RESUMO
Chromatin remodeling complexes (CRCs) use ATP hydrolysis to maintain correct expression profiles, chromatin stability, and inherited epigenetic states. More than 20 CRCs have been described to date, which encompass four large families defined by their ATPase subunits. These complexes and their subunits are conserved from yeast to humans through evolution. Their activities depend on their catalytic subunits which through ATP hydrolysis provide the energy necessary to fulfill cellular functions such as gene transcription, DNA repair, and transposon silencing. These activities take place at the first levels of chromatin compaction, and CRCs have been recognized as essential elements of chromatin dynamics. Recent studies have demonstrated an important role for these complexes in the maintenance of higher order chromatin structure. In this review, we present an overview of the organization of the genome within the cell nucleus, the different levels of chromatin compaction, and importance of the architectural proteins, and discuss the role of CRCs and how their functions contribute to the dynamics of the 3D genome organization.
RESUMO
Understanding the packaging of DNA into chromatin has become a crucial aspect in the study of gene regulatory mechanisms. Heterochromatin establishment and maintenance dynamics have emerged as some of the main features involved in genome stability, cellular development, and diseases. The most extensively studied heterochromatin protein is HP1a. This protein has two main domains, namely the chromoshadow and the chromodomain, separated by a hinge region. Over the years, several works have taken on the task of identifying HP1a partners using different strategies. In this review, we focus on describing these interactions and the possible complexes and subcomplexes associated with this critical protein. Characterization of these complexes will help us to clearly understand the implications of the interactions of HP1a in heterochromatin maintenance, heterochromatin dynamics, and heterochromatin's direct relationship to gene regulation and chromatin organization.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Eucromatina/metabolismo , Heterocromatina/metabolismo , Animais , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Instabilidade Genômica , Humanos , Elementos Isolantes , Filogenia , Ligação Proteica , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre ProteínasRESUMO
BACKGROUND: dADD1 and dXNP proteins are the orthologs in Drosophila melanogaster of the ADD and SNF2 domains, respectively, of the ATRX vertebrate's chromatin remodeler, they suppress position effect variegation phenotypes and participate in heterochromatin maintenance. RESULTS: We performed a search in human cancer databases and found that ATRX protein levels were elevated in more than 4.4% of the samples analyzed. Using the Drosophila model, we addressed the effects of over and under-expression of dADD1 proteins in polytene cells. Elevated levels of dADD1 in fly tissues caused different phenotypes, such as chromocenter disruption and loss of banding pattern at the chromosome arms. Analyses of the heterochromatin maintenance protein HP1a, the dXNP ATPase and the histone post-translational modification H3K9me3 revealed changes in their chromatin localization accompanied by mild transcriptional defects of genes embedded in heterochromatic regions. Furthermore, the expression of heterochromatin embedded genes in null dadd1 organisms is lower than in the wild-type conditions. CONCLUSION: These data indicate that dADD1 overexpression induces chromatin changes, probably affecting the stoichiometry of HP1a containing complexes that lead to transcriptional and architectural changes. Our results place dADD1 proteins as important players in the maintenance of chromatin architecture and heterochromatic gene expression.
Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/metabolismo , Proteínas de Drosophila/metabolismo , Animais , Efeitos da Posição Cromossômica , Proteínas de Drosophila/genética , Drosophila melanogaster , Expressão Gênica , Heterocromatina/metabolismo , Fatores de Transcrição , Proteína Nuclear Ligada ao X/metabolismoRESUMO
Telomeres are important contributors to genome stability, as they prevent linear chromosome end degradation and contribute to the avoidance of telomeric fusions. An important component of the telomeres is the heterochromatin protein 1a (HP1a). Mutations in Su(var)205, the gene encoding HP1a in Drosophila, result in telomeric fusions, retrotransposon regulation loss and larger telomeres, leading to chromosome instability. Previously, it was found that several proteins physically interact with HP1a, including dXNP and dAdd1 (orthologues to the mammalian ATRX gene). In this study, we found that mutations in the genes encoding the dXNP and dAdd1 proteins affect chromosome stability, causing chromosomal aberrations, including telomeric defects, similar to those observed in Su(var)205 mutants. In somatic cells, we observed that dXNP and dAdd1 participate in the silencing of the telomeric HTT array of retrotransposons, preventing anomalous retrotransposon transcription and integration. Furthermore, the lack of dAdd1 results in the loss of HP1a from the telomeric regions without affecting other chromosomal HP1a binding sites; mutations in dxnp also affected HP1a localization but not at all telomeres, suggesting a specialized role for dAdd1 and dXNP proteins in locating HP1a at the tips of the chromosomes. These results place dAdd1 as an essential regulator of HP1a localization and function in the telomere heterochromatic domain.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Instabilidade Genômica , Telômero/genética , Telômero/metabolismo , Animais , Animais Geneticamente Modificados , Homólogo 5 da Proteína Cromobox , Aberrações Cromossômicas , Feminino , Inativação Gênica , Heterocromatina/metabolismo , Perda de Heterozigosidade , Masculino , Mutação , Transporte Proteico , RetroelementosRESUMO
Eukaryotic gene expression is activated by factors that interact within complex machinery to initiate transcription. An important component of this machinery is the DNA repair/transcription factor TFIIH. Mutations in TFIIH result in three human syndromes: xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Transcription and DNA repair defects have been linked to some clinical features of these syndromes. However, how mutations in TFIIH affect specific developmental programmes, allowing organisms to develop with particular phenotypes, is not well understood. Here, we show that mutations in the p52 and p8 subunits of TFIIH have a moderate effect on the gene expression programme in the Drosophila testis, causing germ cell differentiation arrest in meiosis, but no Polycomb enrichment at the promoter of the affected differentiation genes, supporting recent data that disagree with the current Polycomb-mediated repression model for regulating gene expression in the testis. Moreover, we found that TFIIH stability is not compromised in p8 subunit-depleted testes that show transcriptional defects, highlighting the role of p8 in transcription. Therefore, this study reveals how defects in TFIIH affect a specific cell differentiation programme and contributes to understanding the specific syndrome manifestations in TFIIH-afflicted patients.
Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Mutação , Espermatozoides/citologia , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/genética , Animais , Diferenciação Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Perfilação da Expressão Gênica , Masculino , Estabilidade Proteica , Testículo/citologia , Testículo/embriologia , Transcrição GênicaRESUMO
This work examines the cellular localization of holocarboxylase synthetase (HCS) and its association to chromatin during different stages of development of Drosophila melanogaster. While HCS is well known for its role in the attachment of biotin to biotin-dependent carboxylase, it also regulates the transcription of HCS and carboxylases genes by triggering a cGMP-dependent signal transduction cascade. Further, its presence in the nucleus of cells suggests additional regulatory roles, but the mechanism involved has remained elusive. In this study, we show in D. melanogaster that HCS migrates to the nucleus at the gastrulation stage. In polytene chromosomes, it is associated to heterochromatin bands where it co-localizes with histone 3 trimethylated at lysine 9 (H3K9met3) but not with the euchromatin mark histone 3 acetylated at lysine 9 (H3K9ac). Further, we demonstrate the association of HCS with the hsp70 promoter by immunofluorescence and chromatin immuno-precipitation (ChIP) of associated DNA sequences. We demonstrate the occupancy of HCS to the core promoter region of the transcriptionally inactive hsp70 gene. On heat-shock activation of the hsp70 promoter, HCS is displaced and the promoter region becomes enriched with the TFIIH subunits XPD and XPB and elongating RNA pol II, the latter also demonstrated using ChIP assays. We suggest that HCS may have a role in the repression of gene expression through a mechanism involving its trafficking to the nucleus and interaction with heterochromatic sites coincident with H3K9met3.
Assuntos
Carbono-Nitrogênio Ligases/metabolismo , Cromatina/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/enzimologia , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Carbono-Nitrogênio Ligases/genética , Núcleo Celular/enzimologia , Drosophila melanogaster/genética , Proteínas de Choque Térmico HSP72/genética , Células Hep G2 , Histonas/metabolismo , Temperatura Alta , Humanos , Dados de Sequência Molecular , Cromossomos Politênicos/genética , Cromossomos Politênicos/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Transporte Proteico , Alinhamento de SequênciaRESUMO
At the present time research situates differential regulation of gene expression in an increasingly complex scenario based on interplay between genetic and epigenetic information networks, which need to be highly coordinated. Here we describe in a comparative way relevant concepts and models derived from studies on the chicken alpha- and beta-globin group of genes. We discuss models for globin switching and mechanisms for coordinated transcriptional activation. A comparative overview of globin genes chromatin structure, based on their genomic domain organization and epigenetic components is presented. We argue that the results of those studies and their integrative interpretation may contribute to our understanding of epigenetic abnormalities, from beta-thalassemias to human cancer. Finally we discuss the interdependency of genetic-epigenetic components and the need of their mutual consideration in order to visualize the regulation of gene expression in a more natural context and consequently better understand cell differentiation, development and cancer.
Assuntos
Cromatina/química , Epigênese Genética , Globinas/genética , Neoplasias/genética , Transcrição Gênica , Animais , Globinas/química , Globinas/metabolismo , Humanos , Regiões Promotoras Genéticas/genéticaRESUMO
We present the first analysis of the dynamics of the transcription DNA-repair factor TFIIH at the onset of transcription in early Drosophila development. TFIIH is composed of ten polypeptides that are part of two complexes - the core and the CAK. We found that the TFIIH core is initially located in the cytoplasm of syncytial blastoderm embryos, and that after mitotic division ten and until the cellular blastoderm stage, the core moves from the cytoplasm to the nucleus. By contrast, the CAK complex is mostly cytoplasmic during cellularization and during gastrulation. However, both components are positioned at promoters of genes that are activated at transcription onset. Later in development, the CAK complex becomes mostly nuclear and co-localizes in most chromosomal regions with the TFIIH core, but not in all sites, suggesting that the CAK complex could have a TFIIH-independent role in transcription of some loci. We also demonstrate that even though the CAK and the core coexist in the early embryo cytoplasm, they do not interact until they are in the nucleus and suggest that the complete assembly of the ten subunits of TFIIH occurs in the nucleus at the mid-blastula transition. In addition, we present evidence that suggests that DNA helicase subunits XPB and XPD are assembled in the core when they are transported into the nucleus and are required for the onset of transcription.
Assuntos
Núcleo Celular/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Fator de Transcrição TFIIH/metabolismo , Animais , Blástula/citologia , Cromossomos/genética , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Fatores de Transcrição Fushi Tarazu/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos/genética , Modelos Genéticos , Regiões Promotoras Genéticas/genética , Transporte Proteico , Transcrição Gênica , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
It has been demonstrated that the human tumor suppressor p53 has an important role in modulating histone modifications after UV light irradiation. In this work we explored if the p53 Drosophila homologue has a similar role. Taking advantage of the existence of polytene chromosomes in the salivary glands of third instar larvae, we analyzed K9 and K14 H3 acetylation patterns in situ after UV irradiation of wild-type and Dmp53 null flies. As in human cells, after UV damage there is an increase in H3 acetylation in wild-type organisms. In Dmp53 mutant flies, this response is significantly affected at the K9 position. These results are similar to those found in human p53 mutant tumor cells with one interesting difference, only the basal H3 acetylation of K14 is reduced in Dmp53 mutant flies, while the basal H3-K9 acetylation is not affected. This work shows, that the presence of Dmp53 is necessary to maintain normal H3-K14 acetylation levels in Drosophila chromatin and that the function of p53 to maintaining histone modifications, is conserved in Drosophila and humans.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Histonas/metabolismo , Larva/efeitos da radiação , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Animais , Cromossomos/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/efeitos da radiação , Humanos , Larva/metabolismo , Proteína Supressora de Tumor p53/genética , Raios UltravioletaRESUMO
We review several aspects of RNAi and gene silencing with baculovirus. We show that the potency of RNAi in Spodoptera frugiperda (Sf21) insect cells correlates well with the efficiency of transfection of the siRNA. Using a fluorescein-labeled siRNA we found that the siRNA localized in areas surrounding the endoplasmic reticulum (ER). Both long (700 nucleotides long) and small ( approximately 25 nucleotides long) interfering RNAs were equally effective in initiating RNA interference (RNAi), and the duration of the interfering effect was indistinguishable. Even though RNAi in Sf21 cells is very effective, in vitro experiments show that these cells fragment the long dsRNA into siRNA poorly, when compared to HEK cells. Finally, we show that in vivo inhibition of baculovirus infection with dsRNA homologous to genes that are essential for baculovirus infectivity depends strongly on the amount of dsRNA used in the assays. Five hundred nanogram of dsRNA directly injected into the haemolymph of insects prevent animal death to over 95%. In control experiments, over 96% of insects not injected with dsRNA or injected with an irrelevant dsRNA died within a week. These results demonstrate the efficiency of dsRNA for in vivo prevention of a viral infection by virus that is very cytotoxic and lytic in animals.