Modeling Reveals Bistability and Low-Pass Filtering in the Network Module Determining Blood Stem Cell Fate

Combinatorial regulation of gene expression is ubiquitous in eukaryotes with multiple inputs converging on regulatory control elements. The dynamic properties of these elements determine the functionality of genetic networks regulating differentiation and development. Here we propose a method to quantitatively characterize the regulatory output of distant enhancers with a biophysical approach that recursively determines free energies of protein-protein and protein-DNA interactions from experimental analysis of transcriptional reporter libraries. We apply this method to model the Scl-Gata2-Fli1 triad—a network module important for cell fate specification of hematopoietic stem cells. We show that this triad module is inherently bistable with irreversible transitions in response to physiologically relevant signals such as Notch, Bmp4 and Gata1 and we use the model to predict the sensitivity of the network to mutations. We also show that the triad acts as a low-pass filter by switching between steady states only in response to signals that persist for longer than a minimum duration threshold. We have found that the auto-regulation loops connecting the slow-degrading Scl to Gata2 and Fli1 are crucial for this low-pass filtering property. Taken together our analysis not only reveals new insights into hematopoietic stem cell regulatory network functionality but also provides a novel and widely applicable strategy to incorporate experimental measurements into dynamical network models

Coupling between feedback loops in autoregulatory networks affects bistability range, open-loop gain and switching times

Biochemical regulatory networks governing diverse cellular processes such as stress-response, differentiation and cell cycle often contain coupled feedback loops. We aim at understanding how features of feedback architecture, such as the number of loops, the sign of the loops and the type of their coupling, affect network dynamical performance. Specifically, we investigate how bistability range, maximum open-loop gain and switching times of a network with transcriptional positive feedback are affected by additive or multiplicative coupling with another positive- or negative-feedback loop. We show that a network's bistability range is positively correlated with its maximum open-loop gain and that both quantities depend on the sign of the feedback loops and the type of feedback coupling. Moreover, we find that the addition of positive feedback could decrease the bistability range if we control the basal level in the signal-response curves of the two systems. Furthermore, the addition of negative feedback has the capacity to increase the bistability range if its dissociation constant is much lower than that of the positive feedback. We also find that the addition of a positive feedback to a bistable network increases the robustness of its bistability range, whereas the addition of a negative feedback decreases it. Finally, we show that the switching time for a transition from a high to a low steady state increases with the effective fold change in gene regulation. In summary, we show that the effect of coupled feedback loops on the bistability range and switching times depends on the underlying mechanistic details

Emerging properties of animal gene regulatory networks

Gene regulatory networks (GRNs) provide system level explanations of developmental and physiological functions in the terms of the genomic regulatory code. Depending on their developmental functions, GRNs differ in their degree of hierarchy, and also in the types of modular sub-circuit of which they are composed, although there is a commonly employed sub-circuit repertoire. Mathematical modelling of some types of GRN sub-circuit has deepened biological understanding of the functions they mediate. The structural organization of various kinds of GRN reflects their roles in the life process, and causally illuminates both developmental and evolutionary process

Stability and multi-attractor dynamics of a toggle switch based on a two-stage model of stochastic gene expression

A toggle switch consists of two genes that mutually repress each other. This regulatory motif is active during cell differentiation and is thought to act as a memory device, being able to choose and maintain cell fate decisions. In this contribution, we study the stability and dynamics of a two-stage gene expression switch within a probabilistic framework inspired by the properties of the Pu/Gata toggle switch in myeloid progenitor cells. We focus on low mRNA numbers, high protein abundance and monomeric transcription factor binding. Contrary to the expectation from a deterministic description, this switch shows complex multi-attractor dy- namics without autoactivation and cooperativity. Most importantly, the four attractors of the system, which only emerge in a probabilistic two-stage description, can be identified with committed and primed states in cell differentiation. We first study the dynamics of the system and infer the mechanisms that move the system between attractors using both the quasi-potential and the probability flux of the system. Second, we show that the residence times of the system in one of the committed attractors are geometrically distributed and provide an analytical expression of the distribution. Most importantly we find that the mean residence time increases linearly with the mean protein level. Finally, we study the implications of this distribution for the stability of a switch and discuss the influence of the stability on a specific cell differentiation mechanism. Our model explains lineage priming and proposes the need of either high protein numbers or long term modifications such as chromatin remodeling to achieve stable cell fate decisions. Notably we present a system with high protein abundance that nevertheless requires a probabilistic description to exhibit multistability, complex switching dynamics and lineage priming.Comment: to appear in the Biophysical Journa

Engineering of a synthetic quadrastable gene network to approach Waddington landscape and cell fate determination

abstract: The process of cell fate determination has been depicted intuitively as cells travelling and resting on a rugged landscape, which has been probed by various theoretical studies. However, few studies have experimentally demonstrated how underlying gene regulatory networks shape the landscape and hence orchestrate cellular decision-making in the presence of both signal and noise. Here we tested different topologies and verified a synthetic gene circuit with mutual inhibition and auto-activations to be quadrastable, which enables direct study of quadruple cell fate determination on an engineered landscape. We show that cells indeed gravitate towards local minima and signal inductions dictate cell fates through modulating the shape of the multistable landscape. Experiments, guided by model predictions, reveal that sequential inductions generate distinct cell fates by changing landscape in sequence and hence navigating cells to different final states. This work provides a synthetic biology framework to approach cell fate determination and suggests a landscape-based explanation of fixed induction sequences for targeted differentiation

The acylation cycle

Innerhalb von Zellen ist die Verteilung von Proteinen häufig präzise, das bedeutet mit spezifischer Anreicherung in verschiedenen intrazellulären Kompartimenten. Als Ursache solcher Verteilungen wurden bereits Interaktionen, Rezeptoren oder auch Signalsequenzen beschrieben. In dieser Arbeit wird die Entstehung einer räumlich inhomogenen Proteinverteilung innerhalb von Zellen beschrieben – "Acylation Cycle" genannt - die auf einem Reaktions-Diffusions-Prozess basiert, welcher die reversible SPalmitoylierung umfasst. Palmitoylierung läuft am Golgi-Apparat ab, wobei die dort lokalisierten Enzyme kaum oder keine Spezifität für ein konkretes Proteinsubstrat zeigen. Anscheinend benötigen DHHC-Palmitoyltransferasen zur Palmitoylierung lediglich einen membrannahen Cysteinrest. Den gerichteten Transport zur Plasmamembran ermöglicht der sekretorische Weg. Der "Acylation Cycle" wirkt der entropie-getriebenen Homogenisierung palmitoylierter Proteine im Zellvolumen entgegen. Weiterhin wird die Aufenthaltsdauer palmitoylierter Proteine, wie z.B. Ras, an der Plasmamembran durch die Kinetik des "Acylation Cycle" beeinflusst, wodurch auch die Menge an Ras, die dem MAPK-Signalweg zur Verfügung steht, verändert wird. Das Unterbrechen des "Acylation Cycle" durch den neuartigen Inhibitor Palmostatin B bewirkt die durch Entropie getriebene Umverteilung von Ras. Durch Palmostatin B-Behandlung wird die Ras-Signalaktivität nach EGF Stimulierung an der Plasmamembran von der Aktivität am Golgi Apparat entkoppelt. Letztendlich führt die Palmostatin B-Behandlung von mit onkogenem HRasG12V transformierten MDCK Zellen zu einer Unterdrückung ihres konstitutiv-aktiven MAPKSignals, welches zur Zellteilung führen kann und verursacht so die Reversion des Phänotyps zu einem der untransformierten MDCK Zellen sehr ähnlichen. Der "Acylation Cycle" ist ein Phänomen, dem viele Proteine unterworfen sind und bietet daher die Gelegenheit die Modulation von Signalaktivitäten therapeutisch auszunutzen