RESUMO
BACKGROUND: Cell fate decisions are governed by interactions between sequence-specific transcription factors and a dynamic chromatin landscape. Zebrafish offer a powerful system for probing the mechanisms that drive these cell fate choices, especially in the context of early embryogenesis. However, technical challenges associated with conventional methods for chromatin profiling have slowed progress toward understanding the exact relationships between chromatin changes, transcription factor binding, and cellular differentiation during zebrafish embryogenesis. RESULTS: To overcome these challenges, we adapted the chromatin profiling methods Cleavage Under Targets and Release Using Nuclease (CUT&RUN) and CUT&Tag for use in zebrafish and applied these methods to generate high-resolution enrichment maps for H3K4me3, H3K27me3, H3K9me3, RNA polymerase II, and the histone variant H2A.Z using tissue isolated from whole, mid-gastrula stage embryos. Using this data, we identify a subset of genes that may be bivalently regulated during both zebrafish and mouse gastrulation, provide evidence for an evolving H2A.Z landscape during embryo development, and demonstrate the effectiveness of CUT&RUN for detecting H3K9me3 enrichment at repetitive sequences. CONCLUSIONS: Our results demonstrate the power of combining CUT&RUN and CUT&Tag methods with the strengths of the zebrafish system to define emerging chromatin landscapes in the context of vertebrate embryogenesis.
Assuntos
Cromatina , Peixe-Zebra , Animais , Cromatina/genética , Imunoprecipitação da Cromatina , Desenvolvimento Embrionário/genética , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Peixe-Zebra/genéticaRESUMO
Chemical modification of nucleotide bases in DNA provides one mechanism for conveying information in addition to the genetic code. 5-methylcytosine (5mC) represents the most common chemically modified base in eukaryotic genomes. Sometimes referred to simply as DNA methylation, in eukaryotes 5mC is most prevalent at CpG dinucleotides and is frequently associated with transcriptional repression of transposable elements. However, 5mC levels and distributions are variable across phylogenies, and emerging evidence suggests that the functions of DNA methylation may be more diverse and complex than was previously appreciated. We summarize the current understanding of DNA methylation profiles and functions in different eukaryotic lineages.
Assuntos
Metilação de DNA , Eucariotos/genética , Regulação da Expressão Gênica , Epigênese Genética , Genoma , Genômica/métodosRESUMO
The intestinal epithelium forms a barrier protecting the organism from microbes and other proinflammatory stimuli. The integrity of this barrier and the proper response to infection requires precise regulation of powerful immune homing signals such as tumor necrosis factor (TNF). Dysregulation of TNF leads to inflammatory bowel diseases (IBD), but the mechanism controlling the expression of this potent cytokine and the events that trigger the onset of chronic inflammation are unknown. Here, we show that loss of function of the epigenetic regulator ubiquitin-like protein containing PHD and RING finger domains 1 (uhrf1) in zebrafish leads to a reduction in tnfa promoter methylation and the induction of tnfa expression in intestinal epithelial cells (IECs). The increase in IEC tnfa levels is microbe-dependent and results in IEC shedding and apoptosis, immune cell recruitment, and barrier dysfunction, consistent with chronic inflammation. Importantly, tnfa knockdown in uhrf1 mutants restores IEC morphology, reduces cell shedding, and improves barrier function. We propose that loss of epigenetic repression and TNF induction in the intestinal epithelium can lead to IBD onset.
Assuntos
Metilação de DNA , Epigênese Genética/fisiologia , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/embriologia , Peixe-Zebra/embriologia , Animais , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Inflamação/genética , Inflamação/mortalidade , Inflamação/patologia , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Transativadores/genética , Transativadores/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The Gal4-UAS regulatory system of yeast is widely used to modulate gene expression in Drosophila; however, there are limitations to its usefulness in transgenic zebrafish, owing to progressive methylation and silencing of the CpG-rich multicopy upstream activation sequence. Although a modified, less repetitive UAS construct may overcome this problem, it is highly desirable to have additional transcriptional regulatory systems that can be applied independently or in combination with the Gal4/UAS system for intersectional gene expression. The Q transcriptional regulatory system of Neurospora crassa functions similarly to Gal4/UAS. QF is a transcriptional activator that binds to the QUAS upstream regulatory sequence to drive reporter gene expression. Unlike Gal4, the QF binding site does not contain essential CpG dinucleotide sequences that are subject to DNA methylation. The QS protein is a repressor of QF mediated transcriptional activation akin to Gal80. The functionality of the Q system has been demonstrated in Drosophila and Caenorhabditis elegans and we now report its successful application to a vertebrate model, the zebrafish, Danio rerio. Several tissue-specific promoters were used to drive QF expression in stable transgenic lines, as assessed by activation of a QUAS:GFP transgene. The QS repressor was found to dramatically reduce QF activity in injected zebrafish embryos; however, a similar repression has not yet been achieved in transgenic animals expressing QS under the control of ubiquitous promoters. A dual reporter construct containing both QUAS and UAS, each upstream of different fluorescent proteins was also generated and tested in transient assays, demonstrating that the two systems can work in parallel within the same cell. The adoption of the Q system should greatly increase the versatility and power of transgenic approaches for regulating gene expression in zebrafish.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Engenharia Genética/métodos , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados/metabolismo , Regulação da Expressão Gênica/genética , Genes Fúngicos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Neurospora crassa/genética , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
The structural organization of eukaryotic genomes is contingent upon the fractionation of DNA into transcriptionally permissive euchromatin and repressive heterochromatin. However, we have a limited understanding of how these distinct states are first established during animal embryogenesis. Histone 3 lysine 9 trimethylation (H3K9me3) is critical to heterochromatin formation, and bulk establishment of this mark is thought to help drive large-scale remodeling of an initially naive chromatin state during animal embryogenesis. However, a detailed understanding of this process is lacking. Here, we leverage CUT&RUN to define the emerging H3K9me3 landscape of the zebrafish embryo with high sensitivity and temporal resolution. Despite the prevalence of DNA transposons in the zebrafish genome, we found that LTR transposons are preferentially targeted for embryonic H3K9me3 deposition, with different families exhibiting distinct establishment timelines. High signal-to-noise ratios afforded by CUT&RUN revealed new, emerging sites of low-amplitude H3K9me3 that initiated before the major wave of zygotic genome activation (ZGA). Early sites of establishment predominated at specific subsets of transposons and were particularly enriched for transposon sequences with maternal piRNAs and pericentromeric localization. Notably, the number of H3K9me3 enriched sites increased linearly across blastula development, while quantitative comparison revealed a >10-fold genome-wide increase in H3K9me3 signal at established sites over just 30 minutes at the onset of major ZGA. Continued maturation of the H3K9me3 landscape was observed beyond the initial wave of bulk establishment.
RESUMO
The structural organization of eukaryotic genomes is contingent upon the fractionation of DNA into transcriptionally permissive euchromatin and repressive heterochromatin. However, we have a limited understanding of how these distinct states are first established during animal embryogenesis. Histone 3 lysine 9 trimethylation (H3K9me3) is critical to heterochromatin formation and bulk establishment of this mark is thought to help drive large-scale remodeling of an initially naive chromatin state during animal embryogenesis. However, a detailed understanding of this process is lacking. Here, we leverage CUT&RUN to define the emerging H3K9me3 landscape of the zebrafish embryo with high sensitivity and temporal resolution. Despite the prevalence of DNA transposons in the zebrafish genome, we found that LTR transposons are preferentially targeted for embryonic H3K9me3 deposition, with different families exhibiting distinct establishment timelines. High signal-to-noise ratios afforded by CUT&RUN revealed new, emerging sites of low-amplitude H3K9me3 that initiated before the major wave of zygotic genome activation (ZGA). Early sites of establishment predominated at specific subsets of transposons and were particularly enriched for transposon sequences with maternal piRNAs and pericentromeric localization. Notably, the number of H3K9me3 enriched sites increased linearly across blastula development, while quantitative comparison revealed a >10-fold genome-wide increase in H3K9me3 signal at established sites over just 30 minutes at the onset of ZGA. Continued maturation of the H3K9me3 landscape was observed beyond the initial wave of bulk establishment.
RESUMO
Cytosine DNA methylation is a heritable epigenetic mark present in many eukaryotic organisms. Although DNA methylation likely has a conserved role in gene silencing, the levels and patterns of DNA methylation appear to vary drastically among different organisms. Here we used shotgun genomic bisulfite sequencing (BS-Seq) to compare DNA methylation in eight diverse plant and animal genomes. We found that patterns of methylation are very similar in flowering plants with methylated cytosines detected in all sequence contexts, whereas CG methylation predominates in animals. Vertebrates have methylation throughout the genome except for CpG islands. Gene body methylation is conserved with clear preference for exons in most organisms. Furthermore, genes appear to be the major target of methylation in Ciona and honey bee. Among the eight organisms, the green alga Chlamydomonas has the most unusual pattern of methylation, having non-CG methylation enriched in exons of genes rather than in repeats and transposons. In addition, the Dnmt1 cofactor Uhrf1 has a conserved function in maintaining CG methylation in both transposons and gene bodies in the mouse, Arabidopsis, and zebrafish genomes.
Assuntos
Metilação de DNA/genética , Evolução Molecular , Plantas/genética , Animais , Arabidopsis/genética , Éxons/genética , Íntrons/genética , Mutação/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta/genética , Filogenia , Sequências Repetitivas de Ácido Nucleico/genética , Transativadores/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The yeast Gal4/UAS transcriptional activation system is a powerful tool for regulating gene expression in Drosophila and has been increasing in popularity for developmental studies in zebrafish. It is also useful for studying the basis of de novo transcriptional silencing. Fluorescent reporter genes under the control of multiple tandem copies of the upstream activator sequence (UAS) often show evidence of variegated expression and DNA methylation in transgenic zebrafish embryos. To characterize this systematically, we monitored the progression of transcriptional silencing of UAS-regulated transgenes that differ in their integration sites and in the repetitive nature of the UAS. Transgenic larvae were examined in three generations for tissue-specific expression of a green fluorescent protein (GFP) reporter and DNA methylation at the UAS. Single insertions containing four distinct upstream activator sequences were far less susceptible to methylation than insertions containing fourteen copies of the same UAS. In addition, transgenes that integrated in or adjacent to transposon sequence exhibited silencing regardless of the number of UAS sites included in the transgene. Placement of promoter-driven Gal4 upstream of UAS-regulated responder genes in a single bicistronic construct also appeared to accelerate silencing and methylation. The results demonstrate the utility of the zebrafish for efficient tracking of gene silencing mechanisms across several generations, as well as provide useful guidelines for optimal Gal4-regulated gene expression in organisms subject to DNA methylation.
Assuntos
Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Metilação de DNA , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas Recombinantes/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
Site-specific recombinases (SSRs) are powerful tools for genome manipulation, used in diverse organisms including Drosophila melanogaster, mouse, Arabidopsis, zebrafish, and human cultured cells. The integrase from the bacteriophage ΦC31 belongs to the large serine family of integrases, and in contrast to other widely used SSRs such as Cre and Flp, recombination is directional and therefore irreversible. We have developed a vector system for recombinase-mediated cassette exchange (RMCE) in the zebrafish, allowing swapping of the coding sequence in an integrated transgene. Utilizing codon-optimized ΦC31 integrase RNA bearing the 3'UTR from the nanos1 gene, we replaced the egfp coding sequence of an integrated reporter transgene with mCherry coding sequence. Recombination was achieved at high efficiency in both somatic cells and in the germline. We demonstrate an effective approach to RMCE, increasing the repertoire of tools available to manipulate the zebrafish genome.
Assuntos
Bacteriófagos/enzimologia , DNA Nucleotidiltransferases/metabolismo , Integrases/metabolismo , Animais , Bacteriófagos/genética , DNA Nucleotidiltransferases/genética , Células Germinativas , Integrases/genética , Modelos Biológicos , Peixe-ZebraRESUMO
Heterochromatin formation and maintenance is critical for the repression of transcription from repetitive sequences. However, in vivo tools for monitoring heterochromatin mediated repression of repeats in the context of vertebrate development have been lacking. Here we demonstrate that a large concatemeric transgene integration containing the dsRed fluorescent reporter under the control of a ubiquitous promoter recapitulates molecular hallmarks of heterochromatic silencing, and that expression from the transgene array can be reactivated by depletion of known regulators of heterochromatin. We then use this reporter to identify a previously unappreciated role for the zebrafish NSD1 orthologs, Nsd1a and Nsd1b, in promoting heterochromatin mediated repression. Our results provide proof-principle that this transgenic reporter line can be used to rapidly identify genes with potential roles in heterochromatic silencing in the context of a live, vertebrate organism.
RESUMO
Developmental mechanisms regulating gene expression and the stable acquisition of cell fate direct cytodifferentiation during organogenesis. Moreover, it is likely that such mechanisms could be exploited to repair or regenerate damaged organs. DNA methyltransferases (Dnmts) are enzymes critical for epigenetic regulation, and are used in concert with histone methylation and acetylation to regulate gene expression and maintain genomic integrity and chromosome structure. We carried out two forward genetic screens for regulators of endodermal organ development. In the first, we screened for altered morphology of developing digestive organs, while in the second we screed for the lack of terminally differentiated cell types in the pancreas and liver. From these screens, we identified two mutant alleles of zebrafish dnmt1. Both lesions are predicted to eliminate dnmt1 function; one is a missense mutation in the catalytic domain and the other is a nonsense mutation that eliminates the catalytic domain. In zebrafish dnmt1 mutants, the pancreas and liver form normally, but begin to degenerate after 84 h post fertilization (hpf). Acinar cells are nearly abolished through apoptosis by 100 hpf, though neither DNA replication, nor entry into mitosis is halted in the absence of detectable Dnmt1. However, endocrine cells and ducts are largely spared. Surprisingly, dnmt1 mutants and dnmt1 morpholino-injected larvae show increased capacity for pancreatic beta cell regeneration in an inducible model of pancreatic beta cell ablation. Thus, our data suggest that Dnmt1 is dispensable for pancreatic duct or endocrine cell formation, but not for acinar cell survival. In addition, Dnmt1 may influence the differentiation of pancreatic beta cell progenitors or the reprogramming of cells toward the pancreatic beta cell fate.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Pâncreas/citologia , Regeneração/fisiologia , Proteínas de Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sobrevivência Celular , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Células Endócrinas/metabolismo , Imunofluorescência , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Dados de Sequência Molecular , Pâncreas/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Epigenetic regulation of transcriptional silencing is essential for normal development. Despite its importance, in vivo systems for examining gene silencing at cellular resolution have been lacking in developing vertebrates. We describe a transgenic approach that allows monitoring of an epigenetically regulated fluorescent reporter in developing zebrafish and their progeny. Using a self-reporting Gal4-VP16 gene/enhancer trap vector, we isolated tissue-specific drivers that regulate expression of the green fluorescent protein (GFP) gene through a multicopy, upstream activator sequence (UAS). Transgenic larvae initially exhibit robust fluorescence (GFP(high)); however, in subsequent generations, gfp expression is mosaic (GFP(low)) or entirely absent (GFP(off)), despite continued Gal4-VP16 activity. We find that transcriptional repression is heritable and correlated with methylation of the multicopy UAS. Silenced transgenes can be reactivated by increasing Gal4-VP16 levels or in DNA methyltransferase-1 (dnmt1) mutants. Strikingly, in dnmt1 homozygous mutants, reactivation of gfp expression occurs in a reproducible subset of cells, raising the possibility of different sensitivities or alternative silencing mechanisms in discrete cell populations. The results demonstrate the power of the zebrafish system for in vivo monitoring of epigenetic processes using a genetic approach.
Assuntos
Inativação Gênica , Proteínas de Fluorescência Verde/genética , Ativação Transcricional , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Epigênese Genética , Feminino , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , Larva/genética , Larva/metabolismo , Masculino , Microscopia de Fluorescência , Mutação , Transativadores/genética , Transativadores/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Early animal development is characterized by intense reorganization of the embryonic genome, including large-scale changes in chromatin structure and in the DNA and histone modifications that help shape this structure. Particularly profound shifts in the chromatin landscape are associated with the maternal-to-zygotic transition, when the zygotic genome is first transcribed and maternally loaded transcripts are degraded. The accessibility of the early zebrafish embryo facilitates the interrogation of chromatin during this critical window of development, making it an important model for early chromatin regulation. Here, we review our current understanding of chromatin dynamics during early zebrafish development, highlighting new advances as well as similarities and differences between early chromatin regulation in zebrafish and other species.
Assuntos
Cromatina/genética , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Peixe-Zebra , Peixe-Zebra/embriologia , Animais , ZigotoRESUMO
The segregation of eukaryotic genomes into euchromatin and heterochromatin represents a fundamental and poorly understood process. Here, we demonstrate that genome-wide establishment of heterochromatin is triggered by the maternal to zygotic transition (MZT) during zebrafish embryogenesis. We find that prior to MZT, zebrafish lack hallmarks of heterochromatin including histone H3 lysine 9 trimethylation (H3K9me3) and condensed chromatin ultrastructure. Global establishment of heterochromatic features occurs following MZT and requires both activation of the zygotic genome and degradation of maternally deposited RNA. Mechanistically, we demonstrate that zygotic transcription of the micro RNA miR-430 promotes degradation of maternal RNA encoding the chromatin remodeling protein Smarca2, and that clearance of Smarca2 is required for global heterochromatin establishment in the early embryo. Our results identify MZT as a key developmental regulator of heterochromatin establishment during vertebrate embryogenesis and uncover functions for Smarca2 in protecting the embryonic genome against heterochromatinization.
Assuntos
Desenvolvimento Embrionário/genética , Heterocromatina/genética , Peixe-Zebra/embriologia , Animais , Cromatina/genética , Cromatina/metabolismo , Cromatina/ultraestrutura , Embrião não Mamífero/citologia , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/metabolismo , Heterocromatina/ultraestrutura , MicroRNAs/metabolismo , MicroRNAs/fisiologia , Transcrição Gênica , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Ten-eleven translocation (Tet) enzymes (Tet1/2/3) mediate 5-methylcytosine (5mC) hydroxylation, which can facilitate DNA demethylation and thereby impact gene expression. Studied mostly for how mutant isoforms impact cancer, the normal roles for Tet enzymes during organogenesis are largely unknown. By analyzing compound mutant zebrafish, we discovered a requirement for Tet2/3 activity in the embryonic heart for recruitment of epicardial progenitors, associated with development of the atrial-ventricular canal (AVC). Through a combination of methylation, hydroxymethylation, and transcript profiling, the genes encoding the activin A subunit Inhbaa (in endocardium) and Sox9b (in myocardium) were implicated as demethylation targets of Tet2/3 and critical for organization of AVC-localized extracellular matrix (ECM), facilitating migration of epicardial progenitors onto the developing heart tube. This study elucidates essential DNA demethylation modifications that govern gene expression changes during cardiac development with striking temporal and lineage specificities, highlighting complex interactions in multiple cell populations during development of the vertebrate heart.
Assuntos
Dioxigenases/genética , Matriz Extracelular/metabolismo , Coração/fisiopatologia , Organogênese/genética , Proteínas de Peixe-Zebra/genética , Animais , Movimento Celular , Peixe-ZebraRESUMO
The Tet family of methylcytosine dioxygenases (Tet1, Tet2, and Tet3) convert 5-methylcytosine to 5-hydroxymethylcytosine. To date, functional overlap among Tet family members has not been examined systematically in the context of embryonic development. To clarify the potential for overlap among Tet enzymes during development, we mutated the zebrafish orthologs of Tet1, Tet2, and Tet3 and examined single-, double-, and triple-mutant genotypes. Here, we identify Tet2 and Tet3 as the major 5-methylcytosine dioxygenases in the zebrafish embryo and uncover a combined requirement for Tet2 and Tet3 in hematopoietic stem cell (HSC) emergence. We demonstrate that Notch signaling in the hemogenic endothelium is regulated by Tet2/3 prior to HSC emergence and show that restoring expression of the downstream gata2b/scl/runx1 transcriptional network can rescue HSCs in tet2/3 double mutant larvae. Our results reveal essential, overlapping functions for tet genes during embryonic development and uncover a requirement for 5hmC in regulating HSC production.
Assuntos
Dioxigenases/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Dioxigenases/genética , Desenvolvimento Embrionário , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/citologia , Receptores Notch/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
The transition zone is a specialized compartment found at the base of cilia, adjacent to the centriole distal end, where axonemal microtubules are heavily crosslinked to the surrounding membrane to form a barrier that gates the ciliary compartment. A number of ciliopathy molecules have been found to associate with the transition zone, but factors that directly recognize axonemal microtubules to specify transition zone assembly at the cilia base remain unclear. Here, through quantitative centrosome proteomics, we identify an axoneme-associated protein, CEP162 (KIAA1009), tethered specifically at centriole distal ends to promote transition zone assembly. CEP162 interacts with core transition zone components, and mediates their association with microtubules. Loss of CEP162 arrests ciliogenesis at the stage of transition zone assembly. Abolishing its centriolar tethering, however, allows CEP162 to stay on the growing end of the axoneme and ectopically assemble transition zone components at cilia tips. This generates extra-long cilia with strikingly swollen tips that actively release ciliary contents into the extracellular environment. CEP162 is thus an axoneme-recognition protein pre-tethered at centriole distal ends before ciliogenesis to promote and restrict transition zone formation specifically at the cilia base.
Assuntos
Adenosina Trifosfatases/metabolismo , Antígenos de Neoplasias/metabolismo , Axonema/metabolismo , Centríolos/metabolismo , Cílios/metabolismo , Proteínas dos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Células 3T3 , Adenosina Trifosfatases/genética , Animais , Antígenos de Neoplasias/genética , Proteínas de Ciclo Celular , Linhagem Celular , Centrossomo/metabolismo , Proteínas do Citoesqueleto , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Proteínas dos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Proteínas de Neoplasias/genética , Proteômica , Interferência de RNA , RNA Interferente PequenoRESUMO
DNA methylation is crucial for normal development and cellular differentiation in many large-genome eukaryotes. The small tropical freshwater fish Danio rerio (zebrafish) has recently emerged as a powerful system for the study of DNA methylation, especially in the context of development. This review summarizes our current knowledge of DNA methylation in zebrafish and provides evidence for the general conservation of this system with mammals. In addition, emerging strategies are highlighted that use the fish model to address some of the key unanswered questions in DNA methylation research.
Assuntos
Metilação de DNA , Peixe-Zebra/genética , AnimaisRESUMO
The ability to regulate gene expression in a cell-specific and temporally restricted manner provides a powerful means to test gene function, bypass the action of lethal genes, label subsets of cells for developmental studies, monitor subcellular structures, and target tissues for selective ablation or physiological analyses. The galactose-inducible system of yeast, mediated by the transcriptional activator Gal4 and its consensus UAS binding site, has proven to be a highly successful and versatile system for controlling transcriptional activation in Drosophila. It has also been used effectively, albeit in a more limited manner, in the mouse. While zebrafish has lagged behind other model systems in the widespread application of Gal4 transgenic approaches to modulate gene activity during development, recent technological advances are permitting rapid progress. Here we review Gal4-regulated genetic tools and discuss how they have been used in zebrafish as well as their potential drawbacks. We describe some exciting new directions, in large part afforded by the Tol2 transposition system, that are generating valuable new Gal4/UAS reagents for zebrafish research.
Assuntos
Galactose/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Peixe-Zebra/genética , Animais , Proteínas de Ligação a DNA , Drosophila/genética , Camundongos , Organismos Geneticamente Modificados , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Prior studies with transgenic zebrafish confirmed the functionality of the transcription factor Gal4 to drive expression of other genes under the regulation of upstream activator sequences (UAS). However, widespread application of this powerful binary system has been limited, in part, by relatively inefficient techniques for establishing transgenic zebrafish and by the inadequacy of Gal4 to effect high levels of expression from UAS-regulated genes. We have used the Tol2 transposition system to distribute a self-reporting gene/enhancer trap vector efficiently throughout the zebrafish genome. The vector uses the potent, hybrid transcription factor Gal4-VP16 to activate expression from a UAS:eGFP reporter cassette. In a pilot screen, stable transgenic lines were established that express eGFP in reproducible patterns encompassing a wide variety of tissues, including the brain, spinal cord, retina, notochord, cranial skeleton and muscle, and can transactivate other UAS-regulated genes. We demonstrate the utility of this approach to track Gal4-VP16 expressing migratory cells in UAS:Kaede transgenic fish, and to induce tissue-specific cell death using a bacterial nitroreductase gene under UAS control. The Tol2-mediated gene/enhancer trapping system together with UAS transgenic lines provides valuable tools for regulated gene expression and for targeted labeling and ablation of specific cell types and tissues during early zebrafish development.