such as PRC2 and DNA methyl-transferases, and proteins governing chromosome architecture. Deletion of PRC2, Dnmt1/3a/3b or Mecp2 in ESCs leads to an increase in the size of individual mitotic chromosomes, consistent with de-condensation. Similar results were obtained by the experimental cleavage of cohesin. Thus, we identify chromosome-bound factors in pluripotent stem cells during mitosis and reveal that PRC2, DNA methylation and Mecp2 are required to maintain chromosome compaction.


Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , Células-Tronco Embrionárias/metabolismo , Fatores de Transcrição/metabolismo , Animais , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Metilação de DNA/fisiologia , Imunofluorescência , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Proteômica
6.
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
7.
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
8.
Nature ; 584(7822): 635-639, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32674113

RESUMO

In mammals, the acquisition of the germline from the soma provides the germline with an essential challenge: the need to erase and reset genomic methylation1. In the male germline, RNA-directed DNA methylation silences young, active transposable elements2-4. The PIWI protein MIWI2 (PIWIL4) and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of transposable elements3,5. piRNAs are proposed to tether MIWI2 to nascent transposable element transcripts; however, the mechanism by which MIWI2 directs the de novo methylation of transposable elements is poorly understood, although central to the immortality of the germline. Here we define the interactome of MIWI2 in mouse fetal gonocytes undergoing de novo genome methylation and identify a previously unknown MIWI2-associated factor, SPOCD1, that is essential for the methylation and silencing of young transposable elements. The loss of Spocd1 in mice results in male-specific infertility but does not affect either piRNA biogenesis or the localization of MIWI2 to the nucleus. SPOCD1 is a nuclear protein whose expression is restricted to the period of de novo genome methylation. It co-purifies in vivo with DNMT3L and DNMT3A, components of the de novo methylation machinery, as well as with constituents of the NURD and BAF chromatin remodelling complexes. We propose a model whereby tethering of MIWI2 to a nascent transposable element transcript recruits repressive chromatin remodelling activities and the de novo methylation apparatus through SPOCD1. In summary, we have identified a previously unrecognized and essential executor of mammalian piRNA-directed DNA methylation.


Assuntos
Metilação de DNA/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonauta/metabolismo , Montagem e Desmontagem da Cromatina , DNA (Citosina-5-)-Metiltransferases/metabolismo , Elementos de DNA Transponíveis/genética , Feminino , Fertilidade/genética , Inativação Gênica , Genes de Partícula A Intracisternal/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Masculino , Camundongos , RNA Interferente Pequeno/biossíntese , Espermatogênese/genética
9.
PLoS One ; 15(6): e0234641, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32574164

RESUMO

Chondrocytes, comparable to many cells from the connective tissue, dedifferentiate and end up in a similar fibroblastoid cell type, marked by the loss of the specific expression pattern. Here, chondrocytes isolated from osteoarthritic (OA) patients were investigated. The OA chondrocytes used in this work were not affected by the loss of specific gene expression upon cell culture. The mRNA levels of known cartilage markers, such as SOX5, SOX6, SOX9, aggrecan and proteoglycan 4, remained unchanged. Since chondrocytes from OA and healthy tissue show different DNA methylation patterns, the underlying mechanisms of cartilage marker maintenance were investigated with a focus on the epigenetic modification by DNA methylation. The treatment of dedifferentiated chondrocytes with the DNA methyltransferase inhibitor 5-aza-2´-deoxycytidine (5-aza-dC) displayed no considerable impact on the maintenance of marker gene expression observed in the dedifferentiated state, while the chondrogenic differentiation capacity was compromised. On the other hand, the pre-cultivation with 5-aza-dC improved the osteogenesis and adipogenesis of OA chondrocytes. Contradictory to these effects, the DNA methylation levels were not reduced after treatment for four weeks with 1 µM 5-aza-dC. In conclusion, 5-aza-dC affects the differentiation capacity of OA chondrocytes, while the global DNA methylation level remains stable. Furthermore, dedifferentiated chondrocytes isolated from late-stage OA patients represent a reliable cell source for in vitro studies and disease models without the need for additional alterations.


Assuntos
Condrócitos/patologia , Decitabina/farmacologia , Osteoartrite/patologia , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Biomarcadores/metabolismo , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Forma Celular/efeitos dos fármacos , Forma Celular/genética , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Osteoartrite/genética , Osteogênese/efeitos dos fármacos , Osteogênese/genética
10.
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
11.
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
12.
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
13.
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
14.
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
16.
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
18.
J Biomed Sci ; 27(1): 46, 2020 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-32238162

RESUMO

BACKGROUND: Epigenetic silencing of retinoic acid (RA) signaling-related genes have been linked with the pathogenesis and clinical outcome in oral squamous cell carcinoma (OSCC) carcinogenesis. However, the precise mechanisms underlying the abnormal silencing of RA signaling-related genes in OSCC have not been well investigated. METHODS: Using combined analysis of genome-wide gene expression and methylation profile from 40 matched normal-tumor pairs of OSCC specimens, we found a set of retinoid signaling related genes are frequently hypermethylated and downregulated in OSCC patient samples, including alcohol dehydrogenase, iron containing 1 (ADHFE1) and aldehyde dehydrogenase 1 family, member A2 (ALDH1A2), which are the important rate-limiting enzymes in synthesis of RA. The expression of ADHFE1 and ALDH1A2 in OSCC patients was determine by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. The binding sites of miR-30a and miR-379 with DNA methyltransferase 3B (DNMT3B) were predicted using a series of bioinformatic tools, and validated using dual luciferase assay and Western blot analyses. The functions of miR-30a, miR-379, and DNMT3B were accessed by growth and colony formation analyses using gain- and loss-of-function approaches. Chromatin immunoprecipitation (ChIP) was performed to explore the molecular mechanisms by arecoline and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) treatment. RESULTS: We demonstrated that deregulated miR-30a and miR-379 could represent a mechanism for the silencing of ADHFE1 and ALDH1A2 in OSCC through targeting DNMT3B. Ectopic expression of miR-30a and miR-379 could induce re-expression of methylation-silenced ADHFE1 and ALDH1A2, and lead to growth inhibition in oral cancer cells. Furthermore, the dysregulation of the miRNAs and DNMT-3B may result from exposure to tobacco smoking and betel quid chewing. CONCLUSIONS: Our results demonstrate that tobacco smoking and betel quid chewing could repress miR-30a and miR-379, which upregulate the DNMT3B expression, in turn, lead to the hypermethylation of ADHFE1 and ALDH1A genes, consequently, promote the oncogenic activity. These findings highlight the potential use of retinoids in combination with epigenetic modifiers for the prevention or treatment of oral cancer.


Assuntos
Carcinoma de Células Escamosas/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Inativação Gênica , MicroRNAs/genética , Neoplasias Bucais/genética , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Aldeído Desidrogenase 1/genética , Aldeído Desidrogenase 1/metabolismo , Arecolina/química , Carcinogênese/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Redes e Vias Metabólicas , MicroRNAs/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Nitrosaminas/química , Retinal Desidrogenase/genética , Retinal Desidrogenase/metabolismo , Tretinoína/metabolismo
19.
PLoS Genet ; 16(4): e1008599, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32271759

RESUMO

In 1993, Denise Barlow proposed that genomic imprinting might have arisen from a host defense mechanism designed to inactivate retrotransposons. Although there were few examples at hand, she suggested that there should be maternal-specific and paternal-specific factors involved, with cognate imprinting boxes that they recognized; furthermore, the system should build on conserved biochemical factors, including DNA methylation, and maternal control should predominate for imprints. Here, we revisit this hypothesis in the light of recent advances in our understanding of host defense and DNA methylation and in particular, the link with Krüppel-associated box-zinc finger (KRAB-ZF) proteins.


Assuntos
Inativação Gênica , Impressão Genômica , Modelos Genéticos , Retroelementos/genética , Animais , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/metabolismo
20.
Cancer Res ; 80(12): 2612-2627, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32265226

RESUMO

Current cancer treatments are largely based on the genetic characterization of primary tumors and are ineffective for metastatic disease. Here we report that DNA methyltransferase 3B (DNMT3B) is induced at distant metastatic sites and mediates epigenetic reprogramming of metastatic tumor cells. Multiomics analysis and spontaneous metastatic mouse models revealed that DNMT3B alters multiple pathways including STAT3, NFκB, PI3K/Akt, ß-catenin, and Notch signaling, which are critical for cancer cell survival, apoptosis, proliferation, invasion, and colonization. PGE2 and IL6 were identified as critical inflammatory mediators in DNMT3B induction. DNMT3B expression levels positively correlated with human metastatic progression. Targeting IL6 or COX-2 reduced DNMT3B induction and improved chemo or PD1 therapy. We propose a novel mechanism linking the metastatic microenvironment with epigenetic alterations that occur at distant sites. These results caution against the "Achilles heel" in cancer therapies based on primary tumor characterization and suggests targeting DNMT3B induction as new option for treating metastatic disease. SIGNIFICANCE: These findings reveal that DNMT3B epigenetically regulates multiple pro-oncogenic signaling pathways via the inflammatory microenvironment at distant sites, cautioning the clinical approach basing current therapies on genetic characterization of primary tumors.


Assuntos
Neoplasias da Mama/patologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , Dinoprostona/metabolismo , Interleucina-6/metabolismo , Neoplasias Pulmonares/secundário , Animais , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral/transplante , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Progressão da Doença , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Humanos , Interleucina-6/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Camundongos , Receptor de Morte Celular Programada 1/imunologia , Estudo de Prova de Conceito , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
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