RESUMO
Before zygotic genome activation (ZGA), the quiescent genome undergoes reprogramming to transition into the transcriptionally active state. However, the mechanisms underlying euchromatin establishment during early embryogenesis remain poorly understood. Here, we show that histone H4 lysine 16 acetylation (H4K16ac) is maintained from oocytes to fertilized embryos in Drosophila and mammals. H4K16ac forms large domains that control nucleosome accessibility of promoters prior to ZGA in flies. Maternal depletion of MOF acetyltransferase leading to H4K16ac loss causes aberrant RNA Pol II recruitment, compromises the 3D organization of the active genomic compartments during ZGA, and causes downregulation of post-zygotically expressed genes. Germline depletion of histone deacetylases revealed that other acetyl marks cannot compensate for H4K16ac loss in the oocyte. Moreover, zygotic re-expression of MOF was neither able to restore embryonic viability nor onset of X chromosome dosage compensation. Thus, maternal H4K16ac provides an instructive function to the offspring, priming future gene activation.
Assuntos
Histonas/metabolismo , Lisina/metabolismo , Ativação Transcricional/genética , Acetilação , Animais , Sequência de Bases , Segregação de Cromossomos/genética , Sequência Conservada , Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Evolução Molecular , Feminino , Genoma , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Masculino , Mamíferos/genética , Camundongos , Mutação/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Oócitos/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Cromossomo X/metabolismo , Zigoto/metabolismoRESUMO
The idea that epigenetic determinants such as DNA methylation, histone modifications or RNA can be passed to the next generation through meiotic products (gametes) is long standing. Such meiotic epigenetic inheritance (MEI) is fairly common in yeast, plants and nematodes, but its extent in mammals has been much debated. Advances in genomics techniques are now driving the profiling of germline and zygotic epigenomes, thereby improving our understanding of MEI in diverse species. Whereas the role of DNA methylation in MEI remains unclear, insights from genome-wide studies suggest that a previously underappreciated fraction of mammalian genomes bypass epigenetic reprogramming during development. Notably, intergenerational inheritance of histone modifications, tRNA fragments and microRNAs can affect gene regulation in the offspring. It is important to note that MEI in mammals rarely constitutes transgenerational epigenetic inheritance (TEI), which spans multiple generations. In this Review, we discuss the examples of MEI in mammals, including mammalian epigenome reprogramming, and the molecular mechanisms of MEI in vertebrates in general. We also discuss the implications of the inheritance of histone modifications and small RNA for embryogenesis in metazoans, with a particular focus on insights gained from genome-wide studies.
Assuntos
Epigênese Genética/genética , Epigênese Genética/fisiologia , Mamíferos/genética , Animais , Metilação de DNA/genética , Desenvolvimento Embrionário/genética , Epigenômica/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Genoma , Histonas/genética , Humanos , Pequeno RNA não Traduzido/genéticaRESUMO
The global rise in obesity has revitalized a search for genetic and epigenetic factors underlying the disease. We present a Drosophila model of paternal-diet-induced intergenerational metabolic reprogramming (IGMR) and identify genes required for its encoding in offspring. Intriguingly, we find that as little as 2 days of dietary intervention in fathers elicits obesity in offspring. Paternal sugar acts as a physiological suppressor of variegation, desilencing chromatin-state-defined domains in both mature sperm and in offspring embryos. We identify requirements for H3K9/K27me3-dependent reprogramming of metabolic genes in two distinct germline and zygotic windows. Critically, we find evidence that a similar system may regulate obesity susceptibility and phenotype variation in mice and humans. The findings provide insight into the mechanisms underlying intergenerational metabolic reprogramming and carry profound implications for our understanding of phenotypic variation and evolution.
Assuntos
Modelos Animais de Doenças , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Epigênese Genética , Obesidade/genética , Animais , Metabolismo dos Carboidratos , Dieta , Embrião não Mamífero/metabolismo , Cor de Olho , Feminino , Predisposição Genética para Doença , Heterocromatina/metabolismo , Humanos , Masculino , Camundongos , Obesidade/metabolismo , Espermatozoides/metabolismoRESUMO
Constitutive heterochromatin is essential for transcriptional silencing and genome integrity. The establishment of constitutive heterochromatin in early embryos and its role in early fruitfly development are unknown. Lysine 9 trimethylation of histone H3 (H3K9me3) and recruitment of its epigenetic reader, heterochromatin protein 1a (HP1a), are hallmarks of constitutive heterochromatin. Here, we show that H3K9me3 is transmitted from the maternal germline to the next generation. Maternally inherited H3K9me3, and the histone methyltransferases (HMT) depositing it, are required for the organization of constitutive heterochromatin: early embryos lacking H3K9 methylation display de-condensation of pericentromeric regions, centromere-centromere de-clustering, mitotic defects, and nuclear shape irregularities, resulting in embryo lethality. Unexpectedly, quantitative CUT&Tag and 4D microscopy measurements of HP1a coupled with biophysical modeling revealed that H3K9me2/3 is largely dispensable for HP1a recruitment. Instead, the main function of H3K9me2/3 at this developmental stage is to drive HP1a clustering and subsequent heterochromatin compaction. Our results show that HP1a binding to constitutive heterochromatin in the absence of H3K9me2/3 is not sufficient to promote proper embryo development and heterochromatin formation. The loss of H3K9 HMTs and H3K9 methylation alters genome organization and hinders embryonic development.
Assuntos
Proteínas Cromossômicas não Histona , Heterocromatina , Histonas , Animais , Histonas/metabolismo , Histonas/genética , Heterocromatina/metabolismo , Heterocromatina/genética , Metilação , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Homólogo 5 da Proteína Cromobox , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrião não Mamífero/metabolismo , Genoma de Inseto , Desenvolvimento Embrionário/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genéticaRESUMO
Fundamental features of 3D genome organization are established de novo in the early embryo, including clustering of pericentromeric regions, the folding of chromosome arms and the segregation of chromosomes into active (A-) and inactive (B-) compartments. However, the molecular mechanisms that drive de novo organization remain unknown1,2. Here, by combining chromosome conformation capture (Hi-C), chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq), 3D DNA fluorescence in situ hybridization (3D DNA FISH) and polymer simulations, we show that heterochromatin protein 1a (HP1a) is essential for de novo 3D genome organization during Drosophila early development. The binding of HP1a at pericentromeric heterochromatin is required to establish clustering of pericentromeric regions. Moreover, HP1a binding within chromosome arms is responsible for overall chromosome folding and has an important role in the formation of B-compartment regions. However, depletion of HP1a does not affect the A-compartment, which suggests that a different molecular mechanism segregates active chromosome regions. Our work identifies HP1a as an epigenetic regulator that is involved in establishing the global structure of the genome in the early embryo.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Posicionamento Cromossômico , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Genoma de Inseto/genética , Conformação Molecular , Animais , Imunoprecipitação da Cromatina , Cromossomos de Insetos/química , Cromossomos de Insetos/genética , Cromossomos de Insetos/metabolismo , Drosophila melanogaster/citologia , Embrião não Mamífero/citologia , Desenvolvimento Embrionário/genética , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/metabolismo , Hibridização in Situ FluorescenteRESUMO
Embryonic stem cell (ESC) pluripotency is maintained by core transcriptional circuits whereby critical factors sustain their own expression while preventing the expression of genes required for differentiation. Thomson et al. (2011) now show that two core components of the pluripotency circuit, Oct4 and Sox2, are also critical for germ layer fate choice.
RESUMO
Triatomine assassin bugs comprise hematophagous insect vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Although the microbiome of these species has been investigated to some extent, only one virus infecting Triatoma infestans has been identified to date. Here, we describe for the first time seven (+) single-strand RNA viruses (RpV1-7) infecting Rhodnius prolixus, a primary vector of Chagas disease in Central and South America. We show that the RpVs belong to the Iflaviridae, Permutotetraviridae and Solemoviridae and are vertically transmitted from the mothers to the progeny via transovarial transmission. Consistent with this, all the RpVs, except RpV2 that is related to the entomopathogenic Slow bee paralysis virus, established persistent infections in our R. prolixus colony. Furthermore, we show that R. prolixus ovaries express 22-nucleotide viral siRNAs (vsiRNAs), but not viral piRNAs, that originate from the processing of dsRNA intermediates during viral replication of the RpVs. Interestingly, the permutotetraviruses and sobemoviruses display shared pools of vsiRNAs that might provide the basis for a cross-immunity system. The vsiRNAs are maternally deposited in the eggs, where they likely contribute to reduce the viral load and protect the developing embryos. Our results unveil for the first time a complex core virome in R. prolixus and begin to shed light on the RNAi-based antiviral defenses in triatomines.
Assuntos
Doença de Chagas/transmissão , Insetos Vetores/virologia , Vírus de RNA/fisiologia , Rhodnius/virologia , Triatoma/virologia , Trypanosoma cruzi/fisiologia , Viroma , Animais , Feminino , Genoma Viral , Oogênese , Vírus de RNA/classificação , RNA Interferente Pequeno/genética , Coelhos , TranscriptomaRESUMO
Cellular memory is provided by two counteracting groups of chromatin proteins termed Trithorax group (TrxG) and Polycomb group (PcG) proteins. TrxG proteins activate transcription and are perhaps best known because of the involvement of the TrxG protein MLL in leukaemia. However, in terms of molecular analysis, they have lived in the shadow of their more famous counterparts, the PcG proteins. Recent advances have improved our understanding of TrxG protein function and demonstrated that the heterogeneous group of TrxG proteins is of critical importance in the epigenetic regulation of the cell cycle, senescence, DNA damage and stem cell biology.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Epigênese Genética , Animais , DNA/metabolismo , Dano ao DNA , Humanos , Ligação Proteica , Transdução de SinaisRESUMO
Fear-, anxiety- and stress-related disorders are among the most frequent mental disorders. Given substantial rates of insufficient treatment response and often a chronic course, a better understanding of the pathomechanisms of fear-, anxiety- and stress-related disorders is urgently warranted. Epigenetic mechanisms such as histone modifications - positioned at the interface between the biological and the environmental level in the complex pathogenesis of mental disorders - might be highly informative in this context. The current state of knowledge on histone modifications, chromatin-related pharmacology and animal models modified for genes involved in the histone-related epigenetic machinery will be reviewed with respect to fear-, anxiety- and stress-related states. Relevant studies, published until 30th June 2022, were identified using a multi-step systematic literature search of the Pub- Med and Web of Science databases. Animal studies point towards histone modifications (e.g., H3K4me3, H3K9me1/2/3, H3K27me2/3, H3K9ac, H3K14ac and H4K5ac) to be dynamically and mostly brain region-, task- and time-dependently altered on a genome-wide level or gene-specifically (e.g., Bdnf) in models of fear conditioning, retrieval and extinction, acute and (sub-)chronic stress. Singular and underpowered studies on histone modifications in human fear-, anxiety- or stress-related phenotypes are currently restricted to the phenotype of PTSD. Provided consistent validation in human phenotypes, epigenetic biomarkers might ultimately inform indicated preventive interventions as well as personalized treatment approaches, and could inspire future innovative pharmacological treatment options targeting the epigenetic machinery improving treatment response in fear-, anxiety- and stressrelated disorders.
Assuntos
Ansiedade , Código das Histonas , Animais , Humanos , Ansiedade/genética , Ansiedade/tratamento farmacológico , Transtornos de Ansiedade , Medo , Epigênese GenéticaRESUMO
This protocol provides specific details on how to perform Hi-C, the genome-wide version of Chromosome Conformation Capture (3C) followed by high-throughput sequencing, in Drosophila embryos. Hi-C provides a genome-wide population-averaged snapshot of the 3D genome organization within nuclei. In Hi-C, formaldehyde-cross-linked chromatin is enzymatically digested using restriction enzymes; digested fragments are biotinylated and subjected to proximity ligation; ligated fragments are purified using streptavidin followed by paired-end sequencing. Hi-C allows the detection of higher order folding structures such as topologically associated domains (TADs) and active/inactive compartments (A/B compartments, respectively). Performing this assay in developing embryos gives the unique opportunity to investigate dynamic chromatin changes when 3D chromatin structure is established in embryogenesis.
Assuntos
Cromossomos , Drosophila , Animais , Drosophila/genética , Mapeamento Cromossômico/métodos , Cromatina/genética , Núcleo Celular/genéticaRESUMO
CUT&Tag is a method to map the genome-wide distribution of histone modifications and some chromatin-associated proteins. CUT&Tag relies on antibody-targeted chromatin tagmentation and can easily be scaled up or automatized. This protocol provides clear experimental guidelines and helpful considerations when planning and executing CUT&Tag experiments.
Assuntos
Código das Histonas , Histonas , Animais , Histonas/genética , Histonas/metabolismo , Drosophila/genética , Drosophila/metabolismo , Cromatina/genética , GenomaRESUMO
Zygotic genome activation (ZGA) is a crucial step of embryonic development. So far, little is known about the role of chromatin factors during this process. Here, we used an in vivo RNA interference reverse genetic screen to identify chromatin factors necessary for embryonic development in Drosophila melanogaster. Our screen reveals that histone acetyltransferases (HATs) and histone deacetylases are crucial ZGA regulators. We demonstrate that Nejire (CBP/EP300 ortholog) is essential for the acetylation of histone H3 lysine-18 and lysine-27, whereas Gcn5 (GCN5/PCAF ortholog) for lysine-9 of H3 at ZGA, with these marks being enriched at all actively transcribed genes. Nonetheless, these HATs activate distinct sets of genes. Unexpectedly, individual catalytic dead mutants of either Nejire or Gcn5 can activate zygotic transcription (ZGA) and transactivate a reporter gene in vitro. Together, our data identify Nejire and Gcn5 as key regulators of ZGA.
Assuntos
Drosophila melanogaster , Fatores de Transcrição de p300-CBP , Animais , Acetilação , Cromatina , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Histona Acetiltransferases/genética , Lisina/metabolismo , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Small RNAs bound to Argonaute proteins recognize partially or fully complementary nucleic acid targets in diverse gene-silencing processes. A subgroup of the Argonaute proteins--known as the 'Piwi family'--is required for germ- and stem-cell development in invertebrates, and two Piwi members--MILI and MIWI--are essential for spermatogenesis in mouse. Here we describe a new class of small RNAs that bind to MILI in mouse male germ cells, where they accumulate at the onset of meiosis. The sequences of the over 1,000 identified unique molecules share a strong preference for a 5' uridine, but otherwise cannot be readily classified into sequence families. Genomic mapping of these small RNAs reveals a limited number of clusters, suggesting that these RNAs are processed from long primary transcripts. The small RNAs are 26-31 nucleotides (nt) in length--clearly distinct from the 21-23 nt of microRNAs (miRNAs) or short interfering RNAs (siRNAs)--and we refer to them as 'Piwi-interacting RNAs' or piRNAs. Orthologous human chromosomal regions also give rise to small RNAs with the characteristics of piRNAs, but the cloned sequences are distinct. The identification of this new class of small RNAs provides an important starting point to determine the molecular function of Piwi proteins in mammalian spermatogenesis.
Assuntos
Proteínas/metabolismo , RNA/classificação , RNA/metabolismo , Testículo/metabolismo , Animais , Animais Recém-Nascidos , Proteínas Argonautas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Família Multigênica/genética , RNA/genética , Espermatogênese/genética , Fatores de TempoRESUMO
During embryogenesis, the genome shifts from transcriptionally quiescent to extensively active in a process known as Zygotic Genome Activation (ZGA). In Drosophila, the pioneer factor Zelda is known to be essential for the progression of development; still, it regulates the activation of only a small subset of genes at ZGA. However, thousands of genes do not require Zelda, suggesting that other mechanisms exist. By conducting GRO-seq, HiC and ChIP-seq in Drosophila embryos, we demonstrate that up to 65% of zygotically activated genes are enriched for the histone variant H2A.Z. H2A.Z enrichment precedes ZGA and RNA Polymerase II loading onto chromatin. In vivo knockdown of maternally contributed Domino, a histone chaperone and ATPase, reduces H2A.Z deposition at transcription start sites, causes global downregulation of housekeeping genes at ZGA, and compromises the establishment of the 3D chromatin structure. We infer that H2A.Z is essential for the de novo establishment of transcriptional programs during ZGA via chromatin reorganization.
Assuntos
Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Genoma , Histonas/classificação , Histonas/genética , Histonas/metabolismo , Zigoto/metabolismo , Adenosina Trifosfatases , Animais , Divisão Celular , Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Regulação para Baixo , Drosophila , Epigenômica , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Genes Essenciais , Chaperonas de Histonas , Masculino , RNA Polimerase II , Sítio de Iniciação de Transcrição , Ativação TranscricionalRESUMO
In an effort to make transgenesis more flexible and reproducible, we developed a system based on novel 5' and 3' 'gene trap' vectors containing heterospecific Flp recognition target sites and the corresponding 'exchange' vectors allowing the insertion of any DNA sequence of interest into the trapped locus. Flp-recombinase-mediated cassette exchange was demonstrated to be highly efficient in our system, even in the absence of locus-specific selection. The feasibility of constructing a library of ES cell clones using our gene trap vectors was tested and a thousand insertion sites were characterized, following electroporation in ES cells, by RACE-PCR and sequencing. We validated the system in vivo for two trapped loci in transgenic mice and demonstrated that the reporter transgenes inserted into the trapped loci have an expression pattern identical to the endogenous genes. We believe that this system will facilitate in vivo studies of gene function and large-scale generation of mouse models of human diseases, caused by not only loss but also gain of function alleles.
Assuntos
Marcação de Genes/métodos , Vetores Genéticos , Camundongos Transgênicos , Animais , Linhagem Celular , DNA Nucleotidiltransferases/metabolismo , Camundongos , Recombinação GenéticaRESUMO
Gametes carry parental genetic material to the next generation. Stress-induced epigenetic changes in the germ line can be inherited and can have a profound impact on offspring development. However, the molecular mechanisms and consequences of transgenerational epigenetic inheritance are poorly understood. We found that Drosophila oocytes transmit the repressive histone mark H3K27me3 to their offspring. Maternal contribution of the histone methyltransferase Enhancer of zeste, the enzymatic component of Polycomb repressive complex 2, is required for active propagation of H3K27me3 during early embryogenesis. H3K27me3 in the early embryo prevents aberrant accumulation of the active histone mark H3K27ac at regulatory regions and precocious activation of lineage-specific genes at zygotic genome activation. Disruption of the germ line-inherited Polycomb epigenetic memory causes embryonic lethality that cannot be rescued by late zygotic reestablishment of H3K27me3. Thus, maternally inherited H3K27me3, propagated in the early embryo, regulates the activation of enhancers and lineage-specific genes during development.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Elementos Facilitadores Genéticos , Epigênese Genética , Histonas/metabolismo , Herança Materna , Oócitos/metabolismo , Zigoto/metabolismo , Animais , Drosophila melanogaster/genética , Perda do Embrião , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Complexo Repressor Polycomb 2/metabolismoRESUMO
Small molecule drugs and probes are important tools in drug discovery, pharmacology, and cell biology. This is of course also true for epigenetic inhibitors. Important examples for the use of established epigenetic inhibitors are the study of the mechanistic role of a certain target in a cellular setting or the modulation of a certain phenotype in an approach that aims toward therapeutic application. Alternatively, cellular testing may aim at the validation of a new epigenetic inhibitor in drug discovery approaches. Cellular and eventually animal models provide powerful tools for these different approaches but certain caveats have to be recognized and taken into account. This involves both the selectivity of the pharmacological tool as well as the specificity and the robustness of the cellular system. In this article, we present an overview of different methods that are used to profile and screen for epigenetic agents and comment on their limitations. We describe not only diverse successful case studies of screening approaches using different assay formats, but also some problematic cases, critically discussing selected applications of these systems.
Assuntos
Metilação de DNA/efeitos dos fármacos , Descoberta de Drogas , Epigênese Genética , Bibliotecas de Moléculas Pequenas/uso terapêutico , Humanos , Terapia de Alvo MolecularRESUMO
Traditional treatments for breast cancer fail to address therapy-resistant cancer stem-like cells that have been characterized by changes in epigenetic regulators such as the lysine demethylase KDM4. Here, we describe an orally available, selective and potent KDM4 inhibitor (QC6352) with unique preclinical characteristics. To assess the antitumor properties of QC6352, we established a method to isolate and propagate breast cancer stem-like cells (BCSC) from individual triple-negative tumors resected from patients after neoadjuvant chemotherapy. Limiting-dilution orthotopic xenografts of these BCSCs regenerated original patient tumor histology and gene expression. QC6352 blocked BCSC proliferation, sphere formation, and xenograft tumor formation. QC6352 also abrogated expression of EGFR, which drives the growth of therapy-resistant triple-negative breast cancer cells. Our findings validate a unique BCSC culture system for drug screening and offer preclinical proof of concept for KDM4 inhibition as a new strategy to treat triple-negative breast cancer. Cancer Res; 77(21); 5900-12. ©2017 AACR.
Assuntos
Proliferação de Células/genética , Histona Desmetilases com o Domínio Jumonji/genética , Células-Tronco Neoplásicas/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Animais , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Estrutura Molecular , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Interferência de RNA , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This protocol provides specific details on how to perform Chromatin immunoprecipitation (ChIP) from Drosophila embryos. ChIP allows the matching of proteins or histone modifications to specific genomic regions. Formaldehyde-cross-linked chromatin is isolated and antibodies against the target of interest are used to determine whether the target is associated with a specific DNA sequence. This can be performed in spatial and temporal manner and it can provide information about the genome-wide localization of a given protein or histone modification if coupled with deep sequencing technology (ChIP-Seq).
Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/isolamento & purificação , Análise de Sequência de DNA/métodos , Animais , Cromatina/genética , Drosophila/embriologia , Drosophila/genética , Sequenciamento de Nucleotídeos em Larga Escala , Código das Histonas/genética , Histonas/genética , Histonas/isolamento & purificaçãoRESUMO
Several DNA vectors for RNA interference in mammalian cells have been described. These express a short hairpin RNA (shRNA) that is subsequently processed into mature small interfering RNAs (siRNAs). We previously developed the siRNA-expressing vector psiUx based on the polII promoter of the U1 small nuclear RNA gene. Here we describe the conversion of such construct into an inducible system. The starting construct psiUStuff contains a loxP-Stuffer-loxP cassette just upstream the transcription initiation site and does not express the shRNA until the two canonical loxP sites undergo Cre-mediated recombination. If sustained expression of the recombinase is maintained, transcription is repressed and shRNA synthesis is abolished. Therefore, in our system the Cre recombinase exhibits the dual function of activator and repressor allowing the on/off regulation of siRNAs production. Using a Cre recombinase whose transcription is under the control of a tetOn system, we show the temporally controlled expression of an shRNA directed towards the lamin A/C mRNA, as well as the regulated knockdown of its target.