RESUMO
Rett syndrome (RTT) is an inherited neurodevelopmental disorder of females that occurs once in 10,000-15,000 births. Affected females develop normally for 6-18 months, but then lose voluntary movements, including speech and hand skills. Most RTT patients are heterozygous for mutations in the X-linked gene MECP2 (refs. 3-12), encoding a protein that binds to methylated sites in genomic DNA and facilitates gene silencing. Previous work with Mecp2-null embryonic stem cells indicated that MeCP2 is essential for mouse embryogenesis. Here we generate mice lacking Mecp2 using Cre-loxP technology. Both Mecp2-null mice and mice in which Mecp2 was deleted in brain showed severe neurological symptoms at approximately six weeks of age. Compensation for absence of MeCP2 in other tissues by MeCP1 (refs. 19,20) was not apparent in genetic or biochemical tests. After several months, heterozygous female mice also showed behavioral symptoms. The overlapping delay before symptom onset in humans and mice, despite their profoundly different rates of development, raises the possibility that stability of brain function, not brain development per se, is compromised by the absence of MeCP2.
Assuntos
Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA/genética , Proteínas Repressoras , Síndrome de Rett/genética , Animais , Sequência de Bases , Primers do DNA/genética , Proteínas de Ligação a DNA/deficiência , Modelos Animais de Doenças , Feminino , Marcação de Genes , Humanos , Masculino , Proteína 2 de Ligação a Metil-CpG , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/fisiopatologia , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Síndrome de Rett/fisiopatologiaRESUMO
X-chromosome inactivation is the process by which a cell recognizes the presence of two copies of an X chromosome early in the development of XX embryos and chooses one to be active and one to be inactive. Although it is commonly believed that the initiation of X inactivation is random, with an equal probability (50:50) that either X chromosome will be the inactive X in a given cell, significant variation in the proportion of cells with either X inactive is observed both in mice heterozygous for alleles at the Xce locus and among normal human females in the population. Families in which multiple females demonstrate extremely skewed inactivation patterns that are otherwise quite rare in the general population are thought to reflect possible genetic influences on the X-inactivation process. Here we report a rare cytosine to guanine mutation in the XIST minimal promoter that underlies both epigenetic and functional differences between the two X chromosomes in nine females from two unrelated families. All females demonstrate preferential inactivation of the X chromosome carrying the mutation, suggesting that there is an association between alterations in the regulation of XIST expression and X-chromosome inactivation.
Assuntos
Mecanismo Genético de Compensação de Dose , Mutação , RNA não Traduzido , Fatores de Transcrição/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Feminino , Haplótipos , Humanos , Masculino , Linhagem , Regiões Promotoras Genéticas , RNA Longo não Codificante , Receptores Androgênicos/genéticaRESUMO
Mammalian DNA is methylated at many CpG dinucleotides. The biological consequences of methylation are mediated by a family of methyl-CpG binding proteins. The best characterized family member is MeCP2, a transcriptional repressor that recruits histone deacetylases. Our report concerns MBD2, which can bind methylated DNA in vivo and in vitro and has been reported to actively demethylate DNA (ref. 8). As DNA methylation causes gene silencing, the MBD2 demethylase is a candidate transcriptional activator. Using specific antibodies, however, we find here that MBD2 in HeLa cells is associated with histone deacetylase (HDAC) in the MeCP1 repressor complex. An affinity-purified HDAC1 corepressor complex also contains MBD2, suggesting that MeCP1 corresponds to a fraction of this complex. Exogenous MBD2 represses transcription in a transient assay, and repression can be relieved by the deacetylase inhibitor trichostatin A (TSA; ref. 12). In our hands, MBD2 does not demethylate DNA. Our data suggest that HeLa cells, which lack the known methylation-dependent repressor MeCP2, use an alternative pathway involving MBD2 to silence methylated genes.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Histona Desacetilases/fisiologia , Proteínas Repressoras/fisiologia , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Metilação de DNA , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Ratos , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3 , Ativação Transcricional , TransfecçãoRESUMO
To understand how the nucleosome remodeling and deacetylase (NuRD) complex regulates enhancers and enhancer-promoter interactions, we have developed an approach to segment and extract key biophysical parameters from live-cell three-dimensional single-molecule trajectories. Unexpectedly, this has revealed that NuRD binds to chromatin for minutes, decompacts chromatin structure and increases enhancer dynamics. We also uncovered a rare fast-diffusing state of enhancers and found that NuRD restricts the time spent in this state. Hi-C and Cut&Run experiments revealed that NuRD modulates enhancer-promoter interactions in active chromatin, allowing them to contact each other over longer distances. Furthermore, NuRD leads to a marked redistribution of CTCF and, in particular, cohesin. We propose that NuRD promotes a decondensed chromatin environment, where enhancers and promoters can contact each other over longer distances, and where the resetting of enhancer-promoter interactions brought about by the fast decondensed chromatin motions is reduced, leading to more stable, long-lived enhancer-promoter relationships.
Assuntos
Cromatina , Nucleossomos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Regiões Promotoras Genéticas , Elementos Facilitadores GenéticosAssuntos
Metilação de DNA , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína/genética , Homologia de Sequência de AminoácidosRESUMO
Two human genetic diseases have recently been shown to be due to mutations in genes encoding proteins involved in DNA methylation. The phenotypes of these two diseases are surprisingly distinct from each other and provide insights into the functions of DNA methylation in mammals.
Assuntos
Metilação de DNA , Genética Médica , Metilases de Modificação do DNA/metabolismo , Humanos , Mutação , Síndrome de Rett/genética , SíndromeRESUMO
Methylation at the DNA sequence 5'-CpG is required for mouse development. MeCP2 and MBD1 (formerly PCM1) are two known proteins that bind specifically to methylated DNA via a related amino acid motif and that can repress transcription. We describe here three novel human and mouse proteins (MBD2, MBD3, and MBD4) that contain the methyl-CpG binding domain. MBD2 and MBD4 bind specifically to methylated DNA in vitro. Expression of MBD2 and MBD4 tagged with green fluorescent protein in mouse cells shows that both proteins colocalize with foci of heavily methylated satellite DNA. Localization is disrupted in cells that have greatly reduced levels of CpG methylation. MBD3 does not bind methylated DNA in vivo or in vitro. MBD1, MBD2, MBD3, and MBD4 are expressed in somatic tissues, but MBD1 and MBD2 expression is reduced or absent in embryonic stem cells which are known to be deficient in MeCP1 activity. The data demonstrate that MBD2 and MBD4 bind specifically to methyl-CpG in vitro and in vivo and are therefore likely to be mediators of the biological consequences of the methylation signal.
Assuntos
Ilhas de CpG/genética , Metilação de DNA , Proteínas de Ligação a DNA/química , Endodesoxirribonucleases , Histona Desacetilases , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Linhagem Celular , Núcleo Celular/química , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica/genética , Proteínas de Fluorescência Verde , Humanos , Hibridização in Situ Fluorescente , Proteínas Luminescentes/genética , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Fatores de TranscriçãoRESUMO
The nucleosome remodeling deacetylase (NuRD) complex is a highly conserved regulator of chromatin structure and transcription. Structural studies have shed light on this and other chromatin modifying machines, but much less is known about how they assemble and whether stable and functional sub-modules exist that retain enzymatic activity. Purification of the endogenous Drosophila NuRD complex shows that it consists of a stable core of subunits, while others, in particular the chromatin remodeler CHD4, associate transiently. To dissect the assembly and activity of NuRD, we systematically produced all possible combinations of different components using the MultiBac system, and determined their activity and biophysical properties. We carried out single-molecule imaging of CHD4 in live mouse embryonic stem cells, in the presence and absence of one of core components (MBD3), to show how the core deacetylase and chromatin-remodeling sub-modules associate in vivo. Our experiments suggest a pathway for the assembly of NuRD via preformed and active sub-modules. These retain enzymatic activity and are present in both the nucleus and the cytosol, an outcome with important implications for understanding NuRD function.
Assuntos
Histona Desacetilases/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Nucleossomos/metabolismo , Animais , Núcleo Celular/metabolismo , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Citosol/metabolismo , Drosophila/metabolismo , Camundongos , Subunidades Proteicas/metabolismo , Células-Tronco/metabolismoRESUMO
Defects of mismatch repair are thought to be responsible for carcinogenesis in hereditary non-polyposis colorectal cancer and about 15% of sporadic colon cancers. The phenotype is seen as microsatellite instability and is known to be caused either by mutations in mismatch repair genes or by aberrant methylation of these genes stabilizing their downregulation. Lack of repair of microsatellite sequence errors, created during replication, leads to a mutation-prone phenotype. Where mutations occur within mononucleotide tracts within exons they cause translation frameshifts, premature cessation of translation and abnormal protein expression. Such mutations have been observed in the TGFbetaRII, BAX, IGFIIR, MSH3 and MSH6 genes in colon and other cancers. We describe here frameshift mutations affecting the gene for the methyl-CpG binding thymine glycosylase, MBD4, in over 40% of microsatellite unstable sporadic colon cancers. The mutations all appear heterozygous but their location would ensure truncation of the protein between the methyl-CpG binding and glycosylase domains, thus potentially generating a dominant negative effect. It is thus possible that such mutations enhance mutation frequency at other sites in these tumours. A suggestion has been made that MBD4 (MED1) mutations may lead to an increased rate of microsatellite instability but this mechanism appears unlikely due to the nature of mutations we have found.
Assuntos
Pareamento Incorreto de Bases , Neoplasias do Colo/genética , Reparo do DNA/genética , Endodesoxirribonucleases/genética , Mutação da Fase de Leitura , Sequência de Bases , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/cirurgia , Neoplasias Colorretais/genética , Metilação de DNA , Éxons , Regulação Neoplásica da Expressão Gênica , Humanos , Repetições de MicrossatélitesRESUMO
Computed tomography (CT) and intraarterial cerebral angiography are essential methods in early diagnosis of cerebral vascular malformations. In recent years however non-invasive or minimally invasive methods like MR angiography and CT angiography (CTA), which could potentially replace angiography, have been developed. The aim of presented study is to demonstrate our own experience in application of CTA in early diagnosis of cerebral vascular malformations. The material consists of 86 CTA examinations performed shortly after non-traumatic intracranial haemorrhage. Angiographic correlation has been available in 23 patients and surgical one--in 31 cases. CTA studies began with serio-CT to select the optimal time between contrast injection and CT scanning. After that 100-120 ml of non-ionic contrast medium was injected intravenously (5 ml per sec.) and spiral CT acqusition was performed with the delay calculated on the basis of the serio-CT. The obtained images were postprocessed on the workstation using always MIP and in many cases also SSD and VRT reconstructions. Vascular malformations have been diagnosed in 44 of 86 CTA studies including 38 patients with aneurysms (total number 51) and 6 patients with AVMs. In 17 cases the diameter of the aneurysm did not exceed 5 mm. In all surgical cases the CTA diagnosis of the aneurysm has been confirmed. There was however one false-negative case. On the other hand in 7 patients CTA revealed the small aneurysm, despite unclear angiographic appearance. In 2 of 6 patients with CTA suspicion of AVM this diagnosis has been excluded either by angiography or surgery. Comparison of CTA and angiography in 22 aneurysms showed in 17 cases superiority of CTA in evaluation of aneurysmal neck and the relationship between aneurysm and adjacent vessels (especially with VRT and SSD reconstructions). In patients with AVMs however the evaluation of supplying and draining vessels was better with angiography. On the base of our material we can conclude that CTA is very efficient in detecting and evaluating the aneurysms. We believe that CTA can replace angiography if it reveals aneurysm in a site corresponding with location of haemorrhage on CT. In patients with suspicion of AVM value of CTA is doubtful and angiography remains the method of choice.
Assuntos
Aneurisma Intracraniano/diagnóstico por imagem , Malformações Arteriovenosas Intracranianas/diagnóstico por imagem , Angiografia Cerebral , Humanos , Aneurisma Intracraniano/cirurgia , Malformações Arteriovenosas Intracranianas/cirurgia , Intensificação de Imagem Radiográfica , Tomografia Computadorizada por Raios XRESUMO
Aneurysms of the posterior cerebral artery (PCA) are uncommon (about 1% of all intracranial aneurysms) and their neurosurgical treatment is associated with high operative risk. We report on two saccular aneurysms of the P1 segment of the right PCA, combined with occlusion of the C1 segment of the internal carotid artery (ICA) detected during routine diagnostic studies in a 58-year-old patient with slight sensory loss in the left extremities. The aneurysms were embolized during two consecutive sessions, without subsequent complications. Endovascular intervention is an efficient method in the treatment of multiple aneurysms in the P1 segment of the PCA.
RESUMO
Chromatin structure is important for regulating gene expression and for the proper condensation and segregation of chromosomes during cell division. Several human genetic diseases have been found to be due to mutations in genes producing proteins known or suspected to be involved in maintaining or modifying chromatin structure. Here we describe these 'chromatin diseases' and review what is known about the associated chromatin proteins in light of recent advances in the understanding of chromatin components, modification and function.
Assuntos
Cromatina/genética , Doenças Genéticas Inatas/genética , DNA/química , DNA/genética , DNA/metabolismo , Regulação da Expressão Gênica , Inativação Gênica , Histonas/genética , Humanos , Conformação Molecular , Transcrição GênicaRESUMO
Epigenetic gene regulation refers to different states of phenotypic expression caused by differential effects of chromosome or chromatin packaging rather than by differences in DNA sequence. Examples of epigenetic regulation can be found in organisms as diverse as the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, and mammals. Three major types of epigenetic regulation are considered in this review: dosage compensation, imprinting and position effect variegation. While the specific details and mechanisms of each is quite different, they all involve either local or extensive alterations in chromatin structure. A number of genes implicated in epigenetic regulation have been isolated and their products identified as proteins or RNA molecules involved at various levels in DNA, chromatin or chromosome binding. While in general our understanding of mammalian epigenetic phenomena is not as advanced as that in model systems, the detailed molecular and genetic understanding of processes responsible for conditional gene silencing in invertebrate systems provides strong models for consideration of such effects in human and mouse genetics.
Assuntos
Cromatina/genética , Regulação da Expressão Gênica/genética , Animais , Caenorhabditis elegans , Drosophila melanogaster , Evolução Molecular , Humanos , Camundongos , Saccharomyces cerevisiae , SchizosaccharomycesRESUMO
The XIST gene in both humans and mice is expressed exclusively from the inactive X chromosome and is required for X chromosome inactivation to occur early in development. In order to understand transcriptional regulation of the XIST gene, we have identified and characterized the human XIST promoter and two repeated DNA elements that modulate promoter activity. As determined by reporter gene constructs, the XIST minimal promoter is constitutively active at high levels in human male and female cell lines and in transgenic mice. We demonstrate that this promoter activity is dependent in vitro upon binding of the common transcription factors SP1, YY1 and TBP. We further identify two cis -acting repeated DNA sequences that influence reporter gene activity. First, DNA fragments containing a set of highly conserved repeats located within the 5'-end of XIST stimulate reporter activity 3-fold in transiently transfected cell lines. Second, a 450 bp alternating purine-pyrimidine repeat located 25 kb upstream of the XIST promoter partially suppresses promoter activity by approximately 70% in transient transfection assays. These results indicate that the XIST promoter is constitutively active and that critical steps in the X inactivation process must involve silencing of XIST on the active X chromosome by factors that interact with and/or recognize sequences located outside the minimal promoter.
Assuntos
DNA/química , Regiões Promotoras Genéticas , RNA não Traduzido , Fatores de Transcrição/genética , Cromossomo X , Animais , Sequência de Bases , Eletroforese em Gel de Poliacrilamida , Feminino , Células HeLa , Cavalos , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , RNA Longo não Codificante , Sequências Repetitivas de Ácido Nucleico , Homologia de SequênciaRESUMO
The human XIST gene, a candidate for a role in X chromosome inactivation, has recently been cloned and sequenced, yielding a 17 kb cDNA with no apparent significant, conserved open reading frame. In addition, the XIST transcript has been localized within the nucleus to the Barr body by RNA in situ hybridization. This subnuclear localization and lack of any significant protein-coding potential suggest that XIST may act as a functional RNA within the nucleus. In the absence of a conserved open reading frame, we have turned to evolutionary studies as a first step toward elucidating a function for XIST in the process of X inactivation. While probes for XIST detect homologues in numerous eutherians, sequence comparisons require significant gapping and reveal identity levels intermediate between those seen for coding and non-coding regions in other genes. Further, sequence comparison of the most likely candidate open reading frame among several primate species reveals sequence changes not normally associated with protein-coding regions. Other features of XIST are conserved in different species, however, including the position of a major transcription start site and active X chromosome-specific DNA methylation patterns at the gene's 5' end. Finally, a possible molecular basis for differing propensity toward X inactivation between Xce alleles in mouse is investigated by comparing the sequence of the Xist conserved 5' repeats in mouse strains carrying different Xce alleles.
Assuntos
Mecanismo Genético de Compensação de Dose , Genes , Camundongos/genética , Cromossomo X , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Regulação da Expressão Gênica , Humanos , Mamíferos/genética , Metilação , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Primatas/genética , Sequências Repetitivas de Ácido Nucleico , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
DNA methylation is essential for murine development and is implicated in the control of gene expression. MeCP2, MBD1, MBD2, MBD3, and MBD4 comprise a family of mammalian, nuclear proteins related by the presence in each of an amino acid motif called the methyl-CpG binding domain (MBD). Each of these proteins, with the exception of MBD3, is capable of binding specifically to methylated DNA. MeCP2, MBD1 and MBD2 can also repress transcription. We describe the genomic structure and chromosomal localization of the human and murine Mbd1, Mbd2, Mbd3, and Mbd4 genes. We find that the highly similar MBD2 and MBD3 proteins are encoded by genes that map to different chromosomes in humans and mice but show a similar genomic structure. The Mbd1 and Mbd2 genes, in contrast, map together to murine and human Chromosomes (Chrs)18. The Mbd3 and Mbd4 genes map to murine Chrs 10 and 6, respectively, while the human MBD3 and MBD4 genes map to Chrs 19 and 3, respectively.
Assuntos
Metilação de DNA , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases , Animais , Mapeamento Cromossômico , Cromossomos Humanos Par 18/genética , Cromossomos Humanos Par 19/genética , Cromossomos Humanos Par 3/genética , DNA/genética , Primers do DNA/genética , Éxons , Genoma , Humanos , Hibridização in Situ Fluorescente , Íntrons , Camundongos , Especificidade da Espécie , Fatores de TranscriçãoRESUMO
MBD2 and MBD3 are closely related proteins with consensus methyl-CpG binding domains. MBD2 is a transcriptional repressor that specifically binds to methylated DNA and is a component of the MeCP1 protein complex. In contrast, MBD3 fails to bind methylated DNA in murine cells, and is a component of the Mi-2/NuRD corepressor complex. We show by gene targeting that the two proteins are not functionally redundant in mice, as Mbd3-/- mice die during early embryogenesis, whereas Mbd2-/- mice are viable and fertile. Maternal behavior of Mbd2-/- mice is however defective and, at the molecular level, Mbd2-/- mice lack a component of MeCP1. Mbd2-mutant cells fail to fully silence transcription from exogenous methylated templates, but inappropriate activation of endogenous imprinted genes or retroviral sequences was not detected. Despite their differences, Mbd3 and Mbd2 interact genetically suggesting a functional relationship. Genetic and biochemical data together favor the view that MBD3 is a key component of the Mi-2/NuRD corepressor complex, whereas MBD2 may be one of several factors that can recruit this complex to DNA.