RESUMO
Panstrongylus megistus, a potential vector of Chagas disease, currently occupies a wider geographic distribution in Brazil than Triatoma infestans, another member of the hemipteran Reduviidae family and a vector of the same disease. A small heterochromatic body (chromocenter) formed by the Y chromosome is evident in the somatic cells of P. megistus, differing in size and chromosome type contribution from the well-studied chromocenters present in T. infestans. While the overall distribution of histone epigenetic marks differ when comparing the heterochromatin and euchromatin territories in T. infestans, no similar data have been established for other hemipteran reduviids, including P. megistus. In the present work, histone acetylation and methylation marks were investigated in cells of Malpighian tubules of P. megistus 5th instar nymphs using immunocytochemical assays and compared to previously published data for T. infestans. Although similarities between these species were found regarding absence of acetylated H3K9, H4K8 and H4K16, and H3K9me and H3K9me2 in the chromocenter, presence of these marks in euchromatin, and presence of H3K9me3 in the chromocenter, no intimate association of acetylated H4K8 and 18S rDNA was revealed in the chromocenter of P. megistus. The elevated abundance of H3K9me2 marks at the nuclear periphery in P. megistus cells, differing from data for T. infestans, is suggested to reflect differences in the interaction of lamina-associated chromatin domains with the nuclear lamina, methyl-transferase modulation and/or association with the last DNA endoreplication step in 5th instar nymphs, which is a matter for further investigation.
Assuntos
Cromatina/metabolismo , Hemípteros/metabolismo , Histonas/metabolismo , Proteínas de Insetos/metabolismo , Acetilação , Animais , Linhagem Celular , Metilação , Especificidade de ÓrgãosRESUMO
Triatoma infestans, a vector of Chagas' disease, shows several particular cell biology characteristics, including the presence of conspicuous heterochromatic bodies (chromocenters) where DNA methylation has not been previously detected. Whether histone modifications contribute to the condensed state of these bodies has not yet been studied. Here, we investigated epigenetic modifications of histones H3 and H4 and presence of the non-histone heterochromatin protein (HP1-α) in the chromocenters and euchromatin of T. infestans cell nuclei, using immunocytochemistry. The effect of different concentrations of the histone deacetylase inhibitors valproic acid (VPA) and sodium butyrate (NaBt) on chromocenter condensation was visually examined; in VPA-treated specimens, this effect was also analyzed by image analysis. Trimethylated H3K9 signals, which were revealed in chromocenter and non-chromocenter areas, were strongest in chromocenters, whereas selected acetylated histone marks and mono- and dimethylated H3K9 and H4K20 signals were detected only in euchromatin. Weak trimethylated H4K20 signals and variable distribution of HP1-α were detected in chromocenters of part of the cellular population analyzed. Although specific VPA and NaBt treatment conditions affected the heterochromatin condensation pattern, they did not induce a decrease in survival and molting rates of the T. infestans nymphs. The VPA-induced chromatin remodeling was not accompanied by induction of H3K9 acetylation in chromocenters. Present findings regarding histone modifications and effects following VPA or NaBt treatments did not yet solve the question of which factors are responsible for maintenance of the condensed state of chromocenters in T. infestans. A possibility requiring further investigation remains on histone methylation marks and/or non-histone proteins.
Assuntos
Eucromatina/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Proteínas de Insetos/metabolismo , Triatoma/genética , Animais , Doença de Chagas , Montagem e Desmontagem da Cromatina , Cromossomos de Insetos/genética , Cromossomos de Insetos/metabolismo , Vetores de Doenças , Epigênese Genética , Eucromatina/genética , Heterocromatina/genética , Inibidores de Histona Desacetilases/farmacologia , Masculino , Triatoma/citologia , Ácido Valproico/farmacologiaRESUMO
Chromatin organization has been considered to play a major role on aging, by regulating DNA accessibility to transcription and repair machinery. Such organization can be modulated by epigenetic events, such as DNA methylation and histone post-translational modifications. Since changes on gene expression profiles have been described in aged neurons, our aim was to study the age-dependent relationship between structural and epigenetic alterations on chromatin of cortical neurons from mice. For this purpose, isolated neuronal nuclei from mice of two ages were studied by image analysis after cytochemistry, or assessed for chromatin accessibility by enzymatic digestion. Additionally, two epigenetic marks, for open and for densely packed chromatin fibers were quantified. Results indicate epigenetically driven alterations on chromatin organization of cortical neurons with advancing age, whose fibers seem to undergo redistribution and unpackaging. Since increased transcriptional activity is not characteristic of aged neurons, these loosened chromatin fibers may be associated with impaired genome stability, as well as with increased accessibility of repair machinery to a life span damaged DNA.
Assuntos
Envelhecimento/genética , Cromatina/ultraestrutura , Epigênese Genética , Neurônios/fisiologia , Acetilação , Animais , Núcleo Celular/genética , Córtex Cerebral/citologia , Córtex Cerebral/fisiologia , Cromatina/genética , Feminino , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Transcribed sequences have been suggested to be associated with the nuclear matrix, differing from non-transcribing sequences, which have been reported to be contained in DNA loops. However, although a dozen of genes have their expression level affected by aging, data on chromatin-nuclear matrix interactions under this physiological condition are still scarce. In the present study, liver imprints from young, adult and old mice were subjected to FISH (fluorescence in situ hybridization) for 45S rDNA and telomeric sequences, with or without a lysis treatment to produce extended chromatin fibres. There was an increased amount of 45S rDNA sequences located in DNA loops as the animals grow older, while telomeric sequences were always observed in DNA loops irrespective of the animal age. We assume that active rRNA genes associate with the nuclear matrix, while DNA loops contain silent sequences. Transcription of each 45S rDNA repeat unit is suggested to be dependent on its interaction with the nuclear matrix.
Assuntos
Envelhecimento/metabolismo , DNA Ribossômico/metabolismo , Hepatócitos/metabolismo , Matriz Nuclear/metabolismo , Telômero/metabolismo , Animais , Sequência de Bases , Fracionamento Celular , Cromatina/metabolismo , DNA/metabolismo , Hepatócitos/citologia , Hibridização in Situ Fluorescente , Masculino , CamundongosRESUMO
BACKGROUND: Chromatin supraorganization and extensibility, which lead to the formation of extended chromatin fibers (ECF), are affected by starvation and refeeding in adult mouse hepatocytes. It is expected that they could also change with mouse development and aging. METHODS: Methods used involved topochemistry, image analysis, microspectrophotometry, gravity action, and polarization microscopy. RESULTS: Increased nuclear areas and Feulgen-DNA amounts with advancing hepatocyte polyploidy were found with development and aging. A slightly less packed chromatin with more heterogeneously distributed condensation levels was detected in young and old mice. Con-A responsiveness was almost absent in young mice but very deep in aged mice. ECFs formed from nuclei of adult and aged mice but not from nuclei of young mice. The frequency of ECF formation with the long lysis protocol increased with aging. CONCLUSIONS: In young mice, a less packed chromatin state may be associated with more intense gene activity, thus increasing the DNA-nuclear matrix interactions, and inhibiting ECF formation. Reduced DNA-nuclear matrix interactions besides defects in heterochromatin formation may induce higher ECF formation and chromatin unpackaging in old mice. We suggest that differences in Con-A staining relate to different gene activity with advancing development and aging.
Assuntos
Envelhecimento/fisiologia , Cromatina/fisiologia , Hepatócitos/fisiologia , Animais , Hepatócitos/citologia , Masculino , CamundongosRESUMO
BACKGROUND: How much DNA remains in mouse hepatocyte nuclei after extended chromatin fiber (ECF) formation or whether this content varies within the nuclear population is not known. This information could be relevant to understanding chromatin extensibility as related to chromatin organization, possibly associated with variable nuclear activities in hepatocytes. METHODS: A protocol for ECF formation under the gravity action, image analysis of Feulgen-stained unfixed mouse hepatocyte remnants, and DAPI fluorescence were used. RESULTS: Areas, shape, Feulgen-DNA amounts, and chromatin texture were affected in unfixed, lysed nuclei. The Feulgen-DNA values in nuclear remnants represented 37% of the content in fixed, nonlysed nuclei in terms of median values; the coefficient of variation of Feulgen-DNA values in the nuclear remnants was much higher than those in controls. Enhancement in DAPI fluorescence was evident in chromocenters of the fixed nuclei and in remnants and some ECF granules of the unfixed, lysed nuclei. CONCLUSIONS: The DNA content of the nuclear remnants was much more variable than that assumed from known variability in hepatocyte ploidy degrees. The variable constraint to chromatin extrusion from hepatocyte nuclei is hypothesized to depend on variable chromatin organization with possible involvement of nuclear matrix association, transcriptional activities, and AT-rich DNA-containing heterochromatin.
Assuntos
Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , DNA/análise , Hepatócitos/citologia , Citometria por Imagem/métodos , Microscopia de Vídeo/métodos , Animais , Núcleo Celular/química , DNA/ultraestrutura , Desoxirribonucleases , Feminino , Corantes Fluorescentes , Hepatócitos/ultraestrutura , Hidrólise , Indóis , Camundongos , Ploidias , Corantes de RosanilinaRESUMO
A variation of the Concanavalin A (Con-A)-peroxidase labelling method originally described by Kiernan [Localization of alpha-D-glucosyl and alpha-D-mannosyl groups of mucosubstances with Concanavalin A and horseradish peroxidase. Histochemistry 1975;44:39-45] was applied to unsectioned cell preparations, with an emphasis on the nuclear localization of glycoproteins. Mouse liver imprints and chicken blood smears fixed in acetic acid-ethanol solution were studied. Modifications of the method included using increased Con-A concentration, and a range of pH values for the Con-A solutions. The strongest Con-A labelling of both erythrocytes and hepatocytes was obtained after incubation with Con-A at pH 6.5 and with Con-A concentrations at least two-fold greater than those used for tissue sections. These conditions may alter the Con-A conformation, enabling the lectin molecule to enter the cell nucleus and bind to nuclear glycoproteins, thus allowing their localization and quantification.
Assuntos
Concanavalina A/química , Eritroblastos/metabolismo , Glicoproteínas/metabolismo , Hepatócitos/metabolismo , Animais , Galinhas , Eritroblastos/citologia , Hepatócitos/citologia , Peroxidase do Rábano Silvestre/química , Imuno-Histoquímica/métodos , Masculino , CamundongosRESUMO
BACKGROUND: The effect of 48 h of starvation and of 48 h of refeeding subsequent to starvation on chromatin supraorganization and extensibility was studied in hepatocytes from adult mice. METHODS: Methods used involved topochemical assays, image analysis, gravity action, and polarization microscopy. RESULTS: Starvation increased the chromatin packing states, especially in areas of noncondensed chromatin, and induced drastic decreases in concanavalin A reactivity due to nuclear matrix glycoproteins and the frequency of nuclei with chromatin extensibility under gravity. Changes in chromatin packing state were accompanied by shifts of nuclear areas of part of the nuclear population to smaller values but did not affect the respective Feulgen-DNA amounts except for a few nuclei. The extent of chromatin unpackaging, but not of frequency of nuclei with formation of extended chromatin fibers, in starved mice that were refed was greater than in well-fed controls. Refeeding induced increase in Feulgen-DNA amounts and regain and redistribution of concanavalin A-reactive nuclear glycoproteins. However, the duration of refeeding used was probably insufficient to reestablish the stereo arrangement of the chromatin-nuclear matrix and to restore chromatin fluidity to the level seen in well-fed mice. CONCLUSIONS: The changes in the liver cell nuclei associated with starvation and refeeding of adult mice involved chromatin supraorganization, hepatocyte proliferation (refeeding), and the loss, regain, and redistribution of nuclear proteins, especially nuclear matrix components, related to chromatin organization and extensibility. These changes are suggested as favoring the silencing and reactivation of transcriptional activities, depending on the organism's nutritional state.