RESUMO
MethylomeDB (http://epigenomics.columbia.edu/methylomedb/index.html) is a new database containing genome-wide brain DNA methylation profiles. DNA methylation is an important epigenetic mark in the mammalian brain. In human studies, aberrant DNA methylation alterations have been associated with various neurodevelopmental and neuropsychiatric disorders such as schizophrenia, and depression. In this database, we present methylation profiles of carefully selected non-psychiatric control, schizophrenia, and depression samples. We also include data on one mouse forebrain sample specimen to allow for cross-species comparisons. In addition to our DNA methylation data generated in-house, we have and will continue to include published DNA methylation data from other research groups with the focus on brain development and function. Users can view the methylation data at single-CpG resolution with the option of wiggle and microarray formats. They can also download methylation data for individual samples. MethylomeDB offers an important resource for research into brain function and behavior. It provides the first source of comprehensive brain methylome data, encompassing whole-genome DNA methylation profiles of human and mouse brain specimens that facilitate cross-species comparative epigenomic investigations, as well as investigations of schizophrenia and depression methylomes.
Assuntos
Encéfalo/metabolismo , Metilação de DNA , Bases de Dados de Ácidos Nucleicos , Animais , Epigênese Genética , Genômica , Humanos , Camundongos , Interface Usuário-ComputadorRESUMO
Abnormalities of genomic methylation patterns are lethal or cause disease, but the cues that normally designate CpG dinucleotides for methylation are poorly understood. We have developed a new method of methylation profiling that has single-CpG resolution and can address the methylation status of repeated sequences. We have used this method to determine the methylation status of >275 million CpG sites in human and mouse DNA from breast and brain tissues. Methylation density at most sequences was found to increase linearly with CpG density and to fall sharply at very high CpG densities, but transposons remained densely methylated even at higher CpG densities. The presence of histone H2A.Z and histone H3 di- or trimethylated at lysine 4 correlated strongly with unmethylated DNA and occurred primarily at promoter regions. We conclude that methylation is the default state of most CpG dinucleotides in the mammalian genome and that a combination of local dinucleotide frequencies, the interaction of repeated sequences, and the presence or absence of histone variants or modifications shields a population of CpG sites (most of which are in and around promoters) from DNA methyltransferases that lack intrinsic sequence specificity.
Assuntos
Sequência de Bases/fisiologia , Cromatina/química , Cromatina/fisiologia , Metilação de DNA , Animais , Encéfalo/metabolismo , Mama/metabolismo , Cromatina/genética , Mapeamento Cromossômico , Ilhas de CpG/genética , Feminino , Genoma , Histonas/metabolismo , Humanos , Camundongos , Análise de Sequência de DNA , Estudos de Validação como AssuntoRESUMO
Although it is well established that inheritance of mutations in the Brca1 gene significantly increases the chances of developing breast or ovarian cancers, the mechanisms underlying this specific tumor susceptibility remain to be clarified. It is clear that one of the roles of the Brca1 protein is to facilitate cellular responses to DNA damage. We recently reported that Brca1 function is required for appropriate cell cycle arrests after ionizing irradiation in both the S-phase and the G2 phase of the cell cycle. We also found that mutation of serine 1423 in Brca1, a target of Atm phosphorylation, abrogates the G2-M checkpoint but not the ionizing irradiation-induced S-phase checkpoint. Here we demonstrate that mutation of serine 1387 in Brca1, another target of Atm phosphorylation, conversely abrogates the radiation-induced S-phase arrest but does not affect the G2-M checkpoint. Thus, these two posttranslational modifications of Brca1 have two distinct functional roles in the protein. In addition, although mutation of this site abrogates the ionizing irradiation-induced S-phase arrest, it does not adversely affect cell survival after irradiation. This demonstrates that loss of this checkpoint function by itself does not affect cell survival and suggests that some other function of Brca1 alters cell survival after DNA damage.
Assuntos
Proteína BRCA1/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Tolerância a Radiação/fisiologia , Fase S/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteína BRCA1/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular , Dano ao DNA , Proteínas de Ligação a DNA , Fase G2/fisiologia , Fase G2/efeitos da radiação , Genes BRCA1 , Humanos , Mutação , Fosforilação , Tolerância a Radiação/genética , Fase S/efeitos da radiação , Serina/metabolismo , Transfecção , Células Tumorais Cultivadas , Proteínas Supressoras de TumorRESUMO
The solvent kinetic isotope effects (SKIE) on the yeast alpha-glucosidase-catalyzed hydrolysis of p-nitrophenyl and methyl-d-glucopyranoside were measured at 25 degrees C. With p-nitrophenyl-D-glucopyranoside (pNPG), the dependence of k(cat)/K(m) on pH (pD) revealed an unusually large (for glycohydrolases) solvent isotope effect on the pL-independent second-order rate constant, (DOD)(k(cat)/K(m)), of 1.9 (+/-0.3). The two pK(a)s characterizing the pH profile were increased in D(2)O. The shift in pK(a2) of 0.6 units is typical of acids of comparable acidity (pK(a)=6.5), but the increase in pK(a1) (=5.7) of 0.1 unit in going from H(2)O to D(2)O is unusually small. The initial velocities show substrate inhibition (K(is)/K(m) approximately 200) with a small solvent isotope effect on the inhibition constant [(DOD)K(is)=1.1 (+/-0.2)]. The solvent equilibrium isotope effects on the K(is) for the competitive inhibitors D-glucose and alpha-methyl D-glucoside are somewhat higher [(DOD)K(i)=1.5 (+/-0.1)]. Methyl glucoside is much less reactive than pNPG, with k(cat) 230 times lower and k(cat)/K(m) 5 x 10(4) times lower. The solvent isotope effect on k(cat) for this substrate [=1.11 (+/-0. 02)] is lower than that for pNPG [=1.67 (+/-0.07)], consistent with more extensive proton transfer in the transition state for the deglucosylation step than for the glucosylation step.
Assuntos
Isótopos/metabolismo , Solventes/química , Solventes/metabolismo , alfa-Glucosidases/metabolismo , Soluções Tampão , Óxido de Deutério/metabolismo , Inibidores Enzimáticos/química , Estabilidade Enzimática , Glucose/metabolismo , Glucose/farmacologia , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Metilglucosídeos/metabolismo , Metilglucosídeos/farmacologia , Prótons , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Especificidade por Substrato , Temperatura , alfa-Glucosidases/genéticaRESUMO
Clinical studies find that childhood adversity and stressful life events in adulthood increase the risk for major depression and for suicide. The predispositions to either major depression or suicide are thought to depend on genetic risk factors or epigenetic effects. We investigated DNA methylation signatures postmortem in brains of suicides with diagnosis of major depressive disorder. DNA methylation levels were determined at single C-phosphate-G (CpG) resolution sites within ventral prefrontal cortex of 53 suicides and nonpsychiatric controls, aged 16 to 89 years. We found that DNA methylation increases throughout the lifespan. Suicides showed an 8-fold greater number of methylated CpG sites relative to controls (P < 2.2 x 10(-16)), with greater DNA methylation changes over and above the increased methylation observed in normal aging. This increased DNA methylation may be a significant contributor to the neuropathology and psychopathology underlying the risk of suicide in depression.
Los estudíos clínicos han encontrado que la niñez adversa y los acontecimientos vitales estresantes en la adultez aumentan el riesgo para la depresión mayor y para el suícídío. Se piensa que la predísposición tanto para la depresión como para el suicidio depende de factores de ríesgo genético o de efectos epígenéticos. Se investigaron los signos de metilación del ADN en cerebros postmortem de pacíentes suícídas con díagnóstíco de depresíón mayor. Los níveles de metílacíón del ADN se determínaron en resolución única de Cítosinafosfato-Guanina (CfG) en la corteza prefrontal ventral de 53 pacíentes suicidas y de controles no psiquíátricos con edades entre 16 y 89 años. Se encontró que la metílación del ADN aumenta a lo largo de la vída. Los pacientes suícidas mostraron un número 8 veces mayor de sitios CfG metílados en relacíón con los controles (p<2,2x10-16), con cambíos mayores en la metilación del ADN, por sobre lo observado en el envejecímíento normal. Este aumento en la metílación del ADN puede contribuir de manera signifícativa a la neuropatología y a la psicopatología que están a la base del riesgo de suicidio en la depresión.
D'après des études cliniques, le malheur dans l'enfance et les événements de vie stressant à l'âge adulte augmentent le risque de dépression caractérisée (majeure) et de suicide. Les prédispositions à la dépression caractérisée ou au suicide dépendent probablement de facteurs de risque génétiques ou d'effets épigénétiques. Nous avons analysé des signatures postmortem de la méthylation de l'ADN dans des cerveaux de suicidés diagnostiqués comme ayant eu un épisode dépressif caractérisé. Nous avons déterminé les taux de méthylation de l'ADN avec une résolution CpG (C-phosphate-G) à site unique dans le cortex préfrontal ventral de 53 suicidés et témoins non psychiatriques âgés de 16 à 89 ans. Nous avons trouvé que la méthylation de l'ADN augmente tout au long de la vie. Le nombre de sites CpG méthylés étaient multipliés par 8 chez les suicidés par rapport aux témoins (p<2,2x10-16), avec des changements de méthylation de l'ADN plus importants, allant au-delà de l'accroissement de la méthylation observé dans le vieillissement normal. Cette augmentation de la méthylation de l'ADN peut participer significativement à la neuropathologie et à la psychopathologie qui sous-tend le risque de suicide dans la dépression.
Assuntos
Encéfalo/fisiopatologia , Metilação de DNA/fisiologia , Transtornos do Humor/psicologia , Suicídio , Adolescente , Adulto , Idoso , Envelhecimento , Encéfalo/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Promoter silencing by ectopic de novo methylation of tumor suppressor genes has been proposed as comparable or equivalent to inactivating mutations as a factor in carcinogenesis. However, this hypotheses had not previously been tested by high resolution, high-coverage whole-genome methylation profiling in primary carcinomas. We have determined the genomic methylation status of a series of primary mammary carcinomas and matched control tissues by examination of more than 2.7 billion CpG dinucleotides. Most of the tumors showed variable losses of DNA methylation from all sequence compartments, but increases in promoter methylation were infrequent, very small in extent, and were observed largely at CpG-poor promoters. De novo methylation at the promoters of proto-oncogenes and tumor suppressor genes occurred at approximately the same frequency. The findings indicate that tumor suppressor silencing by de novo methylation is much less common than currently believed. We put forward a hypothesis under which the demethylation commonly observed in carcinomas is a manifestation of a defensive system that kills incipient cancer cells.
RESUMO
DNA methylation is essential in brain function and behavior; therefore, understanding the role of DNA methylation in brain-based disorders begins with the study of DNA methylation profiles in normal brain. Determining the patterns and scale of methylation conservation and alteration in an evolutionary context enables the design of focused but effective methylation studies of disease states. We applied an enzymatic-based approach, Methylation Mapping Analysis by Paired-end Sequencing (Methyl-MAPS), which utilizes second-generation sequencing technology to provide an unbiased representation of genome-wide DNA methylation profiles of human and mouse brains. In this large-scale study, we assayed CpG methylation in cerebral cortex of neurologically and psychiatrically normal human postmortem specimens, as well as mouse forebrain specimens. Cross-species human-mouse DNA methylation conservation analysis shows that DNA methylation is not correlated with sequence conservation. Instead, greater DNA methylation conservation is correlated with increasing CpG density. In addition to CpG density, these data show that genomic context is a critical factor in DNA methylation conservation and alteration signatures throughout mammalian brain evolution. We identify key genomic features that can be targeted for identification of epigenetic loci that may be developmentally and evolutionarily conserved and wherein aberrations in DNA methylation patterns can confer risk for disease.
Assuntos
Encéfalo/metabolismo , Ilhas de CpG , Metilação de DNA , Evolução Molecular , Perfilação da Expressão Gênica , Animais , Epigenômica , Feminino , Genoma , Genômica , Humanos , Masculino , Camundongos , Pessoa de Meia-IdadeRESUMO
Comparative genomics of CpG dinucleotides, which are targets of DNA methyltransferases in vertebrate genomes, has been constrained by their evolutionary instability and by the effect of methylation on their mutation rates. We compared the human and chimpanzee genomes to identify DNA sequence signatures correlated with rates of mutation at CpG dinucleotides. The new signatures were used to develop robust comparative genomics of CpG dinucleotides in heterogeneous regions and to identify genomic domains that have anomalous CpG divergence rates. The data showed that there are approximately 200 genomic regions where CpG distributions are far more conserved than predicted. These hyperconserved CpG domains largely coincide with domains bound by Polycomb repressive complex 2 in undifferentiated human embryonic stem cells and are almost exclusively present near genes whose products are involved in the regulation of embryonic development. Several domains were experimentally shown to be unmethylated at different developmental stages. These data indicate that particular evolutionary patterns and distinct sequence properties on scales much larger than standard transcription factor-binding sites may play an important role in Polycomb recruitment and transcriptional regulation of key developmental genes.