RESUMO
Transcription in the early Drosophila blastoderm is coordinated by the collective action of hundreds of enhancers. Many genes are controlled by so-called 'shadow enhancers', which provide resilience to environment or genetic insult, allowing the embryo to robustly generate a precise transcriptional pattern. Emerging evidence suggests that many shadow enhancer pairs do not drive identical expression patterns, but the biological significance of this remains unclear. In this study, we characterize the shadow enhancer pair controlling the gene short gastrulation (sog). We removed either the intronic proximal enhancer or the upstream distal enhancer and monitored sog transcriptional kinetics. Notably, each enhancer differs in sog spatial expression, timing of activation and RNA Polymerase II loading rates. In addition, modeling of individual enhancer activities demonstrates that these enhancers integrate activation and repression signals differently. Whereas activation is due to the sum of the two enhancer activities, repression appears to depend on synergistic effects between enhancers. Finally, we examined the downstream signaling consequences resulting from the loss of either enhancer, and found changes in tissue patterning that can be explained by the differences in transcriptional kinetics measured.
Assuntos
Proteínas de Drosophila , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Drosophila/metabolismo , GastrulaçãoRESUMO
The Drosophila genome activator Vielfaltig (Vfl), also known as Zelda (Zld), is thought to prime enhancers for activation by patterning transcription factors (TFs). Such priming is accompanied by increased chromatin accessibility, but the mechanisms by which this occurs are poorly understood. Here, we analyze the effect of Zld on genome-wide nucleosome occupancy and binding of the patterning TF Dorsal (Dl). Our results show that early enhancers are characterized by an intrinsically high nucleosome barrier. Zld tackles this nucleosome barrier through local depletion of nucleosomes with the effect being dependent on the number and position of Zld motifs. Without Zld, Dl binding decreases at enhancers and redistributes to open regions devoid of enhancer activity. We propose that Zld primes enhancers by lowering the high nucleosome barrier just enough to assist TFs in accessing their binding motifs and promoting spatially controlled enhancer activation if the right patterning TFs are present. We envision that genome activators in general will utilize this mechanism to activate the zygotic genome in a robust and precise manner.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Cromatina/genética , Cromatina/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Estudos de Associação Genética , Proteínas Nucleares , Nucleossomos/genética , Regiões Promotoras Genéticas , Alinhamento de Sequência , Análise de Sequência de DNA , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
Transcription factors and microRNAs (miRNAs) are two important classes of trans-regulators in differential gene expression. Transcription factors occupy cis-regulatory motifs in DNA to activate or repress gene transcription, whereas miRNAs specifically pair with seed sites in target mRNAs to trigger mRNA decay or inhibit translation. Dynamic spatiotemporal expression patterns of transcription factors and miRNAs during development point to their stage- and tissue-specific functions. Recent studies have focused on miRNA functions during development; however, much remains to explore regarding how the expression of miRNAs is initiated and how dynamic miRNA expression patterns are achieved by transcriptional regulatory networks at different developmental stages. Here, we focused on the identification, regulation and function of miRNAs during the earliest stage of Drosophila development, when the maternal-to-zygotic transition (MZT) takes place. Eleven miRNA clusters comprise the first set of miRNAs activated in the blastoderm embryo. The transcriptional activator Zelda is required for their proper activation and regulation, and Zelda binding observed in genome-wide binding profiles is predictive of enhancer activity. In addition, other blastoderm transcription factors, comprising both activators and repressors, the activities of which are potentiated and coordinated by Zelda, contribute to the accurate temporal and spatial expression of these miRNAs, which are known to function in diverse developmental processes. Although previous genetic studies showed no early phenotypes upon loss of individual miRNAs, our analysis of the miR-1; miR-9a double mutant revealed defects in gastrulation, demonstrating the importance of co-activation of miRNAs by Zelda during the MZT.
Assuntos
Padronização Corporal/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , MicroRNAs/genética , Fatores de Transcrição/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Proteínas Nucleares , RNA Mensageiro Estocado/genética , Fatores de Tempo , Fatores de Transcrição/genética , Ativação Transcricional , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismoRESUMO
ERK controls gene expression in development, but mechanisms that link ERK activation to changes in transcription are not well understood. We used high-resolution analysis of signaling dynamics to study transcriptional interpretation of ERK signaling during Drosophila embryogenesis, at a stage when ERK induces transcription of intermediate neuroblasts defective (ind), a gene essential for patterning of the nerve cord. ERK induces ind by antagonizing its repression by Capicua (Cic), a transcription factor that acts as a sensor of receptor tyrosine kinases in animal development and human diseases. A recent study established that active ERK reduces the nuclear levels of Cic, but it remained unclear whether this is required for the induction of Cic target genes. We provide evidence that Cic binding sites within the regulatory DNA of ind control the spatial extent and the timing of ind expression. At the same time, we demonstrate that ERK induces ind before Cic levels in the nucleus are reduced. Based on this, we propose that ERK-dependent relief of gene repression by Cic is a two-step process, in which fast reduction of repressor activity is followed by slower changes in nuclear localization and overall protein levels. This may be a common feature of systems in which ERK induces genes by relief of transcriptional repression.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas HMGB/metabolismo , Proteínas de Homeodomínio/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Repressoras/metabolismo , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Padronização Corporal/fisiologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas HMGB/genética , Proteínas de Homeodomínio/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Microfluídica , Fosforilação/fisiologia , Proteínas Repressoras/genéticaRESUMO
Pattern formation in the developing embryo relies on key regulatory molecules, many of which are distributed in concentration gradients. For example, a gradient of BMP specifies cell fates along the dorsoventral axis in species ranging from flies to mammals. In Drosophila, a gradient of the BMP molecule Dpp gives rise to nested domains of target gene expression in the dorsal region of the embryo; however, the mechanisms underlying the differential response are not well understood, partly owing to an insufficient number of well-studied targets. Here we analyze how the Dpp gradient regulates expression of pannier (pnr), a candidate low-level Dpp target gene. We predicted that the pnr enhancer would contain high-affinity binding sites for the Dpp effector Smad transcription factors, which would be occupied in the presence of low-level Dpp. Unexpectedly, the affinity of Smad sites in the pnr enhancer was similar to those in the Race enhancer, a high-level Dpp target gene, suggesting that the affinity threshold mechanism plays a minimal role in the regulation of pnr. Our results indicate that a mechanism involving a conserved bipartite motif that is predicted to bind a homeodomain factor in addition to Smads and the Brinker repressor, establishes the pnr expression domain. Furthermore, the pnr enhancer has a highly complex structure that integrates cues not only from the dorsoventral axis, but also from the anteroposterior and terminal patterning systems in the blastoderm embryo.
Assuntos
Padronização Corporal/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Eletroforese em Gel de Poliacrilamida , Elementos Facilitadores Genéticos/genética , Hibridização In Situ , Mutagênese , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Smad/metabolismo , Fatores de Transcrição/genéticaRESUMO
Despite years of study, the precise mechanisms that control position-specific gene expression during development are not understood. Here, we analyze an enhancer element from the even skipped (eve) gene, which activates and positions two stripes of expression (stripes 3 and 7) in blastoderm stage Drosophila embryos. Previous genetic studies showed that the JAK-STAT pathway is required for full activation of the enhancer, whereas the gap genes hunchback (hb) and knirps (kni) are required for placement of the boundaries of both stripes. We show that the maternal zinc-finger protein Zelda (Zld) is absolutely required for activation, and present evidence that Zld binds to multiple non-canonical sites. We also use a combination of in vitro binding experiments and bioinformatics analysis to redefine the Kni-binding motif, and mutational analysis and in vivo tests to show that Kni and Hb are dedicated repressors that function by direct DNA binding. These experiments significantly extend our understanding of how the eve enhancer integrates positive and negative transcriptional activities to generate sharp boundaries in the early embryo.
Assuntos
Proteínas de Drosophila/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Cruzamentos Genéticos , Análise Mutacional de DNA , Proteínas de Ligação a DNA/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Genes Reporter , Proteínas de Homeodomínio/fisiologia , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Nucleares , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Transgenes , Técnicas do Sistema de Duplo-HíbridoRESUMO
In all animals, the initial events of embryogenesis are controlled by maternal gene products that are deposited into the developing oocyte. At some point after fertilization, control of embryogenesis is transferred to the zygotic genome in a process called the maternal-to-zygotic transition. During this time, many maternal RNAs are degraded and transcription of zygotic RNAs ensues. There is a long-standing question as to which factors regulate these events. The recent findings that microRNAs and Smaug mediate maternal transcript degradation have shed new light on this aspect of the problem. However, the transcription factor(s) that activate the zygotic genome remain elusive. The discovery that many of the early transcribed genes in Drosophila share a cis-regulatory heptamer motif, CAGGTAG and related sequences, collectively referred to as TAGteam sites raised the possibility that a dedicated transcription factor could interact with these sites to activate transcription. Here we report that the zinc-finger protein Zelda (Zld; Zinc-finger early Drosophila activator) binds specifically to these sites and is capable of activating transcription in transient transfection assays. Mutant embryos lacking zld are defective in cellular blastoderm formation, and fail to activate many genes essential for cellularization, sex determination and pattern formation. Global expression profiling confirmed that Zld has an important role in the activation of the early zygotic genome and suggests that Zld may also regulate maternal RNA degradation during the maternal-to-zygotic transition.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Genoma de Inseto/genética , Fatores de Transcrição/metabolismo , Dedos de Zinco , Zigoto/metabolismo , Animais , Blastoderma/citologia , Blastoderma/embriologia , Blastoderma/metabolismo , Padronização Corporal/genética , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Masculino , Proteínas Nucleares , Estabilidade de RNA , RNA Mensageiro Estocado/genética , RNA Mensageiro Estocado/metabolismo , Processos de Determinação Sexual , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Ativação Transcricional , Zigoto/citologia , Zigoto/crescimento & desenvolvimentoRESUMO
In past years, much attention has focused on the gene networks that regulate early developmental processes, but less attention has been paid to how multiple networks and processes are temporally coordinated. Recently the discovery of the transcriptional activator Zelda (Zld), which binds to CAGGTAG and related sequences present in the enhancers of many early-activated genes in Drosophila, hinted at a mechanism for how batteries of genes could be simultaneously activated. Here we use genome-wide binding and expression assays to identify Zld target genes in the early embryo with the goal of unraveling the gene circuitry regulated by Zld. We found that Zld binds to genes involved in early developmental processes such as cellularization, sex determination, neurogenesis, and pattern formation. In the absence of Zld, many target genes failed to be activated, while others, particularly the patterning genes, exhibited delayed transcriptional activation, some of which also showed weak and/or sporadic expression. These effects disrupted the normal sequence of patterning-gene interactions and resulted in highly altered spatial expression patterns, demonstrating the significance of a timing mechanism in early development. In addition, we observed prevalent overlap between Zld-bound regions and genomic "hotspot" regions, which are bound by many developmental transcription factors, especially the patterning factors. This, along with the finding that the most over-represented motif in hotspots, CAGGTA, is the Zld binding site, implicates Zld in promoting hotspot formation. We propose that Zld promotes timely and robust transcriptional activation of early-gene networks so that developmental events are coordinated and cell fates are established properly in the cellular blastoderm embryo.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Desenvolvimento Embrionário/genética , Redes Reguladoras de Genes , Fatores de Transcrição/genética , Ativação Transcricional/genética , Animais , Sítios de Ligação/genética , Blastoderma/embriologia , Blastoderma/crescimento & desenvolvimento , Padronização Corporal/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/genética , Proteínas Nucleares , Motivos de Nucleotídeos/genética , Regiões Promotoras Genéticas , Ligação Proteica/genética , Processos de Determinação Sexual/genética , Fatores de Transcrição/metabolismo , Zigoto/crescimento & desenvolvimentoRESUMO
The zygote has a daunting task ahead of itself; it must develop from a single cell (fertilized egg) into a fully functioning adult with a multitude of different cell types. In the beginning, the zygote has help from its mother, in the form of gene products deposited into the egg, but eventually, it must rely on its own resources to proceed through development. The transfer of developmental control from the mother to the embryo is called the maternal-to-zygotic transition (MZT). All animals undergo this transition, which is defined by two main processes-the degradation of maternal RNAs and the synthesis of new RNAs from the zygote's own genome. Here, we review the regulation of the MZT in Drosophila, but given the broad conservation of this essential process, much of the regulation is shared among metazoans.
Assuntos
Drosophila , Zigoto , Animais , Zigoto/metabolismo , Drosophila/genética , Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genoma , RNA Mensageiro/genética , RNA/metabolismo , Desenvolvimento Embrionário/genéticaRESUMO
Developmental enhancers bind transcription factors and dictate patterns of gene expression during development. Their molecular evolution can underlie phenotypical evolution, but the contributions of the evolutionary pathways involved remain little understood. Here, using mutation libraries in Drosophila melanogaster embryos, we observed that most point mutations in developmental enhancers led to changes in gene expression levels but rarely resulted in novel expression outside of the native pattern. In contrast, random sequences, often acting as developmental enhancers, drove expression across a range of cell types; random sequences including motifs for transcription factors with pioneer activity acted as enhancers even more frequently. Our findings suggest that the phenotypic landscapes of developmental enhancers are constrained by enhancer architecture and chromatin accessibility. We propose that the evolution of existing enhancers is limited in its capacity to generate novel phenotypes, whereas the activity of de novo elements is a primary source of phenotypic novelty.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Cromatina/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Evolução Molecular , Fenótipo , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Chromatin accessibility is integral to the process by which transcription factors (TFs) read out cis-regulatory DNA sequences, but it is difficult to differentiate between TFs that drive accessibility and those that do not. Deep learning models that learn complex sequence rules provide an unprecedented opportunity to dissect this problem. Using zygotic genome activation in Drosophila as a model, we analyzed high-resolution TF binding and chromatin accessibility data with interpretable deep learning and performed genetic validation experiments. We identify a hierarchical relationship between the pioneer TF Zelda and the TFs involved in axis patterning. Zelda consistently pioneers chromatin accessibility proportional to motif affinity, whereas patterning TFs augment chromatin accessibility in sequence contexts where they mediate enhancer activation. We conclude that chromatin accessibility occurs in two tiers: one through pioneering, which makes enhancers accessible but not necessarily active, and the second when the correct combination of TFs leads to enhancer activation.
RESUMO
The early Drosophila embryo is patterned by graded distributions of maternal transcription factors. Recent studies revealed that pattern formation by these graded signals depends on uniformly expressed transcriptional activators, such as Zelda. Removal of Zelda influences both the timing and the spatial expression domains for most of the genes controlled by maternal gradients. We demonstrate that some of these patterning defects, which range from temporal delay to loss of expression, can be rationalized with the use of a mathematical model based on cooperative binding of graded and uniform factors. This model makes a number of predictions, which we confirm experimentally by analyzing the expression of short gastrulation (sog), a gene that is controlled by a combination of the Dorsal morphogen gradient and Zelda. The proposed model suggests a general mechanism for the formation of nested gene expression domains, which is a hallmark of tissue patterning by morphogen gradients. According to this mechanism, the differential effects of a morphogen on its target genes can depend on their differential sensitivity to uniform factors.
Assuntos
Drosophila melanogaster/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Modelos Biológicos , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrião não Mamífero/citologia , Feminino , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
The early Drosophila embryo provides unique experimental advantages for addressing fundamental questions of gene regulation at multiple levels of organization, from individual gene loci to the entire genome. Using 1.5-h-old Drosophila embryos undergoing the first wave of genome activation,1 we detected â¼110 discrete "speckles" of RNA polymerase II (RNA Pol II) per nucleus, two of which were larger and localized to the histone locus bodies (HLBs).2,3 In the absence of the primary driver of Drosophila genome activation, the pioneer factor Zelda (Zld),1,4,5 70% fewer speckles were present; however, the HLBs tended to be larger than wild-type (WT) HLBs, indicating that RNA Pol II accumulates at the HLBs in the absence of robust early-gene transcription. We observed a uniform distribution of distances between active genes in the nuclei of both WT and zld mutant embryos, indicating that early co-regulated genes do not cluster into nuclear sub-domains. However, in instances whereby transcribing genes did come into close 3D proximity (within 400 nm), they were found to have distinct RNA Pol II speckles. In contrast to the emerging model whereby active genes are clustered to facilitate co-regulation and sharing of transcriptional resources, our data support an "individualist" model of gene control at early genome activation in Drosophila. This model is in contrast to a "collectivist" model, where active genes are spatially clustered and share transcriptional resources, motivating rigorous tests of both models in other experimental systems.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/metabolismo , RNA Polimerase II/genética , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Even though transcriptional repressors are studied with ever-increasing molecular resolution, the temporal aspects of gene repression remain poorly understood. Here, we address the dynamics of transcriptional repression by Capicua (Cic), which is essential for normal development and is commonly mutated in human cancers and neurodegenerative diseases.1,2 We report the speed limit for Cic-dependent gene repression based on live imaging and optogenetic perturbations in the early Drosophila embryo, where Cic was originally discovered.3 Our measurements of Cic concentration and intranuclear mobility, along with real-time monitoring of the activity of Cic target genes, reveal remarkably fast transcriptional repression within minutes of removing an optogenetic de-repressive signal. In parallel, quantitative analyses of transcriptional bursting of Cic target genes support a repression mechanism providing a fast-acting brake on burst generation. This work sets quantitative constraints on potential mechanisms for gene regulation by Cic.
Assuntos
Proteínas de Drosophila , Proteínas HMGB , Proteínas Repressoras , Animais , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Humanos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Connecting the developmental patterning of tissues to the mechanistic control of RNA polymerase II remains a long-term goal of developmental biology. Many key elements have been identified in the establishment of spatial-temporal control of transcription in the early Drosophila embryo, a model system for transcriptional regulation. The dorsal-ventral axis of the Drosophila embryo is determined by the graded distribution of Dorsal (Dl), a homolog of the nuclear factor κB (NF-κB) family of transcriptional activators found in humans [1, 2]. A second maternally deposited factor, Zelda (Zld), is uniformly distributed in the embryo and is thought to act as a pioneer factor, increasing enhancer accessibility for transcription factors, such as Dl [3-9]. Here, we utilized the MS2 live imaging system to evaluate the expression of the Dl target gene short gastrulation (sog) to better understand how a pioneer factor affects the kinetic parameters of transcription. Our experiments indicate that Zld modifies probability of activation, the timing of this activation, and the rate at which transcription occurs. Our results further show that this effective rate increase is due to an increased accumulation of Dl at the site of transcription, suggesting that transcription factor "hubs" induced by Zld [10] functionally regulate transcription.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/genética , Transcrição Gênica , Ativação Transcricional , Animais , Núcleo Celular/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Elementos Facilitadores Genéticos , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The thirteen nuclear cleavages that give rise to the Drosophila blastoderm are some of the fastest known cell cycles [1]. Surprisingly, the fertilized egg is provided with at most one-third of the dNTPs needed to complete the thirteen rounds of DNA replication [2]. The rest must be synthesized by the embryo, concurrent with cleavage divisions. What is the reason for the limited supply of DNA building blocks? We propose that frugal control of dNTP synthesis contributes to the well-characterized deceleration of the cleavage cycles and is needed for robust accumulation of zygotic gene products. In support of this model, we demonstrate that when the levels of dNTPs are abnormally high, nuclear cleavages fail to sufficiently decelerate, the levels of zygotic transcription are dramatically reduced, and the embryo catastrophically fails early in gastrulation. Our work reveals a direct connection between metabolism, the cell cycle, and zygotic transcription.
Assuntos
Ciclo Celular , Drosophila/embriologia , Zigoto/citologia , Animais , Drosophila/citologia , Drosophila/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Zigoto/metabolismoRESUMO
Body size is a tightly regulated phenotype in metazoans that depends on both intrinsic and extrinsic factors. While signaling pathways are known to control organ and body size, the downstream effectors that mediate their effects remain poorly understood. In the nematode Caenorhabditis elegans, a Bone Morphogenetic Protein (BMP)-related signaling pathway is the major regulator of growth and body size. We investigated the transcriptional network through which the BMP pathway regulates body size and identified cuticle collagen genes as major effectors of growth control. We demonstrate that cuticle collagens can act as positive regulators (col-41), negative regulators (col-141), or dose-sensitive regulators (rol-6) of body size. Moreover, we find a requirement of BMP signaling for stage-specific expression of cuticle collagen genes. We show that the Smad signal transducers directly bind conserved Smad-binding elements in regulatory regions of col-141 and col-142, but not of col-41 Hence, cuticle collagen genes may be directly and indirectly regulated via the BMP pathway. Our work thus connects a conserved signaling pathway with its critical downstream effectors, advancing insight into how body size is specified. Since collagen mutations and misregulation are implicated in numerous human genetic disorders and injury sequelae, understanding how collagen gene expression is regulated has broad implications.
Assuntos
Tamanho Corporal/genética , Proteínas Morfogenéticas Ósseas/genética , Colágeno/genética , Redes Reguladoras de Genes/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Regulação da Expressão Gênica no Desenvolvimento , Transdução de Sinais , Fator de Crescimento Transformador beta/genéticaRESUMO
To understand the relationship between an enhancer DNA sequence and quantitative gene expression, thermodynamics-driven mathematical models of transcription are often employed. These "sequence-to-expression" models can describe an incomplete or even incorrect set of regulatory relationships if the parameter space is not searched systematically. Here, we focus on an enhancer of the Drosophila gene ind and demonstrate how a systematic search of parameter space can reveal a more comprehensive picture of a gene's regulatory mechanisms, resolve outstanding ambiguities, and suggest testable hypotheses. We describe an approach that generates an ensemble of ind models; all of these models are technically acceptable solutions to the sequence-to-expression problem in light of wild-type data, and some represent mechanistically distinct hypotheses about the regulation of ind. This ensemble can be restricted to biologically plausible models using requirements gleaned from in vivo perturbation experiments. Biologically plausible models make unique predictions about how specific ind enhancer sequences affect ind expression; we validate these predictions in vivo through site mutagenesis in transgenic Drosophila embryos.
RESUMO
Zygotic genome activation (ZGA) is a major genome programming event whereby the cells of the embryo begin to adopt specified fates. Experiments in Drosophila and zebrafish have revealed that ZGA depends on transcription factors that provide large-scale control of gene expression by direct and specific binding to gene regulatory sequences. Zelda (Zld) plays such a role in the Drosophila embryo, where it has been shown to control the action of patterning signals; however, the mechanisms underlying this effect remain largely unclear. A recent model proposed that Zld binding sites act as quantitative regulators of the spatiotemporal expression of genes activated by Dorsal (Dl), the morphogen that patterns the dorsoventral axis. Here we tested this model experimentally, using enhancers of brinker (brk) and short gastrulation (sog), both of which are directly activated by Dl, but at different concentration thresholds. In agreement with the model, we show that there is a clear positive correlation between the number of Zld binding sites and the spatial domain of enhancer activity. Likewise, the timing of expression could be advanced or delayed. We present evidence that Zld facilitates binding of Dl to regulatory DNA, and that this is associated with increased chromatin accessibility. Importantly, the change in chromatin accessibility is strongly correlated with the change in Zld binding, but not Dl. We propose that the ability of genome activators to facilitate readout of transcriptional input is key to widespread transcriptional induction during ZGA.