RESUMEN
How complex organs coordinate cellular morphogenetic events to achieve three-dimensional (3D) form is a central question in development. The question is uniquely tractable in the late Drosophila pupal retina, where cells maintain stereotyped contacts as they elaborate the specialized cytoskeletal structures that pattern the apical, basal and longitudinal planes of the epithelium. In this study, we combined cell type-specific genetic manipulation of the cytoskeletal regulator Abelson (Abl) with 3D imaging to explore how the distinct cellular morphogenetic programs of photoreceptors and interommatidial pigment cells (IOPCs) organize tissue pattern to support retinal integrity. Our experiments show that photoreceptor and IOPC terminal differentiation is unexpectedly interdependent, connected by an intercellular feedback mechanism that coordinates and promotes morphogenetic change across orthogonal tissue planes to ensure correct 3D retinal pattern. We propose that genetic regulation of specialized cellular differentiation programs combined with inter-plane mechanical feedback confers spatial coordination to achieve robust 3D tissue morphogenesis.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila melanogaster/genética , Proteínas de Drosophila/genética , Pupa , Retroalimentación , Retina , Morfogénesis/genéticaRESUMEN
How complex three-dimensional (3D) organs coordinate cellular morphogenetic events to achieve the correct final form is a central question in development. The question is uniquely tractable in the late Drosophila pupal retina where cells maintain stereotyped contacts as they elaborate the specialized cytoskeletal structures that pattern the apical, basal and longitudinal planes of the epithelium. In this study, we combined cell type-specific genetic manipulation of the cytoskeletal regulator Abelson (Abl) with 3D imaging to explore how the distinct cellular morphogenetic programs of photoreceptors and interommatidial pigment cells coordinately organize tissue pattern to support retinal integrity. Our experiments revealed an unanticipated intercellular feedback mechanism whereby correct cellular differentiation of either cell type can non-autonomously induce cytoskeletal remodeling in the other Abl mutant cell type, restoring retinal pattern and integrity. We propose that genetic regulation of specialized cellular differentiation programs combined with inter-plane mechanical feedback confers spatial coordination to achieve robust 3D tissue morphogenesis.
RESUMEN
Cell state transitions are often triggered by large changes in the concentrations of transcription factors and therefore large differences in their stoichiometric ratios. Whether cells can elicit transitions using modest changes in the ratios of co-expressed factors is unclear. Here, we investigate how cells in the Drosophila eye resolve state transitions by quantifying the expression dynamics of the ETS transcription factors Pnt and Yan. Eye progenitor cells maintain a relatively constant ratio of Pnt/Yan protein, despite expressing both proteins with pulsatile dynamics. A rapid and sustained twofold increase in the Pnt/Yan ratio accompanies transitions to photoreceptor fates. Genetic perturbations that modestly disrupt the Pnt/Yan ratio produce fate transition defects consistent with the hypothesis that transitions are normally driven by a twofold shift in the ratio. A biophysical model based on cooperative Yan-DNA binding coupled with non-cooperative Pnt-DNA binding illustrates how twofold ratio changes could generate ultrasensitive changes in target gene transcription to drive fate transitions. Thus, coupling cell state transitions to the Pnt/Yan ratio sensitizes the system to modest fold-changes, conferring robustness and ultrasensitivity to the developmental program.
Asunto(s)
Proteínas de Drosophila , Factores de Transcripción , Animales , Factores de Transcripción/metabolismo , Drosophila/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Represoras/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas del Ojo/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , ADNRESUMEN
Spatiotemporally precise and robust cell fate transitions, which depend on specific signaling cues, are fundamental to the development of appropriately patterned tissues. The fidelity and precision with which photoreceptor fates are recruited in the Drosophila eye exemplifies these principles. The fly eye consists of a highly ordered array of ~750 ommatidia, each of which contains eight distinct photoreceptors, R1-R8, specified sequentially in a precise spatial pattern. Recruitment of R1-R7 fates requires reiterative receptor tyrosine kinase / mitogen activated protein kinase (MAPK) signaling mediated by the transcriptional effector Pointed (Pnt). However the overall signaling levels experienced by R2-R5 cells are distinct from those experienced by R1, R6 and R7. A relay mechanism between two Pnt isoforms initiated by MAPK activation directs the universal transcriptional response. Here we ask how the generic Pnt response is tailored to these two rounds of photoreceptor fate transitions. We find that during R2-R5 specification PntP2 is coexpressed with a closely related but previously uncharacterized isoform, PntP3. Using CRISPR/Cas9-generated isoform specific null alleles we show that under otherwise wild type conditions, R2-R5 fate specification is robust to loss of either PntP2 or PntP3, and that the two activate pntP1 redundantly; however under conditions of reduced MAPK activity, both are required. Mechanistically, our data suggest that intrinsic activity differences between PntP2 and PntP3, combined with positive and unexpected negative transcriptional auto- and cross-regulation, buffer first-round fates against conditions of compromised RTK signaling. In contrast, in a mechanism that may be adaptive to the stronger signaling environment used to specify R1, R6 and R7 fates, the Pnt network resets to a simpler topology in which PntP2 uniquely activates pntP1 and auto-activates its own transcription. We propose that differences in expression patterns, transcriptional activities and regulatory interactions between Pnt isoforms together facilitate context-appropriate cell fate specification in different signaling environments.
Asunto(s)
Diferenciación Celular/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Sistema de Señalización de MAP Quinasas/genética , Proteínas del Tejido Nervioso/metabolismo , Células Fotorreceptoras de Invertebrados/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Alelos , Animales , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Redes Reguladoras de Genes , Modelos Animales , Proteínas del Tejido Nervioso/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas/genética , Análisis Espacio-Temporal , Factores de Transcripción/genéticaRESUMEN
During development, transcriptional complexes at enhancers regulate gene expression in complex spatiotemporal patterns. To achieve robust expression without spurious activation, the affinity and specificity of transcription factor-DNA interactions must be precisely balanced. Protein-protein interactions among transcription factors are also critical, yet how their affinities impact enhancer output is not understood. The Drosophila transcription factor Yan provides a well-suited model to address this, as its function depends on the coordinated activities of two independent and essential domains: the DNA-binding ETS domain and the self-associating SAM domain. To explore how protein-protein affinity influences Yan function, we engineered mutants that increase SAM affinity over four orders of magnitude. This produced a dramatic subcellular redistribution of Yan into punctate structures, reduced repressive output and compromised survival. Cell-type specification and genetic interaction defects suggest distinct requirements for polymerization in different regulatory decisions. We conclude that tuned protein-protein interactions enable the dynamic spectrum of complexes that are required for proper regulation.
Asunto(s)
ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas del Ojo/metabolismo , Polimerizacion , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Alelos , Animales , Linaje de la Célula , Núcleo Celular/metabolismo , Modelos Biológicos , Mutagénesis/genética , Mutación/genética , Células Fotorreceptoras de Invertebrados/citología , Células Fotorreceptoras de Invertebrados/metabolismo , Agregado de Proteínas , Unión Proteica , Transducción de Señal , Transcripción GenéticaRESUMEN
The acquisition of cellular identity during development depends on precise spatiotemporal regulation of gene expression, with combinatorial interactions between transcription factors, accessory proteins and the basal transcription machinery together translating complex signaling inputs into appropriate gene expression outputs. The opposing repressive and activating inputs of the Drosophila ETS family transcription factors Yan and Pointed orchestrate numerous cell fate transitions downstream of receptor tyrosine kinase signaling, providing one of the premier systems for studying this process. Current models describe the differentiative transition as a switch from Yan-mediated repression to Pointed-mediated activation of common target genes. We describe here a new layer of regulation whereby Yan and Pointed co-occupy regulatory elements to repress gene expression in a coordinated manner, with Pointed being unexpectedly required for the genome-wide occupancy of both Yan and the co-repressor Groucho. Using even skipped as a test-case, synergistic genetic interactions between Pointed, Groucho, Yan and components of the RNA polymerase II pausing machinery suggest that Pointed integrates multiple scales of repressive regulation to confer robustness. We speculate that this mechanism may be used broadly to fine-tune the expression of many genes crucial for development.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas del Ojo/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-ets/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas del Ojo/genética , Proteínas del Tejido Nervioso/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Represoras/genética , Factores de Transcripción/genéticaRESUMEN
Cis-regulatory modules (CRMs) are defined by unique combinations of transcription factor-binding sites. Emerging evidence suggests that the number, affinity, and organization of sites play important roles in regulating enhancer output and, ultimately, gene expression. Here, we investigate how the cis-regulatory logic of a tissue-specific CRM responsible for even-skipped (eve) induction during cardiogenesis organizes the competing inputs of two E-twenty-six (ETS) members: the activator Pointed (Pnt) and the repressor Yan. Using a combination of reporter gene assays and CRISPR-Cas9 gene editing, we suggest that Yan and Pnt have distinct syntax preferences. Not only does Yan prefer high-affinity sites, but an overlapping pair of such sites is necessary and sufficient for Yan to tune Eve expression levels in newly specified cardioblasts and block ectopic Eve induction and cell fate specification in surrounding progenitors. Mechanistically, the efficient Yan recruitment promoted by this high-affinity ETS supersite not only biases Yan-Pnt competition at the specific CRM but also organizes Yan-repressive complexes in three dimensions across the eve locus. Taken together, our results uncover a novel mechanism by which differential interpretation of CRM syntax by a competing repressor-activator pair can confer both specificity and robustness to developmental transitions.
Asunto(s)
Diferenciación Celular/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Miocardio/citología , Proteínas Represoras/metabolismo , Animales , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrión no Mamífero , Elementos de Facilitación Genéticos/genética , Proteínas del Ojo/química , Proteínas del Ojo/genética , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Organogénesis/genética , Unión Proteica , Estructura Cuaternaria de Proteína , Transporte de Proteínas , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Master regulatory transcription factors cooperate in networks to shepherd cells through organogenesis. In the Drosophila eye, a collection of master control proteins known as the retinal determination gene network (RDGN) switches the direction and targets of its output to choreograph developmental transitions, but the molecular partners that enable such regulatory flexibility are not known. We recently showed that two RDGN members, Eyes absent (Eya) and Sine oculis (So), promote exit from the terminal cell cycle known as the second mitotic wave (SMW) to permit differentiation. A search for co-factors identified the ubiquitously expressed Combgap (Cg) as a novel transcriptional partner that impedes cell cycle exit and interferes with Eya-So activity specifically in this context. Here, we argue that Cg acts as a flexible transcriptional platform that contributes to numerous gene expression outcomes by a variety of mechanisms. For example, Cg provides repressive activities that dampen Eya-So output, but not by recruiting Polycomb chromatin-remodeling complexes as it does in other contexts. We propose that master regulators depend on both specifically expressed co-factors that assemble the combinatorial code and broadly expressed partners like Cg that recruit the diverse molecular activities needed to appropriately regulate their target enhancers.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Redes Reguladoras de Genes , Factores de Transcripción/metabolismo , Animales , Ciclo Celular , Drosophila melanogaster/citología , Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/metabolismo , Organogénesis , Retina/embriología , Transcripción GenéticaRESUMEN
A limited collection of signaling networks and transcriptional effectors directs the full spectrum of cellular behaviors that comprise development. One mechanism to diversify regulatory potential is to combine multiple biochemical activities into the same protein. Exemplifying this principle of modularity, Eyes absent (Eya), originally identified as a transcriptional co-activator within the retinal determination gene network (RDGN), also harbors tyrosine and threonine phosphatase activities. Although mounting evidence argues for the importance of Eya's phosphatase activities to mammalian biology, genetic rescue experiments in Drosophila have shown that the tyrosine phosphatase function is dispensable for normal development. In this study, we repeated these rescue experiments in genetically sensitized backgrounds in which the dose of one or more RDGN factor was reduced. Heterozygosity for sine oculis or dachshund, both core RDGN members, compromised the ability of phosphatase-dead eya, but not of the control wild type eya transgene, to rescue the retinal defects and reduced viability associated with eya loss. We speculate that Eya's tyrosine phosphatase activity, although non-essential, confers robustness to RDGN output.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas del Ojo/metabolismo , Redes Reguladoras de Genes , Mutación , Proteínas Tirosina Fosfatasas/metabolismo , Retina/metabolismo , Animales , Drosophila , Proteínas de Drosophila/genética , Proteínas del Ojo/genética , Fertilidad , Heterocigoto , Proteínas Tirosina Fosfatasas/genética , Retina/crecimiento & desarrollo , TransgenesRESUMEN
The transition from proliferation to specification is fundamental to the development of appropriately patterned tissues. In the developing Drosophila eye, Eyes absent (Eya) and Sine oculis (So) orchestrate the progression of progenitor cells from asynchronous cell division to G1 arrest and neuronal specification at the morphogenetic furrow. Here, we uncover a novel role for Eya and So in promoting cell cycle exit in the second mitotic wave (SMW), a synchronized, terminal cell division that occurs several hours after passage of the furrow. We show that Combgap (Cg), a zinc-finger transcription factor, antagonizes Eya-So function in the SMW. Based on the ability of Cg to attenuate Eya-So transcriptional output in vivo and in cultured cells and on meta analysis of their chromatin occupancy profiles, we speculate that Cg limits Eya-So activation of select target genes posterior to the furrow to ensure properly timed mitotic exit. Our work supports a model in which context-specific modulation of transcriptional activity enables Eya and So to promote both entry into and exit from the cell cycle in a distinct spatiotemporal sequence.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/metabolismo , Retina/crecimiento & desarrollo , Retina/metabolismo , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Ciclo Celular , Linaje de la Célula , Proliferación Celular , Supervivencia Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Genes de Insecto , Proteínas de Homeodominio/genética , Mitosis , Mutación , Retina/citología , Factores de Transcripción/genéticaRESUMEN
Transcription factors use both protein-DNA and protein-protein interactions to assemble appropriate complexes to regulate gene expression. Although most transcription factors operate as monomers or dimers, a few, including the E26 transformation-specific family repressors Drosophila melanogaster Yan and its human homolog TEL/ETV6, can polymerize. Although polymerization is required for both the normal and oncogenic function of Yan and TEL/ETV6, the mechanisms by which it influences the recruitment, organization, and stability of transcriptional complexes remain poorly understood. Further, a quantitative description of the DNA occupancy of a polymerizing transcription factor is lacking, and such a description would have broader applications to the conceptually related area of polymerizing chromatin regulators. To expand the theoretical basis for understanding how the oligomeric state of a transcriptional regulator influences its chromatin occupancy and function, we leveraged the extensive biochemical characterization of E26 transformation-specific factors to develop a mathematical model of Yan occupancy at chemical equilibrium. We find that spreading condensation from a specific binding site can take place in a path-independent manner given reasonable values of the free energies of specific and non-specific DNA binding and protein-protein cooperativity. Our calculations show that polymerization confers upon a transcription factor the unique ability to extend occupancy across DNA regions far from specific binding sites. In contrast, dimerization promotes recruitment to clustered binding sites and maximizes discrimination between specific and non-specific sites. We speculate that the association with non-specific DNA afforded by polymerization may enable regulatory behaviors that are well-suited to transcriptional repressors but perhaps incompatible with precise activation.
Asunto(s)
ADN/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas del Ojo/química , Proteínas del Ojo/metabolismo , Modelos Moleculares , Multimerización de Proteína , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Animales , Sitios de Unión , Estructura Cuaternaria de Proteína , Especificidad por SustratoRESUMEN
Among the mechanisms that steer cells to their correct fate during development, master regulatory networks are unique in their sufficiency to trigger a developmental program outside of its normal context. In this review we discuss the key features that underlie master regulatory potency during normal and ectopic development, focusing on two examples, the retinal determination gene network (RDGN) that directs eye development in the fruit fly and the pluripotency gene network (PGN) that maintains cell fate competency in the early mammalian embryo. In addition to the hierarchical transcriptional activation, extensive positive transcriptional feedback, and cooperative protein-protein interactions that enable master regulators to override competing cellular programs, recent evidence suggests that network topology must also be dynamic, with extensive rewiring of the interactions and feedback loops required to navigate the correct sequence of developmental transitions to reach a final fate. By synthesizing the in vivo evidence provided by the RDGN with the extensive mechanistic insight gleaned from the PGN, we highlight the unique regulatory capabilities that continual reorganization into new hierarchies confers on master control networks. We suggest that deeper understanding of such dynamics should be a priority, as accurate spatiotemporal remodeling of network topology will undoubtedly be essential for successful stem cell based therapeutic efforts.
Asunto(s)
Drosophila/crecimiento & desarrollo , Drosophila/genética , Redes Reguladoras de Genes , Mamíferos/genética , Células Madre Pluripotentes/metabolismo , Retina/crecimiento & desarrollo , Retina/metabolismo , Animales , Humanos , Transcripción GenéticaRESUMEN
The transcriptional coactivator and phosphatase eyes absent (Eya) is dynamically compartmentalized between the nucleus and cytoplasm. Although the nuclear transcriptional circuits within which Eya operates have been extensively characterized, understanding of its cytoplasmic functions and interactions remains limited. Our previous work showed that phosphorylation of Drosophila Eya by the Abelson tyrosine kinase can recruit Eya to the cytoplasm and that eya-abelson interactions are required for photoreceptor axons to project to correct layers in the brain. Based on these observations, we postulated that photoreceptor axon targeting might provide a suitable context for identifying the cytoplasmic signaling cascades with which Eya interacts. Using a dose-sensitive eya misexpression background, we performed an RNA interference-based genetic screen to identify suppressors. Included among the top 10 hits were nonreceptor tyrosine kinases and multiple members of the Jak/Stat signaling network (hop, Stat92E, Socs36E, and Socs44A), a pathway not previously implicated in axon targeting. Individual loss-of-function phenotypes combined with analysis of axonal projections in Stat92E null clones confirmed the importance of photoreceptor autonomous Jak/Stat signaling. Experiments in cultured cells detected cytoplasmic complexes between Eya and Hop, Socs36E and Socs44A; the latter interaction required both the Src homology 2 motif in Socs44A and tyrosine phosphorylated Eya, suggesting direct binding and validating the premise of the screen. Taken together, our data provide new insight into the cytoplasmic phosphotyrosine signaling networks that operate during photoreceptor axon guidance and suggest specific points of interaction with Eya.
Asunto(s)
Orientación del Axón/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas del Ojo/genética , Retina/metabolismo , Animales , Axones/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Proteínas de Drosophila/biosíntesis , Drosophila melanogaster/crecimiento & desarrollo , Proteínas del Ojo/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Quinasas Janus/genética , Fosforilación , Células Fotorreceptoras/metabolismo , Retina/crecimiento & desarrollo , Factores de Transcripción STAT/genética , Transducción de Señal/genéticaRESUMEN
Eyes absent (Eya), a protein conserved from plants to humans and best characterized as a transcriptional coactivator, is also the prototype for a novel class of eukaryotic aspartyl protein tyrosine phosphatases. This minireview discusses recent breakthroughs in elucidating the substrates and cellular events regulated by Eya's tyrosine phosphatase function and highlights some of the complexities, new questions, and surprises that have emerged from efforts to understand how Eya's unusual multifunctionality influences developmental regulation and signaling.
Asunto(s)
Proteínas del Ojo/metabolismo , Ojo/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Ojo/química , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Proteínas del Ojo/análisis , Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Tirosina Fosfatasas/análisis , Proteínas Tirosina Fosfatasas/genética , Transducción de Señal , Especificidad por Sustrato , Activación TranscripcionalRESUMEN
Yan is an ETS-domain transcription factor responsible for maintaining Drosophila eye cells in a multipotent state. Yan is at the core of a regulatory network that determines the time and place in which cells transit from multipotency to one of several differentiated lineages. Using a fluorescent reporter for Yan expression, we observed a biphasic distribution of Yan in multipotent cells, with a rapid inductive phase and slow decay phase. Transitions to various differentiated states occurred over the course of this dynamic process, suggesting that Yan expression level does not strongly determine cell potential. Consistent with this conclusion, perturbing Yan expression by varying gene dosage had no effect on cell fate transitions. However, we observed that as cells transited to differentiation, Yan expression became highly heterogeneous and this heterogeneity was transient. Signals received via the EGF Receptor were necessary for the transience in Yan noise since genetic loss caused sustained noise. Since these signals are essential for eye cells to differentiate, we suggest that dynamic heterogeneity of Yan is a necessary element of the transition process, and cell states are stabilized through noise reduction.
Asunto(s)
Diferenciación Celular , Proteínas de Drosophila/metabolismo , Drosophila/embriología , Proteínas del Ojo/metabolismo , Regulación de la Expresión Génica , Proteínas Represoras/metabolismo , Animales , Ojo/embriologíaAsunto(s)
Proteínas de Drosophila/metabolismo , Elementos de Facilitación Genéticos/genética , Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Modelos Biológicos , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Drosophila , Proteínas de Drosophila/genética , Ingeniería Genética/métodos , Proteínas de Homeodominio/genética , Factores de Transcripción/genéticaRESUMEN
Proneural transcription factors drive the generation of specialized neurons during nervous system development, and their dynamic expression pattern is critical to their function. The activation of the proneural gene atonal (ato) in the Drosophila eye disc epithelium represents a critical step in the transition from retinal progenitor cell to developing photoreceptor neuron. We show here that the onset of ato transcription depends on two distant enhancers that function differently in subsets of retinal progenitor cells. A detailed analysis of the crosstalk between these enhancers identifies a critical role for three binding sites for the Retinal Determination factors Eyeless (Ey) and Sine oculis (So). We show how these sites interact to induce ato expression in distinct regions of the eye field and confirm them to be occupied by endogenous Ey and So proteins in vivo. Our study suggests that Ey and So operate differently through the same 3' cis-regulatory sites in distinct populations of retinal progenitors.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Drosophila/embriología , Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Retina/embriología , Células Madre/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Sitios de Unión , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Elementos de Facilitación Genéticos , Proteínas del Ojo/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/genética , Hibridación in Situ , Proteínas del Tejido Nervioso/fisiología , Sistema Nervioso/embriología , Neuronas/metabolismo , Neuronas/fisiología , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Proteínas Represoras/genética , Transcripción GenéticaRESUMEN
The biochemical regulatory network downstream of receptor tyrosine kinase (RTK) signaling is controlled by two opposing ETS family members: the transcriptional activator Pointed (Pnt) and the transcriptional repressor Yan. A bistable switch model has been invoked to explain how pathway activation can drive differentiation by shifting the system from a high-Yan/low-Pnt activity state to a low-Yan/high-Pnt activity state. Although the model explains yan and pnt loss-of-function phenotypes in several different cell types, how Yan and Pointed protein expression dynamics contribute to these and other developmental transitions remains poorly understood. Toward this goal we have used a functional GFP-tagged Pnt transgene (Pnt-GFP) to perform a comparative study of Yan and Pnt protein expression throughout Drosophila development. Consistent with the prevailing model of the Pnt-Yan network, we found numerous instances where Pnt-GFP and Yan adopt a mutually exclusive pattern of expression. However we also observed many examples of co-expression. While some co-expression occurred in cells where RTK signaling is presumed low, other co-expression occurred in cells with high RTK signaling. The instances of co-expressed Yan and Pnt-GFP in tissues with high RTK signaling cannot be explained by the current model, and thus they provide important contexts for future investigation of how context-specific differences in RTK signaling, network topology, or responsiveness to other signaling inputs, affect the transcriptional response.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Proteínas del Ojo/genética , Proteínas del Tejido Nervioso/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Represoras/genética , Transducción de Señal , Factores de Transcripción/genética , Transcripción Genética , Animales , Drosophila melanogaster/embriología , Proteínas Fluorescentes Verdes/genéticaRESUMEN
Long-range integration of transcriptional inputs is critical for gene expression, yet the mechanisms remain poorly understood. We investigated the molecular determinants that confer fidelity to expression of the heart identity gene even-skipped (eve). Targeted deletion of regions bound by the repressor Yan defined two novel enhancers that contribute repressive inputs to stabilize tissue-specific output from a third enhancer. Deletion of any individual enhancer reduced Yan occupancy at the other elements, impacting eve expression, cell fate specification, and cardiac function. These long-range interactions may be stabilized by three-dimensional chromatin contacts that we detected between the elements. Our work provides a new paradigm for chromatin-level integration of general repressive inputs with specific patterning information to achieve robust gene expression.
Asunto(s)
Cromatina/química , Cromatina/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas del Ojo/química , Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Animales , Drosophila melanogaster/química , Drosophila melanogaster/crecimiento & desarrollo , Elementos de Facilitación Genéticos/genética , Proteínas del Ojo/genética , Corazón/crecimiento & desarrollo , Heterocigoto , Unión Proteica , Proteínas Represoras/genética , Eliminación de SecuenciaRESUMEN
Developmental programs are driven by transcription factors that coordinate precise patterns of gene expression. While recent publications have described the importance of coordinated action of transcriptional activators at multiple cis-regulatory modules or enhancers, the contribution of sequence-specific repressors to overall regulation and robustness of gene expression has been difficult to ascertain. The Ets transcriptional repressor Yan functions as part of a conserved network downstream of receptor tyrosine kinase (RTK) signaling in Drosophila. This network displays switch-like responsiveness to RTK signaling, with the transition from a high-Yan to a low-Yan state induced by mitogen-activated protein kinase (MAPK)-mediated phosphorylation and inactivation of Yan. The ability of Yan to self-associate through a conserved sterile α motif (SAM) is essential for Yan's repressive ability, and has been suggested to allow spreading of Yan repressive complexes along chromatin. Such a mechanism has the potential to confer both signal responsiveness and robustness to the Yan network. To explore this spreading model, we compared the genome-wide chromatin binding profiles of wild-type vs. monomeric Yan. Consistent with the starting prediction, we found that wild type chromatin occupancy at genes encoding crucial developmental regulators and core signaling pathway components occurs as clusters of peaks that "spread" over multiple kilobases. However monomeric Yan, which fails to rescue a yan null mutation and displays significantly impaired repressive ability, exhibits a broadly similar occupancy profile to that of wild-type Yan, with multi-kilobase binding at developmentally important genes. This unexpected result suggests that SAM-mediated self-association does not mediate Yan recruitment to DNA or chromatin spreading, and raises the questions of why developmentally important genes require extensive Yan chromatin occupancy and how SAM-mediated polymerization might contribute to active repressive mechanisms in this context. In this Extra View article we discuss potential mechanisms by which Yan self-association and extended chromatin occupancy may contribute to robust regulation of gene expression.