RESUMO
TET proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are excised by mammalian DNA glycosylase TDG, implicating 5mC oxidation in DNA demethylation. Here, we show that the genomic locations of 5fC can be determined by coupling chemical reduction with biotin tagging. Genome-wide mapping of 5fC in mouse embryonic stem cells (mESCs) reveals that 5fC preferentially occurs at poised enhancers among other gene regulatory elements. Application to Tdg null mESCs further suggests that 5fC production coordinates with p300 in remodeling epigenetic states of enhancers. This process, which is not influenced by 5hmC, appears to be associated with further oxidation of 5hmC and commitment to demethylation through 5fC. Finally, we resolved 5fC at base resolution by hydroxylamine-based protection from bisulfite-mediated deamination, thereby confirming sites of 5fC accumulation. Our results reveal roles of active 5mC/5hmC oxidation and TDG-mediated demethylation in epigenetic tuning at regulatory elements.
Assuntos
Citosina/análogos & derivados , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Técnicas Genéticas , Estudo de Associação Genômica Ampla , 5-Metilcitosina/metabolismo , Animais , Citosina/metabolismo , Camundongos , Elementos Reguladores de Transcrição , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
In mammals, cytosine methylation (5mC) is widely distributed throughout the genome but is notably depleted from active promoters and enhancers. While the role of DNA methylation in promoter silencing has been well documented, the function of this epigenetic mark at enhancers remains unclear. Recent experiments have demonstrated that enhancers are enriched for 5-hydroxymethylcytosine (5hmC), an oxidization product of the Tet family of 5mC dioxygenases and an intermediate of DNA demethylation. These results support the involvement of Tet proteins in the regulation of dynamic DNA methylation at enhancers. By mapping DNA methylation and hydroxymethylation at base resolution, we find that deletion of Tet2 causes extensive loss of 5hmC at enhancers, accompanied by enhancer hypermethylation, reduction of enhancer activity, and delayed gene induction in the early steps of differentiation. Our results reveal that DNA demethylation modulates enhancer activity, and its disruption influences the timing of transcriptome reprogramming during cellular differentiation.
Assuntos
Diferenciação Celular/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Citosina/análogos & derivados , Citosina/metabolismo , Proteínas de Ligação a DNA/genética , Dioxigenases , Camundongos , Camundongos Knockout , Oxirredução , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Análise de Sequência de DNA , Transcriptoma/genética , Dedos de Zinco/genéticaRESUMO
Cytosine methylation is an important epigenetic mark, but how the distinctive patterns of DNA methylation arise remains elusive. For the first time, we systematically investigated how these patterns can be imparted by the inherent enzymatic preferences of mammalian de novo DNA methyltransferases in vitro and the extent to which this applies in cells. In a biochemical experiment, we subjected a wide variety of DNA sequences to methylation by DNMT3A or DNMT3B and then applied deep bisulfite sequencing to quantitatively determine the sequence preferences for methylation. The data show that DNMT3A prefers CpG and non-CpG sites followed by a 3'-pyrimidine, whereas DNMT3B favors a 3'-purine. Overall, we show that DNMT3A has a sequence preference for a TNC[G/A]CC context, while DNMT3B prefers TAC[G/A]GC. We extended our finding using publicly available data from mouse Dnmt1/3a/3b triple-knockout cells in which reintroduction of either DNMT3A or DNMT3B expression results in the acquisition of the same enzyme specific signature sequences observed in vitro. Furthermore, loss of DNMT3A or DNMT3B in human embryonic stem cells leads to a loss of methylation at the corresponding enzyme specific signatures. Therefore, the global DNA methylation landscape of the mammalian genome can be fundamentally determined by the inherent sequence preference of de novo methyltransferases.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Células-Tronco Embrionárias/fisiologia , Animais , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Genoma , Humanos , Motivos de Nucleotídeos , Especificidade por Substrato , DNA Metiltransferase 3BRESUMO
Controlling the functional dynamics of DNA within living cells is essential in biomedical research. Epigenetic modifications such as DNA methylation play a key role in this endeavour. DNA methylation can be controlled by genetic means. Yet there are few chemical tools available for the spatial and temporal modulation of this modification. Herein, we present a small-molecule approach to modulate DNA methylation with light. The strategy uses a photo-tuneable version of a clinically used drug (5-aza-2'-deoxycytidine) to alter the catalytic activity of DNA methyltransferases, the enzymes that methylate DNA. After uptake by cells, the photo-regulated molecule can be light-controlled to reduce genome-wide DNA methylation levels in proliferating cells. The chemical tool complements genetic, biochemical, and pharmacological approaches to study the role of DNA methylation in biology and medicine.
Assuntos
Metilação de DNA/genética , Epigênese Genética/genética , HumanosRESUMO
Background: Chronic Kidney Disease (CKD) stands as a formidable health challenge, recognized not only for its growing prevalence but also for its association with elevated mortality rates. Emerging evidence suggests that CKD is inherently linked to inflammatory processes, marking it as an inflammatory disorder. In this landscape, the systemic inflammatory response index (SIRI) emerges as a novel inflammation marker, yet to be applied for assessing the risk of mortality in CKD patients. Objective: This study aims to investigate the prognostic significance of the SIRI in all-cause and cardiovascular disease (CVD) mortality among patients with CKD. Method: This study conducted a retrospective observational study using the National Health and Nutrition Examination Survey (NHANES) database, encompassing data from 1999 to 2018. This analysis included 9,115 CKD patients, categorized based on SIRI quartiles. Key outcomes were all-cause and CVD mortality, analyzed using Kaplan-Meier survival curves, restricted cube splines (RCS) and cox proportional hazards models. Result: In this study of 9,115 CKD patients, the Kaplan-Meier survival analysis revealed a greater incidence of all-cause death among groups with higher SIRI (P-log rank <0.001). In the fully adjusted model (Model 3), each unit increase in SIRI led to a 20% increase in the risk of all-cause mortality. Additionally, higher SIRI quartiles (Q3 and Q4) were associated with increased risk compared to the lowest quartile (Q1) (Q3: HR: 1.16, 95% CI: 1.01-1.34; Q4: HR: 1.63, 95% CI: 1.40-1.90; P for trend <0.001). Similarly, for CVD mortality, each unit increase in SIRI in Model 3 increased the risk by 33%, with Q3 and Q4 showing higher risk than Q1 (Q3: HR: 1.39, 95% CI: 1.11-1.70; Q4: HR: 2.26, 95% CI: 1.72-2.98; P for trend <0.001). Conclusion: SIRI was positively associated with all-cause and CVD mortality in patients with CKD.
RESUMO
DNA hydroxymethylation and its mediated DNA demethylation are critical for multiple cellular processes, for example, nuclear reprogramming, embryonic development, and many diseases. Here, we demonstrate that a vital nutrient ascorbic acid (AA), or vitamin C (Vc), can directly enhance the catalytic activity of Tet dioxygenases for the oxidation of 5-methylcytosine (5mC). As evidenced by changes in intrinsic fluorescence and catalytic activity of Tet2 protein caused by AA and its oxidation-resistant derivatives, we further show that AA can uniquely interact with the C-terminal catalytic domain of Tet enzymes, which probably promotes their folding and/or recycling of the cofactor Fe(2+). Other strong reducing chemicals do not have a similar effect. These results suggest that AA also acts as a cofactor of Tet enzymes. In mouse embryonic stem cells, AA significantly increases the levels of all 5mC oxidation products, particularly 5-formylcytosine and 5-carboxylcytosine (by more than an order of magnitude), leading to a global loss of 5mC (â¼40%). In cells deleted of the Tet1 and Tet2 genes, AA alters neither 5mC oxidation nor the overall level of 5mC. The AA effects are however restored when Tet2 is re-expressed in the Tet-deficient cells. The enhancing effects of AA on 5mC oxidation and DNA demethylation are also observed in a mouse model deficient in AA synthesis. Our data establish a direct link among AA, Tet, and DNA methylation, thus revealing a role of AA in the regulation of DNA modifications.
Assuntos
5-Metilcitosina/metabolismo , Ácido Ascórbico/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/química , Animais , Ácido Ascórbico/química , DNA/química , Metilação de DNA , Dioxigenases , Camundongos , OxirreduçãoRESUMO
Limbal stem cells are essential for continuous corneal regeneration and injury repair. METTL3-catalyzed N6-methyladenosine (m6A) mRNA modifications are involved in many biological processes and play a specific role in stem cell regeneration, while the role of m6A modifications in corneal injury repair remains unknown. In this study, we generated a limbal stem cell-specific METTL3 knockout mouse model and studied the role of m6A in repairing corneal injury caused by alkali burn. The results showed that METTL3 knockout in the limbal stem cells promotes the in vivo cell proliferation and migration, leading to the fast repair of corneal injury. In addition, m6A modification profiling identified stem cell regulatory factors AHNAK and DDIT4 as m6A targets. Our study reveals the essential functions of m6A RNA modification in regulating injury repair and provides novel insights for clinical therapy of corneal diseases.
RESUMO
Estrogen receptor-α (ER) drives tumor development in ER-positive (ER+) breast cancer. The transcription factor GATA3 has been closely linked to ER function, but its precise role in this setting remains unclear. Quantitative proteomics was used to assess changes to the ER complex in response to GATA3 depletion. Unexpectedly, few proteins were lost from the ER complex in the absence of GATA3, with the only major change being depletion of the dioxygenase TET2. TET2 binding constituted a near-total subset of ER binding in multiple breast cancer models, with loss of TET2 associated with reduced activation of proliferative pathways. TET2 knockdown did not appear to change global methylated cytosine (5mC) levels; however, oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) was significantly reduced, and these events occurred at ER enhancers. These findings implicate TET2 in the maintenance of 5hmC at ER sites, providing a potential mechanism for TET2-mediated regulation of ER target genes.
Assuntos
5-Metilcitosina/análogos & derivados , Neoplasias da Mama/enzimologia , Montagem e Desmontagem da Cromatina , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Elementos Facilitadores Genéticos , Receptor alfa de Estrogênio/metabolismo , 5-Metilcitosina/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/genética , Bases de Dados Genéticas , Dioxigenases/genética , Antagonistas do Receptor de Estrogênio/farmacologia , Receptor alfa de Estrogênio/genética , Feminino , Fulvestranto/farmacologia , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Camundongos Endogâmicos NOD , Camundongos SCID , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Control of DNA methylation level is critical for gene regulation, and the factors that govern hypomethylation at CpG islands (CGIs) are still being uncovered. Here, we provide evidence that G-quadruplex (G4) DNA secondary structures are genomic features that influence methylation at CGIs. We show that the presence of G4 structure is tightly associated with CGI hypomethylation in the human genome. Surprisingly, we find that these G4 sites are enriched for DNA methyltransferase 1 (DNMT1) occupancy, which is consistent with our biophysical observations that DNMT1 exhibits higher binding affinity for G4s as compared to duplex, hemi-methylated, or single-stranded DNA. The biochemical assays also show that the G4 structure itself, rather than sequence, inhibits DNMT1 enzymatic activity. Based on these data, we propose that G4 formation sequesters DNMT1 thereby protecting certain CGIs from methylation and inhibiting local methylation.
Assuntos
Metilação de DNA , Quadruplex G , Ilhas de CpG , DNA/metabolismo , Epigenômica , Regulação da Expressão Gênica , Genoma Humano , Humanos , Células K562 , Método de Monte Carlo , Conformação de Ácido Nucleico , Regiões Promotoras GenéticasRESUMO
Ten-eleven translocation (Tet) family proteins convert 5-methylcytosine to 5-hydroxymethylcytosine. We show that mouse embryonic stem cells (ESCs) depleted of Tet1 and/or Tet2 by RNAi exhibit short telomeres and chromosomal instability, concomitant with reduced telomere recombination. Tet1 and Tet2 double-knockout ESCs also display short telomeres but to a lesser extent. Notably, Tet1/2/3 triple-knockout ESCs show heterogeneous telomere lengths and increased frequency of telomere loss and chromosomal fusion. Mechanistically, Tets depletion or deficiency increases Dnmt3b and decreases 5hmC levels, resulting in elevated methylation levels at sub-telomeres. Consistently, knockdown of Dnmt3b or addition of 2i (MAPK and GSK3ß inhibitors), which also inhibits Dnmt3b, reduces telomere shortening, partially rescuing Tet1/2 deficiency. Interestingly, Tet1/2 double or Tet1/2/3 triple knockout in ESCs consistently upregulates Zscan4, which may counteract telomere shortening. Together, Tet enzymes play important roles in telomere maintenance and chromosomal stability of ESCs by modulating sub-telomeric methylation levels.
Assuntos
Instabilidade Cromossômica/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Telômero/metabolismo , Animais , Cromossomos de Mamíferos/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Deleção de Genes , Camundongos , Camundongos Knockout , Recombinação Genética/genética , Encurtamento do Telômero , DNA Metiltransferase 3BRESUMO
TET proteins have been found to play an important role in active demethylation at CpG sites in mammals. There are some reports implicating their functions in removal of DNA methylation imprint at the imprinted regions in the germline. However, it is not well established whether TET proteins can also be involved in demethylation of DNA methylation imprint in embryonic stem (ES) cells. Here we report that loss of TET proteins caused a significant increase in DNA methylation at the Igf2-H19 imprinted region in ES cells. We also observed a variable increase in DNA methylation at the Peg1 imprinted region in the ES clones devoid of TET proteins, in particular in the differentiated ES cells. By contrast, we did not observe a significant increase of DNA methylation imprint at the Peg3, Snrpn and Dlk1-Dio3 imprinted regions in ES cells lacking TET proteins. Interestingly, loss of TET proteins did not result in a significant increase of DNA methylation imprint at the Igf2-H19 and Peg1 imprinted regions in the embryoid bodies (EB). Therefore, TET proteins seem to be differentially involved in maintaining DNA methylation imprint at a subset of imprinted regions in ES cells and EBs.
Assuntos
Células-Tronco Embrionárias/metabolismo , Impressão Genômica , Animais , Proteínas de Ligação ao Cálcio , Ilhas de CpG , Metilação de DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Dioxigenases , Células-Tronco Embrionárias/citologia , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Iodeto Peroxidase/genética , Camundongos , Proteínas/genética , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas Centrais de snRNP/genéticaRESUMO
Tet-mediated DNA oxidation is a recently identified mammalian epigenetic modification, and its functional role in cell-fate transitions remains poorly understood. Here, we derive mouse embryonic fibroblasts (MEFs) deleted in all three Tet genes and examine their capacity for reprogramming into induced pluripotent stem cells (iPSCs). We show that Tet-deficient MEFs cannot be reprogrammed because of a block in the mesenchymal-to-epithelial transition (MET) step. Reprogramming of MEFs deficient in TDG is similarly impaired. The block in reprogramming is caused at least in part by defective activation of key miRNAs, which depends on oxidative demethylation promoted by Tet and TDG. Reintroduction of either the affected miRNAs or catalytically active Tet and TDG restores reprogramming in the knockout MEFs. Thus, oxidative demethylation to promote gene activation appears to be functionally required for reprogramming of fibroblasts to pluripotency. These findings provide mechanistic insight into the role of epigenetic barriers in cell-lineage conversion.
Assuntos
Reprogramação Celular , DNA Glicosilases/fisiologia , Metilação de DNA , Proteínas de Ligação a DNA/fisiologia , Células-Tronco Embrionárias/citologia , Transição Epitelial-Mesenquimal , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Western Blotting , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Dioxigenases , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica , Técnicas Imunoenzimáticas , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Knockout , MicroRNAs/fisiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Vitamin C, a micronutrient known for its anti-scurvy activity in humans, promotes the generation of induced pluripotent stem cells (iPSCs) through the activity of histone demethylating dioxygenases. TET hydroxylases are also dioxygenases implicated in active DNA demethylation. Here we report that TET1 either positively or negatively regulates somatic cell reprogramming depending on the absence or presence of vitamin C. TET1 deficiency enhances reprogramming, and its overexpression impairs reprogramming in the context of vitamin C by modulating the obligatory mesenchymal-to-epithelial transition (MET). In the absence of vitamin C, TET1 promotes somatic cell reprogramming independent of MET. Consistently, TET1 regulates 5-hydroxymethylcytosine (5hmC) formation at loci critical for MET in a vitamin C-dependent fashion. Our findings suggest that vitamin C has a vital role in determining the biological outcome of TET1 function at the cellular level. Given its benefit to human health, vitamin C should be investigated further for its role in epigenetic regulation.