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More and more natural DNA variants are being linked to physiological traits. Yet, understanding what differences they make on molecular regulations remains challenging. Important properties of gene regulatory networks can be captured by computational models. If model parameters can be "personalized" according to the genotype, their variation may then reveal how DNA variants operate in the network. Here, we combined experiments and computations to visualize natural alleles of the yeast GAL3 gene in a space of model parameters describing the galactose response network. Alleles altering the activation of Gal3p by galactose were discriminated from those affecting its activity (production/degradation or efficiency of the activated protein). The approach allowed us to correctly predict that a non‐synonymous SNP would change the binding affinity of Gal3p with the Gal80p transcriptional repressor. Our results illustrate how personalizing gene regulatory models can be used for the mechanistic interpretation of genetic variants.
An approach based on genotype‐specific gene regulatory network models is used to examine the functional consequences of yeast GAL3 sequence variants. This framework can be more generally applied to the mechanistic interpretation of genetic variants.
The principle of the proposed approach is linking genetic variation to informative changes of parameter values of a regulatory network model.
Experimental analyses of the yeast GAL network shows that GAL3 natural variation is sufficient to convert a gradual response into a binary switch.
Dynamic network modeling successfully maps alleles to specific locations of the parameter space, allowing functional inference of DNA polymorphisms.
In the past decade, countless DNA variants have been associated with physiological traits. A major challenge now is to understand how they operate at the molecular level. This is a difficult task because the mechanistic consequences resulting from each variant are not easy to identify. Even when the function of a gene is well documented, investigators need to determine the tissues, cells, or organelles in which a mutant allele makes a biological difference, the developmental stage at which this may happen, the metabolic or regulatory network that may be involved, as well as possible molecular scenarios. A mutation may alter the regulation of transcription or mRNA splicing; the enzymatic activity of the target protein; its rate of production, maturation, or degradation; its intracellular localization; its binding affinity to an interacting partner or the specificity of its molecular interactions. In the vast majority of cases, information from the DNA sequence alone is not sufficient to delimit the perimeter of possible implications.
Systems biology has opened new opportunities to better predict the action of DNA variants. First, "omics" data that are gathered at various levels (DNA, transcripts, proteins, metabolites, etc.) establish relations between target sequences and functional pathways. Information about molecular and genetic interactions, expression profiles, chromatin landscapes, post‐transcriptional and post‐translational regulations can be exploited to derive functional predictions of DNA variants. Various methods have been proposed to do this, such as Bayesian genetic mapping (Gaffney et al, 2012), visualization of SNPs on relational protein networks (Bauer‐Mehren et al, 2009), prioritization based on negative selection (Levenstien & Klein, 2011), inference of miRNA:RNA binding defects (Coronnello et al, 2012), or combinations of lncRNA eQTL‐mapping with DnaseI‐hypersensitivity maps (Guo et al, 2016). In addition, structural data of biomolecules can also highlight functional perturbations in specific domains such as catalytic sites or interaction surfaces (Barenboim et al, 2008; Al‐Numair & Martin, 2013).
Another alternative is to model the quantitative and dynamic properties of molecular reactions and to explore which feature(s) may be affected by a DNA variant. The functional consequences of mutations can then be inferred by considering their impact on specific parameters of the model. In other words, assigning function to a DNA variant may be straightforward after it is linked to parameters of a model. This perspective may also, on the long term, generate developments in personalized medicine: If a model can be personalized according to the patient's genotype, then it can help predict disease progress or treatment outcome and therefore adapt medical care to the patient's specificities. Such an approach nonetheless differs from machine‐learning techniques, which can be efficient for prediction but where parameters are often not interpretable. For it to become reality, the model must be (i) informative on the biological trait of interest and (ii) identifiable (variation of one parameter cannot be exactly compensated by variation of another parameter) and sufficiently constrained (few parameters with limited degrees of freedom) so that fitted parameter values can inform on the patient's specificities. These two requirements antagonize each other regarding the complexity of the model to be used. The former asks for completeness: The molecular control of the trait must be correctly covered by the model, describing known reactions as best as possible. The latter asks for simplicity: If too many parameters are allowed to be adjusted to the data, then the validity of the personalized model is questionable and none of the adjustments are informative. It is therefore important to determine if and how personalizing model parameters can be productive.
For a given molecular network, individuals from natural populations have different genotypes at several nodes (genes) of the network, as well as in numerous external factors that can affect network properties. Such external factors can modify, for example, global translation efficiencies, metabolic states, or pathways that cross‐talk with the network of interest. Adapting model parameters to specific individuals is challenging when so many sources of variation exist. A way to circumvent this difficulty is to study the network experimentally in the context of a more reduced and focused variation. If investigators have access to nearly isogenic individuals that differ only at specific genes of the network, they can then characterize the differences in network behavior that result from these specific allelic differences. The numerous external factors affecting the network can then be ignored or drastically simplified in the model because they are common to all individuals. This way, the parameter space is constrained and only potentially informative parameters are allowed to be adjusted to fit individual‐specific data.
Some model organisms such as the yeast Saccharomyces cerevisiae offer this possibility. They can be manipulated to generate single allelic changes, which provides an ideal framework to link DNA variants to model parameters. In particular, the gene regulatory network controlling the yeast response to galactose (GAL network) is well characterized, both in vivo and in silico. This circuit controls galactose utilization by upregulating the expression of regulatory and metabolic genes in response to extracellular galactose (Sellick et al, 2008). Regulation is based on the transcriptional activator Gal4p, the galactose transporter Gal2p, a signal transducer Gal3p, and the transcriptional inhibitor Gal80p. In addition, the galactokinase Gal1p involved in galactose metabolism is also a co‐inducer of the response (Bhat & Hopper, 1992). This system can display either a gradual induction (where the rate of transcription progressively increases in each cell according to the timing and intensity of the stimulus) or a binary induction (where some cells are rapidly activated and others not). This dual behavior has received a lot of attention, and important molecular features have been elucidated by experimental and theoretical approaches (Biggar & Crabtree, 2001; Hawkins & Smolke, 2006; Song et al, 2010; Apostu & Mackey, 2012). In particular, the dynamic response of a population of cells to galactose can be described by two quantities: (i) The inducibility of the network is defined as the proportion of activated cells in the population and (ii) the amplitude of the response refers to the expression level that is reached by induced cells. Regulatory feedback loops of the network are critical to the switch‐like behavior. They were shown to feed back the dynamics of transcription bursts rather than the levels of expression (Hsu et al, 2012). They regulate the amplitude response by reducing noise in GAL gene expression (Ramsey et al, 2006), they control inducibility by fine‐tuning the timing of the switch (Ramsey et al, 2006), and they participate to the memory of previous inductions (Acar et al, 2005; Kundu & Peterson, 2010). As a consequence, bimodal distributions of expression of the GAL genes can be observed in isogenic populations exposed to intermediate concentrations of inducer (Becskei et al, 2001; Venturelli et al, 2012; Peng et al, 2015), and this population heterogeneity can confer a growth advantage during the transition from glucose to galactose metabolism (diauxic shift) (Venturelli et al, 2015). Interestingly, wild yeast isolates present diverse types of induction dynamics during the diauxic shift, ranging from strictly unimodal to transient bimodal distribution of expression levels (New et al, 2014; Wang et al, 2015). This indicates that natural genetic variation can modify the network dynamics.
The GAL3 gene plays a central role in the network. Its protein product Gal3p is activated by binding to galactose and ATP and then binds as a dimer to Gal80p dimers to release the repression on Gal4p at target promoters (Sellick et al, 2008). The protein is enriched in the cytoplasm prior to stimulation and in the nucleus after the stimulation, although this cytonuclear transfer does not account for the dynamics of activation (Jiang et al, 2009; Egriboz et al, 2011). Expression of GAL3 is itself under Gal4p/Gal80p control (positive feedback). In addition, the sequence of GAL3 differs between natural isolates of S. cerevisiae and this allelic variation was recently associated with different sensitivities of the network to galactose (Lee et al, 2017). There are multiple ways that a GAL3 variant could affect the dynamics of induction: by modifying the production or degradation rates of the Gal3p protein or of its messenger RNA, by changing the affinity of Gal3p to galactose or ATP, by changing the capacity of Gal3p to dimerize, by changing the nucleocytoplasmic ratio of Gal3p molecules, or by changing the affinity of Gal3p to Gal80p. A GAL3 variant may also affect the background expression level of Gal3p prior to stimulation, which is known to be critical for network memory of prior stimulations (Stockwell & Rifkin, 2017). Thus, it is difficult to predict the functional consequence of sequence variation in GAL3.
Using the yeast GAL3 gene as a model framework, we show here that experimental acquisitions combined with network modeling are efficient to predict the effect of sequence variants. The principle of the approach is to link genetic variation to informative changes of parameter values of the model. We show that replacing natural GAL3 alleles can be sufficient to transform a gradual response into a binary activation, and the approach allowed us to distinguish between different types of GAL3 alleles segregating in S. cerevisiae populations: those altering the activation of Gal3p by galactose and those altering the strength with which activated Gal3p alleviates the transcriptional inhibition operated by Gal80p. In particular, our approach was efficient to associate a non‐synonymous SNP with a change of binding affinity for Gal80p.
Natural variation in GAL3 is sufficient to convert a gradual induction into a binary switch
We constructed a panel of yeast strains that were all isogenic to the reference laboratory strain BY, except for GAL3. At this locus, each strain carried an allele that was transferred from a natural strain of the Saccharomyces Genome Resequencing Project (Liti et al, 2009; Appendix Fig S1). All strains of the panel also harbored a PGAL1‐GFP reporter of network activity, where the promoter of the GAL1 gene controlled the expression of a GFP fluorescent protein destabilized by a degradation signal (Mateus & Avery, 2000; Chuffart et al, 2016). GAL1 is a paralogous gene of GAL3 (Hittinger & Carroll, 2007) and transcription at its promoter is commonly used as a proxy of GAL network activity (Acar et al, 2005; Venturelli et al, 2015; Wang et al, 2015). Using flow cytometry, we monitored the dynamics of network activation in each strain (Fig 1). This was done by first culturing cells for 3 h in a medium containing 2% raffinose, a sugar known to be neutral on network activity, adding galactose (0.5% final concentration), and quantifying fluorescence at multiple time points for 4 h. Significant differences in the dynamics of activation were observed between the strains. Those harboring the GAL3NCYC361, GAL3K11, GAL3BY, GAL3DBVPG1788, GAL3DBVPG1853, and GAL3JAY291 alleles displayed a gradual response and all cells of the population were induced and responded with similar rate of expression, maintaining population homogeneity (see example shown in Fig 1A). In contrast, strains harboring the GAL3Y12 and GAL3YJM978 alleles displayed a binary response, with a transient coexistence of induced (ON) and uninduced (OFF) cells in the population (example in Fig 1B).
We quantified induction using two metrics: the mean level of reporter expression in activated cells (response amplitude) and the proportion of activated cells in the population (inducibility of the network). We observed that the response amplitude varied little among the strains, all of them approaching steady state with comparable kinetics (Fig 1C). In contrast, inducibility of the network differed between strains (Fig 1D). As expected, in strains showing a gradual response, the fraction of ON cells increased significantly during the first 2 h of induction, reaching full inducibility (all cells activated) by the end of the experiment. On the opposite, the strains showing a transient binary response displayed reduced inducibility over time. For instance, 21% of GAL3Y12 cells were still not induced after 250 min of stimulation. These results indicate that natural genetic variation in GAL3 is sufficient to modify the inducibility of the network and to convert a gradual response into a binary response, or vice versa.
A quantitative model of inducibility over time
To examine what functional properties of the GAL3 gene could determine a gradual or a binary response, we constructed a dynamic stochastic model of the network (Fig 2A). We based our quantitative model on the following current molecular knowledge, which derives from reference laboratory strains. In absence of galactose, a homodimer of the transcription factor Gal4p is constitutively bound to upstream activation sites (UAS) of promoter regions of GAL genes. However, transcription is inactive because of the homodimeric Gal80p inhibition of Gal4p (Peng & Hopper, 2002; Pilauri et al, 2005). When intracellular galactose binds Gal3p, it changes conformation and associates with Gal80p dimers (Lavy et al, 2012), thereby releasing Gal80p from promoters and allowing Gal4p‐mediated transcriptional activation. It was initially thought that activated Gal3p sequestered Gal80p in the cytoplasm, preventing it from its inhibitory role in the nucleus (Peng & Hopper, 2002). Later studies revised this view by showing that Gal3p molecules were not exclusively cytoplasmic (Jiang et al, 2009) and that forcing Gal3p to be mostly nuclear did not alter the kinetics of induction (Jiang et al, 2009). In addition, the slowness of the nucleocytoplasmic translocation of Gal80p, which depends both on transport rates and on the Gal4p:Gal80p dissociation rate, contrasts with the fast induction of transcription (Egriboz et al, 2011). This implies a direct role of Gal3p in promoting the dissociation of Gal80p from UAS. In addition, the galactokinase Gal1p (a paralog of Gal3p) can also act as a co‐inducer of the regulatory circuit, presumably using similar mechanisms as Gal3p (Venturelli et al, 2012).
Our model covers the mRNA and protein species of three major players of GAL network induction: GAL1, GAL3, and GAL80, as well as of the reporter gene. We considered that promoters of each GAL gene could switch between an ON state (full transcription) and an OFF state (leaky transcription) at rates that depended on the concentration of Gal80 dimers, activated Gal3p dimers, and activated Gal1p dimers. The model is provided (computer Code EV1), and a detailed description of it is given in Materials and Methods and in Appendix Text S1. Most of the parameters of the model were fixed at values obtained from previous studies (Appendix Tables S1 and S2).
Stochastic simulations reproduce the two types of induction observed experimentally
We first explored if our model captured the two types of responses of allele‐replacement strains (i.e., binary and gradual). We ran stochastic simulations (Gillespie, 1977) that accounted for intrinsic and extrinsic sources of noise (see Appendix Text S1). We observed that tuning the parameters related to GAL3, while keeping all other parameters constant, was sufficient to modify inducibility and to obtain either a gradual (Fig 2B) or a binary (Fig 2C) response of the network at a given concentration of galactose. In the gradual system, the simulated single‐cell trajectories were all similar; in the binary system, the simulated single‐cell trajectories bifurcated, with a subset of cells having a stochastic lagging time before responding. The single‐cell value of this lag time is directly correlated with the number of potential inducer proteins (Gal1p and Galp3p) present in the cell just before induction (Appendix Fig S2). This is in very good agreement with recent single‐cell experiments on galactose induction (Stockwell & Rifkin, 2017). Note that a binary response is not necessarily a signature of steady‐state bistability (Hermsen et al, 2011) but may represent a transient regime converging to a monostable ON state at equilibrium (see Appendix Text S1).
We then studied the response predicted by the model when stimulating the network with various concentrations of galactose while keeping model parameters constant (Appendix Fig S3). Inducibility increased with the concentration of galactose, with low concentrations causing a binary induction and high concentrations causing a gradual one.
Two parameters related to GAL3 control network behavior
A detailed analysis of the model showed that inducibility of the system was mainly controlled by the average values of promoter switching rates kon and koff at the time of induction (see Materials and Methods, Appendix Text S1, and Figs S2 and S4). Rates koff depend only on GAL80 and are therefore invariant to GAL3 allelic variation. Rates kon depend on GAL3 in two ways: via Gal3p*, the amount of galactose‐activated Gal3p, and via K3, which corresponds to an effective concentration encompassing the dissociation constants of the Gal3p‐Gal80p interaction and of Gal3p dimerization (see Appendix Text S1). Gal3p* is determined by the level of Gal3p and by parameter Kgal, which represents the typical concentration of galactose needed to efficiently activate Gal3p. While Kgal was identifiable, several other GAL3‐related parameters, such as those controlling the level of Gal3p, were not and we grouped them into a meta‐parameter, ρGal3, which we termed the strength of GAL3. ρGal3 corresponds to the invert ratio between K3 and the mean concentration of Gal3p at the time of induction, which depends on the leaky transcription rate, the translation rate and the degradation rates of GAL3 mRNA and protein product.
This formalism made the network sensitive to only two identifiable GAL3‐related parameters, Kgal and ρGal3. At a fixed concentration of galactose induction, high ρGal3 values correspond to high numbers of Gal3p dimers that can rapidly be activated to release Gal80 repression. The model predicted that high values of ρGal3 would generate a gradual response (Appendix Fig S5A) because the number of potential activators was high enough in each cell to rapidly trigger the GAL1/GAL3‐mediated positive feedback loop. In contrast, low values of ρGal3 would generate a binary response (Appendix Fig S5B) because the number of activators is more stochastic, with many cells having too few initial Gal1p or Gal3p dimers to directly trigger the response. These cells need a lag time before fast activation (Fig 2B and C, and Appendix Fig S2). The other important parameter, Kgal, corresponds to a threshold of galactose concentration below which induction was limited and favoured a binary response, and above which induction was efficient and favoured a gradual response (Appendix Fig 3C). In summary, both ρGal3 and Kgal values can determine whether the network adopts a gradual or a binary response at a given concentration of galactose induction.
Linking GAL3 alleles to specific parameter values
We first examined if our model could capture a known functional alteration of the GAL3 gene. The ATP and galactose binding pocket of Gal3p was previously described (Lavy et al, 2012). It contains an aromatic cage that encircles the adenine nucleotide of ATP. A mutation targeting this cage should therefore reduce the affinity of Gal3p for ATP. Galactose is localized more internally than ATP, suggesting that the sequential activation of Gal3p starts first with galactose binding and then ATP binding. We analyzed how parameters of our model should be affected by a mutation reducing Gal3p:ATP affinity, and we found that Kgal should increase, that ρGal3 should decrease, and that the relative change to wild‐type values should be more pronounced for Kgal (Appendix Text S1). In contrast, if ATP binds prior to galactose, then Kgal should also increase but ρGal3 should remain unaffected. Using Crispr/Cas9, we replaced in the BY reference strain the W117 residue of the cage by either an alanine or a threonine, and we monitored the dynamics of transcriptional activation of the corresponding mutants (Appendix Fig S6A–C). The non‐conservative W117T mutation fully abolished the response, even at very high concentrations of galactose. The W117A mutation profoundly reduced activation and caused a binary response. We then inferred parameters ρGal3 and Kgal for the wild‐type strain and for the W117A mutant. This was done by selecting a set of parameters that minimized a global chi‐squared score of deviation between the measured and predicted fractions of induced cells at different times after induction and for the different galactose concentrations (for details, see Materials and Methods and Appendix Text S1). Inferred Kgal value was about 10 times higher for the mutant than for the wild type, and ρGal3 was reduced by about threefold (Appendix Fig S6D). This fully agreed with the expected sequence of activation of Gal3p (galactose first, ATP second).
We then used our model to study natural GAL3 alleles. We measured the transcriptional response of the GAL3BY, GAL3Y12, and GAL3YJM978 strains at different galactose concentrations (0.05, 0.1, and 0.5%). This confirmed that inducibility increased with galactose concentration (Fig 3A). We used these experimental data as above to infer parameters ρGal3 and Kgal for each of the three strains. To evaluate the usefulness of the inferred parameter values, we used the fitted model to predict the behavior of each strain at a galactose concentration that was not used for model training (0.2%) (Fig 3B). Finally, to test model predictions, we experimentally monitored GAL3BY, GAL3Y12, and GAL3YJM978 induction at 0.2% galactose. Without any additional fitting procedure, we observed that inducibility (fraction of activated cells over time) differed between strains in a way that was entirely consistent with model predictions. Thus, the differences among parameter values assigned to the different natural GAL3 alleles are relevant outside the specific experimental conditions used for parameters estimation.
Natural GAL3 alleles map to distinct locations of the parameter space
We sought to classify GAL3 alleles based on the parameter values assigned to them. We made experimental measurements on two additional strains (GAL3NCYC361 and GAL3DBVPG1788), and we determined best‐fit ρGal3 and Kgal values to them as for the three strains described above. Appendix Fig S7 shows these data and the corresponding fitted models. Figure 4A and B shows the obtained parameters, ρGal3 and KGal, normalized by the corresponding values of our reference strain GAL3BY. Different data points represent results obtained by applying the inference process to models with different GAL3‐independent parameters (see Appendix Text S1).
The fold change of a parameter between two different strains is indicative of the functional nature of the genetic variations between the two GAL3 alleles. In agreement with model predictions (Appendix Fig S3), we observed that more gradual strains (GAL3NCYC361 and GAL3DBVPG1788) display a high GAL3 strength ρGal3 and a low "typical" galactose concentration KGal. Interestingly, we observed that ρGal3 and KGal can be decorrelated. In particular, although both GAL3YJM978 and GAL3Y12 strains were binary responders at all galactose concentrations tested, the model attributed this behavior to different functional effects: a low sensitivity to galactose (high Kgal) for the Gal3 protein originating from YJM978 and a reduced strength of the GAL3 gene originating from Y12. Thus, the induction specificities of the strains can be attributed to distinct GAL3‐related parameters.
To address the direct relationship between the network properties (gradual or binary response) and the GAL3‐related parameters, we positioned each of the tested strains within a phenotypic landscape according to their relative ρGal3 and KGal parameters (Fig 4C). According to our model, ρGal3 and KGal parameters are sufficient to predict the behavior (gradual or binary) associated with a given GAL3 allele at a given concentration of galactose. As an illustration of these predictions, we specifically observed the dynamics of transcriptional activation of the network for the strain GAL3DBVPG1788 (Appendix Fig S8). The position of the GAL3DBVPG1788 allele on the phenotypic landscape corresponded to a transient binary activation at low concentration ([gal] = 0.05%) converted into a gradual response at higher concentration ([gal] = 0.1% and [gal] = 0.5%). Importantly, mathematical analysis of the system at steady state indicated that all strains should reach a monostable ON state at equilibrium (Fig 4D and Appendix Text S1). Thus, although a binary regime of induction is observed in some strains, this is a transient regime that should eventually convergence to monostability after a very long time (> 10 h).
Variation in induction dynamics is consistent with variation in diauxic shift decision
The physiological relevance of the GAL network regulation is to switch from the consumption of glucose (the preferred carbon source) to the consumption of galactose when glucose supply is running out. This diauxic switch is controlled not only by galactose induction but also by glucose‐mediated repression. When both sugars are present, their relative concentration ratio determines whether cells activate the switch or not (New et al, 2014; Escalante‐Chong et al, 2015; Venturelli et al, 2015). At some ratio values, only a fraction of the cells are induced, even at steady state. Given this dual regulation, the propensity of a strain to activate GAL metabolism can be quantified by measuring the fraction of induced cells after a prolonged period (8 h) of simultaneous induction (by galactose) and repression (by glucose). If this measurement is repeated at a given concentration of galactose and various concentrations of glucose, a useful score can be computed (called "decision threshold" hereafter): the concentration of glucose needed to maintain half the population of cells in the repressed (OFF) state (Fig 5A). A high decision threshold corresponds to an early activation of GAL genes during the diauxic shift.
A previous study identified GAL3 as an important genetic determinant for this decision: The concentration ratio at which cells turn GAL expression ON differs between strains harboring different natural alleles of GAL3 (Lee et al, 2017). We asked if this variation, visible in the steady‐state exposure to both glucose and galactose, was correlated with the variation observed on the dynamics of network induction upon exposure to galactose only. We chose four strains that showed different decision thresholds because of different GAL3 alleles (Lee et al, 2017; Fig 5B), and we monitored their dynamics of induction at three different concentrations of galactose (with no glucose). We then used our model to assign ρGal3 and KGal parameter values to each strain. Experimental data and model fitting are shown in Fig 5B and Appendix Fig S9. We used the inferred parameter values to visualize the four strains in the parameter space where binary and gradual responses upon stimulation at [gal] = 0.25% are delimited (Fig 5D). Remarkably, the properties of induction dynamics in absence of glucose were fully consistent with the decision threshold during diauxic shift from glucose to galactose. Strains having a low decision threshold, such as GAL3YJM421, displayed a transient binary response, and strain GAL3BC187 had a high decision threshold and responded gradually. Coordinates of strains in the parameter space indicate that ρGal3 values are highly informative on the decision threshold (Fig 5D). Thus, mapping allelic variation to dynamic parameters of induction is also useful to understand trade‐offs that are observed at steady state.
A quantitative parameter change predicts a role of H352D SNP on Gal3:Gal80 complex formation
We noticed that, at position 352 of the Gal3p protein, all natural strains harbored an aspartic acid, whereas the reference laboratory strain BY harbored a histidine. This aspartic acid was also conserved in Saccharomyces mikatae, Saccharomyces paradoxus, and Saccharomyces uvarum protein sequences (Cherry et al, 2012). Given the prevalence of this aspartic acid, we hypothesized that a single H352D amino acid change could have consequences on Gal3p regulatory function.
To test this, we generated an artificial GAL3BY‐H352D allele by introducing the H352D mutation in the GAL3BY strain and we monitored the dynamics of induction of the resulting strain. At similar concentrations of galactose, induction was faster for the modified strain than for the original strain (compare Fig 6A with 2A). We then used our model to make functional predictions. We fitted our model to experimental data of induction as described above for natural alleles. Induction dynamics of the modified strain were fully explained by preserving parameter KGal and increasing ρGal3 (Fig 6B). This suggested that the H352D mutation did not affect activation of Gal3p by galactose but rather the strength of Gal3p, which summarizes six biochemical features: the basal level of GAL3 transcription prior to induction, its translation and degradation rate, the degradation rate of its coding mRNA, its capacity to homodimerize, and the affinity of activated Gal3p for Gal80p.
How the implicated SNP could change either the leaky transcription level prior to induction or the transcription rate during induction is difficult to imagine. In addition, the amino acid change was not surrounded by any particular peptide motif, nor was it located at the extremity of the protein. This did not support for an effect on translation or degradation rates. Thus, the most plausible interpretation of the parameter change of the model was that the H352D modification would increase either the capacity of Gal3p* to dimerize or the affinity of the Gal3p* dimer for Gal80p.
To explore these possibilities, we analyzed the structure of the heterotetramer [Gal3p*]2‐[Gal80p]2 that was previously solved (Lavy et al, 2012). We made three important observations. First, His352 is located at the binding interface of the Gal3p* dimer with the Gal80p dimer (Fig 6C), and distant from the pocket containing galactose and ATP. Secondly, it is spatially close to the Gal80p site where the acidic domain of Gal4p is known to bind (Thoden et al, 2008). Finally, the Gal80p dimer exhibits a positive electrostatic surface potential in the vicinity of Gal3p‐His352, suggesting that the replacement of the neutral His352 by a negatively charged aspartic acid would stabilize the Gal3p*‐Gal80p complex. Stabilization refers here to a gain in thermodynamic stability relative to the Gal4p‐Gal80p complex, or in other words, to a decrease in Gibbs free energy change (ΔGsub) for the substitution of the Gal4p dimer by the Gal3p dimer as binding partner of the Gal80p dimer. A molecular dynamics simulation of the Asp352 mutant (in a model system of the Gal3p*‐Gal80 complex) indicates that two positively charged amino acids, Gal3p‐Arg362 and Gal80p‐Lys287, are able to form direct salt bridges with Asp352 (Fig 6D). These attractive interactions of Asp352 with its environment are, however, expected to be partially canceled out by repulsive interactions with the less proximate, negatively charged amino acids Gal3p‐Glu363 and Gal80p‐Glu348 (Fig 6D). Also, the polar solution (water + counter ions) could partially reduce the stabilization effect of the H352D mutation because residue 352 is better solvated in the Gal3p* dimer than in the Gal3p*‐Gal80p tetramer. Thus, to quantify a possible stabilization effect of the H352D mutation, we computed the change in the Gibbs free energy difference, ∆∆Gsub = ∆GsubD352 − ∆GsubH352, with the aid of the thermodynamic cycle depicted in Fig 6E. The actual free energy calculations (see Materials and Methods) yielded ∆∆Gsub = −2.8 ± 0.9 kcal/mol, which indicates that the H352D mutation indeed increases the thermodynamic stability of the Gal3p*‐Gal80p complex with respect to the Gal4p‐Gal80p complex. Thus, as predicted by the dynamic model of network induction, the H352D mutation increases the cellular response by facilitating the formation of the complex.
We experimentally monitored the induction dynamics of the yeast GAL network in the context of natural genetic variation at the GAL3 gene. This revealed that GAL3 natural variation is sufficient to convert a gradual induction into a binary one. We built a stochastic model of the network and used it to link GAL3 alleles to functional network parameters. This approach discriminated alleles that increased the strength of activated Gal3p (e.g., of strains NCYC361 and DBVPG1788) from alleles that desensitized Gal3p to galactose activation (e.g., of strain YJM978). Alleles showing different glucose/galactose trade‐offs at equilibrium displayed different dynamics of induction, and they were associated with different strength of activated Gal3p. Our approach also predicted a functional effect of a single non‐synonymous SNP that was validated by atomistic simulations of the binding interface between Gal3p and Gal80p dimers. These results provide further details on the yeast GAL system and, perhaps more importantly, they constitute a proof‐of‐concept of the feasibility and usefulness of linking genetic variants to model parameters.
Genetic variability of the yeast GAL network
Our in vivo and in silico analyses of the induction kinetics of yeast GAL activation reveal properties of this system and how it is sensitive to genetic variation. The fact that an allelic exchange in GAL3 was sufficient to convert a gradual response into a transient binary one is important because it shows that, although binary induction requires a polygenic architecture of transcriptional regulation, whether it takes place or not can be dictated by the genotype of a single gene.
Previously, several computational models of the network have been proposed, usually in an effort to understand the properties of the system at steady state (Acar et al, 2005; Apostu & Mackey, 2012; Venturelli et al, 2012). Particularly, they highlighted the important role of Gal3p‐, Gal1p‐, and Gal80p‐mediated feedback loops. Our in silico analysis suggests that the gradual or binary kinetic response is mainly controlled by the initial number of repressors (Gal80p) and inducers (Gal1p and Gal3p), the efficacy of galactose to activate the inducers, and the efficiency of the activated inducers to release the effect of repressors. In particular, a low mean number of inducers at the time of induction may lead to high cell‐to‐cell variability in their actual number. Cells with few inducers (as compared to repressors) display a lag time before responding, leading to a binary response pattern at the population level. This prediction from our model is fully consistent with recent experiments that tracked the induction of the network at the single‐cell level and showed that the initial concentrations of Gal1p and Gal3p are predictive of the transient bimodal response (Stockwell & Rifkin, 2017). We also observed that feedback loops were important to control the strength of cell‐to‐cell variability before induction (Gal80‐mediated negative feedback) and the duration of lag times (Gal3/Gal1‐mediated positive feedbacks), which agrees with the previous observation that disabling the Gal80p and Gal3p feedback loops can transform a gradual response into a binary one (Ramsey et al, 2006). Our results on GAL3 genetic variants also complement previous genetic manipulations of the feedback loops, where their effect on bimodality was tested by modulating promoter activities (Acar et al, 2005; Ramsey et al, 2006; Venturelli et al, 2012; Peng et al, 2015). Here, we showed that a non‐synonymous variant affecting Gal3p:Gal80p interaction directly affects the dynamics of transient bimodality. This is a novel experimentally based observation that is totally coherent with the conclusions of Venturelli et al (2012) who showed computationally that steady‐state bimodality of the network could rely on protein–protein binding affinities.
We also observed that genetic variations at GAL3 could affect its propensity to be activated by galactose/ATP binding. In particular, the GAL3YJM978 allele was associated with increased values of the KGal parameter (more galactose needed for its activation). This allele harbored three non‐synonymous SNPs: M179I, R312I, and H352D. As shown above, H352D is found in all natural alleles that we tested and it therefore does not explain a change in KGal specifically for GAL3YJM978. According to the structure of the Gal3p:Gal80p tetramer complexed with galactose and ATP (Lavy et al, 2012), the other two polymorphic sites do not map close to the pocket containing the ligands. Met179 is located at the surface of the complex, distant from any binding interface and distant from the bound galactose (30 Å) and ATP (25 Å). The mutational effect of the rather conservative amino acid change (methionine to isoleucine) on the KGal parameter might therefore be negligible. In contrast, the non‐conservative arginine to isoleucine mutation at site 312 could influence KGal in several ways: First, the positively charged arginine contributes favorably to the binding of the negatively charged ATP through long‐range electrostatic interactions. The charge‐neutral Ile312 variant lacks this favorable interaction and may have lower affinity for ATP, thereby penalizing activation by the two ligands. Second, residues Arg312 of the two Gal3p units are in direct contact with each other, and the non‐conservative R312I change may affect the dimerization of Gal3p. Lavy et al (2012) reported that, in absence of galactose, Gal3p is monomeric in solution and adopts an open conformation that differs from the conformation generating the Gal3p:Gal3p dimeric interface found upon interaction with Gal80p. If the R312I modification alters Gal3p dimerization, this could modify the overall activation by galactose because these processes are coupled.
We observed that genetic variation of the strength of activated Gal3p (ρGal3), estimated from the dynamic properties of network activation, was correlated with variation of the glucose/galactose trade‐off at steady state. This implies that the two traits co‐evolve in natural populations of S. cerevisiae. Given the relatively short time scale of network induction, mild differences in the dynamics of activation alone are unlikely to cause fitness differences unless environmental galactose concentrations are highly dynamic. In contrast, variation in the sensitivity of the network to the ratio of external sugars corresponds to the triggering of an adaptive metabolic process, which is highly related to fitness even for slow environmental changes. The induction dynamics that we observed on short time scales are probably not themselves under selection, but they provide valuable information on the molecular mechanism affecting a fitness‐related trait operating on longer time scales.
The H352D variant is interesting in this regard. At this position in Gal3p, a histidine residue was found in all laboratory strains (BY4741, CEN.PK, D273‐10B, FL100, FY1679, JK9‐3d, SEY6210, W303, X2180‐1A, YPH499), while nearly all natural isolates as well as distant species possess an aspartic acid. Our results showed the importance of this aspartic acid for interaction with Gal80p, which suggests that its conservation in wild population results from purifying selection. The presence of slightly deleterious mutations in laboratory strains is well known. Examples from the reference strain BY/S288c include mutations in AMN1 (Yvert et al, 2003), BUL2 (Kwan et al, 2011), ERC1 (Fehrmann et al, 2013), FLO8 (Kron, 1997), GPA1 (Yvert et al, 2003), and HAP1 (Gaisne et al, 1999). These mutations likely resulted from a release of purifying selection caused by strong population bottlenecks when propagating yeast on petri dishes. As for the genes listed above, the implication for GAL3 is that most mechanistic studies refer to a "wild‐type" protein that is in fact a slightly hypomorphic allele not found in nature.
We also noted cases where the specificities of a GAL3 allele in the context of the BY strain did not reflect the properties of the donor strain. An extreme example of this was the GAL3DBVPG1853 allele which improved the response of the BY strain (Fig 1D) while the DBVPG1853 strain itself did not respond at all to galactose (not shown), presumably because of genetic defects in other genes. Another example was the GAL3Y12 allele which, when introduced in the BY strain, conferred a binary induction that was milder than the binary phenotype of the Y12 strain itself (Appendix Fig S10). This illustrates the importance of other loci than GAL3 that also contribute to the dynamics of network induction. Background‐specific effects are common and should be taken into account when interpreting the functional impact of natural alleles in their original strain context (Gerke et al, 2010).
Linking DNA variants to model parameters: feasibility and potential
We developed our approach using a model system, the yeast GAL network, which was an ideal context for investigation: Molecular players were well known, important network properties had been previously described, genetic engineering could be used to study the effect of a single gene in an otherwise isogenic background, and experimental measurements were relatively cheap. If network modeling had provided no added value in such a context, it would be hard to imagine how it could be useful in more complex frameworks. We report that it did: Observing different dynamics experimentally was not sufficient to make functional inferences, but combining data and modeling was. The concept is therefore fruitful and it is interesting now to consider how it can be extended to other biological systems.
First, it is important to realize that inference is based on the wealth of information contained in the dynamics of activation. Evidently, studying the system at equilibrium would not be sufficient. Mapping DNA variants to model parameters is therefore promising for systems where time‐course data are available.
Second, even in the simple context of our study, not all parameters of the model were identifiable and it was necessary to aggregate several of them into a meta‐parameter (ρGal3). We admit that this constitutes a limit of the approach: When the H352D SNP was linked to this meta‐parameter, additional assumptions were needed to infer biochemical effects. Similar difficulties will likely be encountered in other systems and the identifiability and sensitivity analysis of the model are therefore crucial to determine the nature of biological information that can be retrieved by the approach.
Third, our method here was to infer function and then to validate a prediction by exploring the structural data of a protein complex. Depending on the system under consideration and the data available, it may be judicious to reverse the approach: scanning protein structures first in order to identify variants modifying binding affinities and then studying these variants specifically using experimental measurements and model fitting. This way, a parameter change is first inferred from structural data and a dynamic model of the network then allows one to predict its phenotypic effect. The SAAP database (Al‐Numair & Martin, 2013), which registers structurally relevant variants of human proteins, may constitute a very helpful resource to do this.
Fourth, while we based our approach on cell population distributions, tracking the response dynamics of individual cells over time is also possible (Stockwell & Rifkin, 2017) and can provide more information on the network response. In other contexts, such methods had been very useful to infer parameters associated with individual cells (Llamosi et al, 2016). A variant may then be associated with one parameter by a whole distribution of values, which likely carries more information than a single scalar value as presented here. In addition, such time‐lapse acquisitions can provide high temporal resolution of gene induction (Aymoz et al, 2016).
Fifth, additional work is now needed to extend the approach to more than one gene. At the level of an entire network, the overall genotype of the individual is a combination of alleles. The number of such combinatorial genotypes of the network segregating in natural populations can be very large and mapping this diversity to the parameter space would be very interesting. In particular, models accounting for genetic changes might predict and explain genetic interactions (epistasis) within the network. The challenge to achieve this will likely reside in the number of free parameters: If the genotype is allowed to vary at too many genes, parameters cannot be constrained efficiently. Mapping variants one gene at a time, as we did here, and then in combination would maintain this necessary constraint while evaluating epistasis. A more difficult task would be to infer the contribution of genes that are external to the network while nonetheless affecting its behavior (e.g., by modifying widely transcription rates or the stability of proteins, or cross‐talks with other networks). Studying these factors by our approach is only possible after they are identified and connected to the network. Their identification can be obtained by genetic mapping. For example, we recently identified a locus on yeast chromosome V that affects the variability of the GAL response at transient times of activation (Chuffart et al, 2016). Once identified, these factors must be integrated in the network model, which may be a complex task.
Network modeling is expected to help the development of personalized medicine and the fact that it is possible, in a yeast system, to personalize model parameters according to DNA variants is encouraging. Can the approach described here be applied to human variants? This requires overcoming several difficulties that could be avoided in our framework. First, most regulatory networks of human systems are incompletely known. Second, most of these networks comprise numerous genes, implying many model parameters and, possibly, too many degrees of freedom for adjustments and identifiability issues. The first task is therefore a careful identifiability and sensitivity analysis of the model and, as much as possible, a reduction of its complexity. The work of Zhao et al (2015) is encouraging in this regard. The authors studied the mitochondrial outer membrane permeabilization network controlling entry in apoptosis. Their model comprised ~50 parameters and ~20 molecular species, but the network critical behavior (bifurcation point) was sensitive to less than half of the parameters. The authors then searched for enrichment of cancer mutations in protein domains involved in molecular interactions and they used molecular dynamics simulations to estimate the affinity changes caused by these mutations. Interestingly, most mutations that were predicted to affect sensitive parameters of the model caused a significant change of affinity in the expected direction, illustrating that the model was able to highlight relevant vulnerabilities. Similarly, Nijhout et al (2015) studied a model of the folate‐mediated one carbon metabolism system. They reported that human mutations that strongly perturb enzymatic activities could have little phenotypic effect if they targeted parameters that are poorly sensitive. Another type of difficulties when studying human networks are experimental limitations: Manipulating human cells needs more time and funds than manipulating yeast; replacing alleles of specific genes is possible via CRISPR/Cas9 editing but the large physical size of human genes as well as the functional redundancy between paralogs can be problematic; and setting up dynamic experimental acquisitions is often not straightforward. Thus, applying our approach to a minimal network in human cells compatible with genetic editing and time‐series acquisitions will probably constitute an important step in the near future.
Materials and Methods
Yeast strains and plasmids
The strains used in this study are listed in Appendix Table S3. Oligonucleotides are listed in Appendix Table S4. We used the strain BY4711 (GY145, isogenic to s288c) as BY reference strain. The PGAL1GFP reporter cassette was obtained from plasmid pGY338 previously described (Chuffart et al, 2016). pGY338 was linearized by NheI and integrated at the HIS3 locus of BY4711 to create strains GY1648 and GY1649, two independent transformants. To replace endogenous GAL3BY allele by natural variants in GY1648 strain, we PCR‐amplified the TRP1‐GAL3 locus of natural wild isolates using primers 1D28 and 1D56. The endogenous locus was then replaced by in vivo homologous recombination and positive transformants were selected on SD‐TRP plates. GAL3NCYC361, GAL3K11, GAL3Y12, GAL3DBVPG1788, GAL3DBVPG1853, GAL3YJM978, GAL3JAY291 were PCR‐amplified from NCYC3451, NCYC3452, NCYC3445, NCYC3311, NCYC3313, NCYC3458 [wild isolates from the Saccaromyces Genome Resequencing Project, SGRP (Louis & Durbin, 2007; Liti et al, 2009)] and JAY291 (Argueso et al, 2009), respectively. The strains used to characterize the effect of natural variants on galactose response were GY1648, GY1689, GY1692, GY1695, GY1698, GY1704, GY1707, and GY1713, all isogenic to S288c except for GAL3BY, GAL3NCYC361, GAL3K11, GAL3Y12, GAL3DBVPG1788, GAL3DBVPG1853, GAL3YJM978, GAL3JAY291, respectively. Strains genotype was verified by PCR and either high‐resolution melting curves, restriction fragment‐length polymorphism typing, or sequencing. The TRP1‐GAL3 locus from BY strain was PCR‐amplified with primers 1M95 and 1M96 and cloned into HpaI‐linearized plasmid pALREP (Fehrmann et al, 2013) by homologous recombination in yeast, generating plasmid pGY409. The mutated GAL3BY‐H352D allele was synthesized by GeneScript and subcloned into pGY409 using MscI‐BstEII restriction sites, generating plasmid pGY418. The TRP1‐GAL3BY‐H352D locus was PCR‐amplified from pGY418 using primers 1D28 and 1D56 and transformed into GY1649 to create strain GY2009. Genotype was validated by PCR and sequencing. Strains of Fig 5 were MPJ125‐E06 (GAL3BY), MPJ143‐H01 (GAL3YJM428), MPJ143‐F01 (GAL3YJM421), and MPJ125‐A07 (GAL3BC187), which were described in another study (Lee et al, 2017); they all derived from a S288c hoΔ::GAL1pr‐YFP‐mTagBFP2‐kanMX4; gal3∆::hphNT1 parental strain. Strains GY2180 and GY2181 carrying mutations GAL3W117A and GAL3W117T, respectively, were obtained using CRISPR/Cas9: Oligonucleotides 1P29 and 1P30 were annealed and cloned in the SwaI, BclI sites of the pML104 plasmid (Laughery et al, 2015) to produce pGY514. Repair‐templates were produced by PCR on pGY474 (a plasmid containing the wild‐type GAL3 gene from strain BY) using primers 1P22 and 1J63 (for W117A) or 1P23 and 1J63 (for W117T). Strain GY1566 (Chuffart et al, 2016) was co‐transformed with pGY514 and the repair template and plated on −URA selection medium. Transformants were verified by PCR with 1P21 and 1J63 followed by Sanger sequencing. They were cultured on YPD plates, and plasmid loss was verified by replica‐plating on −URA plates.
Galactose response measurements
Liquid cultures in synthetic medium with 2% raffinose (yeast nitrogen base w/o amino acids 6.7 g/l, raffinose 2%, dropout Mix 2 g/l, adjusted to pH = 5.8) were inoculated with a single colony and incubated overnight, then diluted to OD600 = 0.1 (synthetic medium, 2% raffinose) and grown for 3–6 h. The galactose induction experiments were carried out in 96‐well sterile microplates using a Freedom EVO200 liquid handler (Tecan) equipped with a 96‐channel pipetting head (MCA), a high precision 8‐channel pipetting arm (LiHa), a robotic manipulator arm (RoMa), and a MOI‐6 incubator (Tecan). All robotic steps were programmed in Evoware v220.127.116.11 (Tecan). Cells were resuspended in synthetic medium with 2% raffinose and the appropriate galactose concentration (0.01, 0.1, 0.2, and 0.5%) and grown for the desired time (from 0 to 250 min). Cells were then washed with PBS1X, incubated for 8 min in 2% paraformaldehyde (PFA) at room temperature, followed by 12 min of incubation in PBS supplemented with glycine 0.1 M at room temperature and finally resuspended in PBS. They were then analyzed on a FACSCalibur (BD Biosciences) flow cytometer to record 10,000 cells per sample. Each set of data is representative of the results of two independent experiments (each comprising three technical replicates).
Flow cytometry data were analyzed using the flowCore package from Bioconductor (Hahne et al, 2009). Cells of homogeneous size were dynamically gated as follows: (i) removal of events with saturated signals (FSC, SSC, or FL1 = 1023 or = 0), (ii) correction by subtracting the mean(FL1) at t = 0 to each FL1 values, (iii) computation of a density kernel of FSC, SSC values to define a perimeter of peak density containing 60% of events, (iv) cell gating using this perimeter, (v) removal of samples containing less than 3,000 cells at the end of the procedure, and (vi) correction of the data according to an eventual experimental bias during cytometer acquisitions. For the twelve time points (0, 10, 20, 30, 40, 60, 80, 100, 130, 160, 205, and 250 min) experimental design, the time course for a given strain was acquired on different plates on the flow cytometer. In order to correct an eventual plate effect, we systematically included 24 replicates on each plate acquired on flow cytometer. We then tested the fixed effect of plates using an ANOVA. The FL1 values of each cell were subsequently corrected according to the plate offset of the ANOVA. For the six time points (0, 30, 60, 80, 130, and 210 min) experimental design, all the time points being acquired on the same experimental plate, we did not apply the normalization filter. The GFP expression values presented here in arbitrary units were the FL1 signal of the retained cells (normalized for the plate effect, if required).
Analysis of flow cytometry distributions
All statistical analyses were done using R (version 3.2.4).
Calculation of the response amplitude.
The response amplitude A was defined as the mean of PGAL1GFP expression in activated cells. First, for each strain, at each time point, we determined by eye if the PGAL1GFP distribution was unimodal or bimodal . If the distribution was unimodal, we calculated: A = μALL. Otherwise, bimodal distributions were considered as mixtures of two normal distributions, such as: , with A = μON. We used the function mixtools::normalmixEM() to calculate A for mixture distributions.
Calculation of inducibility.
Inducibility was defined as the proportion of ON cells in the population. The threshold t between OFF and ON cells was calculated as follows: (i) a subset of OFF cells (all cells acquired at t = 0 min) and ON cells (activated cells belonging to unimodal distributions, acquired at the latest time point of the experiments) was defined for each experiments, (ii) the mean and standard deviation were extracted from each OFF and ON normal distributions using the function mixtools::normalmixEM(), (iii) these parameters were used to determine t such as , with XON the observed fluorescence FL1 in ON_cells and XOFF the observed fluorescence FL1 in OFF_cells, (iv) we finally calculated for each time point, for each strain.
Stochastic modeling
We model the stochastic gene expression of GAL1, GAL3, GAL80, and of the reporter gene (under a GAL1 promoter). For each gene, we account for the status of the promoter (ON/OFF) and for the production and degradation of mRNAs and proteins. In addition, for the reporter gene, we account for the maturation of the fluorescent protein. The promoter switching rate from ON to OFF for gene i is driven by GAL80p: with ni the number of strong GAL4p binding sites in the promoter. We assume that GAL80p represses transcription via its dimerized form (with K80 encompassing the dimer dissociation constant). The promoter switching rate from OFF to ON is driven by GAL3p and Gal1p: with the number of activated proteins at a given galactose concentration [gal] (Kgal being the galactose dissociation constant). Here also, we assume that activated Gal3p and Gal1p are mainly found as dimers. K1 and K3 encompass the dimer dissociation constants as well as the affinity of activated proteins for Gal80p. For a detailed description of the model, see Appendix Text S1. Most of the parameters of the model (except K1, K3, K80, and Kgal) were fixed based on the literature (see Appendix Table S1 in Appendix Text S1). The model had 7 GAL3‐dependent parameters: α3 (leaky transcription rate), γ3 (translation rate), β3 (mRNA degradation rate), μ3 (protein degradation/dilution rate), Δα3 (full transcription rate), K3, and Kgal. The phenotypic response of a strain (gradual vs. binary) at a given galactose concentration mainly depends on Kgal and on the strength of GAL3 defined by ρGal3 = α3γ3/(β3μ3K3) (see main text, Appendix Text S1 and Appendix Fig S12). For a given set of parameters, the stochastic dynamics of galactose induction was simulated using the stochastic simulation algorithm from Gillespie (Gillespie, 1977). The system was first allowed to reach steady state at [gal] = 0. At t = 0, galactose is introduced and the parallel—independent—evolution of 5,000 cells is monitored during 250 min of real time. The model is provided in computer Code EV1.
Parameter inference
For a fixed set of GAL3‐independent parameters, predictions for various values of GAL3‐dependent parameters ρGal3 and Kgal were performed at three different galactose concentrations (0.05, 0.1, and 0.5%). Parameters were sampled from a 2D logarithmic‐grid encompassing the region of interest. Then, for each strain, a global chi‐squared score between the experimental data and the corresponding model predictions integrating the three concentrations were minimized to infer ρGal3 and Kgal. Uncertainties on the parameters reflect the size of the sampling parameter grid. Parameter inference was repeated six times for different values of GAL3‐independent parameters (see Appendix Text S1).
Molecular dynamics simulations for free energy calculations were carried out as described in Appendix Text S2 and Fig S11.
Data availability
All flow cytometry raw data files can be downloaded from http://flowrepository.org under accession number FR‐FCM‐ZY6Y.
We thank Orsolya Symmons for discussions, Olivier Gandrillon for critical reading of the manuscript, Nicolas Rochette for sequence analysis, Sandrine Mouradian and SFR Biosciences Gerland‐Lyon Sud (UMS344/US8) for access to flow cytometers and technical assistance, the Pôle Scientifique de Modélisation Numérique and the CIMENT infrastructure (supported by the Rhône‐Alpes region, Grant CPER07_13 CIRA) for computing resources, BioSyL Federation and Ecofect LabEx (ANR‐11‐LABX‐0048) for inspiring scientific events, developers of R, bioconductor and Ubuntu for their software, and two anonymous reviewers for their comments. This work was supported by the European Research Council under the European Union's Seventh Framework Programme FP7/2007‐2013 Grant Agreement no 281359. DJ acknowledges program AGIR of University Grenoble‐Alpes and the Institut Rhône‐Alpin des Systèmes Complexes for funding. M. Spr. was supported by grants NSF 1349248 and RO1 GM120122‐01 from the National Science Foundation (USA).
Author contributions
Performed experiments: MR, HD‐B, EF, ME, AB; Contributed analysis tools: MR, FC, DJ, and GY; Contributed reagents: MSpr; Developed and evaluated pilot versions of the model: FC and FP; Conceived, implemented and used the model: DJ; Performed molecular dynamics simulations: MSpi; Interpreted results: MR, MSpr, MSpi, DJ and GY; Conceived and designed the study: GY; Wrote the paper: MR, DJ and GY.
Conflict of interest
The authors declare that they have no conflict of interest.
Expanded View
Appendix [msb177803-sup-0001-Appendix.pdf]
Code EV1 [msb177803-sup-0002-CodeEV1.zip]
FundingH2020 | H2020 Priority Excellent Science | H2020 European Research Council (ERC)http://dx.doi.org/10.13039/100010663 SiGHT 281359
This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
- © 2018 The Authors. Published under the terms of the CC BY 4.0 license


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- Published: Monday, 23 January 2017 14:12
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July 22, 2013
No metropolitan area has a skyline as iconic or an infrastructure as intricate as New York City. Given the volume of people who traverse Gotham's streets, descend upon its tourism sites, partake of its cultural offerings, and rub elbows on its subways, the five boroughs present a constant architectural puzzle. How do developers meet the swelling needs of burgeoning neighbourhoods? As tastes change and economic centres expand, how can architects offer something new that will provide maximum utility as well as enduring visual inspiration? These quandaries are the obsession of the world's top architectural minds.
Rick Bell, FAIA, is the Executive Director of AIANY, NYC's chapter of the American institute of Architects. Working from the Center for Architecture – a fascinating spot near Washington Square Park with revolving exhibitions for anyone interested in the city's changing landscape – Bell spends significant time weighing the aforementioned questions and offering his perspective to the global design community. "New York continues to be aspirational – not just in terms of height," says Bell. "There's a big push for connectivity. I think of the Standard hotel that straddles the High Line, built by architect Todd Schliemann. That structure defines the changing attitude of the city – bringing value to everything along its perimeter."
Bell points out that the newly completed 1776-foot spire atop 1 WTC at the resurrected World Trade Center site is a soaring example of the city's expressionistic bent – as is Frank Gehry's Beekman Tower in Lower Manhattan and Renzo Piano's home for The New York Times. Grimshaw Architects' city bus shelters and work on the Queens Museum of Art have contributed a streamlined vision of future New York. Meanwhile, the site that has Bell most captivated is Fulton Center (scheduled to open mid-2014), also conceived and executed by Grimshaw. The center will see 12 subway lines and the PATH train to New Jersey converge beneath four storeys and a glass atrium, outfitted with significant office and retail space and serving more than 300,000 commuters. "The Fulton Center speaks to New York's identity as a "˜portal city,'" says Bell.
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While New York continues to assert itself against the sky in new and proud ways, the most exciting work seems to be taking place on the street, below ground, and on the drafting room table.
Header photo: New York Skyline at night © Songquan Deng, 2013. Used under license from Shutterstock.com
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Have you visited New York recently? Which of the city's architectural masterpieces are your favourites?
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U.S. trade with China is soaring. In the last 15 years, China has become one of the most important manufacturing zones for U.S. corporations, especially apparel companies. Attracted by cheap labor, and a business-friendly climate, companies like Ann Taylor, Kathie Lee/WalMart, Liz Claiborne and Ralph Lauren have now moved much of their production away from the textile mills of the American south and into the free trade zones of South China.
Now Chinese labor activists are slowly pulling away the wall of secrecy that has long stymied any investigation into the new apparel factories of China.
- Sally Chun, the research director of the China Labour Bulletin, a publication that documents worker and labor rights issues in China.
- Apo Leong, of the Asia Monitor Resource Center, a human rights and labor advocacy group based in Hong Kong, China.
- Charlie Kernaghan, the executive director of the National Labor Committee In Support of Worker and Human Rights, a labor research and advocacy group based in New York City.
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Symantec: Mac OS X Viruses and Security
Researchers and engineers who are working in the security field must have strong constitutions especially when it comes to weathering negative backlash and tired conspiracy theories whenever security and Mac OS X are mentioned in the same breath.
With that in mind, in an effort to improve the quality of the dialogue, I would like to discuss some important issues regarding Mac OS X and security. Symantec Security Response Weblog: Mac OS X: Viruses and Security
Back to: PC Security, privacy news


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|A New Dynamism for Research at University of Buea: Promotion of Research Collaboration and Research Uptake
|Friday, 16 October 2015 13:33
The University of Buea's mission for research is to conduct research for sustainable development to provide scientific evidence and evidence-based practices that enhance the quality of life of Cameroonians. Both fundamental and applied research is done to address issues of relevance to industry, the local, national and global communities. Sama Awa-Mengi Levai outlines the University's approach to Research and its promotion of Research Uptake.
The University of Buea (UB) is a young, but dynamic and active research university that is dedicated to excellence and ensuring relevance of its research and training to the community. It is one of nine public Universities in Cameroon and mentor to 14 private higher education institutions. The University has grown rapidly since it went operational in 1993.
It opened its doors to 780 students in four establishments and had a staff strength of less than 10. Today the University of Buea has seven Faculties, two colleges and one school, running 52 departments and 181 programmes. The student population stands at over 18,000 with an academic staff strength of over 400.
"This year, the University is working towards a new dynamism for research and the promotion of collaboration for Research Uptake"
UB's Research Planning for the 2015/2016 Academic Year
It's a tradition that the University of Buea organises research planning days at the beginning of each academic year. These days offer an opportunity for the university to streamline its research activities according to the univerity's spelled out vision for that academic year. This year, the university is working towards a new dynamism for research and the promotion of collaboration for Research Uptake. Research at the University of Buea, though innovative, has, in most cases not been taken up by the community. This may have been for reasons such as lack of attractive modes of communication and la ack of resources (both financial and human). Against this backdrop, University of Buea intends to highlight the uptake component of research and make research findings available and beneficial to those for whom it is intended.
At the start of the planning days, Deputy Vice-Chancellor in charge of Research, Cooperation and Relations with the Business World, Professor Fonkeng Epah George noted in his keynote address entitled: 'Creating an enabling environment for enhanced collaboration on research uptake at UB', that the focus of this year's research planning is to evaluate research projects for their suitability to promote Research Uptake in the institution.
University staff members were treated to presentations on elements of research project management,, including 'Relevance and Getting it Right, Research Uptake and Evaluation: Strategic Approaches' and 'Research Uptake in Practice, a typical case study on the WECARD-CORAF project'. There were also workshops and plenary sessions on Research Collaboration and Resource Mobilisation that focused on 'Stakes and Challenges', 'Strategies and Mechanisms', 'Grant and Proposal Writing for Emerging Researchers' and 'Scientific Writing and Publications for Emerging researchers'.
The over 200 post-graduate students present at the event were reminded of the fundamentals of the requirements of the Thesis Guide of the University, so that the requirements of academic writing form a strong base for subsequent research and writing for Research Uptake.
"research being undertaken at UB contributes toward research-based solutions to important national issues"
* Courtesy: Research and Publications Office, University of Buea
An examination of the subject of these projects shows that the research being undertaken at UB contributes toward research-based solutions to important national issues.
The management of the University of Buea takes a strategic approach to building and sustaining an enabling environment for the growth of research to meet the needs of the community and solve problems plaguing Cameroon as a whole, and focus on Research Uptake as an integral part of the research process.
Sama Awa-Mengi Levai is the University of Buea's DRUSSA Research Uptake Communicator


text: Match the outfits with lovely vests or jackets and a pair of comfy shoes. For accessories pick a tiara made of roses, precious jewelry, a casual scarf, a pair of glasses, amazing bags and a cup of coffee. When you are happy with the outfit, you can complete her look with a gorgeous make-up. Use a bold eye shadow in a cute color combo, mascara to open up her eyes, blush on her cheeks and a bright lipstick. Enjoy Barbie Weekend Outfit game!


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Posts Tagged 'FIDA'
Go Ape!
When was the last time you climbed a tree? When was the last time you swung of a tree? Do you know what it feels like to slide down a wire dangling from a tree and landing in mud? These questions and many more were answered when the finalists went Go Ape!
After a long journey that lasted over ten hours, add to it, the jet lag that always effects you when you travel against time, the last thing you want is to wake the following morning and get down to work right away. The pressure is always mad at the beginning; very reason why the FIDA grand finale made its beginning with a refreshing day-out at the Go Ape, Woburn.
As the entity says about itself, if the word, "Go Ape" were to be in the dictionary, it would be described as 'tree-top adventure'. With tree-top high wires, crossings including walkways, bridges and tunnels made of wood, rope and super strong wire and wind-in-your-face zip wires, the adventures takes a path amidst the woods, giving one a mixture of nature treatment and adventure.
Exciting, though it may seem, the activity is highly risk oriented. To ensure the safety of the apes, they were given safety training at the beginning, followed by a small demonstration before they were finally let to go through the course by themselves. With over 20 crossings over 3 trails and finish on a high (literally), with one awesome zip wire, rejuvenation is guaranteed. Having proven their ability with the karts, the finalists went on to show that they had a few more tricks up their sleeves as well. Further, they also proved their ability to adapt to a given situation when on being stuck, all they required was a little guidance on how to get along and the theory was put into practice right away.
Come evening, the finalists made their presence at the Force India's factory situated in Silverstone. They were given a tour through the facilities by Mick and Damien, who went on to explain in depth the various aspects involved in the making and running of a Formula One car. What's more, the contestants even got an opportunity to stretch their legs in those very Force India's Formula One cars! They were then presented driving suits, shoes and gloves, customized to suit them.
With a relaxing adventure in the morning followed by the painting of a colorful dream in the evening, the contestants would sure be in the best of their spirits to bring the dream to life.
Please follow the following link to take a peak at the original post.
Lights, Camera, Action.
The final day the one from a billion wildcard round was far from a normal day. The day began for us with an 18 year old kid setting the track on fire with a blistering speed of 23.737. Karting since he was an 8 year old kid, Parth was oozing with experience and it was clearly showing in his time as he gained time with every completed lap. A first year B.Com student from Kohlapur, he is a formula 4 racer. What's more he also happens to be the national champion for three continous years from 2008 to 2010. Being asked what it was that got him into racing, he says its his craze for speed. Growing up watching F1, he adds, "The more you watch, the more you get into it".
Support or no, I am racing..
Letting an interest to travel down the path of professional career, has always been a debatable issue. Atleast from the parents' angle, you would say. The parent-children battle, over the choice of career has had a very long history, with all possible permutations long dealt with. In recent times we have had various movies on the same. However its easier said than done, which brings us back to the parents' view, in turn, the debate. On the other hand, there are parents who are very supportive, doing every little thing they can to support their child's interest. Support or no support, we have talent pour in, which brings us back to day 2 of the wildcard round. The second day saw just one competitor for Arya in the form of Srinath. Hailing from Hyderabad, a standard 9 student, Srinath has been karting from the time he was in 7th. However, that didn't seem to help him much as he failed to cut through Arya's timing. If the other competitor, Shamir clocked a 35.27, Srinath came closer by clocking 27.8, which still was over three seconds from beating Arya.
On the other hand, Mohit, the topper in the 17-19 group, at the end of day one, faced quite some competition. First of them was Akshay from Ahmadabad, an automobile engineering student, he managed to clock a 24.637. Rinaldo, a Goan, who has been into karting for three to four years went on the track soon after. Speaking to us, he said his dad was ok with his karting, whereas his mom was not happy with it. On finding out he had clocked a 25.121, he looked very disappointed and said it was one of his worst performances.
Next on the list was Viklesh, a Hyderabadi, mechanical engineering student, who got into karting hardly a week ago. For a person with hardly any experience to clock a 26.44, definitely not bad, you would say. Following him, to go on the track were Taran and Ahmed, who managed to clock 26.375 and 25.783 respectively. Another mechanical engineering student, hailing from Delhi, Taran says his parents find the sport dangerous. However they have no objection to his participation, which is evident from his presence, but they ask him to be safe.
The last of the lot in the 17-19 group was Yamir, an NRI whose parents are from Bengaluru, looked to be a promising talent. Being into the racing field for a few years and having experience of being a rotax racer, the American drove professionally. However, the best of his efforts failed to get him qualified as he managed to clock a 24.39, losing the top spot by half a second.
At the end of day 2 however, both Arya and Mohit still remain unbeaten. By the look of it, the time set looks strong enough to make it through tomorrow as well. However, the possibility of a surprise break in can never be ruled out. What the final day has got in store for us, we can only wait and watch.
A short day with quickies
'Wish I were a year older, wish I were a month younger, so on and so forth..' These thoughts, and many more similar ones were answered to, when The One From A Billion hunt decided to bring out a wild card round. As per the initial hunting regulations set up, the One from a billion was to come from the age group of 14 to 17 which meant the 12 to 14 age group would fall off as a result of early cut-off and the 17 to 19 age group would get chopped due to a late cut-off. The wildcard round takes form to amend just that. Unlike other wildcards, which usually include a knocked out competitor making it back to the challenge, this one was different. A tweak in the age limit, ensuring the inclusion of the 12 to 14 as well as the 17 to 19 age group, by opening up a slot each in both the groups, thus making the number of finalists to a hundred.
However, as all good things come with a catch, so does this one. A catch that is more of a glitch, a rule put in to ensure fairness to the contestants already done with. The catch goes on to say that, in order to be selected, the contestant is required not only to be the topper in his/her group but also has to beat the timing set by the 14th best contestant at the track.
The avid followers would recall reading about a mention of yellow line which lead to a cave that was used by Neanderthals to decide who'd lead the tribe, by karting. The wildcard round happened to stumble upon the same cave to find that amongst the structures installed as a part of the hall of fame, various happened to now have their nose pierced. No kidding, a clean pierce.
The light of the day came in the form of Mohit Ahuja, who also happens to have set the fastest lap timing for the day. Another Mumbaikar, he is been racing since eleven years, participating in various national and international events. He left behind a clear signature by beating the fastest time for the day by 0.2 seconds.


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Phone :+91-(522)-2772450/2772550 Search
My group's interest in the malaria parasite is driven by the desire to understand (a) the molecular workings and functions of the relict plastid (apicoplast) of Plasmodium, (b) mechanisms of protein translation employed by Plasmodium organelles and (c) human genetic factors and susceptibility to severe P. falciparum malaria in endemic and non-endemic regions of India. We use cellular and molecular biology tools to investigate the essentiality of specific apicoplast proteins in housekeeping and metabolic functions, determine their biochemical interactions, and explore the possibility of these serving as specific drug targets against malaria. Exploration of the organisation and replication of the apicoplast genome, its translation machinery, interactions of antibiotics with apicoplast and mitochondrial ribosomes, regulation of DNA repair and organellar transcription, and delineation of the SUF pathway of [Fe-S] cluster biosynthesis engages us. In collaboration with clinicians, we also look at variation in genes encoding immune regulatory and cell adhesion molecules in Indian populations and their association with susceptibility/resistance to falciparum malaria in the country.


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Buba Galadima, chairman of the "reformed" All Progressives Congress (R-APC), has asked a federal high court in Abuja to annul the convention of the ruling party and declare offices of the national working committee (NWC) executives "null and void".
He also asked the court to allow members of the R-APC preside over the affairs of the APC on interim basis.
Galadima said since the APC did not conduct a valid convention, those parading themselves as national officials were doing so illegally.
He asked the court to grant him and members of his faction control of the party.
Among his requests in the documents seen by TheCable are: "A DECLARATION that there being no valid election at the National Convention of the All Progressives Congress (APC) on the 23rd day of June 2018 at the Eagle Square, Abuja, thereby leaving a vacuum, the reform-minded FACTION of APC known as the REFORMED ALL PROGRESSIVES CONGRESS (R-APC) validly constituted the Plaintiffs and the other National Officers and State Executives of APC to fill the vacuum and act in interim capacities as officers of the APC.
"AN ORDER of perpetual injunction restraining the 1st – 52nd Defendants from parading themselves or claiming to be officers of the All Progressives Congress (APC) or performing any function as officers of the Party in any capacity whatsoever as their elections and swearing in as officers of APC are invalid, null and void.
"AN ORDER of perpetual injunction restraining the 53rd Defendant from holding out the 1st – 52nd Defendants as either National Officers, members of the National Working Committee , Zonal Executives or State Executives of the All progressives Congress (APC).
"AN ORDER of perpetual injunction restraining the 53rd Defendant from harassing, intimidating, suspending or expelling any of or all the Plaintiffs from the All Progressives Congress; or taking any steps whatsoever to harass, intimidate, suspend or expel or abridge or terminate the Plaintiffs' membership, rights and entitlements in the All Progressives Congress.
"AN ORDER directing the 54th Defendant to recognize the Plaintiffs who are members of the reform-minded FACTION of the All Progressives Congress (APC) as the current interim National Officers, members of the National Working Committee, Zonal Executives or State Executives of the All progressives Congress (APC) and accord them all rights and privileges appertaining to the respective offices pending the holding of valid elections by the Party."
On Wednesday, the R-APC chairman wrote to the Independent National Electoral Commission (INEC), seeking a cancellation of the election of Adams Oshiomhole, national chairman of the APC.
He alleged that the election which produced Oshiomhole was flawed.
But in a swift response, the ruling party said INEC lacked the powers to cancel what it did not conduct.
Galadima and some aggrieved members of the ruling party formed a breakaway faction a week ago.
On Monday, the group entered an alliance with Peoples Democratic Party (PDP) and 37 political parties to unseat President Muhammadu Buhari in 2019.
The ruling party has dismissed the threats of the group.


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Product 14/193
International shipping
Extra SmallBody Width: 40.5cm (16 inches)Body Length: 67.5cm (26.5 inches)
SmallBody Width: 43cm (17 inches)Body Length: 68.5cm (27 inches)
MediumBody Width: 45.5cm (18 inches)Body Length: 69.5cm (27 inches)
LargeBody Width: 48cm (19 inches)Body Length: 70.5cm (28 inches)
100% Cotton (150 GSM)Shoulder to shoulder tape to prevent stretchingFabric pre-shrunk to minimise shrinkageDouble needle bottom hem and sleevesExtra long body and side seamed for a tailored fit
Care instructions:
Care instructions vary by the product, please look for the care instruction label inside the garment. Please ensure you do not iron directly on top of the animal print of any item.
Black or White
There is no kitten around in this slim fit tee, its time to teach that old dog some new tricks!


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Find Skype user accounts with #southafrica hashtag.
This section lists all the latest Skype #southafrica usernames for 2019.
Explore Skype user photo gallery and discover Skype #southafrica stories.
Meet interesting people on Skype and gain new friends and followers.
Do you want to make new friends or get more followers on Skype? Do you want to be listed on Skype #southafrica?
Join now and add your username and discover interesting people on Skype.
The #southafrica hashtag is an interest that the following listed Skype user accounts likes.
Add #southafrica in your likes hashtag list if you want to be recommended to like-minded Skype users.


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5 Star Lanzarote Hotels
The island of Lanzarote and the other Canary islands are one of the most popular all year round holiday destintions. Regardless of whether you are looking for an all inclusive five star hotel, or even self catering hotel in Lanzarote, we are sure to have the hotel for you.
Choose a five star hotel located in all your favourite Lanzarote holiday resorts such as Playa Blanca, Costa Teguise, and Puerto del Carmen.
Popular 5 Star Hotels in LanzaroteWhen it comes to the high end properties, we understand that many people are looking for great value holidays and we have a great range on offer at affordable prices. Search our website for lavish 5 star Lanzarote hotels and resorts which are equipped with the latest state of the art amenities, a packed schedule of available recreational and leisure activities, stunning gourmet cuisine and flawless facilities. We have handpicked some of favourite five star hotels on this beautiful island.
5* Princesa Yaiza
Great For All Ages
This amazing 5* hotel offers great facilities for all the family, Kikoland provides a great range of activities for the kids.Take me there
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This 5* hotel overlooking the sea, offers visitors all the services and convenience imaginable in a luxury hotel.Take me there
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Adults Only Hotel
This luxururious 5* boutique hotel is perfect for a relaxing break, with fine dining cuisine and exceptional service.Take me there
Popular Lanzarote holiday resorts
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Playa Blanca
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Situated on the south coast, Playa Blanca was originally a fishing village and is now a firm favourite with holidaymakers.Take me there
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One of the most popular beach resorts in Lanzarote, strewn with bars and restaurants perfect for a relaxing holiday in the sun.Take me there


text: CHRIS KLUWE DEALS HIS NUMBER TO DONOVAN McNABB Minnesota Vikings Chat 5 = Donovan McNabb's old jersey number. 5 = Chris Kluwe's old jersey number. 4 = Brett Favre's old jersey number. 4 = Chris Kluwe's new jersey number. THOUGHT: You have likely already heard that the Minnesota Vikings traded with Washington for quarterback Donovan McNabb. He will wear the same … [Read more...]


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This isn't an easy article to write, simply because it's pretty personal. But I've learned that in order to help others who are coping with the loss of a loved one, I need to be more open. So, with that, I want to admit that a few months after my brother, Eddie Au, had passed I decided to seek some counseling.
Now, I wasn't at a stage of severe depression nor feeling like I was losing my mind. What I was feeling, with no better way to put it was, cloudy. I noticed I would want to sleep more, wasn't as excitable, less motivated to exercise, and felt like I wasn't processing thoughts as quickly as I once did. Ultimately, I knew I wasn't being myself and I hated it.
I was extremely hesitant about going for bereavement counseling because of all the negative connotations that seem to follow it. But in the end, it came down to me wanting to become better and stronger. And I'll tell you this now, when I have told people that I've gone, they don't look at me with pity nor shock. It just makes sense. Let's be honest, a majority of the population would probably do a lot better with some form of counseling =).
I want to make it clear that my family and friends were available to me. However, I must say, that there's something different about talking to a person whom you know are binded by law to keep everything you say to them private. And since the company I worked for provided insurance coverage for counseling, I figured it couldn't hurt to at least try.
After the first session, I continued going weekly for just over a year. I realized that each visit helped because it provided me an outlet to talk about what I was going through, even though a few visits felt repetitive and uneventful.
I believe a lot of people sink into depression because they feel they're alone in the matter and that nobody cares about what they're going through. And that's the biggest mistake they make. You need to open up!. If you don't have the means to seek professional counseling, then at least talk to family, friends, or join a grief counseling forum to tell your story.
People experience the loss of a loved one every single day. Each person may experience it differently, but you should know that you're not alone. And there is no shame in asking for some help every now and then.