RESUMEN
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.
Asunto(s)
Aberraciones Cromosómicas , Metilación de ADN , ADN Polimerasa Dirigida por ADN/genética , Epigénesis Genética , Síndromes Mielodisplásicos/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , ADN Polimerasa Dirigida por ADN/metabolismo , Femenino , Humanos , Inmunohistoquímica , Cariotipificación , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/enzimología , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto JovenRESUMEN
Simultaneous measurement of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) at the single-nucleotide level can be obtained by combining data from DNA processing methods including traditional bisulfite (BS), oxidative bisulfite (oxBS), or Tet-assisted (TAB) bisulfite conversion. Array-based technologies have been widely used in this task, due to their time and cost efficiency. For methylation studies using BS data, many protocols and related packages have been suggested in the literature to deal with limitations and confounders that arise from array data. In this chapter, we illustrate how the reader can make small adjustments to these protocols to obtain estimates of methylation and hydroxymethylation proportions.
Asunto(s)
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , Biología Computacional/métodos , Metilación de ADN , ADN/análisis , ADN/química , Epigénesis Genética , Sulfitos/química , ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Oxidación-ReducciónRESUMEN
Maternal malnutrition remains one of the major adversities affecting brain development and long-term mental health outcomes, increasing the risk to develop anxiety and depressive disorders. We have previously shown that malnutrition-induced anxiety-like behaviours can be rescued by a social and sensory stimulation (enriched environment) in male mice. Here, we expand these findings to adult female mice and profiled genome-wide ventral hippocampal 5hmC levels related to malnutrition-induced anxiety-like behaviours and their rescue by an enriched environment. This approach revealed 508 differentially hydroxymethylated genes associated with protein malnutrition and that several genes (N = 34) exhibited a restored 5hmC abundance to control levels following exposure to an enriched environment, including genes involved in neuronal functions like dendrite outgrowth, axon guidance, and maintenance of neuronal circuits (e.g. Fltr3, Itsn1, Lman1, Lsamp, Nav, and Ror1) and epigenetic mechanisms (e.g. Hdac9 and Dicer1). Sequence motif predictions indicated that 5hmC may be modulating the binding of transcription factors for several of these transcripts, suggesting a regulatory role for 5hmC in response to perinatal malnutrition and exposure to an enriched environment. Together, these findings establish a role for 5hmC in early-life malnutrition and reveal genes linked to malnutrition-induced anxious behaviours that are mitigated by an enriched environment.
Asunto(s)
Metilación de ADN , Desnutrición , 5-Metilcitosina/análogos & derivados , Animales , Epigénesis Genética , Femenino , Masculino , RatonesRESUMEN
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.
Asunto(s)
5-Metilcitosina/análogos & derivados , Diferenciación Celular/genética , Metilación de ADN/genética , Factor Nuclear 4 del Hepatocito/genética , Oxigenasas de Función Mixta/genética , Proteínas Proto-Oncogénicas/genética , 5-Metilcitosina/metabolismo , Desmetilación del ADN , Regulación del Desarrollo de la Expresión Génica/genética , Genoma/genética , Hepatocitos/metabolismo , Humanos , Regiones Promotoras Genéticas/genética , Células Madre/metabolismoRESUMEN
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.
Asunto(s)
5-Metilcitosina/análogos & derivados , ADN (Citosina-5-)-Metiltransferasas/genética , Proteínas de Unión al ADN/genética , Proteína 2 de Unión a Metil-CpG/genética , Proteínas Proto-Oncogénicas/genética , Glándulas Salivales Menores/metabolismo , Síndrome de Sjögren/genética , 5-Metilcitosina/metabolismo , Adulto , Anciano , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Citocinas/inmunología , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , ADN Metiltransferasa 3A , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Dioxigenasas/genética , Dioxigenasas/inmunología , Dioxigenasas/metabolismo , Epigénesis Genética , Femenino , Expresión Génica , Regulación de la Expresión Génica , Humanos , Inflamación/genética , Inflamación/metabolismo , Labio , Masculino , Proteína 2 de Unión a Metil-CpG/metabolismo , Persona de Mediana Edad , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/inmunología , Oxigenasas de Función Mixta/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Glándulas Salivales Menores/citología , Glándulas Salivales Menores/inmunología , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/metabolismo , Adulto Joven , ADN Metiltransferasa 3BRESUMEN
Myelodysplastic syndromes (MDS) are myeloid malignancies characterized by ineffective hematopoiesis, dysplasia, peripheral cytopenia and increased risk of progression to acute myeloid leukemia. Refractory cytopenia of childhood (RCC) is the most common subtype of pediatric MDS and has overlapping clinical features with viral infections and autoimmune disorders. Mutations in TET2 gene are found in about 20-25% of adult MDS and are associated with a decrease in 5-hydroxymethylcytosine (5-hmC) content. TET2 deregulation and its malignant potential were reported in adult but not in pediatric MDS. We evaluated the gene expression and the presence of mutations in TET2 gene in 19 patients with RCC. TET2 expression level was correlated with 5-hmC amount in DNA and possible regulatory epigenetic mechanisms. One out of 19 pediatric patients with RCC was a carrier of a TET2 mutation. TET2 expression and 5-hmC levels were decreased in patients when compared to a disease-free group. Lower expression was not associated to the presence of mutation or with the status of promoter methylation, but a significant correlation with microRNA-22 expression was found. These findings suggested that TET2 downregulation and low levels of 5-hmC are inversely related to miR-22 expression. The existence of a regulatory loop between microRNA-22 and TET2 may play a role in MDS pathogenesis.
Asunto(s)
Citosina/análogos & derivados , Proteínas de Unión al ADN/biosíntesis , Regulación de la Expresión Génica/genética , MicroARNs/genética , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , 5-Metilcitosina/análogos & derivados , Adolescente , Estudios de Casos y Controles , Niño , Preescolar , Citosina/biosíntesis , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Dioxigenasas , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Masculino , Mutación , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas/genética , TranscriptomaRESUMEN
Prenatal exposure to neurotoxicants such as lead (Pb) may cause stable changes in the DNA methylation (5mC) profile of the fetal genome. However, few studies have examined its effect on the DNA de-methylation pathway, specifically the dynamic changes of the 5-hydroxymethylcytosine (5hmC) profile. Therefore, in this study, we investigate the relationship between Pb exposure and 5mC and 5hmC modifications during early development. To study the changes in the 5hmC profile, we use a novel modification of the Infinium™ HumanMethylation450 assay (Illumina, Inc.), which we named HMeDIP-450K assay, in an in vitro human embryonic stem cell model of Pb exposure. We model Pb exposure-associated 5hmC changes as clusters of correlated, adjacent CpG sites, which are co-responding to Pb. We further extend our study to look at Pb-dependent changes in high density 5hmC regions in umbilical cord blood DNA from 48 mother-infant pairs from the Early Life Exposure in Mexico to Environmental Toxicants (ELEMENT) cohort. For our study, we randomly selected umbilical cord blood from 24 male and 24 female children from the 1st and 4th quartiles of Pb levels. Our data show that Pb-associated changes in the 5hmC and 5mC profiles can be divided into sex-dependent and sex-independent categories. Interestingly, differential 5mC sites are better markers of Pb-associated sex-dependent changes compared to differential 5hmC sites. In this study we identified several 5hmC and 5mC genomic loci, which we believe might have some potential as early biomarkers of prenatal Pb exposure.
Asunto(s)
Islas de CpG/efectos de los fármacos , Citosina/análogos & derivados , Exposición a Riesgos Ambientales/efectos adversos , Células Madre Embrionarias Humanas/efectos de los fármacos , Plomo/efectos adversos , Cordón Umbilical/efectos de los fármacos , 5-Metilcitosina/análogos & derivados , Línea Celular , Citosina/química , Citosina/metabolismo , Metilación de ADN/efectos de los fármacos , Sangre Fetal/efectos de los fármacos , Humanos , México , Análisis de Secuencia de ADN , Factores SexualesRESUMEN
In this study, we investigated the effects of oxidative stress and hypermethylation through lipid peroxidation and DNA methylation, respectively, in erythrocytes of Oreochromis niloticus exposed to environmental complex mixture of water from Cubatão do Sul River throughout the year. This river is the source of drinking water for the region of Florianópolis, the capital of Santa Catarina State, Brazil. Lipid peroxidation was quantified by the rate of malondialdehyde (MDA) formation, and DNA methylation was quantified by the rate of 5-methyldeoxycytosine (m(5)dC) formation. In all studied sites, the river water samples caused metabolic changes in O. niloticus. MDA formation rates were significantly different when compared to the negative control (except for samples from Site 1 during spring 2010, summer 2011 and fall 2011). All samples (except Site 1, spring 2010) induced increases in the m(5)dC formation rates, and at the end of the study, the values were near the values found in the positive control (potassium dichromate 2.5mg/L). The results showed that samples of environmental complex mixtures of water from Cubatão do Sul River are capable of inducing high levels of oxidative damage and hypermethylation in O. niloticus.
Asunto(s)
Cíclidos/sangre , Metilación de ADN/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Ríos/química , Contaminantes Químicos del Agua/toxicidad , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animales , Brasil , Cíclidos/genética , Cíclidos/metabolismo , Monitoreo del Ambiente , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Malondialdehído/metabolismo , Contaminantes Químicos del Agua/análisisRESUMEN
Bipolar disorder (BD) is a major public health problem characterized by progressive functional impairment. A number of clinical variables have been associated with progression of the disease, most notably number of affective episodes and presence of psychotic symptoms, both of which correlate with greater cognitive impairment, lower response rates for lithium, and possibly lower levels of neurotrophic factors. Oxidative damage to cytosine and guanosine (8-OHdG) has been described as a modulator of DNA methylation, but the extent of DNA oxidative damage involvement in BD remains unclear. The aim of this study was to evaluate the extent of DNA oxidative damage to 8-OHdG and 5-methylcytosine (5-HMec), as well as global methylation (5-Mec), in BD patients and healthy controls. Potential association with clinical variables was also investigated. DNA levels of 8-OHdG, 5-HMec and 5-Mec were measured in 50 BD type I patients and 50 healthy controls. DNA 8-OHdG levels were higher in BD patients compared to healthy controls and found to be positively influenced by number of previous manic episodes. BD subjects had lower levels of 5-HMec compared to controls, whereas this measure was not influenced by the clinical features of BD. Number of manic episodes was correlated with higher levels of 8-OHdG, but not of 5-Mec or 5-HMec. Lower demethylation activity (5-HMec) but no difference in global 5-Mec levels was observed in BD. This finding suggests that oxidative damage to 8-OHdG might be a potential marker of disease progression, although further prospective cross-sectional studies to confirm neuroprogression in BD are warranted.
Asunto(s)
Trastorno Bipolar/metabolismo , Trastorno Bipolar/fisiopatología , Metilación de ADN/fisiología , ADN/metabolismo , Oxidación-Reducción , 5-Metilcitosina/análogos & derivados , 8-Hidroxi-2'-Desoxicoguanosina , Adolescente , Adulto , Citosina/análogos & derivados , Citosina/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Análisis Multivariante , Estrés Oxidativo/fisiología , Adulto JovenRESUMEN
In this study, lipid peroxidation and DNA methylation were observed in erythrocytes of Oreochromis niloticus exposed to soluble fractions of textile, metal-mechanic and pulp and paper industrial waste, after a period of 48 h. Lipid peroxidation was quantified by the rate of malondialdehyde (MDA) and DNA methylation was quantified by the rate of 5-methyldeoxycytosine (m5dC). Soluble fractions of textile industrial waste caused metabolic changes for all studied samples. In organisms exposed to samples TX1 and TX2 (textile waste) MDA rates were 132.36 and 140.28 nM MDA/mg protein, respectively, while in control organism the MDA rates were 27.5 nM MDA/mg protein. All samples from soluble fractions of textile industrial waste induced increases in m5dC rates, increases varied between 300 percent and 700 percent when compared to the control organism. All the organisms exposed to soluble fractions of metal-mechanic industrial waste presented increases between 360 percent and 600 percent in the rates of MDA, and one of them (sample MM3) induced an increase of 180 percent in the rate of m5dC, when compared to control. Although a significant increase was not observed in the MDA rate of fish exposed to the soluble fractions of pulp and paper industrial waste, there was an increase of 460 percent in the rate of m5dC in one of the samples (sample PP2), when compared to control. The results showed that the soluble fractions of these industrial wastes are capable of inducing oxidative damage and altering the DNA methylation of O. niloticus. Thus, the MDA and m5dC rates demonstrated to be effective biomarkers of exposure, which could be used to evaluate the toxicity of soluble fractions of industrial solid waste.
Asunto(s)
Metilación de ADN/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Residuos Industriales/efectos adversos , Peroxidación de Lípido/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animales , Biomarcadores/metabolismo , Cíclidos , Eritrocitos/metabolismo , Eritrocitos/fisiología , Malondialdehído/metabolismo , Estrés OxidativoRESUMEN
Ayu17-449, a novel gene in mice, has been identified as a tumor-suppressor gene in myeloid malignancy; its product catalyzes the conversion of 5-methylcytosine of DNA to 5-hydroxymethylcytosine. However, in vivo, its functional target genes and biological function have remained unclear. Based on the assumption that alterations in the expression of the Ayu17-449 gene affect the expression of other related genes, we screened a microarray of altered gene expression in Ayu17-449(-/-) and Ayu17-449(+/+) mice. We identified 4049 genes with altered expression, including 1296 up-regulated (fold change ≥2) and 2753 down-regulated (fold change ≤0.5) genes in knockout mice compared with control mice. We then used qRT-PCR and RT-PCR to validate the chip data. Gene ontology and pathway analysis were performed on these altered genes. We found that these altered genes are functional genes in the complement and coagulation cascades, metabolism, biosynthesis, transcriptional regulation, proteolysis, and intracellular signaling pathways, such as the peroxisome proliferator-activated-receptor signaling pathway, the TNF-α-NF-κB pathway, the Notch signaling pathway, the MAPK signaling pathway, and the insulin signaling pathway. The results of our genome-wide comprehensive study could be helpful for comprehending the underlying functional mechanisms of the Ayu17-449 gene in mammals.