Low Dimensionality in Gene Expression Data Enables the Accurate Extraction of Transcriptional Programs from Shallow Sequencing

A tradeoff between precision and throughput constrains all biological measurements, including sequencing-based technologies. Here, we develop a mathematical framework that defines this tradeoff between mRNA-sequencing depth and error in the extraction of biological information. We find that transcriptional programs can be reproducibly identified at 1% of conventional read depths. We demonstrate that this resilience to noise of “shallow” sequencing derives from a natural property, low dimensionality, which is a fundamental feature of gene expression data. Accordingly, our conclusions hold for ∼350 single-cell and bulk gene expression datasets across yeast, mouse, and human. In total, our approach provides quantitative guidelines for the choice of sequencing depth necessary to achieve a desired level of analytical resolution. We codify these guidelines in an open-source read depth calculator. This work demonstrates that the structure inherent in biological networks can be productively exploited to increase measurement throughput, an idea that is now common in many branches of science, such as image processing

Synergistic dual positive feedback loops established by molecular sequestration generate robust bimodal response

Feedback loops are ubiquitous features of biological networks and can produce significant phenotypic heterogeneity, including a bimodal distribution of gene expression across an isogenic cell population. In this work, a combination of experiments and computational modeling was used to explore the roles of multiple feedback loops in the bimodal, switch-like response of the Saccharomyces cerevisiae galactose regulatory network. Here, we show that bistability underlies the observed bimodality, as opposed to stochastic effects, and that two unique positive feedback loops established by Gal1p and Gal3p, which both regulate network activity by molecular sequestration of Gal80p, induce this bimodality. Indeed, systematically scanning through different single and multiple feedback loop knockouts, we demonstrate that there is always a concentration regime that preserves the system’s bimodality, except for the double deletion of GAL1 and the GAL3 feedback loop, which exhibits a graded response for all conditions tested. The constitutive production rates of Gal1p and Gal3p operate as bifurcation parameters because variations in these rates can also abolish the system’s bimodal response. Our model indicates that this second loss of bistability ensues from the inactivation of the remaining feedback loop by the overexpressed regulatory component. More broadly, we show that the sequestration binding affinity is a critical parameter that can tune the range of conditions for bistability in a circuit with positive feedback established by molecular sequestration. In this system, two positive feedback loops can significantly enhance the region of bistability and the dynamic response time

Discriminating between rival biochemical network models: three approaches to optimal experiment design

Background: The success of molecular systems biology hinges on the ability to use computational models to design predictive experiments, and ultimately unravel underlying biological mechanisms. A problem commonly encountered in the computational modelling of biological networks is that alternative, structurally different models of similar complexity fit a set of experimental data equally well. In this case, more than one molecular mechanism can explain available data. In order to rule out the incorrect mechanisms, one needs to invalidate incorrect models. At this point, new experiments maximizing the difference between the measured values of alternative models should be proposed and conducted. Such experiments should be optimally designed to produce data that are most likely to invalidate incorrect model structures. Results: In this paper we develop methodologies for the optimal design of experiments with the aim of discriminating between different mathematical models of the same biological system. The first approach determines the 'best' initial condition that maximizes the L2 (energy) distance between the outputs of the rival models. In the second approach, we maximize the L2-distance of the outputs by designing the optimal external stimulus (input) profile of unit L2-norm. Our third method uses optimized structural changes (corresponding, for example, to parameter value changes reflecting gene knock-outs) to achieve the same goal. The numerical implementation of each method is considered in an example, signal processing in starving Dictyostelium amœbæ. Conclusions: Model-based design of experiments improves both the reliability and the efficiency of biochemical network model discrimination. This opens the way to model invalidation, which can be used to perfect our understanding of biochemical networks. Our general problem formulation together with the three proposed experiment design methods give the practitioner new tools for a systems biology approach to experiment design. </p

Population Diversification in a Yeast Metabolic Program Promotes Anticipation of Environmental Shifts

Delineating the strategies by which cells contend with combinatorial changing environments is crucial for understanding cellular regulatory organization. When presented with two carbon sources, microorganisms first consume the carbon substrate that supports the highest growth rate (e.g., glucose) and then switch to the secondary carbon source (e.g., galactose), a paradigm known as the Monod model. Sequential sugar utilization has been attributed to transcriptional repression of the secondary metabolic pathway, followed by activation of this pathway upon depletion of the preferred carbon source. In this work, we demonstrate that although Saccharomyces cerevisiae cells consume glucose before galactose, the galactose regulatory pathway is activated in a fraction of the cell population hours before glucose is fully consumed. This early activation reduces the time required for the population to transition between the two metabolic programs and provides a fitness advantage that might be crucial in competitive environments

Msn2 coordinates a stoichiometric gene expression program.

BackgroundMany cellular processes operate in an "analog" regime in which the magnitude of the response is precisely tailored to the intensity of the stimulus. In order to maintain the coherence of such responses, the cell must provide for proportional expression of multiple target genes across a wide dynamic range of induction states. Our understanding of the strategies used to achieve graded gene regulation is limited.ResultsIn this work, we document a relationship between stress-responsive gene expression and the transcription factor Msn2 that is graded over a large range of Msn2 concentrations. We use computational modeling and in vivo and in vitro analyses to dissect the roots of this relationship. Our studies reveal a simple and general strategy based on noncooperative low-affinity interactions between Msn2 and its cognate binding sites as well as competition over a large number of Msn2 binding sites in the genome relative to the number of Msn2 molecules.ConclusionsIn addition to enabling precise tuning of gene expression to the state of the environment, this strategy ensures colinear activation of target genes, allowing for stoichiometric expression of large groups of genes without extensive promoter tuning. Furthermore, such a strategy enables precise modulation of the activity of any given promoter by addition of binding sites without altering the qualitative relationship between different genes in a regulon. This feature renders a given regulon highly "evolvable.

Low Dimensionality in Gene Expression Data Enables the Accurate Extraction of Transcriptional Programs from Shallow Sequencing

A tradeoff between precision and throughput constrains all biological measurements, including sequencing-based technologies. Here, we develop a mathematical framework that defines this tradeoff between mRNA-sequencing depth and error in the extraction of biological information. We find that transcriptional programs can be reproducibly identified at 1% of conventional read depths. We demonstrate that this resilience to noise of “shallow” sequencing derives from a natural property, low dimensionality, which is a fundamental feature of gene expression data. Accordingly, our conclusions hold for ∼350 single-cell and bulk gene expression datasets across yeast, mouse, and human. In total, our approach provides quantitative guidelines for the choice of sequencing depth necessary to achieve a desired level of analytical resolution. We codify these guidelines in an open-source read depth calculator. This work demonstrates that the structure inherent in biological networks can be productively exploited to increase measurement throughput, an idea that is now common in many branches of science, such as image processing

Multicellular Architecture of Malignant Breast Epithelia Influences Mechanics

Cell–matrix and cell–cell mechanosensing are important in many cellular processes, particularly for epithelial cells. A crucial question, which remains unexplored, is how the mechanical microenvironment is altered as a result of changes to multicellular tissue structure during cancer progression. In this study, we investigated the influence of the multicellular tissue architecture on mechanical properties of the epithelial component of the mammary acinus. Using creep compression tests on multicellular breast epithelial structures, we found that pre-malignant acini with no lumen (MCF10AT) were significantly stiffer than normal hollow acini (MCF10A) by 60%. This difference depended on structural changes in the pre-malignant acini, as neither single cells nor normal multicellular acini tested before lumen formation exhibited these differences. To understand these differences, we simulated the deformation of the acini with different multicellular architectures and calculated their mechanical properties; our results suggest that lumen filling alone can explain the experimentally observed stiffness increase. We also simulated a single contracting cell in different multicellular architectures and found that lumen filling led to a 20% increase in the “perceived stiffness” of a single contracting cell independent of any changes to matrix mechanics. Our results suggest that lumen filling in carcinogenesis alters the mechanical microenvironment in multicellular epithelial structures, a phenotype that may cause downstream disruptions to mechanosensing

De novo design of bioactive protein switches.

Allosteric regulation of protein function is widespread in biology, but is challenging for de novo protein design as it requires the explicit design of multiple states with comparable free energies. Here we explore the possibility of designing switchable protein systems de novo, through the modulation of competing inter- and intramolecular interactions. We design a static, five-helix 'cage' with a single interface that can interact either intramolecularly with a terminal 'latch' helix or intermolecularly with a peptide 'key'. Encoded on the latch are functional motifs for binding, degradation or nuclear export that function only when the key displaces the latch from the cage. We describe orthogonal cage-key systems that function in vitro, in yeast and in mammalian cells with up to 40-fold activation of function by key. The ability to design switchable protein functions that are controlled by induced conformational change is a milestone for de novo protein design, and opens up new avenues for synthetic biology and cell engineering

Module-Based Analysis of Robustness Tradeoffs in the Heat Shock Response System

Biological systems have evolved complex regulatory mechanisms, even in situations where much simpler designs seem to be sufficient for generating nominal functionality. Using module-based analysis coupled with rigorous mathematical comparisons, we propose that in analogy to control engineering architectures, the complexity of cellular systems and the presence of hierarchical modular structures can be attributed to the necessity of achieving robustness. We employ the Escherichia coli heat shock response system, a strongly conserved cellular mechanism, as an example to explore the design principles of such modular architectures. In the heat shock response system, the sigma-factor σ(32) is a central regulator that integrates multiple feedforward and feedback modules. Each of these modules provides a different type of robustness with its inherent tradeoffs in terms of transient response and efficiency. We demonstrate how the overall architecture of the system balances such tradeoffs. An extensive mathematical exploration nevertheless points to the existence of an array of alternative strategies for the existing heat shock response that could exhibit similar behavior. We therefore deduce that the evolutionary constraints facing the system might have steered its architecture toward one of many robustly functional solutions

Homeostatic adaptation to endoplasmic reticulum stress depends on Ire1 kinase activity

Uncoupling of Ire1’s RNAse and kinase activities reveals that its auto-phosphorylation is important for resolution of the unfolded protein response. (See also a related paper by Chawla et al. in this issue)