RESUMO
The Drosophila brain is a frequently used model in neuroscience. Single-cell transcriptome analysis1-6, three-dimensional morphological classification7 and electron microscopy mapping of the connectome8,9 have revealed an immense diversity of neuronal and glial cell types that underlie an array of functional and behavioural traits in the fly. The identities of these cell types are controlled by gene regulatory networks (GRNs), involving combinations of transcription factors that bind to genomic enhancers to regulate their target genes. Here, to characterize GRNs at the cell-type level in the fly brain, we profiled the chromatin accessibility of 240,919 single cells spanning 9 developmental timepoints and integrated these data with single-cell transcriptomes. We identify more than 95,000 regulatory regions that are used in different neuronal cell types, of which 70,000 are linked to developmental trajectories involving neurogenesis, reprogramming and maturation. For 40 cell types, uniquely accessible regions were associated with their expressed transcription factors and downstream target genes through a combination of motif discovery, network inference and deep learning, creating enhancer GRNs. The enhancer architectures revealed by DeepFlyBrain lead to a better understanding of neuronal regulatory diversity and can be used to design genetic driver lines for cell types at specific timepoints, facilitating their characterization and manipulation.
Assuntos
Drosophila , Regulação da Expressão Gênica , Animais , Encéfalo/metabolismo , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes/genética , Fatores de Transcrição/metabolismoRESUMO
The homologous genes GTPBP1 and GTPBP2 encode GTP-binding proteins 1 and 2, which are involved in ribosomal homeostasis. Pathogenic variants in GTPBP2 were recently shown to be an ultra-rare cause of neurodegenerative or neurodevelopmental disorders (NDDs). Until now, no human phenotype has been linked to GTPBP1. Here, we describe individuals carrying bi-allelic GTPBP1 variants that display an identical phenotype with GTPBP2 and characterize the overall spectrum of GTP-binding protein (1/2)-related disorders. In this study, 20 individuals from 16 families with distinct NDDs and syndromic facial features were investigated by whole-exome (WES) or whole-genome (WGS) sequencing. To assess the functional impact of the identified genetic variants, semi-quantitative PCR, western blot, and ribosome profiling assays were performed in fibroblasts from affected individuals. We also investigated the effect of reducing expression of CG2017, an ortholog of human GTPBP1/2, in the fruit fly Drosophila melanogaster. Individuals with bi-allelic GTPBP1 or GTPBP2 variants presented with microcephaly, profound neurodevelopmental impairment, pathognomonic craniofacial features, and ectodermal defects. Abnormal vision and/or hearing, progressive spasticity, choreoathetoid movements, refractory epilepsy, and brain atrophy were part of the core phenotype of this syndrome. Cell line studies identified a loss-of-function (LoF) impact of the disease-associated variants but no significant abnormalities on ribosome profiling. Reduced expression of CG2017 isoforms was associated with locomotor impairment in Drosophila. In conclusion, bi-allelic GTPBP1 and GTPBP2 LoF variants cause an identical, distinct neurodevelopmental syndrome. Mutant CG2017 knockout flies display motor impairment, highlighting the conserved role for GTP-binding proteins in CNS development across species.
Assuntos
Proteínas de Ligação ao GTP , Microcefalia , Malformações do Sistema Nervoso , Transtornos do Neurodesenvolvimento , Animais , Humanos , Drosophila melanogaster/genética , GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP/genética , Transtornos do Neurodesenvolvimento/genética , Fenótipo , Proteínas de Drosophila/genéticaRESUMO
During neuronal development, extensive changes to chromatin states occur to regulate lineage-specific gene expression. The molecular factors underlying the repression of non-neuronal genes in differentiated neurons are poorly characterised. The Mi2/NuRD complex is a multiprotein complex with nucleosome remodelling and histone deacetylase activity. Whilst NuRD has previously been implicated in the development of nervous system tissues, the precise nature of the gene expression programmes that it coordinates is ill-defined. Furthermore, evidence from several species suggests that Mi-2 may be incorporated into multiple complexes that may not possess histone deacetylase activity. We show that Mi-2 activity is required for suppressing ectopic expression of germline genes in neurons independently of HDAC1/NuRD, whilst components of NuRD, including Mi-2, regulate neural gene expression to ensure proper development of the larval nervous system. We find that Mi-2 binding in the genome is dynamic during neuronal maturation, and Mi-2-mediated repression of ectopic gene expression is restricted to the early stages of neuronal development, indicating that Mi-2/NuRD is required for establishing stable neuronal transcriptomes during the early stages of neuronal differentiation.
Assuntos
Expressão Ectópica do Gene , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Histona Desacetilases/metabolismo , Cromatina/genética , NucleossomosRESUMO
The epidermis of Caenorhabditis elegans is an essential tissue for survival because it contributes to the formation of the cuticle barrier as well as facilitating developmental progression and animal growth. Most of the epidermis consists of the hyp7 hypodermal syncytium, the nuclei of which are largely generated by the seam cells, which exhibit stem cell-like behaviour during development. How seam cell progenitors differ transcriptionally from the differentiated hypodermis is poorly understood. Here, we introduce Targeted DamID (TaDa) in C. elegans as a method for identifying genes expressed within a tissue of interest without cell isolation. We show that TaDa signal enrichment profiles can be used to identify genes transcribed in the epidermis and use this method to resolve differences in gene expression between the seam cells and the hypodermis. Finally, we predict and functionally validate new transcription and chromatin factors acting in seam cell development. These findings provide insights into cell type-specific gene expression profiles likely associated with epidermal cell fate patterning.
Assuntos
Células Epidérmicas/citologia , Células Epidérmicas/metabolismo , Perfilação da Expressão Gênica/métodos , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Diferenciação Celular , Linhagem da Célula , Cromatina/genética , Cromatina/metabolismo , Epiderme/crescimento & desenvolvimento , Epiderme/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The interaction of proteins and RNA with chromatin underlies the regulation of gene expression. The ability to profile easily these interactions is fundamental for understanding chromatin biology in vivo DNA adenine methyltransferase identification (DamID) profiles genome-wide protein-DNA interactions without antibodies, fixation or protein pull-downs. Recently, DamID has been adapted for applications beyond simple assaying of protein-DNA interactions, such as for studying RNA-chromatin interactions, chromatin accessibility and long-range chromosome interactions. Here, we provide an overview of DamID and introduce improvements to the technology, discuss their applications and compare alternative methodologies.
Assuntos
Cromatina/metabolismo , Metilação de DNA , Técnicas Genéticas , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Animais , Sítios de Ligação/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Genômica , Humanos , Camundongos , Ligação Proteica , Ribossomos/metabolismo , Análise de Sequência de DNARESUMO
CTP synthase (CTPsyn) is essential for the biosynthesis of pyrimidine nucleotides. It has been shown that CTPsyn is incorporated into a novel cytoplasmic structure which has been termed the cytoophidium. Here, we report that Myc regulates cytoophidium formation during Drosophila oogenesis. We have found that Myc protein levels correlate with cytoophidium abundance in follicle epithelia. Reducing Myc levels results in cytoophidium loss and small nuclear size in follicle cells, while overexpression of Myc increases the length of cytoophidia and the nuclear size of follicle cells. Ectopic expression of Myc induces cytoophidium formation in late stage follicle cells. Furthermore, knock-down of CTPsyn is sufficient to suppress the overgrowth phenotype induced by Myc overexpression, suggesting CTPsyn acts downstream of Myc and is required for Myc-mediated cell size control. Taken together, our data suggest a functional link between Myc, a renowned oncogene, and the essential nucleotide biosynthetic enzyme CTPsyn.
Assuntos
Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Animais , Tamanho Celular , Drosophila melanogaster/citologia , Feminino , Técnicas de Silenciamento de Genes , Folículo Ovariano/citologia , Folículo Ovariano/enzimologia , FenótipoRESUMO
Determining the mechanisms of enzymatic regulation is central to the study of cellular metabolism. Regulation of enzyme activity via polymerization-mediated strategies has been shown to be widespread, and plays a vital role in mediating cellular homeostasis. In this review, we begin with an overview of the filamentation of CTP synthase, which forms filamentous structures termed cytoophidia. We then highlight other important examples of the phenomenon. Moreover, we discuss recent data relating to the regulation of enzyme activity by compartmentalization into cytoophidia. Finally, we hypothesize potential roles for enzyme filament formation in the regulation of metabolism, development and disease.
Assuntos
Carbono-Nitrogênio Ligases/metabolismo , Multimerização Proteica/fisiologia , Animais , Carbono-Nitrogênio Ligases/química , Eucariotos/enzimologia , HumanosRESUMO
The transcription factor Shavenbaby (Svb), the only member of the OvoL family in Drosophila, controls the fate of various epithelial embryonic cells and adult stem cells. Post-translational modification of Svb produces two protein isoforms, Svb-ACT and Svb-REP, which promote adult intestinal stem cell renewal or differentiation, respectively. To define Svb mode of action, we used engineered cell lines and develop an unbiased method to identify Svb target genes across different contexts. Within a given cell type, Svb-ACT and Svb-REP antagonistically regulate the expression of a set of target genes, binding specific enhancers whose accessibility is constrained by chromatin landscape. Reciprocally, Svb-REP can influence local chromatin marks of active enhancers to help repressing target genes. Along the intestinal lineage, the set of Svb target genes progressively changes, together with chromatin accessibility. We propose that Svb-ACT-to-REP transition promotes enterocyte differentiation of intestinal stem cells through direct gene regulation and chromatin remodeling.
RESUMO
Critical periods are windows of heightened plasticity occurring during neurodevelopment. Alterations in neural activity during these periods can cause long-lasting changes in the structure, connectivity, and intrinsic excitability of neurons, which may contribute to the pathology of neurodevelopmental disorders. However, endogenous regulators of critical periods remain poorly defined. Here, we study this issue using a fruit fly (Drosophila) model of an early-onset movement disorder caused by BK potassium channel gain of function (BK GOF). Deploying a genetic method to place robust expression of GOF BK channels under spatiotemporal control, we show that adult-stage neuronal expression of GOF BK channels minimally disrupts fly movement. In contrast, limiting neuronal expression of GOF BK channels to a short window during late neurodevelopment profoundly impairs locomotion and limb kinematics in resulting adult flies. During this critical period, BK GOF perturbs synaptic localization of the active zone protein Bruchpilot and reduces excitatory neurotransmission. Conversely, enhancing neural activity specifically during development rescues motor defects in BK GOF flies. Collectively, our results reveal a critical developmental period for limb control in Drosophila that is influenced by BK channels and suggest that BK GOF causes movement disorders by disrupting activity-dependent aspects of synaptic development.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Canais de Potássio Ativados por Cálcio de Condutância Alta , Locomoção , Animais , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Drosophila melanogaster/fisiologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Neurônios/metabolismo , Neurônios/fisiologiaRESUMO
Retinoblastoma (RB) proteins are highly conserved transcriptional regulators that play important roles during development by regulating cell-cycle gene expression. RBL2 dysfunction has been linked to a severe neurodevelopmental disorder. However, to date, clinical features have only been described in six individuals carrying five biallelic predicted loss of function (pLOF) variants. To define the phenotypic effects of RBL2 mutations in detail, we identified and clinically characterized a cohort of 28 patients from 18 families carrying LOF variants in RBL2 , including fourteen new variants that substantially broaden the molecular spectrum. The clinical presentation of affected individuals is characterized by a range of neurological and developmental abnormalities. Global developmental delay and intellectual disability were uniformly observed, ranging from moderate to profound and involving lack of acquisition of key motor and speech milestones in most patients. Frequent features included postnatal microcephaly, infantile hypotonia, aggressive behaviour, stereotypic movements and non-specific dysmorphic features. Common neuroimaging features were cerebral atrophy, white matter volume loss, corpus callosum hypoplasia and cerebellar atrophy. In parallel, we used the fruit fly, Drosophila melanogaster , to investigate how disruption of the conserved RBL2 orthologueue Rbf impacts nervous system function and development. We found that Drosophila Rbf LOF mutants recapitulate several features of patients harboring RBL2 variants, including alterations in the head and brain morphology reminiscent of microcephaly, and perturbed locomotor behaviour. Surprisingly, in addition to its known role in controlling tissue growth during development, we find that continued Rbf expression is also required in fully differentiated post-mitotic neurons for normal locomotion in Drosophila , and that adult-stage neuronal re-expression of Rbf is sufficient to rescue Rbf mutant locomotor defects. Taken together, this study provides a clinical and experimental basis to understand genotype-phenotype correlations in an RBL2 -linked neurodevelopmental disorder and suggests that restoring RBL2 expression through gene therapy approaches may ameliorate aspects of RBL2 LOF patient symptoms.
RESUMO
The processes that drive naive multipotent stem cells towards fully differentiated fates are increasingly well understood. However, once differentiated, the mechanisms and molecular factors involved in maintaining differentiated states and associated transcriptomes are less well studied. Neurons are a post-mitotic cell-type with highly specialised functions that largely lack the capacity for renewal. Therefore, neuronal cell identities and the transcriptional states that underpin them are locked into place by active mechanisms that prevent lineage reversion/dedifferentiation and repress cell cycling. Furthermore, individual neurons may be very long-lived, so these mechanisms must be sufficient to ensure the fidelity of neuronal transcriptomes over long time periods. This Review aims to provide an overview of recent progress in understanding how neuronal cell fate and associated gene expression are maintained and the transcriptional regulators that are involved. Maintenance of neuronal fate and subtype specification are discussed, as well as the activating and repressive mechanisms involved. The relevance of these processes to disease states, such as brain cancers and neurodegeneration is outlined. Finally, outstanding questions and hypotheses in this field are proposed.
Assuntos
Neurônios , Neurônios/metabolismo , Diferenciação Celular/genéticaRESUMO
The ability to control transgene expression, both spatially and temporally, is essential for studying model organisms. In Drosophila, spatial control is primarily provided by the GAL4/UAS system, whilst temporal control relies on a temperature-sensitive GAL80 (which inhibits GAL4) and drug-inducible systems. However, these are not ideal. Shifting temperature can impact on many physiological and behavioural traits, and the current drug-inducible systems are either leaky, toxic, incompatible with existing GAL4-driver lines, or do not generate effective levels of expression. Here, we describe the auxin-inducible gene expression system (AGES). AGES relies on the auxin-dependent degradation of a ubiquitously expressed GAL80, and therefore, is compatible with existing GAL4-driver lines. Water-soluble auxin is added to fly food at a low, non-lethal, concentration, which induces expression comparable to uninhibited GAL4 expression. The system works in both larvae and adults, providing a stringent, non-lethal, cost-effective, and convenient method for temporally controlling GAL4 activity in Drosophila.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Animais Geneticamente Modificados , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Expressão Gênica , Ácidos Indolacéticos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Targeted DamID (TaDa) is an increasingly popular method of generating cell-type-specific DNA-binding profiles in vivo. Although sensitive and versatile, TaDa requires the generation of new transgenic fly lines for every protein that is profiled, which is both time-consuming and costly. Here, we describe the FlyORF-TaDa system for converting an existing FlyORF library of inducible open reading frames (ORFs) to TaDa lines via a genetic cross, with recombinant progeny easily identifiable by eye color. Profiling the binding of the H3K36me3-associated chromatin protein MRG15 in larval neural stem cells using both FlyORF-TaDa and conventional TaDa demonstrates that new lines generated using this system provide accurate and highly reproducible DamID-binding profiles. Our data further show that MRG15 binds to a subset of active chromatin domains in vivo. Courtesy of the large coverage of the FlyORF library, the FlyORF-TaDa system enables the easy creation of TaDa lines for 74% of all transcription factors and chromatin-modifying proteins within the Drosophila genome.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Cromatina , Proteínas Cromossômicas não Histona , DNA , Metilação de DNA , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Ligação ProteicaRESUMO
Coordination of stem cell function by local and niche-derived signals is essential to preserve adult tissue homeostasis and organismal health. The vasculature is a prominent component of multiple stem cell niches. However, its role in adult intestinal homeostasis remains largely understudied. Here we uncover a previously unrecognised crosstalk between adult intestinal stem cells in Drosophila and the vasculature-like tracheal system, which is essential for intestinal regeneration. Following damage to the intestinal epithelium, gut-derived reactive oxygen species activate tracheal HIF-1α and bidirectional FGF/FGFR signalling, leading to reversible remodelling of gut-associated terminal tracheal cells and intestinal stem cell proliferation following damage. Unexpectedly, reactive oxygen species-induced adult tracheal plasticity involves downregulation of the tracheal specification factor trachealess (trh) and upregulation of IGF2 messenger RNA-binding protein (IGF2BP2/Imp). Our results reveal an intestine-vasculature inter-organ communication programme that is essential to adapt the stem cell response to the proliferative demands of the intestinal epithelium.
Assuntos
Adaptação Fisiológica/fisiologia , Células-Tronco Adultas/metabolismo , Homeostase/fisiologia , Células-Tronco/metabolismo , Animais , Drosophila/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Nicho de Células-Tronco/fisiologiaRESUMO
Condensin complexes are essential for mitotic chromosome assembly and segregation during cell divisions, however, little is known about their functions in post-mitotic cells. Here we report a role for the condensin I subunit Cap-G in Drosophila neurons. We show that, despite not requiring condensin for mitotic chromosome compaction, post-mitotic neurons express Cap-G. Knockdown of Cap-G specifically in neurons (from their birth onwards) results in developmental arrest, behavioural defects, and dramatic gene expression changes, including reduced expression of a subset of neuronal genes and aberrant expression of genes that are not normally expressed in the developing brain. Knockdown of Cap-G in mature neurons results in similar phenotypes but to a lesser degree. Furthermore, we see dynamic binding of Cap-G at distinct loci in progenitor cells and differentiated neurons. Therefore, Cap-G is essential for proper gene expression in neurons and plays an important role during the early stages of neuronal development.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Mitose/genética , Complexos Multiproteicos/metabolismo , Neurônios/citologia , Adenosina Trifosfatases/genética , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Masculino , Complexos Multiproteicos/genéticaRESUMO
During development eukaryotic gene expression is coordinated by dynamic changes in chromatin structure. Measurements of accessible chromatin are used extensively to identify genomic regulatory elements. Whilst chromatin landscapes of pluripotent stem cells are well characterised, chromatin accessibility changes in the development of somatic lineages are not well defined. Here we show that cell-specific chromatin accessibility data can be produced via ectopic expression of E. coli Dam methylase in vivo, without the requirement for cell-sorting (CATaDa). We have profiled chromatin accessibility in individual cell-types of Drosophila neural and midgut lineages. Functional cell-type-specific enhancers were identified, as well as novel motifs enriched at different stages of development. Finally, we show global changes in the accessibility of chromatin between stem-cells and their differentiated progeny. Our results demonstrate the dynamic nature of chromatin accessibility in somatic tissues during stem cell differentiation and provide a novel approach to understanding gene regulatory mechanisms underlying development.
Assuntos
Diferenciação Celular , Cromatina/metabolismo , Drosophila/embriologia , Epigênese Genética , Células-Tronco Pluripotentes/fisiologia , Animais , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Coloração e RotulagemRESUMO
The interaction of proteins with chromatin is fundamental for several essential cellular processes. During the development of an organism, genes must to be tightly regulated both temporally and spatially. This is achieved through the action of chromatin-binding proteins such as transcription factors, histone modifiers, nucleosome remodelers, and lamins. Furthermore, protein-DNA interactions are important in the adult, where their perturbation can lead to disruption of homeostasis, metabolic dysregulation, and diseases such as cancer. Understanding the nature of these interactions is of paramount importance in almost all areas of molecular biological research. In recent years, DNA adenine methyltransferase identification (DamID) has emerged as one of the most comprehensive and versatile methods available for profiling protein-DNA interactions on a genomic scale. DamID has been used to map a variety of chromatin-binding proteins in several model organisms and has the potential for continued adaptation and application in the field of genomic biology. WIREs Dev Biol 2016, 5:25-37. doi: 10.1002/wdev.205 For further resources related to this article, please visit the WIREs website.
Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Técnicas de Sonda Molecular , Animais , Cromatina/fisiologia , Cromatina/ultraestrutura , Imunoprecipitação da Cromatina , Epigênese Genética , Humanos , Anotação de Sequência Molecular , Ligação Proteica , DNA Metiltransferases Sítio Específica (Adenina-Específica)/fisiologiaRESUMO
The essential metabolic enzyme CTP synthase (CTPsyn) can be compartmentalised to form an evolutionarily-conserved intracellular structure termed the cytoophidium. Recently, it has been demonstrated that the enzymatic activity of CTPsyn is attenuated by incorporation into cytoophidia in bacteria and yeast cells. Here we demonstrate that CTPsyn is regulated in a similar manner in Drosophila tissues in vivo. We show that cytoophidium formation occurs during nutrient deprivation in cultured cells, as well as in quiescent and starved neuroblasts of the Drosophila larval central nervous system. We also show that cytoophidia formation is reversible during neurogenesis, indicating that filament formation regulates pyrimidine synthesis in a normal developmental context. Furthermore, our global metabolic profiling demonstrates that CTPsyn overexpression does not significantly alter CTPsyn-related enzymatic activity, suggesting that cytoophidium formation facilitates metabolic stabilisation. In addition, we show that overexpression of CTPsyn only results in moderate increase of CTP pool in human stable cell lines. Together, our study provides experimental evidence, and a mathematical model, for the hypothesis that inactive CTPsyn is incorporated into cytoophidia.