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1.
In Vivo ; 37(1): 445-453, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36593050

RESUMO

BACKGROUND/AIM: DNA methylation is the most studied epigenetic modification in cancer. Ten-eleven translocation enzymes (TET) catalyze the oxidation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) in the DNA. In the current research, we aimed to evaluate the role of 5-hmC and TET enzymes in non-small cell lung cancer (NSCLC) patients and their possible association with outcomes. PATIENTS AND METHODS: ELISA was used to measure the 5-hmC levels in genomic DNA and qRT-PCR was used to evaluate TET1, TET2, and TET3 mRNAs expression levels in NSCLC tissues and their paired normal controls. RESULTS: The levels of 5-hmC were significantly lower in NSCLC tissues than in normal tissues, with a mean ±SD of 0.28±0.37 vs. 1.84±0.58, respectively (t=22.77, p<0.0001), and this reduction was correlated with adverse clinical features. In addition, all TET genes were significantly down-regulated in NSCLC tissues in comparison to their matched normal tissues. The mean±SD level of TET1-mRNA was 38.48±16.38 in NSCLC vs. 80.65±11.25 in normal tissues (t=21.33, p<0.0001), TET2-mRNA level in NSCLC was 5.25±2.78 vs. 9.52±1.01 in normal tissues (t=14.48, p<0.0001), and TET3-mRNA level in NSCLC was 5.21±2.8 vs. 9.51±0.86 in normal tissues (t=14.75, p<0.0001). Downregulation of TET genes was correlated with poor clinical features. CONCLUSION: 5-HmC levels as well as TET1, TET2, and TET3 mRNA levels were reduced in NSCLC tissues. The reduced levels of 5-hmC and TET mRNAs were associated with adverse clinical features, suggesting that the level of 5-hmC may serve as a valuable prognostic biomarker for NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Dioxigenases , Neoplasias Pulmonares , Humanos , 5-Metilcitosina , Citosina/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Metilação de DNA/genética , Epigênese Genética , Expressão Gênica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo
2.
Int J Mol Sci ; 24(2)2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36675240

RESUMO

The mammalian DNA methylation landscape is established and maintained by the combined activities of the two key epigenetic modifiers, DNA methyltransferases (DNMT) and Ten-eleven-translocation (TET) enzymes. Once DNMTs produce 5-methylcytosine (5mC), TET proteins fine-tune the DNA methylation status by consecutively oxidizing 5mC to 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives. The 5mC and oxidized methylcytosines are essential for the maintenance of cellular identity and function during differentiation. Cytosine modifications with DNMT and TET enzymes exert pleiotropic effects on various aspects of hematopoiesis, including self-renewal of hematopoietic stem/progenitor cells (HSPCs), lineage determination, differentiation, and function. Under pathological conditions, these enzymes are frequently dysregulated, leading to loss of function. In particular, the loss of DNMT3A and TET2 function is conspicuous in diverse hematological disorders, including myeloid and lymphoid malignancies, and causally related to clonal hematopoiesis and malignant transformation. Here, we update recent advances in understanding how the maintenance of DNA methylation homeostasis by DNMT and TET proteins influences normal hematopoiesis and malignant transformation, highlighting the potential impact of DNMT3A and TET2 dysregulation on clonal dominance and evolution of pre-leukemic stem cells to full-blown malignancies. Clarification of the normal and pathological functions of DNA-modifying epigenetic regulators will be crucial to future innovations in epigenetic therapies for treating hematological disorders.


Assuntos
Dioxigenases , Doenças Hematológicas , Neoplasias , Animais , Humanos , Citosina , Epigênese Genética , Diferenciação Celular/genética , Transformação Celular Neoplásica/genética , Metilação de DNA , Neoplasias/genética , Doenças Hematológicas/genética , 5-Metilcitosina/metabolismo , DNA/metabolismo , Dioxigenases/genética , Mamíferos/metabolismo
3.
Mol Cancer ; 22(1): 5, 2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36627693

RESUMO

BACKGROUND: Accumulated evidence highlights the significance of the crosstalk between epigenetic and epitranscriptomic mechanisms, notably 5-methylcytosine (5mC) and N6-methyladenosine (m6A). Herein, we conducted a widespread analysis regarding the crosstalk between 5mC and m6A regulators in hepatocellular carcinoma (HCC). METHODS: Pan-cancer genomic analysis of the crosstalk between 5mC and m6A regulators was presented at transcriptomic, genomic, epigenetic, and other multi-omics levels. Hub 5mC and m6A regulators were summarized to define an epigenetic and epitranscriptomic module eigengene (EME), which reflected both the pre- and post-transcriptional modifications. RESULTS: 5mC and m6A regulators interacted with one another at the multi-omic levels across pan-cancer, including HCC. The EME scoring system enabled to greatly optimize risk stratification and accurately predict HCC patients' clinical outcomes and progression. Additionally, the EME accurately predicted the responses to mainstream therapies (TACE and sorafenib) and immunotherapy as well as hyper-progression. In vitro, 5mC and m6A regulators cooperatively weakened apoptosis and facilitated proliferation, DNA damage repair, G2/M arrest, migration, invasion and epithelial-to-mesenchymal transition (EMT) in HCC cells. The EME scoring system was remarkably linked to potential extrinsic and intrinsic immune escape mechanisms, and the high EME might contribute to a reduced copy number gain/loss frequency. Finally, we determined potential therapeutic compounds and druggable targets (TUBB1 and P2RY4) for HCC patients with high EME. CONCLUSIONS: Our findings suggest that HCC may result from a unique synergistic combination of 5mC-epigenetic mechanism mixed with m6A-epitranscriptomic mechanism, and their crosstalk defines therapeutic response and pharmacogenomic landscape.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , 5-Metilcitosina , Apoptose , Farmacogenética , Linhagem Celular Tumoral , Pontos de Checagem da Fase G2 do Ciclo Celular , Progressão da Doença
4.
Anal Chim Acta ; 1239: 340636, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36628742

RESUMO

DNA methylation is intensively studied in medical science. Current HPLC methods for quantification of global DNA methylation involve digestion of a DNA sample and HPLC determination of both cytosine (C) and 5-methylcytosine (5mC) so that percentage of 5mC in total cytosine can be calculated as DNA methylation level. Herein we report a novel HPLC method based on a one-pot fluorescence tagging and depyrimidination reaction between DNA and chloroacetaldehyde (CAA) for highly sensitive quantification of global DNA methylation. In the one-pot reaction, C and 5mC residues in a DNA sequence react with CAA, forming fluorescent etheno-adducts that are then released from the sequence through depyrimidination. Interestingly, etheno-5mC (ε-5mC) is ∼20 times more fluorescent than ε-C and other ε-nucleobases resulting from the reaction, which greatly facilitates the quantification. Further, due to the tagging-induced increase in structural aromaticity, ε-nucleobases are far more separable by HPLC than intact nucleobases. The proposed HPLC method with fluorescence detection (HPLC-FD) is quick (i.e., < 1h per assay) and highly sensitive with a detection limit of 0.80 nM (or 250 fg on column) for 5mC. Using the method, DNA samples isolated from yeast, HCT-116 cells, and tissues were analyzed. Global DNA methylation was measured to be in the range from 0.35% to 2.23% in the samples analyzed. This sensitive method allowed accurate analyses of minute DNA samples (∼100 ng) isolated from milligrams of tissues.


Assuntos
5-Metilcitosina , Metilação de DNA , 5-Metilcitosina/análise , Citosina , Cromatografia Líquida de Alta Pressão/métodos , DNA/análise
6.
Int J Mol Sci ; 24(2)2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36675131

RESUMO

Scrapie is a neurodegenerative disorder belonging to the group of transmissible spongiform encephalopathies or prion diseases, which are caused by an infectious isoform of the innocuous cellular prion protein (PrPC) known as PrPSc. DNA methylation, one of the most studied epigenetic mechanisms, is essential for the proper functioning of the central nervous system. Recent findings point to possible involvement of DNA methylation in the pathogenesis of prion diseases, but there is still a lack of knowledge about the behavior of this epigenetic mechanism in such neurodegenerative disorders. Here, we evaluated by immunohistochemistry the 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) levels in sheep and mouse brain tissues infected with scrapie. Expression analysis of different gene coding for epigenetic regulatory enzymes (DNMT1, DNMT3A, DNMT3B, HDAC1, HDAC2, TET1, and TET2) was also carried out. A decrease in 5mC levels was observed in scrapie-affected sheep and mice compared to healthy animals, whereas 5hmC displayed opposite patterns between the two models, demonstrating a decrease in 5hmC in scrapie-infected sheep and an increase in preclinical mice. 5mC correlated with prion-related lesions in mice and sheep, but 5hmC was associated with prion lesions only in sheep. Differences in the expression changes of epigenetic regulatory genes were found between both disease models, being differentially expressed Dnmt3b, Hdac1, and Tet1 in mice and HDAC2 in sheep. Our results support the evidence that DNA methylation in both forms, 5mC and 5hmC, and its associated epigenetic enzymes, take part in the neurodegenerative course of prion diseases.


Assuntos
Encéfalo , Príons , Scrapie , Animais , Camundongos , 5-Metilcitosina/metabolismo , Encéfalo/metabolismo , Doenças Priônicas/genética , Doenças Priônicas/metabolismo , Príons/genética , Príons/metabolismo , Scrapie/genética , Scrapie/metabolismo , Ovinos , Metilação de DNA/genética , Metilação de DNA/fisiologia , Epigênese Genética/genética , Epigênese Genética/fisiologia , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo
7.
Anal Chem ; 95(2): 1556-1565, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36563112

RESUMO

DNA methylation (5-methylcytosine, 5mC) is the most important epigenetic modification in mammals. Deciphering the roles of 5mC relies on the quantitative detection of 5mC at the single-base resolution. Bisulfite sequencing (BS-seq) is the most often employed technique for mapping 5mC in DNA. However, bisulfite treatment may cause serious degradation of input DNA due to the harsh reaction conditions. Here, we engineered the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3C (A3C) protein to endow the engineered A3C (eA3C) protein with differential deamination activity toward cytosine and 5mC. By the virtue of the unique property of eA3C, we proposed an engineered A3C sequencing (EAC-seq) method for the bisulfite-free and quantitative mapping of 5mC in DNA at the single-base resolution. In EAC-seq, the eA3C protein can deaminate C but not 5mC, which is employed to differentiate C and 5mC in sequencing. Using the EAC-seq method, we quantitatively detected 5mC in genomic DNA of lung cancer tissue. In contrast to the harsh reaction conditions of BS-seq, which could lead to significant degradation of DNA, the whole procedure of EAC-seq is carried out under mild conditions, thereby preventing DNA damage. Taken together, the EAC-seq approach is bisulfite-free and straightforward, making it an invaluable tool for the quantitative detection of 5mC in limited DNA at the single-base resolution.


Assuntos
5-Metilcitosina , Citidina Desaminase , Metilação de DNA , Humanos , 5-Metilcitosina/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Citosina , DNA/genética , DNA/metabolismo , Epigênese Genética , Análise de Sequência de DNA/métodos , Sulfitos/metabolismo
8.
Nat Genet ; 55(1): 100-111, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36539616

RESUMO

Generation of functional transcripts requires transcriptional initiation at regular start sites, avoiding production of aberrant and potentially hazardous aberrant RNAs. The mechanisms maintaining transcriptional fidelity and the impact of spurious transcripts on cellular physiology and organ function have not been fully elucidated. Here we show that TET3, which successively oxidizes 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and other derivatives, prevents aberrant intragenic entry of RNA polymerase II pSer5 into highly expressed genes of airway smooth muscle cells, assuring faithful transcriptional initiation at canonical start sites. Loss of TET3-dependent 5hmC production in SMCs results in accumulation of spurious transcripts, which stimulate the endosomal nucleic-acid-sensing TLR7/8 signaling pathway, thereby provoking massive inflammation and airway remodeling resembling human bronchial asthma. Furthermore, we found that 5hmC levels are substantially lower in human asthma airways compared with control samples. Suppression of spurious transcription might be important to prevent chronic inflammation in asthma.


Assuntos
5-Metilcitosina , Asma , Humanos , 5-Metilcitosina/metabolismo , Imunidade Inata/genética , Inflamação/genética , Asma/genética , Metilação de DNA
9.
Nat Genet ; 55(1): 130-143, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36539615

RESUMO

In mammals, DNA 5-hydroxymethylcytosine (5hmC) is involved in methylation reprogramming during early embryonic development. Yet, to what extent 5hmC participates in genome-wide methylation reprogramming remains largely unknown. Here, we characterize the 5hmC landscapes in mouse early embryos and germ cells with parental allele specificity. DNA hydroxymethylation was most strongly correlated with DNA demethylation as compared with de novo or maintenance methylation in zygotes, while 5hmC was targeted to particular de novo methylated sites in postimplantation epiblasts. Surprisingly, DNA replication was also required for 5hmC generation, especially in the female pronucleus. More strikingly, aberrant nuclear localization of Dnmt1/Uhrf1 in mouse zygotes due to maternal deficiency of Nlrp14 led to defects in DNA-replication-coupled passive demethylation and impaired 5hmC deposition, revealing the divergency between genome-wide 5-methylcytosine (5mC) maintenance and Tet-mediated oxidation. In summary, our work provides insights and a valuable resource for the study of epigenetic regulation in early embryo development.


Assuntos
5-Metilcitosina , Metilação de DNA , Animais , Feminino , Camundongos , 5-Metilcitosina/metabolismo , Metilação de DNA/genética , Epigênese Genética , Desenvolvimento Embrionário/genética , Zigoto/metabolismo , Mamíferos , DNA/genética , DNA/metabolismo , Citosina/metabolismo
10.
Science ; 378(6623): 983-989, 2022 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-36454826

RESUMO

Neurons harbor high levels of single-strand DNA breaks (SSBs) that are targeted to neuronal enhancers, but the source of this endogenous damage remains unclear. Using two systems of postmitotic lineage specification-induced pluripotent stem cell-derived neurons and transdifferentiated macrophages-we show that thymidine DNA glycosylase (TDG)-driven excision of methylcytosines oxidized with ten-eleven translocation enzymes (TET) is a source of SSBs. Although macrophage differentiation favors short-patch base excision repair to fill in single-nucleotide gaps, neurons also frequently use the long-patch subpathway. Disrupting this gap-filling process using anti-neoplastic cytosine analogs triggers a DNA damage response and neuronal cell death, which is dependent on TDG. Thus, TET-mediated active DNA demethylation promotes endogenous DNA damage, a process that normally safeguards cell identity but can also provoke neurotoxicity after anticancer treatments.


Assuntos
Quebras de DNA de Cadeia Simples , Desmetilação do DNA , Reparo do DNA , Elementos Facilitadores Genéticos , Células-Tronco Pluripotentes Induzidas , Neurônios , Timina DNA Glicosilase , Diferenciação Celular , Neurônios/enzimologia , 5-Metilcitosina/metabolismo , Humanos , Transdiferenciação Celular
11.
Sci Rep ; 12(1): 19583, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36380112

RESUMO

Covalent modifications of standard DNA/RNA nucleobases affect epigenetic regulation of gene expression by modulating interactions between nucleic acids and protein readers. We derive here the absolute binding free energies and analyze the binding modalities between key modified nucleobases 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and N6-methyladenine (m6A) and all non-prolyl/non-glycyl protein side chains using molecular dynamics simulations and umbrella sampling in both water and methanol, the latter mimicking the low dielectric environment at the dehydrated nucleic-acid/protein interfaces. We verify the derived affinities by comparing against a comprehensive set of high-resolution structures of nucleic-protein complexes involving 5mC. Our analysis identifies protein side chains that are highly tuned for detecting cytosine methylation as a function of the environment and can thus serve as microscopic readers of epigenetic marks. Conversely, we show that the relative ordering of sidechain affinities for 5hmC and m6A does not differ significantly from those for their precursor bases, cytosine and adenine, respectively, especially in the low dielectric environment. For those two modified bases, the effect is more nuanced and manifests itself primarily at the level of absolute changes in the binding free energy. Our results contribute towards establishing a quantitative foundation for understanding, predicting and modulating the interactions between modified nucleic acids and proteins at the atomistic level.


Assuntos
5-Metilcitosina , Epigênese Genética , 5-Metilcitosina/metabolismo , Citosina/metabolismo , Proteínas/metabolismo , Metilação de DNA , DNA/química
12.
Anal Chem ; 94(47): 16402-16410, 2022 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-36383421

RESUMO

It is universally recognized that the quantification of DNA hydroxymethylation at random gene sequences still remains challenging. Herein, the highly sensitive identifying strategy of 5-hydroxymethylcytosine (5-hmC) without sequence specificity was achieved with a novel electrochemiluminescence (ECL) biosensor, which deftly integrated metal-organic framework (MOF)-derived amorphous MnOxSy nanoflowers (MnOxSy NFs) as a bifunctional co-reaction accelerator and cross-shaped DNA tracks as a well-regulated signal switch. Specifically, the target recognition process of 5-hmC was performed through specific chemical modification, where the hydroxymethyl sites were first aminated and then labeled with a 5'-carboxyl-functioned DNA walker, thus forming the target labeled DNA walker (5-ghmC-walker). Subsequently, the cross-shaped DNA tracks were ingeniously designed to endow the 5-ghmC-walker with continuous mechanical motion due to the long periodic linear alignment structure and well-regulated highly ordered interfaces. Furthermore, as a bifunctional co-reaction accelerator synthesized by in situ Mn-MOF template-sacrificing strategy, the MnOxSy NFs could promote the reduction of both dissolved O2 and S2O82-, remarkably boosting the ECL intensity of a peroxydisulfate (S2O82-) solution by 5.2 times compared to the pure S2O82- solution. Benefiting from specific target recognition and a dual-pathway strategy for boosting ECL, the proposed ECL platform can quantify 5-hmC with a wide linear range of 1 fM-1 nM and a low detection limit of 0.29 fM. This simple, highly sensitive strategy without sequence specificity provides a powerful platform for 5-hmC detection in the epigenetic study and disease pathogenesis.


Assuntos
Técnicas Biossensoriais , Estruturas Metalorgânicas , Técnicas Eletroquímicas , 5-Metilcitosina , DNA , Medições Luminescentes , Limite de Detecção
13.
Adv Exp Med Biol ; 1389: 111-136, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36350508

RESUMO

Cytosine methylation at the C5-position-generating 5-methylcytosine (5mC)-is a DNA modification found in many eukaryotic organisms, including fungi, plants, invertebrates, and vertebrates, albeit its levels vary greatly in different organisms. In mammals, cytosine methylation occurs predominantly in the context of CpG dinucleotides, with the majority (60-80%) of CpG sites in their genomes being methylated. DNA methylation plays crucial roles in the regulation of chromatin structure and gene expression and is essential for mammalian development. Aberrant changes in DNA methylation and genetic alterations in enzymes and regulators involved in DNA methylation are associated with various human diseases, including cancer and developmental disorders. In mammals, DNA methylation is mediated by two families of DNA methyltransferases (Dnmts), namely Dnmt1 and Dnmt3 proteins. Over the last three decades, genetic manipulations of these enzymes, as well as their regulators, in mice have greatly contributed to our understanding of the biological functions of DNA methylation in mammals. In this chapter, we discuss genetic studies on mammalian Dnmts, focusing on their roles in embryogenesis, cellular differentiation, genomic imprinting, and human diseases.


Assuntos
DNA (Citosina-5-)-Metiltransferases , Metilação de DNA , Humanos , Camundongos , Animais , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferase 1 , Metilases de Modificação do DNA/genética , 5-Metilcitosina , Mamíferos/genética , Mamíferos/metabolismo , DNA/metabolismo
14.
Adv Exp Med Biol ; 1389: 239-267, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36350513

RESUMO

Mammalian DNA methylation mainly occurs at the carbon-C5 position of cytosine (5mC). TET enzymes were discovered to successively oxidize 5mC to 5-hydromethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Ten-eleven translocation (TET) enzymes and oxidized 5mC derivatives play important roles in various biological and pathological processes, including regulation of DNA demethylation, gene transcription, embryonic development, and oncogenesis. In this chapter, we will discuss the discovery of TET-mediated 5mC oxidation and the structure, function, and regulation of TET enzymes. We start with brief descriptions of the mechanisms of TET-mediated 5mC oxidation and TET-dependent DNA demethylation. We then discuss the TET-mediated epigenetic reprogramming in pluripotency maintenance and embryogenesis, as well as in tumorigenesis and neural system. We further describe the structural basis for substrate recognition and preference in TET-mediated 5mC oxidation. Finally, we summarize the chemical molecules and interacting proteins that regulate TET's activity.


Assuntos
5-Metilcitosina , Citosina , Animais , 5-Metilcitosina/química , Metilação de DNA , Oxirredução , Desenvolvimento Embrionário , Mamíferos/metabolismo
15.
Adv Exp Med Biol ; 1389: 295-315, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36350515

RESUMO

The modification of DNA bases is a classic hallmark of epigenetics. Four forms of modified cytosine-5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine-have been discovered in eukaryotic DNA. In addition to cytosine carbon-5 modifications, cytosine and adenine methylated in the exocyclic amine-N4-methylcytosine and N6-methyladenine-are other modified DNA bases discovered even earlier. Each modified base can be considered a distinct epigenetic signal with broader biological implications beyond simple chemical changes. Since 1994, several crystal structures of proteins and enzymes involved in writing, reading, and erasing modified bases have become available. Here, we present a structural synopsis of writers, readers, and erasers of the modified bases from prokaryotes and eukaryotes. Despite significant differences in structures and functions, they are remarkably similar regarding their engagement in flipping a target base/nucleotide within DNA for specific recognitions and/or reactions. We thus highlight base flipping as a common structural framework broadly applied by distinct classes of proteins and enzymes across phyla for epigenetic regulations of DNA.


Assuntos
5-Metilcitosina , Metilação de DNA , DNA , 5-Metilcitosina/química , Citosina/química , DNA/metabolismo , Epigênese Genética , Eucariotos/genética , Eucariotos/metabolismo
16.
Adv Exp Med Biol ; 1389: 395-469, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36350519

RESUMO

DNA methylation is the most studied epigenetic modification, and altered DNA methylation patterns have been identified in cancer and more recently also in many other complex diseases. Furthermore, DNA methylation is influenced by a variety of environmental factors, and the analysis of DNA methylation patterns might allow deciphering previous exposures. A number of techniques to study DNA methylation either genome-wide or at specific loci have been devised using a limited number of principles for differentiating the methylation state: (1) methylation-sensitive/dependent restriction enzymes, (2) antibody or methyl-binding protein-based enrichment, or (3) chemical or enzymatic conversion, (4) direct sequence readout. Second-generation sequencing has largely replaced microarrays as a readout platform and is also becoming more popular for locus-specific DNA methylation analysis. In this chapter, the currently used methods for both genome-wide and locus-specific analysis of 5-methylcytosine as well as its oxidative derivatives such as 5-hydroxymethylcytosine are reviewed in detail and advantages and limitations of each approach are discussed. Furthermore, emerging technologies avoiding PCR amplification and allowing a direct readout of DNA methylation are summarized, together with novel applications, such as the detection of DNA methylation in single cells or in circulating cell-free DNA.


Assuntos
5-Metilcitosina , Metilação de DNA , Metilação de DNA/genética , Análise de Sequência de DNA/métodos , Genoma , Epigênese Genética , Reação em Cadeia da Polimerase
17.
Cell Rep ; 41(6): 111612, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36351399

RESUMO

DNA methylation has emerged as a critical modulator of neuronal plasticity and cognitive function. Notwithstanding, the role of enzymes that demethylate DNA remain to be fully explored. Here, we report that loss of ten-eleven translocation methylcytosine dioxygenase 2 (Tet2), which catalyzes oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), in adult neurons enhances cognitive function. In the adult mouse hippocampus, we detected an enrichment of Tet2 in neurons. Viral-mediated neuronal overexpression and RNA interference of Tet2 altered dendritic complexity and synaptic-plasticity-related gene expression in vitro. Overexpression of neuronal Tet2 in adult hippocampus, and loss of Tet2 in adult glutamatergic neurons, resulted in differential hydroxymethylation associated with genes involved in synaptic transmission. Functionally, overexpression of neuronal Tet2 impaired hippocampal-dependent memory, while loss of neuronal Tet2 enhanced memory. Ultimately, these data identify neuronal Tet2 as a molecular target to boost cognitive function.


Assuntos
Dioxigenases , Proteínas Proto-Oncogênicas , Animais , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ligação a DNA/metabolismo , 5-Metilcitosina/metabolismo , Dioxigenases/genética , Metilação de DNA/genética , Cognição , Neurônios/metabolismo , Hipocampo/metabolismo
18.
Proc Natl Acad Sci U S A ; 119(42): e2123338119, 2022 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-36240321

RESUMO

5-methylcytosine (m5C) is one of the most prevalent modifications of RNA, playing important roles in RNA metabolism, nuclear export, and translation. However, the potential role of RNA m5C methylation in innate immunity remains elusive. Here, we show that depletion of NSUN2, an m5C methyltransferase, significantly inhibits the replication and gene expression of a wide range of RNA and DNA viruses. Notably, we found that this antiviral effect is largely driven by an enhanced type I interferon (IFN) response. The antiviral signaling pathway is dependent on the cytosolic RNA sensor RIG-I but not MDA5. Transcriptome-wide mapping of m5C following NSUN2 depletion in human A549 cells revealed a marked reduction in the m5C methylation of several abundant noncoding RNAs (ncRNAs). However, m5C methylation of viral RNA was not noticeably altered by NSUN2 depletion. In NSUN2-depleted cells, the host RNA polymerase (Pol) III transcribed ncRNAs, in particular RPPH1 and 7SL RNAs, were substantially up-regulated, leading to an increase of unshielded 7SL RNA in cytoplasm, which served as a direct ligand for the RIG-I-mediated IFN response. In NSUN2-depleted cells, inhibition of Pol III transcription or silencing of RPPH1 and 7SL RNA dampened IFN signaling, partially rescuing viral replication and gene expression. Finally, depletion of NSUN2 in an ex vivo human lung model and a mouse model inhibits viral replication and reduces pathogenesis, which is accompanied by enhanced type I IFN responses. Collectively, our data demonstrate that RNA m5C methylation controls antiviral innate immunity through modulating the m5C methylome of ncRNAs and their expression.


Assuntos
Interferon Tipo I , Viroses , 5-Metilcitosina/metabolismo , Animais , Antivirais , Proteína DEAD-box 58/metabolismo , Humanos , Imunidade Inata/genética , Interferon Tipo I/genética , Interferons , Ligantes , Camundongos , RNA Polimerase III , Replicação Viral/genética
19.
Anal Chem ; 94(44): 15489-15498, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36280344

RESUMO

DNA methylation (5-methylcytosine, 5mC) is the most prevalent epigenetic modification that is predominantly found in CG dinucleotides in mammalian genomes. In-depth investigation of the functions of 5mC heavily relies on the quantitative measurement of 5mC at single-base resolution in genomes. Here, we proposed a methyltransferase-directed labeling with APOBEC3A (A3A) deamination sequencing (MLAD-seq) method for the single-base resolution and quantitative detection of 5mC in DNA. In MLAD-seq, a mutant of DNA methyltransferase, M.MpeI-N374K, is utilized to selectively transfer a carboxymethyl group to the 5 position of cytosine in the CG dinucleotide to form 5-carboxymethylcytosine (5camC) using carboxy-S-adenosyl-l-methionine (caSAM) as the cofactor. After A3A treatment, 5camC is resistant to the deamination and base pairs with guanine. Thus, the cytosines in CG sites are read as C in sequencing. On the contrary, the methyl group in 5mC inhibits its carboxymethylcytosine by M.MpeI-N374K and therefore is readily deaminated by A3A to produce thymine that pairs with adenine and is read as T in sequencing. The differential readouts from C and 5mC in the MLAD-seq enable the single-base resolution mapping of 5mC in CG sites in DNA. With the developed MLAD-seq method, we observed the hypermethylation in the promoter region of retinoic acid receptor ß (RARB) gene from human nonsmall cell lung tumor tissue. Compared to harsh reaction conditions in bisulfite sequencing that could lead to significant degradation of DNA, the whole procedure of MLAD-seq is carried out under mild conditions, which will avoid DNA damage. Thus, MLAD-seq is more suitable in the scenario where only limited input DNA is available. Taken together, the MLAD-seq offers a valuable tool for bisulfite-free, single-base resolution and quantitative detection of 5mC in limited DNA.


Assuntos
5-Metilcitosina , Metiltransferases , Animais , Humanos , Desaminação , Análise de Sequência de DNA/métodos , Sulfitos , Epigênese Genética , DNA/genética , Citosina , Metilação de DNA , Mamíferos
20.
Nat Commun ; 13(1): 6230, 2022 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-36266342

RESUMO

TET (Ten-Eleven Translocation) dioxygenases effect DNA demethylation through successive oxidation of the methyl group of 5-methylcytosine (5mC) in DNA. In humans and in mouse models, TET loss-of-function has been linked to DNA damage, genome instability and oncogenesis. Here we show that acute deletion of all three Tet genes, after brief exposure of triple-floxed, Cre-ERT2-expressing mouse embryonic stem cells (mESC) to 4-hydroxytamoxifen, results in chromosome mis-segregation and aneuploidy; moreover, embryos lacking all three TET proteins showed striking variation in blastomere numbers and nuclear morphology at the 8-cell stage. Transcriptional profiling revealed that mRNA encoding a KH-domain protein, Khdc3 (Filia), was downregulated in triple TET-deficient mESC, concomitantly with increased methylation of CpG dinucleotides in the vicinity of the Khdc3 gene. Restoring KHDC3 levels in triple Tet-deficient mESC prevented aneuploidy. Thus, TET proteins regulate Khdc3 gene expression, and TET deficiency results in mitotic infidelity and genome instability in mESC at least partly through decreased expression of KHDC3.


Assuntos
Aneuploidia , Proteínas de Ligação a DNA , Dioxigenases , Células-Tronco Embrionárias Murinas , Animais , Camundongos , 5-Metilcitosina/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , DNA/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/metabolismo
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