RESUMO
Rhythmic gene expression is found throughout the central nervous system. This harmonized regulation can be dependent on- and independent of- the master regulator of biological clocks, the suprachiasmatic nucleus (SCN). Substantial oscillatory activity in the brain's reward system is regulated by dopamine. While light serves as a primary time-giver (zeitgeber) of physiological clocks and synchronizes biological rhythms in 24-h cycles, nonphotic stimuli have a profound influence over circadian biology. Indeed, reward-related activities (e.g., feeding, exercise, sex, substance use, and social interactions), which lead to an elevated level of dopamine, alters rhythms in the SCN and the brain's reward system. In this chapter, we will discuss the influence of the dopaminergic reward pathways on circadian system and the implication of this interplay on human health.
Assuntos
Ritmo Circadiano , Núcleo Supraquiasmático , Relógios Biológicos , Dopamina , Humanos , RecompensaRESUMO
The endogenous NMDA receptor (NMDAR) agonist D-aspartate occurs transiently in the mammalian brain because it is abundant during embryonic and perinatal phases before drastically decreasing during adulthood. It is well established that postnatal reduction of cerebral D-aspartate levels is due to the concomitant onset of D-aspartate oxidase (DDO) activity, a flavoenzyme that selectively degrades bicarboxylic D-amino acids. In the present work, we show that d-aspartate content in the mouse brain drastically decreases after birth, whereas Ddo mRNA levels concomitantly increase. Interestingly, postnatal Ddo gene expression is paralleled by progressive demethylation within its putative promoter region. Consistent with an epigenetic control on Ddo expression, treatment with the DNA-demethylating agent, azacitidine, causes increased mRNA levels in embryonic cortical neurons. To indirectly evaluate the effect of a putative persistent Ddo gene hypermethylation in the brain, we used Ddo knock-out mice (Ddo(-/-)), which show constitutively suppressed Ddo expression. In these mice, we found for the first time substantially increased extracellular content of d-aspartate in the brain. In line with detrimental effects produced by NMDAR overstimulation, persistent elevation of D-aspartate levels in Ddo(-/-) brains is associated with appearance of dystrophic microglia, precocious caspase-3 activation, and cell death in cortical pyramidal neurons and dopaminergic neurons of the substantia nigra pars compacta. This evidence, along with the early accumulation of lipufuscin granules in Ddo(-/-) brains, highlights an unexpected importance of Ddo demethylation in preventing neurodegenerative processes produced by nonphysiological extracellular levels of free D-aspartate.
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Envelhecimento , Encéfalo/metabolismo , D-Aspartato Oxidase/metabolismo , Ácido D-Aspártico/metabolismo , Neurônios/fisiologia , Regiões Promotoras Genéticas/genética , Fatores Etários , Animais , Animais Recém-Nascidos , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Encéfalo/citologia , Morte Celular/genética , D-Aspartato Oxidase/genética , Decitabina , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Crohn's disease (CD) is a chronic inflammation of the intestinal mucosa, characterized by periods of acute recurrence and remission. Depending on the specific region affected, CD is classified as ileal CD or colonic CD. It is largely accepted that the intestinal microbiota is involved in the onset of the pathology. Indeed, a reduced immune tolerance to components of the intestinal commensal microbiota and inflammation of the intestinal barrier typifies patients with CD. Several studies have shown defective expression of intestinal antimicrobial peptides (AMPs) in patients with CD compared to controls, particularly defensins. A reduction in α-defensins is observed in ileal CD, while ß-defensins are increased in colonic CD. In addition to an immunological basis, the disease is frequently associated with genetic alterations including mutations of NOD2 gene. Several therapeutic strategies to circumvent the dysfunction observed in CD are currently under investigation. These include the use of delivery systems to administer endogenous AMPs and the engineering of peptidomimetics that could ameliorate the severity of CD. In this review, the role defensins play in CD and the strategies aimed at overcoming bacterial resistance will be discussed.
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Doença de Crohn/metabolismo , Doença de Crohn/microbiologia , Defensinas/metabolismo , Mucosa Intestinal/microbiologia , Microbiota , Animais , Peptídeos Catiônicos Antimicrobianos/química , Doença de Crohn/imunologia , Defensinas/imunologia , Modelos Animais de Doenças , Farmacorresistência Bacteriana , Humanos , Íleo/metabolismo , Íleo/microbiologia , Sistema Imunitário , Inflamação , Mucosa Intestinal/imunologia , Intestino Delgado/metabolismo , Intestino Delgado/microbiologia , Camundongos , alfa-Defensinas , beta-DefensinasRESUMO
BACKGROUND: CpG sites in an individual molecule may exist in a binary state (methylated or unmethylated) and each individual DNA molecule, containing a certain number of CpGs, is a combination of these states defining an epihaplotype. Classic quantification based approaches to study DNA methylation are intrinsically unable to fully represent the complexity of the underlying methylation substrate. Epihaplotype based approaches, on the other hand, allow methylation profiles of cell populations to be studied at the single molecule level. For such investigations, next-generation sequencing techniques can be used, both for quantitative and for epihaplotype analysis. Currently available tools for methylation analysis lack output formats that explicitly report CpG methylation profiles at the single molecule level and that have suited statistical tools for their interpretation. RESULTS: Here we present ampliMethProfiler, a python-based pipeline for the extraction and statistical epihaplotype analysis of amplicons from targeted deep bisulfite sequencing of multiple DNA regions. CONCLUSIONS: ampliMethProfiler tool provides an easy and user friendly way to extract and analyze the epihaplotype composition of reads from targeted bisulfite sequencing experiments. ampliMethProfiler is written in python language and requires a local installation of BLAST and (optionally) QIIME tools. It can be run on Linux and OS X platforms. The software is open source and freely available at http://amplimethprofiler.sourceforge.net .
Assuntos
Ilhas de CpG/genética , D-Aspartato Oxidase/genética , Metilação de DNA , DNA/química , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Software , Animais , DNA/análise , DNA/genética , Trato Gastrointestinal/metabolismo , Humanos , Camundongos , Análise de Sequência de DNA/métodos , Sulfitos/químicaRESUMO
One of the most fascinating aspects of the field of epigenetics is the emerging ability of environmental factors to trigger epigenetic changes in eukaryotic cells, thus contributing to transient or stable, and potentially heritable, changes in gene expression program in the absence of alteration in DNA sequence. Epigenetic response may result in cell adaptation to environmental stimuli or, in some instances, may contribute to generation or progression of different kind of diseases. A paradigmatic case of disease that is accompanied by multiple epigenetic alterations is gastric cancer, among other relevant examples. In turn, Helicobacter pylori (Hp) infection has been associated as a leading cause of gastric cancer. One possible hypothesis is that Hp-gastric cell interaction initiates an epigenetic reprogramming of host cell genome that may favor tumorigenesis. Accordingly, an abundance of experimental evidence indicates that several epigenetic alterations underlie the gastric cancerogenesis process and that these alterations represent one of the major hallmarks of gastric cancer. However, several critical questions remain unanswered: Does Hp directly provoke epigenetic alterations? Which mechanisms underlie these phenomena? Based on currently available data, it is often arduous to discriminate between the epigenetic modifications directly triggered by Hp-gastric cell interaction and those alterations that are mediated by inflammation process or by many other molecular and genetic events occurring during the gastric cancer progression. We will review our present knowledge of epigenetic modifications and alterations proven to occur in host cells as a direct consequence of Hp infection.
Assuntos
Epigênese Genética , Células Epiteliais/microbiologia , Regulação da Expressão Gênica , Helicobacter pylori/fisiologia , Interações Hospedeiro-Patógeno , Neoplasias Gástricas/microbiologia , HumanosRESUMO
Degeneration of dopaminergic neurons leads to Parkinson's disease (PD), characterized by reduced levels of striatal dopamine (DA) and impaired voluntary movements. DA replacement is achieved by levodopa treatment which in long-term causes involuntary movements or dyskinesia. Dyskinesia is linked to the pulsatile activation of D1 receptors of the striatal medium spiny neurons (MSNs) forming the direct output pathway (dMSNs). The contribution of DA stimulation of D2R in MSNs of the indirect pathway (iMSNs) is less clear. Using the 6-hydroxydopamine model of PD, here we show that loss of DA-mediated inhibition of these neurons intensifies levodopa-induced dyskinesia (LID) leading to reprogramming of striatal gene expression. We propose that the motor impairments characteristic of PD and of its therapy are critically dependent on D2R-mediated iMSNs activity. D2R signaling not only filters inputs to the striatum but also indirectly regulates dMSNs mediated responses.
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FXYD1 is a key protein controlling ion channel transport. FXYD1 exerts its function by regulating Na+/K+-ATPase activity, mainly in brain and cardiac tissues. Alterations of the expression level of the FXYD1 protein cause diastolic dysfunction and arrhythmias in heart and decreased neuronal dendritic tree and spine formation in brain. Moreover, FXYD1, a target of MeCP2, plays a crucial role in the pathogenesis of the Rett syndrome, a neurodevelopmental disorder. Thus, the amount of FXYD1 must be strictly controlled in a tissue specific manner and, likely, during development. Epigenetic modifications, particularly DNA methylation, represent the major candidate mechanism that may regulate Fxyd1 expression. In the present study, we performed a comprehensive DNA methylation analysis and mRNA expression level measurement of the two Fxyd1 transcripts, Fxyd1a and Fxyd1b, in brain and heart tissues during mouse development. We found that DNA methylation at Fxyd1a increased during brain development and decreased during heart development along with coherent changes in mRNA expression levels. We also applied ultra-deep methylation analysis to detect cell to cell methylation differences and to identify possible distinct methylation profile (epialleles) distribution between heart and brain and in different developmental stages. Our data indicate that the expression of Fxyd1 transcript isoforms inversely correlates with DNA methylation in developing brain and cardiac tissues suggesting the existence of a temporal-specific epigenetic program. Moreover, we identified a clear remodeling of epiallele profiles which were distinctive for single developmental stage both in brain and heart tissues.
Assuntos
Proteínas de Membrana , Fosfoproteínas , Animais , Encéfalo/metabolismo , Metilação de DNA , Epigênese Genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosfoproteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Molecular differentiation between benign (follicular thyroid adenoma, FTA) and malignant (follicular thyroid carcinoma, FTC) thyroid neoplasms is challenging. Here, we explored the genome-wide DNA methylation profile of FTA (n.10) and FTC (n.11) compared to normal thyroid (NT) (n.7) tissues. FTC featured 3,564 differentially-methylated CpGs (DMCpG), most (84%) of them hypermethylated, with respect to normal controls. At the principal component analysis (PCA), the methylation profile of FTA occupied an intermediate position between FTC and normal tissue. A large fraction (n. 2,385) of FTC-associated DMCpG were related (intragenic or within 1500 bp from the transcription start site) to annotated genes (n. 1,786). FTC-hypermethylated genes were enriched for targets of the Polycomb transcriptional repressor complex and the specific histone H3 marks (H3K4me2/me3-H3K27me3) found in chromatin domains known as "bivalent". Transcriptome profiling by RNAseq showed that 7.9% of the DMCpGs-associated genes were differentially expressed in FTC compared to NT, suggesting that altered DNA methylation may contribute to their altered expression. Overall, this study suggests that perturbed DNA methylation, in particular hypermethylation, is a component of the molecular mechanisms leading to the formation of FTC and that DNA methylation profiling may help differentiating FTCs from their benign counterpart.
Assuntos
Adenocarcinoma Folicular/genética , Metilação de DNA , Neoplasias da Glândula Tireoide/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Glândula Tireoide/metabolismoRESUMO
BACKGROUND: Programmed epigenetic modifications occurring at early postnatal brain developmental stages may have a long-lasting impact on brain function and complex behavior throughout life. Notably, it is now emerging that several genes that undergo perinatal changes in DNA methylation are associated with neuropsychiatric disorders. In this context, we envisaged that epigenetic modifications during the perinatal period may potentially drive essential changes in the genes regulating brain levels of critical neuromodulators such as D-serine and D-aspartate. Dysfunction of this fine regulation may contribute to the genesis of schizophrenia or other mental disorders, in which altered levels of D-amino acids are found. We recently demonstrated that Ddo, the D-aspartate degradation gene, is actively demethylated to ultimately reduce D-aspartate levels. However, the role of epigenetics as a mechanism driving the regulation of appropriate D-ser levels during brain development has been poorly investigated to date. METHODS: We performed comprehensive ultradeep DNA methylation and hydroxymethylation profiling along with mRNA expression and HPLC-based D-amino acids level analyses of genes controlling the mammalian brain levels of D-serine and D-aspartate. DNA methylation changes occurring in specific cerebellar cell types were also investigated. We conducted high coverage targeted bisulfite sequencing by next-generation sequencing and single-molecule bioinformatic analysis. RESULTS: We report consistent spatiotemporal modifications occurring at the Dao gene during neonatal development in a specific brain region (the cerebellum) and within specific cell types (astrocytes) for the first time. Dynamic demethylation at two specific CpG sites located just downstream of the transcription start site was sufficient to strongly activate the Dao gene, ultimately promoting the complete physiological degradation of cerebellar D-serine a few days after mouse birth. High amount of 5'-hydroxymethylcytosine, exclusively detected at relevant CpG sites, strongly evoked the occurrence of an active demethylation process. CONCLUSION: The present investigation demonstrates that robust and selective demethylation of two CpG sites is associated with postnatal activation of the Dao gene and consequent removal of D-serine within the mouse cerebellum. A single-molecule methylation approach applied at the Dao locus promises to identify different cell-type compositions and functions in different brain areas and developmental stages.
Assuntos
Cerebelo/crescimento & desenvolvimento , D-Aminoácido Oxidase/genética , Metilação de DNA , Serina/metabolismo , Ativação Transcricional , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Animais Recém-Nascidos , Cerebelo/metabolismo , Ilhas de CpG , Ácido D-Aspártico/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Camundongos , Análise de Sequência de DNA/métodos , Imagem Individual de Molécula/métodosRESUMO
The spatio-temporal regulation of genes involved in the synthesis and degradation of D-serine and D-aspartate such as serine racemase (SR), D-amino acid oxidase (DAO), G72 and D-aspartate oxidase (DDO), play pivotal roles in determining the correct levels of these D-amino acids in the human brain. Here we provide a comprehensive analysis of mRNA expression and DNA methylation status of these genes in post-mortem samples from hippocampus, dorsolateral prefrontal cortex, and cerebellum from patients with schizophrenia and non-psychiatric controls. DNA methylation analysis was performed at an ultradeep level, measuring individual epialleles frequency by single molecule approach. Differential CpG methylation and expression was detected across different brain regions, although no significant correlations were found with diagnosis. G72 showed the highest CpG and non-CpG methylation degree, which may explain the repression of G72 transcription in the brain regions considered here. Conversely, in line with the sustained SR mRNA expression in the analyzed areas, very low methylation levels were detected at this gene's regulatory regions. Furthermore, for DAO and DDO, our single-molecule methylation approach demonstrated that analysis of epiallele distribution was able to detect differences in DNA methylation representing area-specific methylation signatures, which are likely not detectable with targeted or genome-wide classic methylation analyses.
Assuntos
Encéfalo/metabolismo , Ácido D-Aspártico/metabolismo , Metilação de DNA/genética , Mudanças Depois da Morte , Esquizofrenia/genética , Serina/metabolismo , Alelos , Estudos de Casos e Controles , D-Aminoácido Oxidase/genética , D-Aminoácido Oxidase/metabolismo , D-Aspartato Oxidase/genética , Epigênese Genética , Humanos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Periodontitis is one of the most common oral inflammatory diseases, and results in connective tissue degradation and gradual tooth loss. It manifests with formation of periodontal pockets, in which anaerobic and Gramnegative bacteria proliferate rapidly. Consequently, alteration of the subgingival microbiota is considered the primary etiologic agent of periodontitis. Previous studies have reported that smokers are at increased risk of periodontal disease, in both prevalence and severity, indicating that smoking is a risk factor for the onset and progression of the pathology. In the present study, 16S rRNA sequencing was employed to assess the subgingival microbiota in 6 smoker patients with chronic periodontitis, 6 nonsmoker patients with chronic periodontitis and 8 healthy controls. The results demonstrated significant alterations in the microbial structure of periodontitis patients. High relative abundance of Parvimonans, Desulfubulbus, Paludibacter, Haemophilus, and Sphaerochaeta genera characterized subgingival microbiota of periodontitis patients, both smokers and nonsmokers. Due to the high precision and sensitivity of the 16S rRNA sequencing method, analysis for lowabundant genera (including Pedobacter, Granulicatella, Paracoccus, Atopobium, Bifidobacterium, Coprococcus, Oridobacteriu, Peptococcus, Oscillospira and Akkermansia) was feasible, and revealed novel phylotypes associated with periodontitis. Of note, a major microbial community alteration was evident in smoker patients, suggesting an association between smoking and severity of subgingival dysbiosis. The present study confirmed that chronic periodontitis is a polymicrobial disease where changes in the equilibrium of subgingival microbiota contribute to severity of pathology.
Assuntos
Periodontite Crônica/complicações , Periodontite Crônica/microbiologia , Disbiose/complicações , Disbiose/microbiologia , Fumar/efeitos adversos , Adulto , Periodontite Crônica/etiologia , Disbiose/etiologia , Feminino , Humanos , Masculino , Microbiota , Pessoa de Meia-Idade , RNA Ribossômico 16S/genética , Fatores de Risco , Adulto JovemRESUMO
It is long acknowledged that the N-methyl d-aspartate receptor co-agonist, d-serine, plays a crucial role in several N-methyl d-aspartate receptor-mediated physiological and pathological processes, including schizophrenia. Besides d-serine, another free d-amino acid, d-aspartate, is involved in the activation of N-methyl d-aspartate receptors acting as an agonist of this receptor subclass, and is abundantly detected in the developing human brain. Based on the hypothesis of N-methyl d-aspartate receptor hypofunction in the pathophysiology of schizophrenia and considering the ability of d-aspartate and d-serine to stimulate N-methyl d-aspartate receptor-dependent transmission, in the present work we assessed the concentration of these two d-amino acids in the post-mortem dorsolateral prefrontal cortex and hippocampus of patients with schizophrenia and healthy subjects. Moreover, in this cohort of post-mortem brain samples we investigated the spatiotemporal variations of d-aspartate and d-serine. Consistent with previous work, we found that d-aspartate content was selectively decreased by around 30% in the dorsolateral prefrontal cortex, but not in the hippocampus, of schizophrenia-affected patients, compared to healthy subjects. Interestingly, such selective reduction was associated to greater (around 25%) cortical activity of the enzyme responsible for d-aspartate catabolism, d-aspartate oxidase. Conversely, no significant changes were found in the methylation state and transcription of DDO gene in patients with schizophrenia, compared to control individuals, as well as in the expression levels of serine racemase, the major enzyme responsible for d-serine biosynthesis, which also catalyzes aspartate racemization. These results reveal the potential involvement of altered d-aspartate metabolism in the dorsolateral prefrontal cortex as a factor contributing to dysfunctional N-methyl d-aspartate receptor-mediated transmission in schizophrenia.
RESUMO
Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles.
Assuntos
Dano ao DNA , Metilação de DNA , Reparo do DNA , Sequência de Bases , Humanos , SulfitosRESUMO
Alterations of microbiota-gut-brain axis have been invoked in the pathogenesis of autism spectrum disorders (ASD). Mouse models could represent an excellent tool to understand how gut dysbiosis and related alterations may contribute to autistic phenotype. In this study we paralleled gut microbiota (GM) profiles, behavioral characteristics, intestinal integrity and immunological features of colon tissues in BTBR T + tf/J (BTBR) inbred mice, a well established animal model of ASD. Sex differences, up to date poorly investigated in animal models, were specifically addressed. Results showed that BTBR mice of both sexes presented a marked intestinal dysbiosis, alterations of behavior, gut permeability and immunological state with respect to prosocial C57BL/6j (C57) strain. Noticeably, sex-related differences were clearly detected. We identified Bacteroides, Parabacteroides, Sutterella, Dehalobacterium and Oscillospira genera as key drivers of sex-specific gut microbiota profiles associated with selected pathological traits. Taken together, our findings indicate that alteration of GM in BTBR mice shows relevant sex-associated differences and supports the use of BTBR mouse model to dissect autism associated microbiota-gut-brain axis alteration.
Assuntos
Transtorno do Espectro Autista/patologia , Microbioma Gastrointestinal , Animais , Transtorno do Espectro Autista/metabolismo , Bactérias/genética , Bactérias/isolamento & purificação , Comportamento Animal , Colo/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Intestinos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Permeabilidade , Fenótipo , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Análise de Sequência de DNA , Fatores SexuaisRESUMO
We performed ultra-deep methylation analysis at single molecule level of the promoter region of developmentally regulated D-Aspartate oxidase (Ddo), as a model gene, during brain development and embryonic stem cell neural differentiation. Single molecule methylation analysis enabled us to establish the effective epiallele composition within mixed or pure brain cell populations. In this framework, an epiallele is defined as a specific combination of methylated CpG within Ddo locus and can represent the epigenetic haplotype revealing a cell-to-cell methylation heterogeneity. Using this approach, we found a high degree of polymorphism of methylated alleles (epipolymorphism) evolving in a remarkably conserved fashion during brain development. The different sets of epialleles mark stage, brain areas, and cell type and unravel the possible role of specific CpGs in favoring or inhibiting local methylation. Undifferentiated embryonic stem cells showed non-organized distribution of epialleles that apparently originated by stochastic methylation events on individual CpGs. Upon neural differentiation, despite detecting no changes in average methylation, we observed that the epiallele distribution was profoundly different, gradually shifting toward organized patterns specific to the glial or neuronal cell types. Our findings provide a deep view of gene methylation heterogeneity in brain cell populations promising to furnish innovative ways to unravel mechanisms underlying methylation patterns generation and alteration in brain diseases.
Assuntos
Encéfalo/embriologia , Diferenciação Celular/genética , D-Aspartato Oxidase/genética , Epigênese Genética , Células-Tronco Neurais/fisiologia , Animais , Animais Recém-Nascidos , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Células Cultivadas , Ilhas de CpG , D-Aspartato Oxidase/metabolismo , Metilação de DNA , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Polimorfismo Genético , GravidezRESUMO
DNA methylation is often analyzed by reporting the average methylation degree of each cytosine. In this study, we used a single molecule methylation analysis in order to look at the methylation conformation of individual molecules. Using D-aspartate oxidase as a model gene, we performed an in-depth methylation analysis through the developmental stages of 3 different mouse tissues (brain, lung, and gut), where this gene undergoes opposite methylation destiny. This approach allowed us to track both methylation and demethylation processes at high resolution. The complexity of these dynamics was markedly simplified by introducing the concept of methylation classes (MCs), defined as the number of methylated cytosines per molecule, irrespective of their position. The MC concept smooths the stochasticity of the system, allowing a more deterministic description. In this framework, we also propose a mathematical model based on the Markov chain. This model aims to identify the transition probability of a molecule from one MC to another during methylation and demethylation processes. The results of our model suggest that: 1) both processes are ruled by a dominant class of phenomena, namely, the gain or loss of one methyl group at a time; and 2) the probability of a single CpG site becoming methylated or demethylated depends on the methylation status of the whole molecule at that time.
Assuntos
Ilhas de CpG/genética , Citosina/metabolismo , Metilação de DNA/genética , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Mucosa Gástrica/metabolismo , Humanos , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Camundongos , Modelos Teóricos , Estômago/crescimento & desenvolvimentoRESUMO
Bacterial lipopolysaccharide (LPS) induces release of inflammatory mediators both in immune and epithelial cells. We investigated whether changes of epigenetic marks, including selected histone modification and DNA methylation, may drive or accompany the activation of COX-2 gene in HT-29 human intestinal epithelial cells upon exposure to LPS. Here we describe cyclical histone acetylation (H3), methylation (H3K4, H3K9, H3K27) and DNA methylation changes occurring at COX-2 gene promoter overtime after LPS stimulation. Histone K27 methylation changes are carried out by the H3 demethylase JMJD3 and are essential for COX-2 induction by LPS. The changes of the histone code are associated with cyclical methylation signatures at the promoter and gene body of COX-2 gene.
Assuntos
Ciclo-Oxigenase 2/genética , Metilação de DNA/genética , Células Epiteliais/enzimologia , Histonas/metabolismo , Intestinos/citologia , Lipopolissacarídeos/farmacologia , Ilhas de CpG/genética , Ciclo-Oxigenase 2/metabolismo , Ativação Enzimática/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Células HT29 , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina/metabolismo , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismoRESUMO
In this study we assessed the BDNF promoter IV methylation state of a large genomic region surrounding promoter IV and evaluated BDNF transcript IV expression from prefrontal cortex and striatum of 15 schizophrenic and 15 control subjects. In prefrontal cortex, a single CpG site at -93, appeared to be undermethylated in patients׳group. BDNF mRNA levels in frontal cortex and striatum were variable among individuals but did not associate with disease.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Metilação de DNA/genética , Córtex Pré-Frontal/metabolismo , Esquizofrenia/genética , Adulto , Idoso de 80 Anos ou mais , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Feminino , Lobo Frontal/metabolismo , Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Córtex Pré-Frontal/patologia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , Esquizofrenia/patologia , Estatística como Assunto , Adulto JovemRESUMO
Re-induction of fetal genes and/or re-expression of postnatal genes represent hallmarks of pathological cardiac remodeling, and are considered important in the progression of the normal heart towards heart failure (HF). Whether epigenetic modifications are involved in these processes is currently under investigation. Here we hypothesized that histone chromatin modifications may underlie changes in the gene expression program during pressure overload-induced HF. We evaluated chromatin marks at the promoter regions of the sarcoplasmic reticulum Ca2+ATPase (SERCA-2A) and ß-myosin-heavy chain (ß-MHC) genes (Atp2a2 and Myh7, respectively) in murine hearts after one or eight weeks of pressure overload induced by transverse aortic constriction (TAC). As expected, all TAC hearts displayed a significant reduction in SERCA-2A and a significant induction of ß-MHC mRNA levels. Interestingly, opposite histone H3 modifications were identified in the promoter regions of these genes after TAC, including H3 dimethylation (me2) at lysine (K) 4 (H3K4me2) and K9 (H3K9me2), H3 trimethylation (me3) at K27 (H3K27me3) and dimethylation (me2) at K36 (H3K36me2). Consistently, a significant reduction of lysine-specific demethylase KDM2A could be found after eight weeks of TAC at the Atp2a2 promoter. Moreover, opposite changes in the recruitment of DNA methylation machinery components (DNA methyltransferases DNMT1 and DNMT3b, and methyl CpG binding protein 2 MeCp2) were found at the Atp2a2 or Myh7 promoters after TAC. Taken together, these results suggest that epigenetic modifications may underlie gene expression reprogramming in the adult murine heart under conditions of pressure overload, and might be involved in the progression of the normal heart towards HF.
Assuntos
Epigênese Genética , Insuficiência Cardíaca/genética , Cadeias Pesadas de Miosina/genética , Pressão , Regiões Promotoras Genéticas , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Cromatina/metabolismo , Perfilação da Expressão Gênica , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Lisina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Cadeias Pesadas de Miosina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genéticaRESUMO
In order to supplement the cytopathological assessment of thyroid tumors, there is a need for new markers to correctly diagnose malignant thyroid lesions and avoid unnecessary and potentially harmful therapies for patients. The immunohistochemical expression of galectin-3 is currently considered to be the most accurate stand-alone marker for thyroid cancer diagnosis. The aim of this study was to establish whether the methylation state of the galectin-3 gene is a candidate molecular marker for thyroid malignancy. Thyroid specimens from 50 patients were analyzed, including 5 normal thyroid, 3 goiters, 39 papillary and 3 anaplastic thyroid carcinoma cases. High-resolution methylation analyses was performed to investigate the methylation state of a large genomic region (from -89 to +408) encompassing the galectin-3 transcriptional start site. Within this region, 5 CpG sites (nucleotide positions +134, +137, +142, +147 and +156) were observed to be differentially methylated among the samples and were further analyzed by the quantitative pyrosequencing technique. The hypomethylation of the +134, +137, +142, +147 and +156 CpG sites was observed to be markedly associated with cancer. Although the methylation degree of each single site was highly variable in non-neoplastic tissues, the average methylation state of the 5 CpG sites clearly distinguished cancer from the nonneoplastic thyroid tissues.