RESUMO
Gene regulation is central to cellular function. Yet, despite decades of work, we lack quantitative models that can predict how transcriptional control emerges from molecular interactions at the gene locus. Thermodynamic models of transcription, which assume that gene circuits operate at equilibrium, have previously been employed with considerable success in the context of bacterial systems. However, the presence of ATP-dependent processes within the eukaryotic transcriptional cycle suggests that equilibrium models may be insufficient to capture how eukaryotic gene circuits sense and respond to input transcription factor concentrations. Here, we employ simple kinetic models of transcription to investigate how energy dissipation within the transcriptional cycle impacts the rate at which genes transmit information and drive cellular decisions. We find that biologically plausible levels of energy input can lead to significant gains in how rapidly gene loci transmit information but discover that the regulatory mechanisms underlying these gains change depending on the level of interference from noncognate activator binding. When interference is low, information is maximized by harnessing energy to push the sensitivity of the transcriptional response to input transcription factors beyond its equilibrium limits. Conversely, when interference is high, conditions favor genes that harness energy to increase transcriptional specificity by proofreading activator identity. Our analysis further reveals that equilibrium gene regulatory mechanisms break down as transcriptional interference increases, suggesting that energy dissipation may be indispensable in systems where noncognate factor interference is sufficiently large.
Assuntos
Eucariotos , Células Eucarióticas , Redes Reguladoras de Genes , Cinética , Termodinâmica , Fatores de Transcrição/genéticaRESUMO
Predicting how interactions between transcription factors and regulatory DNA sequence dictate rates of transcription and, ultimately, drive developmental outcomes remains an open challenge in physical biology. Using stripe 2 of the even-skipped gene in Drosophila embryos as a case study, we dissect the regulatory forces underpinning a key step along the developmental decision-making cascade: the generation of cytoplasmic mRNA patterns via the control of transcription in individual cells. Using live imaging and computational approaches, we found that the transcriptional burst frequency is modulated across the stripe to control the mRNA production rate. However, we discovered that bursting alone cannot quantitatively recapitulate the formation of the stripe and that control of the window of time over which each nucleus transcribes even-skipped plays a critical role in stripe formation. Theoretical modeling revealed that these regulatory strategies (bursting and the time window) respond in different ways to input transcription factor concentrations, suggesting that the stripe is shaped by the interplay of 2 distinct underlying molecular processes.
Assuntos
Drosophila/fisiologia , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário/fisiologia , Fatores de Transcrição/metabolismo , Animais , Núcleo Celular , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Masculino , Modelos Biológicos , RNA Mensageiro , Transcrição GênicaRESUMO
Transcription often occurs in bursts as gene promoters switch stochastically between active and inactive states. Enhancers can dictate transcriptional activity in animal development through the modulation of burst frequency, duration, or amplitude. Previous studies observed that different enhancers can achieve a wide range of transcriptional outputs through the same strategies of bursting control. For example, despite responding to different transcription factors, all even-skipped enhancers increase transcription by upregulating burst frequency and amplitude while burst duration remains largely constant. These shared bursting strategies suggest that a unified molecular mechanism constraints how enhancers modulate transcriptional output. Alternatively, different enhancers could have converged on the same bursting control strategy because of natural selection favoring one of these particular strategies. To distinguish between these two scenarios, we compared transcriptional bursting between endogenous and ectopic gene expression patterns. Because enhancers act under different regulatory inputs in ectopic patterns, dissimilar bursting control strategies between endogenous and ectopic patterns would suggest that enhancers adapted their bursting strategies to their trans-regulatory environment. Here, we generated ectopic even-skipped transcription patterns in fruit fly embryos and discovered that bursting strategies remain consistent in endogenous and ectopic even-skipped expression. These results provide evidence for a unified molecular mechanism shaping even-skipped bursting strategies and serve as a starting point to uncover the realm of strategies employed by other enhancers.
RESUMO
YAP is a transcriptional regulator that controls pluripotency, cell fate, and proliferation. How cells ensure the selective activation of YAP effector genes is unknown. This knowledge is essential to rationally control cellular decision-making. Here we leverage optogenetics, live-imaging of transcription, and cell fate analysis to understand and control gene activation and cell behavior. We reveal that cells decode the steady-state concentrations and timing of YAP activation to control proliferation, cell fate, and expression of the pluripotency regulators Oct4 and Nanog. While oscillatory YAP inputs induce Oct4 expression and proliferation optimally at frequencies that mimic native dynamics, cellular differentiation requires persistently low YAP levels. We identify the molecular logic of the Oct4 dynamic decoder, which acts through an adaptive change sensor. Our work reveals how YAP levels and dynamics enable multiplexing of information transmission for the regulation of developmental decision-making and establishes a platform for the rational control of these behaviors.
Assuntos
Optogenética , Células-Tronco , Diferenciação Celular/genética , Proliferação de Células/genética , Comunicação CelularRESUMO
Three-dimensional eukaryotic genome organization provides the structural basis for gene regulation. In Drosophila melanogaster, genome folding is characterized by somatic homolog pairing, where homologous chromosomes are intimately paired from end to end; however, how homologs identify one another and pair has remained mysterious. Recently, this process has been proposed to be driven by specifically interacting 'buttons' encoded along chromosomes. Here, we turned this hypothesis into a quantitative biophysical model to demonstrate that a button-based mechanism can lead to chromosome-wide pairing. We tested our model using live-imaging measurements of chromosomal loci tagged with the MS2 and PP7 nascent RNA labeling systems. We show solid agreement between model predictions and experiments in the pairing dynamics of individual homologous loci. Our results strongly support a button-based mechanism of somatic homolog pairing in Drosophila and provide a theoretical framework for revealing the molecular identity and regulation of buttons.
Assuntos
Pareamento Cromossômico , Cromossomos , Modelos Genéticos , Animais , Pareamento Cromossômico/genética , Pareamento Cromossômico/fisiologia , Cromossomos/química , Cromossomos/genética , Cromossomos/metabolismo , Drosophila melanogaster , Embrião não Mamífero , Feminino , Genoma de Inseto/genética , Masculino , Microscopia ConfocalRESUMO
We used live imaging to visualize the transcriptional dynamics of the Drosophila melanogaster even-skipped gene at single-cell and high-temporal resolution as its seven stripe expression pattern forms, and developed tools to characterize and visualize how transcriptional bursting varies over time and space. We find that despite being created by the independent activity of five enhancers, even-skipped stripes are sculpted by the same kinetic phenomena: a coupled increase of burst frequency and amplitude. By tracking the position and activity of individual nuclei, we show that stripe movement is driven by the exchange of bursting nuclei from the posterior to anterior stripe flanks. Our work provides a conceptual, theoretical and computational framework for dissecting pattern formation in space and time, and reveals how the coordinated transcriptional activity of individual nuclei shapes complex developmental patterns.
Assuntos
Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Animais , Proteínas de Drosophila , Drosophila melanogaster/embriologia , Engenharia Genética , Proteínas de Homeodomínio , Morfogênese/genética , Regiões Promotoras Genéticas , Recombinação Genética , Fatores de TranscriçãoRESUMO
Eukaryotic transcription generally occurs in bursts of activity lasting minutes to hours; however, state-of-the-art measurements have revealed that many of the molecular processes that underlie bursting, such as transcription factor binding to DNA, unfold on timescales of seconds. This temporal disconnect lies at the heart of a broader challenge in physical biology of predicting transcriptional outcomes and cellular decision-making from the dynamics of underlying molecular processes. Here, we review how new dynamical information about the processes underlying transcriptional control can be combined with theoretical models that predict not only averaged transcriptional dynamics, but also their variability, to formulate testable hypotheses about the molecular mechanisms underlying transcriptional bursting and control.
Assuntos
Modelos Genéticos , Transcrição Gênica , Animais , Humanos , Cinética , Fatores de TempoRESUMO
Information from developmental signaling pathways must be accurately decoded to generate transcriptional outcomes. In the case of Notch, the intracellular domain (NICD) transduces the signal directly to the nucleus. How enhancers decipher NICD in the real time of developmental decisions is not known. Using the MS2-MCP system to visualize nascent transcripts in single cells in Drosophila embryos, we reveal how two target enhancers read Notch activity to produce synchronized and sustained profiles of transcription. By manipulating the levels of NICD and altering specific motifs within the enhancers, we uncover two key principles. First, increased NICD levels alter transcription by increasing duration rather than frequency of transcriptional bursts. Second, priming of enhancers by tissue-specific transcription factors is required for NICD to confer synchronized and sustained activity; in their absence, transcription is stochastic and bursty. The dynamic response of an individual enhancer to NICD thus differs depending on the cellular context.