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1.
PLoS One ; 15(2): e0229144, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084194

RESUMO

The multi-domain protein UHRF1 is essential for DNA methylation maintenance and binds DNA via a base-flipping mechanism with a preference for hemi-methylated CpG sites. We investigated its binding to hemi- and symmetrically modified DNA containing either 5-methylcytosine (mC), 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), or 5-carboxylcytosine (caC). Our experimental results indicate that UHRF1 binds symmetrically carboxylated and hybrid methylated/carboxylated CpG dyads in addition to its previously reported substrates. Complementary molecular dynamics simulations provide a possible mechanistic explanation of how the protein could differentiate between modification patterns. First, we observe different local binding modes in the nucleotide binding pocket as well as the protein's NKR finger. Second, both DNA modification sites are coupled through key residues within the NKR finger, suggesting a communication pathway affecting protein-DNA binding for carboxylcytosine modifications. Our results suggest a possible additional function of the hemi-methylation reader UHRF1 through binding of carboxylated CpG sites. This opens the possibility of new biological roles of UHRF1 beyond DNA methylation maintenance and of oxidised methylcytosine derivates in epigenetic regulation.


Assuntos
5-Metilcitosina/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ilhas de CpG/genética , Citosina/análogos & derivados , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT/química , Citosina/metabolismo , Epigênese Genética , Camundongos , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos , Especificidade por Substrato , Ubiquitina-Proteína Ligases/química
2.
Chemosphere ; 241: 124989, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31590028

RESUMO

Here we reported the stress responses of nutrient deprivation and extended observation of autophagy, apoptosis, and DNA methylation in zebrafish embryonic fibroblast (ZF4) cells. Our results showed that serum deprivation resulted in the changes of cell shape and adherent ability, the suppressed cell growth and viability, and the inhibited proliferation and cell cycle. Besides, the appearance of lysosome and autophagosome/autolysosome with significantly increased expression of mRNAs (ulk1a, becn1, atg12, sqstm1, maplc3, and lamp1) and proteins (Atg12, Becn1, Sqstm1, and Lamp1) indicate the autophagic activity was boosted at initial stage but relatively weakened at 48 h of serum starvation. When autophagy no longer mitigate for the stress, cell apoptosis detected by the mRNA expression of caspases, Bcl-2/Bax expression, and Annexin V/PI was gradually enhanced to execute the death plan upon prolonged starvation process. Furthermore, the methyl group metabolism was increased in accordance with autophagic activity and was suppressed by enhanced apoptotic activity. These data suggested that the recycle activity induced by autophagy could compensate the substrates and reactions of DNA transmethylation, which obviously increased 5-methylcytosine (5 mC) level in ZF4 cells. In summary, our results discovered the cellular responses under prolonged serum starvation stress and elaborated the switch from autophagy to apoptosis and corresponding correlation with 5 mC level changes in teleost fish in vitro.


Assuntos
Nutrientes/deficiência , Estresse Fisiológico , Peixe-Zebra/metabolismo , 5-Metilcitosina/metabolismo , Animais , Apoptose/fisiologia , Autofagia/fisiologia , Caspases/metabolismo , Linhagem Celular , Proliferação de Células , Lisossomos/metabolismo , Inanição
3.
Int J Cancer ; 146(1): 115-122, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31211411

RESUMO

Epigenetic transformations may provide early indicators for cancer and other disease. Specifically, the amount of genomic 5-hydroxymethylcytosine (5-hmC) was shown to be globally reduced in a wide range of cancers. The integration of this global biomarker into diagnostic workflows is hampered by the limitations of current 5-hmC quantification methods. Here we present and validate a fluorescence-based platform for high-throughput and cost-effective quantification of global genomic 5-hmC levels. We utilized the assay to characterize cancerous tissues based on their 5-hmC content, and observed a pronounced reduction in 5-hmC level in various cancer types. We present data for glioblastoma, colorectal cancer, multiple myeloma, chronic lymphocytic leukemia and pancreatic cancer, compared to corresponding controls. Potentially, the technique could also be used to follow response to treatment for personalized treatment selection. We present initial proof-of-concept data for treatment of familial adenomatous polyposis.


Assuntos
5-Metilcitosina/análogos & derivados , Biomarcadores Tumorais/metabolismo , Epigênese Genética , Ensaios de Triagem em Larga Escala/métodos , Neoplasias/genética , 5-Metilcitosina/metabolismo , Animais , Análise Custo-Benefício , Fluorescência , Ensaios de Triagem em Larga Escala/economia , Humanos , Camundongos , Neoplasias/classificação , Estudo de Prova de Conceito
4.
Int J Cancer ; 146(2): 373-387, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31211412

RESUMO

Loss of 5-hydroxymethylcytosine (5hmC) has been associated with mutations of the ten-eleven translocation (TET) enzymes in several types of cancer. However, tumors with wild-type TET genes can also display low 5hmC levels, suggesting that other mechanisms involved in gene regulation might be implicated in the decline of this epigenetic mark. Here we show that DNA hypermethylation and loss of DNA hydroxymethylation, as well as a marked reduction of activating histone marks in the TET3 gene, impair TET3 expression and lead to a genome-wide reduction in 5hmC levels in glioma samples and cancer cell lines. Epigenetic drugs increased expression of TET3 in glioblastoma cells and ectopic overexpression of TET3 impaired in vitro cell growth and markedly reduced tumor formation in immunodeficient mice models. TET3 overexpression partially restored the genome-wide patterns of 5hmC characteristic of control brain samples in glioblastoma cell lines, while elevated TET3 mRNA levels were correlated with better prognosis in glioma samples. Our results suggest that epigenetic repression of TET3 might promote glioblastoma tumorigenesis through the genome-wide alteration of 5hmC.


Assuntos
Neoplasias Encefálicas/genética , Carcinogênese/genética , Dioxigenases/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Biópsia , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Metilação de DNA , Regulação para Baixo , Glioblastoma/mortalidade , Glioblastoma/patologia , Código das Histonas/genética , Humanos , Camundongos , Prognóstico , RNA Mensageiro/metabolismo , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Environ Pollut ; 255(Pt 2): 113318, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31610501

RESUMO

Bisphenol A (BPA), as synthetic monomer used in the production of polycarbonate plastic and epoxy resins, has endocrine disruptor properties and high risk on human health. Epigenetic alterations could act an important role in BPA-induced toxicity, but its mechanism has not been fully understood. We investigated the effects of BPA on gene expression of chromatin modifying enzymes, promoter methylation of tumor suppressor genes and histone modifications in human prostate carcinoma cells (PC-3). IC50 value of BPA was determined as 217 and 190 µM in PC-3 cells by MTT and NRU tests, respectively. We revealed an increase in global levels of 5-methylcytocine and 5-hydroxymethylcytocine at 10 µM of BPA for 96 h. We observed a significant increase on promoter DNA methylation and decrease on gene expression of p16 gene while no change was observed for Cyclin D2 and Rassf1. Significant changes were observed in global histone modifications (H3K9ac, H3K9me3, H3K27me3, and H4K20me3) in PC-3 cells. According to these results, we investigated wide-range epigenetic modifications using PCR arrays. After 96 h BPA exposure, chromatin modifying enzymes including KDM5B and NSD1 were significantly downregulated. Also, promoter methylation of tumor suppressor genes including BCR, GSTP1, LOX, MGMT, NEUROG1, PDLIM4, PTGS2, PYCARD, TIMP3, TSC2 and ZMYDN10 altered significantly. ChIP results showed that H3K9ac, H3K9me3 and H3K27me3 modifications on p16 gene showed significant increases after 1 and 10 µM of BPA exposure. In conclusion, epigenetic signatures such as DNA methylation and histone modifications could be proposed as molecular biomarkers of BPA-induced prostate cancer progression.


Assuntos
Compostos Benzidrílicos/toxicidade , Metilação de DNA/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Código das Histonas/efeitos dos fármacos , Fenóis/toxicidade , Neoplasias da Próstata/induzido quimicamente , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Linhagem Celular Tumoral , Ciclina D2/biossíntese , Ciclina D2/genética , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Inibidor p16 de Quinase Dependente de Ciclina/genética , Regulação para Baixo/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Células PC-3 , Regiões Promotoras Genéticas , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Processamento de Proteína Pós-Traducional , Inibidor Tecidual de Metaloproteinase-3 , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genética
6.
DNA Cell Biol ; 38(12): 1460-1469, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31657619

RESUMO

Multiple factors, including genetic and epigenetic fluctuations, have been linked to gastric cancer formation and progression. Cytosine methylation (5mC) has been recognized as a critical epigenetic mark in the mammalian genome. The recent discovery of 5-hydroxymethylcytosine (5hmC), which is generated by ten-eleven translocation (TET) enzymes, provides new perspectives to understand DNA methylation-related plasticity. In this study, we show that gastric tumors display significant loss of 5hmC. Using matched distant normal, peripheral, and tumor primary tissues, we performed genome-wide profiling of 5hmC and identified differentially hydroxymethylated regions (DhMRs) specifically associated with gastric tumors. Gene ontology analyses indicated that DhMRs (both loss of 5hmC and gain of 5hmC) were enriched among the genes involved in specific pathways. Interestingly, the binding motif of hypoxia-inducible factor 1 (HIF1) is enriched among both peripheral and tumor DhMRs, while the Myc-binding motif is specifically enriched among only tumor DhMRs. Tumor progression analyses revealed a unique set of DhMRs that correlate with tumor progression. These data together suggest that alteration of 5hmC could potentially contribute to the tumorigenesis of gastric tumors.


Assuntos
5-Metilcitosina/análogos & derivados , Adenocarcinoma/patologia , Metilação de DNA , Epigênese Genética , Genoma Humano , Neoplasias Gástricas/patologia , 5-Metilcitosina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Estudos de Casos e Controles , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo
7.
Int J Mol Sci ; 20(19)2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31546611

RESUMO

Methylation of cytosine (5-meC) is a critical epigenetic modification in many eukaryotes, and genomic DNA methylation landscapes are dynamically regulated by opposed methylation and demethylation processes. Plants are unique in possessing a mechanism for active DNA demethylation involving DNA glycosylases that excise 5-meC and initiate its replacement with unmodified C through a base excision repair (BER) pathway. Plant BER-mediated DNA demethylation is a complex process involving numerous proteins, as well as additional regulatory factors that avoid accumulation of potentially harmful intermediates and coordinate demethylation and methylation to maintain balanced yet flexible DNA methylation patterns. Active DNA demethylation counteracts excessive methylation at transposable elements (TEs), mainly in euchromatic regions, and one of its major functions is to avoid methylation spreading to nearby genes. It is also involved in transcriptional activation of TEs and TE-derived sequences in companion cells of male and female gametophytes, which reinforces transposon silencing in gametes and also contributes to gene imprinting in the endosperm. Plant 5-meC DNA glycosylases are additionally involved in many other physiological processes, including seed development and germination, fruit ripening, and plant responses to a variety of biotic and abiotic environmental stimuli.


Assuntos
5-Metilcitosina/metabolismo , Desmetilação do DNA , DNA Glicosilases/metabolismo , DNA de Plantas/genética , Plantas/enzimologia , DNA Glicosilases/química , Metilação de DNA , DNA de Plantas/química , Endosperma/metabolismo , Regulação da Expressão Gênica de Plantas , Instabilidade Genômica/genética , Óvulo Vegetal/metabolismo , Pólen/metabolismo , Estresse Fisiológico/genética
8.
Nat Commun ; 10(1): 4296, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541098

RESUMO

Here we develop a methylation editing toolbox, Casilio-ME, that enables not only RNA-guided methylcytosine editing by targeting TET1 to genomic sites, but also by co-delivering TET1 and protein factors that couple methylcytosine oxidation to DNA repair activities, and/or promote TET1 to achieve enhanced activation of methylation-silenced genes. Delivery of TET1 activity by Casilio-ME1 robustly alters the CpG methylation landscape of promoter regions and activates methylation-silenced genes. We augment Casilio-ME1 to simultaneously deliver the TET1-catalytic domain and GADD45A (Casilio-ME2) or NEIL2 (Casilio-ME3) to streamline removal of oxidized cytosine intermediates to enhance activation of targeted genes. Using two-in-one effectors or modular effectors, Casilio-ME2 and Casilio-ME3 remarkably boost gene activation and methylcytosine demethylation of targeted loci. We expand the toolbox to enable a stable and expression-inducible system for broader application of the Casilio-ME platforms. This work establishes a platform for editing DNA methylation to enable research investigations interrogating DNA methylomes.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desmetilação do DNA , Reparo do DNA , RNA Guia/metabolismo , 5-Metilcitosina/metabolismo , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , DNA Glicosilases/metabolismo , Metilação de DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Edição de Genes , Células HEK293 , Humanos , Oxigenases de Função Mista/genética , Oxirredução , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Análise de Sequência de RNA
9.
Biochemistry (Mosc) ; 84(8): 851-869, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31522668

RESUMO

The review summarizes the data on pro- and eukaryotic RNA (C5-cytosine) methyltransferases. The structure, intracellular location, RNA targets, and catalytic mechanisms of these enzymes, as well as the functional role of methylated cytosine residues in RNA are presented. The functions of RNA (C5-cytosine) methyltransferases unassociated with their methylation activity are discussed. Special attention is given to the similarities and differences in the structures and mechanisms of action of RNA and DNA methyltransferases. The data on the association of mutations in the RNA (C5-cytosine) methyltransferases genes and human diseases are presented.


Assuntos
5-Metilcitosina/metabolismo , Metiltransferases/química , Metiltransferases/fisiologia , Processamento Pós-Transcricional do RNA/fisiologia , 5-Metilcitosina/química , Sequência de Aminoácidos/genética , Animais , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/fisiologia , Humanos , Metilação , Metiltransferases/genética , Camundongos , Mutação , Filogenia , Estrutura Secundária de Proteína , RNA de Transferência/genética , RNA de Transferência/metabolismo , tRNA Metiltransferases
10.
Nucleic Acids Res ; 47(19): e119, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31418020

RESUMO

The current methods for quantifying genome-wide 5-methylcytosine (5mC) oxides are still scarce, mostly restricted with two limitations: assay sensitivity is seriously compromised with cost, assay time and sample input; epigenetic information is irreproducible during polymerase chain reaction (PCR) amplification without bisulfite pretreatment. Here, we propose a novel Polymerization Retardation Isothermal Amplification (PRIA) strategy to directly amplify the minute differences between epigenetic bases and others by arranging DNA polymerase to repetitively pass large electron-withdrawing groups tagged 5mC-oxides. We demonstrate that low abundant 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) in genomic DNA can be accurately quantified within 10 h with 100 ng sample input on a laboratory real-time quantitative PCR instrument, and even multiple samples can be analyzed simultaneously in microplates. The global levels of 5hmC and 5fC in mouse and human brain tissues, rat hippocampal neuronal tissue, mouse kidney tissue and mouse embryonic stem cells were quantified and the observations not only confirm the widespread presence of 5hmC and 5fC but also indicate their significant variation in different tissues and cells. The strategy is easily performed in almost all research and medical laboratories, and would provide the potential capability to other candidate modifications in nucleotides.


Assuntos
5-Metilcitosina/isolamento & purificação , Metilação de DNA/genética , DNA Polimerase Dirigida por DNA/genética , Epigenômica/métodos , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Citosina/análogos & derivados , Citosina/metabolismo , DNA/genética , Genoma/genética , Humanos , Camundongos , Óxidos/química , Reação em Cadeia da Polimerase , Polimerização , Ratos
11.
Mol Cell ; 75(6): 1188-1202.e11, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31399345

RESUMO

The maternal-to-zygotic transition (MZT) is a conserved and fundamental process during which the maternal environment is converted to an environment of embryonic-driven development through dramatic reprogramming. However, how maternally supplied transcripts are dynamically regulated during MZT remains largely unknown. Herein, through genome-wide profiling of RNA 5-methylcytosine (m5C) modification in zebrafish early embryos, we found that m5C-modified maternal mRNAs display higher stability than non-m5C-modified mRNAs during MZT. We discovered that Y-box binding protein 1 (Ybx1) preferentially recognizes m5C-modified mRNAs through π-π interactions with a key residue, Trp45, in Ybx1's cold shock domain (CSD), which plays essential roles in maternal mRNA stability and early embryogenesis of zebrafish. Together with the mRNA stabilizer Pabpc1a, Ybx1 promotes the stability of its target mRNAs in an m5C-dependent manner. Our study demonstrates an unexpected mechanism of RNA m5C-regulated maternal mRNA stabilization during zebrafish MZT, highlighting the critical role of m5C mRNA modification in early development.


Assuntos
5-Metilcitosina/metabolismo , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário/fisiologia , Estabilidade de RNA/fisiologia , RNA Mensageiro Estocado/metabolismo , Peixe-Zebra/embriologia , Animais , Células HeLa , Humanos , Camundongos , RNA Mensageiro Estocado/genética , Peixe-Zebra/genética
12.
Cell Host Microbe ; 26(2): 217-227.e6, 2019 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-31415754

RESUMO

How the covalent modification of mRNA ribonucleotides, termed epitranscriptomic modifications, alters mRNA function remains unclear. One issue has been the difficulty of quantifying these modifications. Using purified HIV-1 genomic RNA, we show that this RNA bears more epitranscriptomic modifications than the average cellular mRNA, with 5-methylcytosine (m5C) and 2'O-methyl modifications being particularly prevalent. The methyltransferase NSUN2 serves as the primary writer for m5C on HIV-1 RNAs. NSUN2 inactivation inhibits not only m5C addition to HIV-1 transcripts but also viral replication. This inhibition results from reduced HIV-1 protein, but not mRNA, expression, which in turn correlates with reduced ribosome binding to viral mRNAs. In addition, loss of m5C dysregulates the alternative splicing of viral RNAs. These data identify m5C as a post-transcriptional regulator of both splicing and function of HIV-1 mRNA, thereby affecting directly viral gene expression.


Assuntos
5-Metilcitosina/farmacologia , Regulação Viral da Expressão Gênica , HIV-1/genética , RNA Viral/metabolismo , Transcriptoma , 5-Metilcitosina/metabolismo , Linfócitos T CD4-Positivos , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Metiltransferases/farmacologia , Processamento de RNA , RNA Mensageiro/metabolismo , RNA Viral/efeitos dos fármacos , Vírion , Replicação Viral/efeitos dos fármacos
13.
Ecotoxicol Environ Saf ; 183: 109461, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31377519

RESUMO

The present work investigated the changes in DNA methylation pattern of Tenebrio molitor mitochondria genome at different development stages, which was fed with polyurethane foam as a sole diet. Polyurethane foam could influence the global methylation levels in mitochondria DNA of Tenebrio molitor. Different leves of 5-methylcytosine appeared at CpG and non-CpG sites of Tenebrio molitor mtDNA while they were fed with polyurethane foam: 10 CpG and 49 non-CpG sites at larval stage, 4 CpG and 31 non-CpG sites at pupa stage, 7 CpG and 56 non-CpG sites at adult stage in general. Moreover, we observed the decreased levels of ATP generation with the mitochondria DNA methylation variation. The results demonstrated that mitochondria DNA gene could be methylated in response to environmental pollutants to modulate stage-specific functions. Moreover, mtDNA methylation of polyurethane-foam-feeding Tenebrio molitor existed discrepancy in the developmental stage. The tentative methylation mechanism of mtDNA might be that polyurethane foam induced oxidative stress and increased the permeability of mitochondrial membranes, which resulted in transmethylase entry into mitochondria.


Assuntos
DNA Mitocondrial/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Epigênese Genética/efeitos dos fármacos , Metamorfose Biológica/efeitos dos fármacos , Poliuretanos/toxicidade , Tenebrio/efeitos dos fármacos , 5-Metilcitosina/metabolismo , Animais , Metilação de DNA/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/genética , Metamorfose Biológica/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Pupa/efeitos dos fármacos , Pupa/genética , Tenebrio/genética , Tenebrio/crescimento & desenvolvimento
15.
Nucleic Acids Res ; 47(16): 8734-8745, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31287866

RESUMO

Post-transcriptional modifications in mitochondrial tRNAs (mt-tRNAs) play critical roles in mitochondrial protein synthesis, which produces respiratory chain complexes. In this study, we took advantage of mass spectrometric analysis to map 5-methylcytidine (m5C) at positions 48-50 in eight mouse and six human mt-tRNAs. We also confirmed the absence of m5C in mt-tRNAs isolated from Nsun2 knockout (KO) mice, as well as from NSUN2 KO human culture cells. In addition, we successfully reconstituted m5C at positions 48-50 of mt-tRNA in vitro with NSUN2 protein in the presence of S-adenosylmethionine. Although NSUN2 is predominantly localized to the nucleus and introduces m5C into cytoplasmic tRNAs and mRNAs, structured illumination microscopy clearly revealed NSUN2 foci inside mitochondria. These observations provide novel insights into the role of NSUN2 in the physiology and pathology of mitochondrial functions.


Assuntos
5-Metilcitosina/metabolismo , Metiltransferases/genética , Mitocôndrias/genética , Processamento Pós-Transcricional do RNA , RNA Mitocondrial/genética , RNA de Transferência/genética , Animais , Sistemas CRISPR-Cas , Fibroblastos/metabolismo , Fibroblastos/patologia , Edição de Genes , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Metilação , Metiltransferases/deficiência , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Conformação de Ácido Nucleico , Fosforilação Oxidativa , Cultura Primária de Células , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mitocondrial/metabolismo , RNA de Transferência/metabolismo , S-Adenosilmetionina/metabolismo
16.
Nucleic Acids Res ; 47(16): 8720-8733, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31276587

RESUMO

Expression of human mitochondrial DNA is indispensable for proper function of the oxidative phosphorylation machinery. The mitochondrial genome encodes 22 tRNAs, 2 rRNAs and 11 mRNAs and their post-transcriptional modification constitutes one of the key regulatory steps during mitochondrial gene expression. Cytosine-5 methylation (m5C) has been detected in mitochondrial transcriptome, however its biogenesis has not been investigated in details. Mammalian NOP2/Sun RNA Methyltransferase Family Member 2 (NSUN2) has been characterized as an RNA methyltransferase introducing m5C in nuclear-encoded tRNAs, mRNAs and microRNAs and associated with cell proliferation and differentiation, with pathogenic variants in NSUN2 being linked to neurodevelopmental disorders. Here we employ spatially restricted proximity labelling and immunodetection to demonstrate that NSUN2 is imported into the matrix of mammalian mitochondria. Using three genetic models for NSUN2 inactivation-knockout mice, patient-derived fibroblasts and CRISPR/Cas9 knockout in human cells-we show that NSUN2 is necessary for the generation of m5C at positions 48, 49 and 50 of several mammalian mitochondrial tRNAs. Finally, we show that inactivation of NSUN2 does not have a profound effect on mitochondrial tRNA stability and oxidative phosphorylation in differentiated cells. We discuss the importance of the newly discovered function of NSUN2 in the context of human disease.


Assuntos
5-Metilcitosina/metabolismo , Eczema/genética , Transtornos do Crescimento/genética , Deficiência Intelectual/genética , Metiltransferases/genética , Microcefalia/genética , Processamento Pós-Transcricional do RNA , RNA Mitocondrial/genética , RNA de Transferência/genética , Animais , Sistemas CRISPR-Cas , Eczema/metabolismo , Eczema/patologia , Facies , Fibroblastos/metabolismo , Fibroblastos/patologia , Edição de Genes , Técnicas de Inativação de Genes , Transtornos do Crescimento/metabolismo , Transtornos do Crescimento/patologia , Células HEK293 , Humanos , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Metilação , Metiltransferases/deficiência , Camundongos , Camundongos Knockout , Microcefalia/metabolismo , Microcefalia/patologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Conformação de Ácido Nucleico , Fosforilação Oxidativa , Cultura Primária de Células , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mitocondrial/metabolismo , RNA de Transferência/metabolismo
17.
Nat Cell Biol ; 21(8): 978-990, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31358969

RESUMO

Although 5-methylcytosine (m5C) is a widespread modification in RNAs, its regulation and biological role in pathological conditions (such as cancer) remain unknown. Here, we provide the single-nucleotide resolution landscape of messenger RNA m5C modifications in human urothelial carcinoma of the bladder (UCB). We identify numerous oncogene RNAs with hypermethylated m5C sites causally linked to their upregulation in UCBs and further demonstrate YBX1 as an m5C 'reader' recognizing m5C-modified mRNAs through the indole ring of W65 in its cold-shock domain. YBX1 maintains the stability of its target mRNA by recruiting ELAVL1. Moreover, NSUN2 and YBX1 are demonstrated to drive UCB pathogenesis by targeting the m5C methylation site in the HDGF 3' untranslated region. Clinically, a high coexpression of NUSN2, YBX1 and HDGF predicts the poorest survival. Our findings reveal an unprecedented mechanism of RNA m5C-regulated oncogene activation, providing a potential therapeutic strategy for UCB.


Assuntos
5-Metilcitosina/metabolismo , Regulação da Expressão Gênica/genética , Metiltransferases/genética , Neoplasias da Bexiga Urinária/genética , Proteína 1 de Ligação a Y-Box/genética , Animais , Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/metabolismo , Humanos , Camundongos , RNA Mensageiro/genética , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia
18.
Artif Cells Nanomed Biotechnol ; 47(1): 2706-2714, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31271297

RESUMO

Cervical cancer is one of the most common gynecological tumors in females. DNA methylation alteration is a type of epigenetic decoration that controls the gene transcriptional regulation and is essential for the pathological progression of cervical cancer to reflect the prognosis and therapeutic sensitivity in clinical practice. Beyond DNA methylation, DNA hydroxymethylation considered as a more stable biomarker draws the outline of the reversible cycle from DNA methylation and demethylation. However, the landscape of 5-hydroxymethylcytosine (5hmC) distributed in the genome is never characterized in cervical cancer. In this study, we presented the whole 5-methylcytosine (5mC) and 5hmC profile in cervical cancer of I-IIa and IIb to IV stages and cervicitis tissues as control by dot plot assay and immunohistochemistry. We observed that the total 5mC was up-regulated while 5hmC was down-regulated in cervical cancer group compared to the control group. Furthermore, we investigated the distribution of 5mC and 5hmC on genomic DNA by MeDIP- and hMeDIP-Seq. 53 differential methylation/hydromethylation regions (DMRs/DHMRs) displayed a continuously increasing or decreasing trend of 5mC or 5hmC from cervicitis to I-IIa and from I-IIa to IIb-IV stages of cervical cancer. Thirty-seven DMRs and DHMRs have a similar variation trend while the other 8 have the opposite trend compared between CSCC and cervicitis. Moreover, the DMR/DHMR associated genes were closely related to Wnt, MAPK, Rap1 and other important signaling pathways. Finally, 5hmC beyond 5mC at the genes such as ACTG1, SALL3, DNAJA3, SERPINB6, CDC14B and CALN1 were considered as the putative novel hallmarks for cervical cancer diagnosis and prognosis. Altogether, this study first describes the DNA hydroxymethylation atlas of cervical cancer and shows a list of novel genes transcriptionally regulated by DNA methylation and hydroxymethylation.


Assuntos
Metilação de DNA , Neoplasias do Colo do Útero/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Adulto , Feminino , Genômica , Humanos , Transcrição Genética , Neoplasias do Colo do Útero/metabolismo
19.
Nat Commun ; 10(1): 2550, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186410

RESUMO

The presence and absence of RNA modifications regulates RNA metabolism by modulating the binding of writer, reader, and eraser proteins. For 5-methylcytosine (m5C) however, it is largely unknown how it recruits or repels RNA-binding proteins. Here, we decipher the consequences of m5C deposition into the abundant non-coding vault RNA VTRNA1.1. Methylation of cytosine 69 in VTRNA1.1 occurs frequently in human cells, is exclusively mediated by NSUN2, and determines the processing of VTRNA1.1 into small-vault RNAs (svRNAs). We identify the serine/arginine rich splicing factor 2 (SRSF2) as a novel VTRNA1.1-binding protein that counteracts VTRNA1.1 processing by binding the non-methylated form with higher affinity. Both NSUN2 and SRSF2 orchestrate the production of distinct svRNAs. Finally, we discover a functional role of svRNAs in regulating the epidermal differentiation programme. Thus, our data reveal a direct role for m5C in the processing of VTRNA1.1 that involves SRSF2 and is crucial for efficient cellular differentiation.


Assuntos
5-Metilcitosina/metabolismo , Metilação de DNA , Células Epidérmicas/citologia , Metiltransferases/metabolismo , RNA/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Diferenciação Celular , Linhagem Celular , Citosina/metabolismo , Células Epidérmicas/metabolismo , Células HEK293 , Células HeLa , Células-Tronco Embrionárias Humanas/citologia , Humanos , Metiltransferases/genética , RNA/genética , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo
20.
Oncol Res Treat ; 42(6): 309-318, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31055566

RESUMO

DNA methylation plays significant roles in a variety of biological and pathological processes including mammalian development, genomic imprinting, retrotransposon silencing, and X-chromosome inactivation. Recent discoveries indicated that ten-eleven translocation (TET) family of dioxygenases can convert 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC). The TET family includes three members: TET1, TET2, and TET3. With increasing evidence, more and more biological and pathological processes in which 5-hmC and TET family serve unparalleled biological roles are noticed, for example, DNA demethylation and transcriptional regulation of different target genes, which are involved in many human diseases, especially hematologic malignancies, resembling chronic myelomonocytic leukemia, myelodysplastic syndromes, and so on. In this review, we focus on the diverse functions of TET family and the novel epigenetic marks, 5-mC and 5-hmC, in hematologic malignancies. This review will provide valuable insights into the potential targets of hematologic malignancies. Further understanding of the normal and pathological functions of TET family may provide new methods to develop novel epigenetic therapies for treating hematologic malignancies.


Assuntos
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Epigênese Genética , Neoplasias Hematológicas/genética , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Metilação de DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dioxigenases/química , Dioxigenases/genética , Humanos , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Mutação , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética
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