RESUMO
AIMS: DNA methylation has its distribution influenced by DNA demethylation processes with the catalytic conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Myelodysplastic syndrome (MDS) has been associated with epigenetic dysregulation of genes related to DNA repair system, chronic immune response and cell cycle. METHODS: We evaluated the tissue DNA methylation/hydroxymethylation in bone marrow trephine biopsies of 73 patients with MDS, trying to correlate with the mRNA expression of 21 genes (POLH, POLL, REV3L, POLN, POLQ, POLI, POLK, IRF-1, IRF-2, IRF-3, IRF-4, IRF-5, IRF6, IRF-7, IRF-8,IRF-9, MAD2, CDC20, AURKA, AURKB and TPX2). RESULTS: The M-score (5mC) was significantly higher in patients with chromosomal abnormalities than patients with normal karyotype (95% CI -27.127779 to -2.368020; p=0.022). We observed a higher 5mC/5hmC ratio in patients classified as high-risk subtypes compared with low-risk subtypes (95% CI -72.922115 to -1.855662; p=0.040) as well as patients with hypercellular bone marrow compared with patients with normocellular/hypocellular bone marrow (95% CI -69.189259 to -0.511828; p=0.047) and with the presence of dyserythropoiesis (95% CI 17.077703 to 51.331388; p=0.001). DNA pols with translesion activity are significantly influenced by methylation. As 5mC immunoexpression increases, the expressions of POLH (r=-0.816; r2 =0.665; p=0.000), POLQ (r=-0.790; r2=0.624; p=0.001), PCNA (r=-0.635; r2=0.403; p=0.020), POLK (r=-0.633; r2=0.400; p=0.036 and REV1 (r=-0.578; r2=0.334; p=0.049) decrease. CONCLUSIONS: Our results confirm that there is an imbalance in the DNA methylation in MDS, influencing the development of chromosomal abnormalities which may be associated with the low expression of DNA polymerases with translesion synthesis polymerases activity.
Assuntos
Aberrações Cromossômicas , Metilação de DNA , DNA Polimerase Dirigida por DNA/genética , Epigênese Genética , Síndromes Mielodisplásicas/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , DNA Polimerase Dirigida por DNA/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Cariotipagem , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/enzimologia , Reação em Cadeia da Polimerase em Tempo Real , Adulto JovemRESUMO
In zebrafish, nicotine is known to regulate sensitivity to psychostimulants via epigenetic mechanisms. Little however is known about the regulation of addictive-like behavior by DNA methylation processes. To evaluate the influence of DNA methylation on nicotine-induced conditioned place preference (CPP), zebrafish were exposed to methyl supplementation through oral L-methionine (Met) administration. Met was found to reduce dramatically nicotine-induced CPP as well as behaviors associated with drug reward. The reduction was associated with the upregulation of DNA methyltransferases (DNMT1 and 3) as well as with the downregulation of methyl-cytosine dioxygenase-1 (TET1) and of nicotinic receptor subunits. Met also increased the expression of histone methyltransferases in nicotine-induced CPP groups. It reversed the nicotine-induced reduction in the methylation at α7 and NMDAR1 gene promoters. Treatment with the DNMT inhibitor 5-aza-2'-deoxycytidine (AZA) was found to reverse the effects of Met in structures of the reward pathway. Interestingly, Met did not modify the amount of the phospho-form of CREB (pCREB), a key factor establishing nicotine conditioning, whereas AZA increased pCREB levels. Our data suggest that nicotine-seeking behavior is partially dependent on DNA methylation occurring probably at specific gene loci, such as α7 and NMDAR1 receptor gene promoters. Overall, they suggest that Met should be considered as a potential therapeutic drug to treat nicotine addiction.
Assuntos
Comportamento Animal/fisiologia , Comportamento de Escolha , Suplementos Nutricionais , Metionina/farmacologia , Nicotina/farmacologia , Peixe-Zebra/fisiologia , 5-Metilcitosina/metabolismo , Animais , Azacitidina/farmacologia , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Condicionamento Clássico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Epigênese Genética/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Recompensa , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Indirect somatic embryogenesis (ISE) establishment for Coffea species started in the 1970s. Since then, intraspecific variations in the morphogenic pathway have been reported, even in the common environmental condition in vitro. Several authors have suggested that these variations are the result of genetic, epigenetic, and/or physiological events, highlighting the need for investigations to know the causes. Along these lines, this study aimed to investigate and describe, for the first time, the global 5-methylcytosine and physiological changes that occur in the cells of the aggregate suspensions of Coffea canephora during proliferation and somatic embryo regeneration steps. The cell proliferation step was characterized by increase in cell mass in all subcultures; relatively low mean values of global 5-methylcytosine (5-mC%), abscisic acid (ABA), and indole-3-acetic acid (IAA); high mean value of 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor); and increase followed by decrease in spermidine (Spd, a polyamine) level. Therefore, these epigenetic and physiologic aspects promoted the cell proliferation, which is fundamental for ISE. In turn, the somatic embryo regeneration was correlated with global 5-mC% and physiological changes. The competence acquisition, determination, and cell differentiation steps were marked by increases in mean values of 5-mC%, IAA and ABA, and decreases in ACC and Spd, evincing that these changes are the triggers for regeneration and maturation of somatic embryos. Therefore, dynamic and coordinated epigenetic and physiologic changes occur in the cells of the aggregate suspensions during the C. canephora ISE in liquid system.
Assuntos
5-Metilcitosina/metabolismo , Coffea/metabolismo , Proteínas de Plantas/metabolismo , Técnicas de Embriogênese Somática de Plantas/métodosRESUMO
DNA methyltransferases (DNMTs) play an essential role in DNA methylation and transcriptional regulation in the genome. DNMTs, along with other poorly studied elements, modulate the dynamic DNA methylation patterns of embryonic and adult cells. We summarize the current knowledge on the molecular mechanism of DNMTs' functional targeting to maintain genome-wide DNA methylation patterns. We focus on DNMTs' intrinsic characteristics, transcriptional regulation, and post-transcriptional modifications. Furthermore, we focus special attention on the DNMTs' specificity for target sites, including key cis-regulatory factors such as CpG content, common motifs, transcription factors (TF) binding sites, lncRNAs, and histone marks to regulate DNA methylation. We also review how complexes of DNMTs/TFs or DNMTs/lncRNAs are involved in DNA methylation in specific genome regions. Understanding these processes is essential because the spatiotemporal regulation of DNA methylation modulates gene expression in health and disease.
Assuntos
Metilação de DNA , Metilases de Modificação do DNA/genética , Código das Histonas/genética , RNA Longo não Codificante/genética , Fatores de Transcrição/genética , 5-Metilcitosina/metabolismo , Animais , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilases de Modificação do DNA/metabolismo , Humanos , Regiões Promotoras GenéticasRESUMO
A basic question linked to differential patterns of gene expression is how cells reach different fates despite using the same DNA template. Since 5-hydroxymethylcytosine (5hmC) emerged as an intermediate metabolite in active DNA demethylation, there have been increasing efforts to elucidate its function as a stable modification of the genome, including a role in establishing such tissue-specific patterns of expression. Recently we described TET1-mediated enrichment of 5hmC on the promoter region of the master regulator of hepatocyte identity, HNF4A, which precedes differentiation of liver adult progenitor cells in vitro. Here, we studied the genome-wide distribution of 5hmC at early in vitro differentiation of human hepatocyte-like cells. We found a global increase in 5hmC as well as a drop in 5-methylcytosine after one week of in vitro differentiation from bipotent progenitors, at a time when the liver transcript program is already established. 5hmC was overall higher at the bodies of overexpressed genes. Furthermore, by modifying the metabolic environment, an adenosine derivative prevents 5hmC enrichment and impairs the acquisition of hepatic identity markers. These results suggest that 5hmC could be a marker of cell identity, as well as a useful biomarker in conditions associated with cell de-differentiation such as liver malignancies.
Assuntos
5-Metilcitosina/análogos & derivados , Diferenciação Celular/genética , Metilação de DNA/genética , Fator 4 Nuclear de Hepatócito/genética , Oxigenases de Função Mista/genética , Proteínas Proto-Oncogênicas/genética , 5-Metilcitosina/metabolismo , Desmetilação do DNA , Regulação da Expressão Gênica no Desenvolvimento/genética , Genoma/genética , Hepatócitos/metabolismo , Humanos , Regiões Promotoras Genéticas/genética , Células-Tronco/metabolismoRESUMO
BACKGROUND: Malar melasma has a chronic and recurrent character that may be related to epigenetic changes. OBJECTIVE: To recognize the expression and DNA methylation of DNA methyltransferases (DNMTs) in malar melasma and perilesional skin, as well as the changes in DNMTs after their treatment with sunscreen in combination with 4% niacinamide, 0.05% retinoic acid, or placebo. METHODS: Thirty female patients were clinically evaluated for the expression of DNMT1 and DNMT3b using real-time PCR and immunofluorescence. These initial results were compared to results after eight weeks of treatment with sunscreen in combination with niacinamide, retinoic acid, or placebo. RESULTS: The relative expression of DNMT1 was significantly elevated in melasma compared with unaffected skin in all subjects, indicating DNA hypermethylation. After treatment, it was decreased in all groups: niacinamide (7 versus 1; p<0.01), retinoic acid (7 versus 2; p<0.05), and placebo (7 versus 3; p<0.05), which correlates with clinical improvement. DNMT3b was not overexpressed in lesional skin but reduced in all groups. CONCLUSIONS: We found DNA hypermethylation in melasma lesions. Environmental factors such as solar radiation may induce cellular changes that trigger hyperpigmentation through the activation of pathways regulated by epigenetic modifications. However, limiting or decreasing DNA methylation through sunscreen, niacinamide, and retinoic acid treatments that provide photoprotection and genetic transcription can counteract this.
Assuntos
Metilases de Modificação do DNA/metabolismo , Melanose/tratamento farmacológico , Melanose/enzimologia , Niacinamida/uso terapêutico , Protetores Solares/uso terapêutico , Tretinoína/uso terapêutico , 5-Metilcitosina/metabolismo , Adulto , Metilação de DNA , Metilases de Modificação do DNA/genética , Epiderme/efeitos dos fármacos , Epiderme/patologia , Feminino , Fluorescência , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Placebos , Protetores Solares/farmacologiaRESUMO
Single-stranded model oligodeoxyribonucleotides, each containing a single protonatable base-cytosine, adenine, guanine, or 5-methylcytosine-centrally located in a background of non-protonatable thymine residues, were acid-titrated in aqueous solution, with UV monitoring. The basicity of the central base was shown to depend on the type of the central base and its nearest neighbours and to rise with increasing oligonucleotide length and decreasing ionic strength of the solution. More complex model oligonucleotides, each containing a centrally located 5-methylcytosine base, were comparatively evaluated in single-stranded and double-stranded form, by UV spectroscopy and high-field NMR. The N3 protonation of the 5-methylcytosine moiety in the double-stranded case occurred at much lower pH, at which the duplex was already experiencing general dissociation, than in the single-stranded case. The central guanine:5-methylcytosine base pair remained intact up to this point, possibly due to an unusual alternative protonation on O2 of the 5-methylcytosine moiety, already taking place at neutral or weakly basic pH, as indicated by UV spectroscopy, thus suggesting that 5-methylcytosine sites in double-stranded DNA might be protonated to a significant extent under physiological conditions.
Assuntos
DNA de Cadeia Simples , Oligodesoxirribonucleotídeos , 5-Metilcitosina/metabolismo , Adenina/metabolismo , Sequência de Bases , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Guanina/metabolismo , Concentração de Íons de Hidrogênio , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , Concentração Osmolar , Prótons , Timina/metabolismoRESUMO
The grasses of the Lolium-Festuca complex show a prominent role in world agricultural scenario. Several studies have demonstrated that the plasticity of 45S rDNA sites has been recently associated with the possible fragility of the loci. Often, these fragile sites were observed as extended sites and gaps in metaphases. This organization can be evaluated in relation to their transcriptional activity/accessibility through epigenetic changes. Thus, this study aimed to investigate the relationship of the 5-methylcytosine and histone H3 lysine-9 dimethylation in different conformations of 45S rDNA sites in interphase nuclei and in metaphase chromosomes of L. perenne, L. multiflorum and F. arundinacea. The FISH technique using 45S rDNA probes was performed sequentially after the immunolocalization. The sites showed predominantly the following characteristics in the interphase nuclei: intra- and perinucleolar position, decondensed or partially condensed and hypomethylated and hyper/hypomethylated status. Extranucleolar sites were mainly hypermethylated for both epigenetic marks. The 45S rDNA sites with gaps identified in metaphases were always hypomethylated, which justifies it decondensed and transcriptional state. The frequency of sites with hypermethylated gaps was very low. The structural differences observed in these sites are directly related to the assessed epigenetic marks, justifying the different conformations throughout the cell cycle.
Assuntos
Festuca/genética , Lolium/genética , RNA Ribossômico/genética , 5-Metilcitosina/metabolismo , Ciclo Celular , Núcleo Celular , Sítios Frágeis do Cromossomo , Cromossomos de Plantas/genética , Metilação de DNA , DNA Ribossômico/genética , Epigênese Genética/genética , Epigenômica/métodos , Festuca/citologia , Hibridização in Situ Fluorescente/métodos , Interfase/genética , Lolium/citologia , MetáfaseRESUMO
Here, we determined the 5-hydroxymethylcytosine (5hmC), 5-methylcytosine (5mC), Ten Eleven Translocation (TETs), and DNA methyltransferases (DNMTs) levels in epithelial and inflammatory cells of labial salivary glands (LSG) from Sjögren's syndrome (SS)-patients and the effect of cytokines on HSG cells. LSG from SS-patients, controls and HSG cells incubated with cytokines were analysed. Levels of 5mC, 5hmC, DNMTs, TET2 and MeCP2 were assessed by immunofluorescence. In epithelial cells from SS-patients, an increase in TET2, 5hmC and a decrease in 5mC and MeCP2 were observed, additionally, high levels of 5mC and DNMTs and low levels of 5hmC were detected in inflammatory cells. Cytokines increased TET2 and 5hmC and decreased 5mC levels. Considering that the TET2 gene.promoter contains response elements for transcription factors activated by cytokines, together to in vitro results suggest that changes in DNA hydroxymethylation, resulting from altered levels of TET2 are likely to be relevant in the Sjögren's syndrome etiopathogenesis.
Assuntos
5-Metilcitosina/análogos & derivados , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Ligação a DNA/genética , Proteína 2 de Ligação a Metil-CpG/genética , Proteínas Proto-Oncogênicas/genética , Glândulas Salivares Menores/metabolismo , Síndrome de Sjogren/genética , 5-Metilcitosina/metabolismo , Adulto , Idoso , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Citocinas/imunologia , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/genética , Dioxigenases/imunologia , Dioxigenases/metabolismo , Epigênese Genética , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/metabolismo , Lábio , Masculino , Proteína 2 de Ligação a Metil-CpG/metabolismo , Pessoa de Meia-Idade , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/imunologia , Oxigenases de Função Mista/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Glândulas Salivares Menores/citologia , Glândulas Salivares Menores/imunologia , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/metabolismo , Adulto Jovem , DNA Metiltransferase 3BRESUMO
The central dogma of molecular biology statically says that the information flows from DNA to messenger RNA to protein. But the recent advances in mass spectrometry and high throughput technology have helped the scientists to view RNA as little more than a courier of genetic information encoded in the DNA. The dynamics of RNA modifications in coding and non-coding RNAs are just emerging as a carrier of non-genetic information, uncovering a new layer of complexity in the regulation of gene expression and protein translation. In this review, we summarize about the current knowledge of N6-methyladenosine (m6A), N1-methyladenosine (m1A), 5-methylcytosine (m5C) and pseudouridine (Ψ) modifications in RNA, and described how these RNA modifications are implicated in early animal development and in several human diseases.