RESUMO
Aberrant DNA methylation can lead to genome destabilization and to deregulated gene expression. Recently, 5-hydroxymethylcytosine (5hmC), derived from oxidation of 5-methylcytosine (5mC) by the Ten-Eleven Translocation (TET) enzymes, has been detected. 5hmC is now considered as a new epigenetic DNA modification with relevant roles in cell homeostasis regulating DNA demethylation and transcription. Our aim was to investigate possible changes in the DNA methylation/demethylation machinery in MS. We assessed the expression of enzymes involved in DNA methylation/demethylation in peripheral blood mononuclear cells (PBMCs) from 40 subjects with MS and 40 matched healthy controls. We performed also, DNA methylation analysis of specific promoters and analysis of global levels of 5mC and 5hmC. We show that TET2 and DNMT1 expression is significantly down-regulated in MS PBMCs and it is associated with aberrant methylation of their promoters. Furthermore, 5hmC is decreased in MS PBMCs, probably as a result of the diminished TET2 level.
Assuntos
Citosina/análogos & derivados , Proteínas de Ligação a DNA/biossíntese , Leucócitos Mononucleares/metabolismo , Esclerose Múltipla/sangue , Esclerose Múltipla/genética , Proteínas Proto-Oncogênicas/biossíntese , 5-Metilcitosina/análogos & derivados , Adulto , Estudos de Casos e Controles , Citosina/sangue , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/sangue , Proteínas de Ligação a DNA/genética , Dioxigenases , Regulação para Baixo , Feminino , Expressão Gênica , Humanos , Masculino , Esclerose Múltipla/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/sangue , Proteínas Proto-Oncogênicas/genéticaRESUMO
Eukaryotic genomes have complex spatial organization in the nucleus. The factors and the mechanisms involved in this organization remain an enigma. Among the many proteins implicated in such a role, the ubiquitous Zn-finger protein CTCF stands out. Here we summarize the evidence placing CTCF in the enviable position of a master organizer of the genome. CTCF can form loops in cis, and can bridge sequences located on different chromosomes in trans. The thousands of CTCF binding sites, identified in recent genome-wide localization studies, and their distribution along the genome further support a crucial role of CTCF as a chromatin organizer.
Assuntos
Genoma , Proteínas Repressoras/química , Proteínas Repressoras/genética , Animais , Sítios de Ligação , Fator de Ligação a CCCTC , Estudo de Associação Genômica Ampla , Impressão Genômica , Humanos , Elementos Isolantes , Conformação Proteica , Proteínas Repressoras/metabolismo , Transcrição Gênica , Cromossomo XRESUMO
In human placenta, the DNA of all subfractions of the third level of chromatin organization exhibits similar values of the methylcytosine-to-cytosine ratio. The tightly bound form of DNA methyltransferase is mostly recovered in the 'stripped loop' fraction, although, on the basis of the DNA content, the 'stripped loops' and the 'stripped matrix' appear to possess a similar amount of the enzyme. DNA methyltransferase activity is instead totally absent from the 'digested matrix', i.e., from the fraction remaining after digestion of the 'stripped matrix' with DNAase I. Upon addition of exogenous DNA methyltransferase, however, the DNA of this fraction, which is only 1% (in weight) of the total chromatin DNA and which has a length of approx. 9 kbp, can readily undergo methylation.
Assuntos
Cromatina/análise , DNA (Citosina-5-)-Metiltransferases/análise , Placenta/enzimologia , 5-Metilcitosina , Cromatografia Líquida de Alta Pressão , Citosina/análogos & derivados , Citosina/análise , DNA/análise , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Desoxirribonuclease I , Eletroforese , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Gravidez , Proteínas/análiseRESUMO
Sucrose density gradient centrifugation in the presence or absence of Na-EDTA and at different ionic strengths allows one to obtain well-defined nucleosome subpopulations the DNA of which, examined by gas chromatography-mass spectrometry, is in all cases hypermethylated as compared to spacer regions, but to a different extent for the different subpopulations. The various nucleosomes differ also in their content of histones and of high-mobility-group proteins, as well as in the levels of RNA polymerase activity associated with them. Such data suggest that these nucleosome subpopulations originate from chromatin fractions differently involved in gene expression.
Assuntos
Citosina/análogos & derivados , DNA/genética , Regulação da Expressão Gênica , Nucleossomos/fisiologia , 5-Metilcitosina , Animais , Citosina/metabolismo , DNA/metabolismo , Rim , SuínosRESUMO
Histones (from calf thymus or from human placenta), if renatured in the presence of EDTA, caused a severe inhibition of in vitro methylation of double-stranded DNA (from Micrococcus luteus) by human placenta DNA methyltransferase. The absence of EDTA during the histone renaturation procedure abolished--at least in the 'physiological' range of the histones/DNA ratio--the inhibition. The H1 component was responsible for this inhibition, no effect being exerted by the other histones. H1 preparations were more effective if renatured in the presence of EDTA--90% inhibition being reached at a 0.3:1 (w/w) H1/DNA ratio. It seems likely that the requirement for the presence of EDTA during the renaturation process is correlated to its ability to induce a fairly stable ordered conformation of the histones, although this effect could also be shown with the 'inactive' H2a, H2b and H3 components, and was instead less evident with histone H1. The restriction to histone H1 of the ability to inhibit enzymic DNA methylation may account for the lower methylation levels present in the internucleosomal DNA of mammalian chromatin.
Assuntos
Cromatina/metabolismo , Metilases de Modificação do DNA/antagonistas & inibidores , DNA Bacteriano/metabolismo , Histonas/metabolismo , Animais , Bovinos , Dicroísmo Circular , Metilases de Modificação do DNA/metabolismo , Humanos , Metilação , Micrococcus/genética , Placenta/enzimologia , Espectrofotometria UltravioletaRESUMO
After removal, by high-salt extraction, of the loosely-bound components present in human placenta chromatin, tightly-bound cationic proteins could be solubilized, by acid extraction, from the 'stripped' chromatin, as well as from the 'stripped' loops or from the 'digested matrix'. These acid-soluble tightly-bound proteins are, in terms of apparent molecular mass and immunoreactivity, quite similar to the 'typical', loosely-bound histones, and, similarly to their 'loosely-bound' counterparts, they can be subdivided in distinct H1-, H2A-, H2B-, H3- and H4-like components, the 'digested matrix' being however characterized by the absence of tightly-bound H1. These tightly-bound histones, at variance from the 'typical' ones, readily find a right-handed helical conformation upon renaturation by progressive dialyses. The H1 components strongly differ also in their effects on enzymic DNA methylation: while 'typical' H1 has a strong inhibitory effect, its tightly-bound counterpart exerts a slight but definite stimulation.
Assuntos
Cromatina/metabolismo , Metilases de Modificação do DNA/metabolismo , Histonas/metabolismo , Cromatina/química , Dicroísmo Circular , Histonas/química , Histonas/isolamento & purificação , Humanos , Metilação , Conformação de Ácido Nucleico , Espectrofotometria UltravioletaRESUMO
H1 exerts a specific in vitro inhibitory effect on enzymic DNA methylation. The experiments reported in this paper were undertaken in order to assess whether the lower methylation level found in internucleosomal DNA compared to core DNA is the in vivo consequence of the well-known localization of this histone in the linker region, as opposed to a possible deficiency of CpG dinucleotides in linker DNA. The methyl-accepting ability of H1-depleted oligonucleosomes from human placenta and of the corresponding core particles were assayed by addition of purified DNA methyltransferase, using S-adenosylmethionine as the methyl group donor. We have found that approx. 80% of newly-incorporated methyl groups are localized in linker DNA, which is indeed a good potential substrate for enzymic DNA methylation. Addition of quasi-physiological amounts of H1 to H1-depleted oligonucleosomes markedly reduced their methyl-accepting ability, while exerting a re-condensing effect on these particles, as revealed by the distortions of their circular dichroism spectra.
Assuntos
Cromatina/metabolismo , Histonas/fisiologia , DNA/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Humanos , Metilação , Nucleossomos/metabolismo , PlacentaRESUMO
Dynamic alterations in chromatin structure mediated by postsynthetic histone modifications and DNA methylation constitute a major regulatory mechanism in DNA functioning. DNA methylation has been implicated in transcriptional silencing, in part by inducing chromatin condensation. To understand the methylation-dependent chromatin structure, we performed atomic force microscope (AFM) studies of fibers isolated from cultured cells containing normal or elevated levels of m5C. Chromatin fibers were reconstituted on control or methylated DNA templates in the presence or absence of linker histone. Visual inspection of AFM images, combined with quantitative analysis of fiber structural parameters, suggested that DNA methylation induced fiber compaction only in the presence of linker histones. This conclusion was further substantiated by biochemical results.
Assuntos
Cromatina/metabolismo , Metilação de DNA , Células 3T3 , Animais , Sequência de Bases , Centrifugação com Gradiente de Concentração , Cromatina/química , Cromatina/ultraestrutura , Citosina/metabolismo , DNA/genética , DNA/metabolismo , Histonas/química , Histonas/metabolismo , Camundongos , Microscopia de Força Atômica , Dados de Sequência Molecular , Nucleossomos/química , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Moldes GenéticosRESUMO
The inhibitory effect that H1 histone exerts on the in vitro DNA methylation process, catalysed by mammalian DNA methyltransferase, together with the relative hypomethylation of linker DNA in eukaryotic cells chromatin, suggest that this hypomethylated state of linker DNA can be of importance in allowing or regulating H1-dependent chromatin condensation. In native oligonucleosomes (olnu), i.e., in chromatin fragments consisting of 5-20 nucleosomes each, there was a correlation between the effects of H1 on the DNA ellipticity at 280 nm and the in vitro assayed methyl-accepting ability. The same was true in H1-depleted or in H1-reconstituted preparations. Artificial methylation caused olnu DNA to lose its ability to allow cooperative H1-H1 interactions under ionic strength conditions similar to those known to affect the transition of the 10-nm filament to the 30-nm chromatin fiber. These results suggest that hypomethylation of linker DNA plays a role in the H1-H1 interactions that are needed for solenoid condensation.
Assuntos
Cromatina/fisiologia , DNA/química , DNA/metabolismo , Mutagênese Insercional , Animais , Cromatina/ultraestrutura , Dicroísmo Circular , DNA/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Feminino , Histonas/metabolismo , Humanos , Mamíferos , Metilação , Nucleossomos/fisiologia , Nucleossomos/ultraestrutura , Placenta/metabolismo , GravidezRESUMO
Upon HPLC fractionation of human placenta or calf thymus H1 histone preparations, only some fractions enriched in the H1e-c variants were able to exert a severe inhibition on in vitro enzymatic DNA methylation. These fractions, though similar to the other variants in interacting with genomic DNA, were also the only ones which could bind CpG-rich ds-oligodeoxyribonucleotides (oligos). Both the 6-CpG ds-oligo and the DNA purified from chromatin fractions enriched in 'CpG islands' were good competitors for the binding of H1e-c to the 6meCpG ds-oligo. This ability to bind any DNA sequence and to suppress the enzymatic methylation in any sequence containing CpG dinucleotides suggests, for these particular H1 variants, a possible role in maintaining CpG island DNA and linker DNA at low methylation levels.
Assuntos
DNA/metabolismo , Variação Genética , Histonas/genética , Histonas/metabolismo , Oligodesoxirribonucleotídeos/química , Animais , Sequência de Bases , Bovinos , Cromatina/química , Cromatina/metabolismo , DNA/química , DNA/isolamento & purificação , Fosfatos de Dinucleosídeos , Feminino , Histonas/isolamento & purificação , Humanos , Metilação , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/metabolismo , Placenta/metabolismo , Gravidez , Timo/metabolismoRESUMO
Enzymes which affect histone acetylation status have been shown to play an important role in determining transcriptional activity in chromatin through conformational modification of its structure. Since the timely presence of such enzymes may be of critical importance, our experiments were designed to determine whether the level of expression of HDAC1 is cell cycle dependent and/or affected by a high cell density. Our results show that in mouse fibroblasts the expression of mHDAC1 is neither affected by cell cycle phases nor by cell density. In contrast, the expression of several hHDACs including hHDAC1 were affected in a cell density dependent fashion in the human prostate adenocarcinoma cell line PC3, paralleling our previously published findings in the hepatocellular carcinoma derived cell line Hep3B. Differential recruitment of HDAC mRNAs suggests that these enzymes may play unique roles in different cell types and under different environmental conditions (i.e., exposure to various cell densities and cell-cell contacts). Our study has implications for the proposed use of HDAC inhibitors in the treatment of human malignancy, highlighting issues of drug action selectivity in tissues and potential secondary effects.
Assuntos
Contagem de Células , Ciclo Celular/fisiologia , Fibroblastos/citologia , Histona Desacetilases/metabolismo , Células Tumorais Cultivadas/patologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Northern Blotting , Butiratos/farmacologia , Divisão Celular , Sondas de DNA , Fibroblastos/metabolismo , Histona Desacetilase 1 , Histona Desacetilases/genética , Humanos , Masculino , Camundongos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ribonuclease Pancreático/metabolismo , Células Tumorais Cultivadas/metabolismoRESUMO
We have previously demonstrated that a significant percentage of poly(ADPR) polymerase is present, as a tightly-bound form, at the third level of chromatin organisation defined by chromosomal loops and nuclear matrix. The present work is focused on the study of poly(ADP-ribosyl)ation of proteins present in these nuclear subfractions. It has been shown that, due to the action of poly(ADPR) polymerase, the ADP-ribose moiety of [14C]NAD is transferred to both loosely-bound and tightly-bound chromosomal proteins, which in consequence are modified by chain polymers of ADP-ribose of different lengths. Moreover, histone-like proteins seem to be ADP-ribosylated in chromosomal loops and nuclear matrix associated regions of DNA loops (MARS). A hypothesis can be put forward that the ADP-ribosylation system is functionally related to the nuclear processes, actively coordinated by the nuclear matrix.
Assuntos
Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Testículo/metabolismo , Animais , Antígenos Nucleares , Masculino , Modelos Biológicos , Ligação Proteica , RatosRESUMO
The tissue specificity of a particular class of non-histone chromatin proteins characterized by strong bonds to DNA, tightly bound non-histone chromatin proteins (t.b. NHCp), was investigated. In connection with its possible role in the cell differentiation, its distribution in chromatin fractions obtained by digestion with DNAase II and precipitation with MgCl2 was studied. It was shown by bidimensional gel electrophoresis that some proteins of this class are tissue specific for pig liver compared to kidney chromatin. For both tissues, most tissue specific proteins were found to be peculiar to the chromatin fraction that remains insoluble after prolonged digestion with DNAase II. The proteins found in this chromatin fraction are common to the nuclear matrix proteins and, considering the chromatin organization, may be involved in the attachment point for loops in the axial matrix structure. It is possible that proteins of this class, directly or indirectly, play an important role in the regulation of transcription and differentiation in eukaryotes, by modulating the supercoiled state of DNA.
Assuntos
Cromatina/análise , Proteínas Cromossômicas não Histona/isolamento & purificação , Endodesoxirribonucleases , Rim/análise , Fígado/análise , Animais , Antígenos Nucleares , Proteínas Cromossômicas não Histona/metabolismo , Nucleoproteínas/análise , Especificidade de Órgãos , Solubilidade , Frações Subcelulares/análise , Suínos , Fatores de TempoAssuntos
Antibióticos Antineoplásicos/farmacologia , Antifúngicos/farmacologia , Carcinoma de Ehrlich/metabolismo , Carcinoma Hepatocelular/metabolismo , Ácidos Graxos/farmacologia , Lactonas/farmacologia , Animais , Isótopos de Carbono , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Isótopos do Cromo , Técnicas de Cultura , Replicação do DNA/efeitos dos fármacos , DNA de Neoplasias/biossíntese , Glutamatos/metabolismo , Concentração de Íons de Hidrogênio , Neoplasias Hepáticas , Regeneração Hepática/efeitos dos fármacos , Masculino , Camundongos , Neoplasias Experimentais/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Potássio , Ratos , Estrofantinas/farmacologia , Timidina/metabolismo , Trítio , Azul Tripano/metabolismoAssuntos
Mitocôndrias Musculares/enzimologia , Fosfolipases/farmacologia , Succinato Desidrogenase/metabolismo , Animais , Soluções Tampão , Bovinos , Clostridium perfringens/enzimologia , Depressão Química , Coração/efeitos dos fármacos , Técnicas In Vitro , Cetonas/antagonistas & inibidores , Mitocôndrias Musculares/efeitos dos fármacos , Miocárdio/enzimologia , Fosfatidilcolinas/farmacologia , Fosfolipídeos , Serpentes , Solubilidade , Succinato Desidrogenase/antagonistas & inibidores , Succinato Desidrogenase/isolamento & purificação , Tiofenos/farmacologia , Fatores de Tempo , PeçonhasRESUMO
Quantitative data from experiments of gene expression are often normalized through levels of housekeeping genes transcription by assuming that expression of these genes is highly uniform. This practice is being questioned as it becomes increasingly clear that the level of housekeeping genes expression may vary considerably in certain biological samples. To date, the validation of reference genes in aging has received little attention and suitable reference genes have not yet been defined. Our aim was to evaluate the expression stability of frequently used reference genes in human peripheral blood mononuclear cells with respect to aging. Using quantitative RT-PCR, we carried out an extensive evaluation of five housekeeping genes, i.e. 18s rRNA, ACTB, GAPDH, HPRT1 and GUSB, for stability of expression in samples from donors in the age range 35-74 years. The consistency in the expression stability was quantified on the basis of the coefficient of variation and two algorithms termed geNorm and NormFinder. Our results indicated GUSB be the most suitable transcript and 18s the least for accurate normalization in PBMCs. We also demonstrated that aging is a confounding factor with respect to stability of 18s, HPRT1 and ACTB expression, which were particularly prone to variability in aged donors.
Assuntos
Envelhecimento/genética , Expressão Gênica , Genes , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/normas , Adulto , Idoso , Algoritmos , Humanos , Hipoxantina Fosforribosiltransferase/metabolismo , Leucócitos Mononucleares/metabolismo , Pessoa de Meia-Idade , RNA Mensageiro/genética , Padrões de Referência , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosAssuntos
Metilases de Modificação do DNA/metabolismo , Proteínas de Ligação a DNA/análise , Histonas , Nucleossomos/análise , Placenta/análise , Animais , Composição de Bases , Proteínas de Ligação a DNA/farmacologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Rim/análise , Metilação , Nucleossomos/ultraestrutura , Gravidez , SuínosRESUMO
In vivo and in vitro experiments carried out on L929 mouse fibroblasts suggested that the poly(ADP-ribosyl) ation process acts somehow as a protecting agent against full methylation of CpG dinucleotides in genomic DNA. Since CpG islands, which are found almost exclusively at the 5'-end of housekeeping genes, are rich in CpG dinucleotides, which are the target of mammalian DNA methyltransferase, we examined the possibility that the poly(ADP-ribosyl)ation reaction is involved in maintaining the unmethylated state of these DNA sequences. Experiments were conducted by two different strategies, using either methylation-dependent restriction enzymes on purified genomic DNA or a sequence-dependent restriction enzyme on an aliquot of the same DNA, previously modified by a bisulfite reaction. With the methylation-dependent restriction enzymes, it was observed that the "HpaII tiny fragments" greatly decreased when the cells were preincubated with 3-aminobenzamide, a well known inhibitor of poly(ADP-ribose) polymerase. The other experimental approach allowed us to prove that, as a consequence of the inhibition of the poly(ADP-ribosyl)ation process, an anomalous methylation pattern could be evidenced in the CpG island of the promoter fragment of the Htf9 gene, amplified from DNA obtained from fibroblasts preincubated with 3-aminobenzamide. These data confirm the hypothesis that, at least for the Htf9 promoter region, an active poly(ADP-ribosyl)ation protects the unmethylated state of the CpG island.