- Research Article
- Open Access
Inferring gene regulatory networks from single-cell data: a mechanistic approach
- Ulysse Herbach^{1, 2, 3}View ORCID ID profile,
- Arnaud Bonnaffoux^{1, 2, 4},
- Thibault Espinasse^{3} and
- Olivier Gandrillon^{1, 2}Email author
https://doi.org/10.1186/s12918-017-0487-0
© The Author(s) 2017
- Received: 12 May 2017
- Accepted: 9 November 2017
- Published: 21 November 2017
Abstract
Background
The recent development of single-cell transcriptomics has enabled gene expression to be measured in individual cells instead of being population-averaged. Despite this considerable precision improvement, inferring regulatory networks remains challenging because stochasticity now proves to play a fundamental role in gene expression. In particular, mRNA synthesis is now acknowledged to occur in a highly bursty manner.
Results
We propose to view the inference problem as a fitting procedure for a mechanistic gene network model that is inherently stochastic and takes not only protein, but also mRNA levels into account. We first explain how to build and simulate this network model based upon the coupling of genes that are described as piecewise-deterministic Markov processes. Our model is modular and can be used to implement various biochemical hypotheses including causal interactions between genes. However, a naive fitting procedure would be intractable. By performing a relevant approximation of the stationary distribution, we derive a tractable procedure that corresponds to a statistical hidden Markov model with interpretable parameters. This approximation turns out to be extremely close to the theoretical distribution in the case of a simple toggle-switch, and we show that it can indeed fit real single-cell data. As a first step toward inference, our approach was applied to a number of simple two-gene networks simulated in silico from the mechanistic model and satisfactorily recovered the original networks.
Conclusions
Our results demonstrate that functional interactions between genes can be inferred from the distribution of a mechanistic, dynamical stochastic model that is able to describe gene expression in individual cells. This approach seems promising in relation to the current explosion of single-cell expression data.
Keywords
- Single-cell transcriptomics
- Gene network inference
- Multiscale modelling
- Piecewise-deterministic Markov processes
Background
Inferring regulatory networks from gene expression data is a longstanding question in systems biology [1], with an active community developing many possible solutions. So far, almost all studies have been based on population-averaged data, which historically used to be the only possible way to observe gene expression. Technologies now allow us to measure mRNA levels in individual cells [2–4], a revolution in terms of precision. However, the network reconstruction task paradoxically remains more challenging than ever.
The main reason is that the variability in gene expression unexpectedly stands at a large distance from a trivial, limited perturbation around the population mean. It is now clear indeed that this variability can have functional significance [5–7] and should therefore not be ignored when dealing with gene network inference. In particular, as the mean is not sufficient to account for a population of cells, a deterministic model – e.g. ordinary differential equation (ODE) systems, often used in inference [8, 9] – is unlikely to faithfully inform about an underlying gene regulatory network. Whether such a deterministic approach could still be a valid approximation or not is a difficult question that may require some biological insight into the system under consideration [10]. Another key aspect when considering individual cells is that they generally have to be killed for measurements: from a statistical point of view, temporal single-cell data therefore should not be seen as a set of time series, but rather snapshots, i.e. independent samples from a time series of distributions.
On the other hand, single-cell data give the opportunity of moving one step further toward a more accurate physical description of gene expression. Molecular processes of gene expression are overall now well understood, in particular transcription, but precisely how stochasticity emerges is still somewhat of a conundrum. Harnessing variability in single-cell data is expected to allow for the identification of critical parameters and also to provide hints about the basic molecular processes involved [11, 12]. Moreover, the variability arising from perturbations in cell populations is often crucial for network reconstruction to succeed [13, 14] as the deterministic inference problem suffers from intrinsic limitations [15]. From this point of view, the same information is expected to be contained in the variability between cells in single-cell data. Some of the few existing single-cell inference methods follow this path, for example using asynchronous Boolean network models [16] or generating pseudo time series [9, 17]. In this article, we use a mechanistic approach in the sense that every part of our model has an explicit physical interpretation. Importantly, mRNA observations are not used as a proxy for proteins since both are explicitly modeled.
Besides, mechanistic models that are accurate enough to describe gene expression at the single-cell level usually do not consider interactions between genes. For example, the so-called “two-state” (aka random telegraph) model has been successfully used with single-cell RNA-seq data [18], but the joint distribution of a set of genes contains much more information than the marginal kinetics of individual genes: our aim is to exploit this information while keeping the mechanistic point of view.
Namely, we propose to view the inference as a fitting procedure for a mechanistic gene network model. Whereas the goal here is not to achieve global predictability performances (e.g. as in [19]), our framework makes it possible to explicitly implement many biological hypotheses, and to test them by going back and forth between simulations and experiments. The main point of this article is to show that a tractable statistical model for network inference from single-cell data can be derived through successive relevant approximations. Finally, we demonstrate that our approach is capable of extracting enough information out of in silico-simulated noisy single-cell data to correctly infer the structures of various two-gene networks.
Methods
In this part, we aim at deriving a tractable statistical model from a mechanistic one. We will use the two-state model for gene expression to build a “network of two-state models” by making the promoter switching rates depend on protein levels. Then, successive relevant simplifications will lead to an explicit approximation of a statistical likelihood.
A simple mechanistic model for gene regulatory networks
Basic block: stochastic expression of a single gene
Chemical reactions defining the two-state model. The rate constants are usually abbreviated to rates as they correspond to actual reactions rates when only one molecule of reactant is present. In the stochastic setting, these rates are in fact propensities, i.e. probabilities per unit of time
Reaction | Rate constant | Interpretation |
---|---|---|
G→G ^{∗} | k _{on} | gene activation |
G ^{∗}→G | k _{off} | gene inactivation |
G ^{∗}→G ^{∗}+M | s _{0} | transcription |
M→M+P | s _{1} | translation |
\(M \to \varnothing \) | d _{0} | mRNA degradation |
\(P \to \varnothing \) | d _{1} | protein degradation |
In practice, the formulas involve hypergeometric series that are not straightforward to use in a statistical inference framework. Besides, these series essentially arise from the fact that such a discrete model has to enumerate all potential collisions between molecules (the stochastic mass-action assumption in the master equation). It is therefore natural to consider keeping only the most important source of noise, that is, keeping a molecular representation for rare species but describing abundant species at a higher level where molecular noise averages out to continuous quantities. A quick look at reactions in Table 1 indicates that the only rare species are G and G ^{∗}, with quantities [G] and [G ^{∗}] being equal to 0 or 1 molecule and satisfying the conservation relation [G]+[G ^{∗}]=1. The other two, M and P, are not conserved quantities in the model and reach a much wider range in biological situations [31], meaning that saturation constants s _{0}/d _{0} and s _{1}/d _{1} are much larger than 1 molecule.
This system simply switches between two ordinary differential equations, depending on the value of the two-state continuous-time Markov process E(t), making it a Piecewise-Deterministic Markov Process (PDMP) [32]. From a mathematical perspective, model (1) rigorously approximates the original molecular model when s _{0}/d _{0} and s _{1}/d _{1} are large enough [33, 34] and interestingly, it has already been implicitly considered in the biological literature [22, 23]. Note also that the stationary distribution of mRNA is a scaled Beta distribution that is exactly the one of the Beta-Poisson mixture in the discrete model [28]. Similarly to a recent approach for a two-gene toggle switch [35], we will use (1) as a basic building block for gene networks.
When both k _{on}≪k _{off} and d _{0}≪k _{off}, mRNA is transcribed by bursts, i.e. during short periods which make the mRNA quantity stay far from saturation. Hence, the amount transcribed within each burst is approximately proportional to the burst duration, whose mean is 1/k _{off} by definition: this justifies the quantity s/k _{off} often being called “burst size” or “burst amplitude”. Furthermore, promoter active periods are much shorter than inactive ones so they can be seen as instantaneous, justifying the name “burst frequency” for the inverse of the mean inactive time k _{on}. We place ourselves in this situation as it often occurs in experiments [22, 23, 36–38]. Note however that these two notions are not clearly defined when relations k _{on}≪k _{off} and d _{0}≪k _{off} do not hold.
Adding interactions between genes: the network model
for which applying the law of mass action directly gives k _{on,i }(P _{1},…,P _{ n })=θ _{ i,0}+θ _{ i,j } P _{ j }. In a regulatory network (Fig. 2 b), it would correspond to adding a directed edge from gene j to gene i, with θ _{ i,0} the basal parameter of gene i, and θ _{ i,j } the strength of activation of gene i by protein P _{ j }. We emphasize that the action of P _{ j } on the promoter G _{ i } is not necessarily direct. For example, P _{ j } can instead indirectly modulate the amount/activity of a transcription factor: we suppose in this article that such hidden reactions are fast enough regarding gene expression dynamics so that protein P _{ j } is a relevant proxy for the transcription factor. Moreover, although we assume here that interactions can only happen at the level of k _{on,i } and k _{off,i }, mainly for identifiability purposes, it is also possible to make d _{1,i } and s _{1,i } depend on proteins without fundamentally changing the mathematical approach (e.g. see [39, 40]).
still omitting the dependence of k _{on,i } and k _{off,i } on (P _{1}(t),…,P _{ n }(t)) for clarity. This form enlightens the fact that s _{0,i } and s _{1,i } are just scaling constants: given a path (E _{ i },M _{ i },P _{ i })_{ i } of system (4), one can go back to the physical path by simply multiplying M _{ i } by (s _{0,i }/d _{0,i }) and P _{ i } by (s _{0,i }/d _{0,i })×(s _{1,i }/d _{1,i }).
Therefore, we get a general network model where each link between two genes is directed and has an explicit biochemical interpretation in terms of molecular interactions. The previous example is very simplistic but one can use virtually any model of chromatin dynamics to derive a form for k _{on,i } and k _{off,i }, involving hit-and-run reactions, sequential binding, etc. [41]. Such aspects are still far from being completely understood [42–45] and this simple network model can hopefully be used to assess biological hypotheses. In the next part, we will introduce a more sophisticated interaction form based on an underlying probabilistic model, which is both “statistics-friendly” and interpretable as a non-equilibrium steady state of chromatin environment [43].
Some known mathematical results
Thanks to some recent theoretical results [40, 46], simple sufficient conditions on k _{on,i } and k _{off,i } ensure that the PDMP network model (4) is actually well-defined and that the overall joint distribution of (E _{ i },M _{ i },P _{ i })_{ i } converges as t→+∞ to a unique stationary distribution, which will be the basis of our statistical approach. Namely, we assume in this article that k _{on,i } and k _{off,i } are continuous functions of (P _{1},…,P _{ n }) and that they are greater than some positive constants. These conditions are satisfied in most interesting cases, including the above toy example (3) when θ _{ i,0}>0.
where P(t)=(P _{1}(t),…,P _{ n }(t)). The diffusion limit, which keeps a residual noise, can also be rigorously derived [48]. Unsurprisingly, one recovers the traditional way of modelling gene regulatory networks with Hill-type interaction functions. Equation 5 is useful to get an insight into the behaviour of the system (4) for given k _{on,i } and k _{off,i }, yet it should be used with caution. Indeed, the d _{0,i }/d _{1,i } ratio has been shown to span a high range, averaging out to the value d _{0,i }/d _{1,i }≈5 in mammalian cells [31], for which taking the limit d _{0,i }≫d _{1,i } is not obvious. This is consistent with recent single-cell experiments showing a high variability of both mRNA and protein levels between cells [37]. In that sense, the PDMP model is much more robust than its deterministic/diffusion counterpart while keeping a similar level of mathematical complexity, which motivates our approach.
Simulation
Deriving a tractable statistical model
We will now adopt a statistical perspective in order to deal with gene network inference, considering a set of observed cells. If they are evolving in the same environment for a long enough time, we can reasonably assume that their mRNA and protein levels follow the stationary distribution of an underlying gene network: this distribution can be used as a statistical likelihood for the cells. Furthermore assuming no cell-cell interactions (which may of course depend on the experimental context), we obtain a standard statistical problem with independent samples. Since the stationary distribution of the stochastic network model (4) is well-defined but a priori not analytically tractable, we will derive an explicit approximation and then reduce our inference problem to a traditional likelihood-based estimation. We will do so in two cases: when there is no self-interaction, and for a specific form of auto-activation.
Separating mRNA and protein timescales
corresponding to the single-gene model [28, 39] with constant parameters k _{on,i }(P) and k _{off,i }(P). Numerically, this approximation works well even for moderate values of d _{0,i }, such as d _{0,i }=5×d _{1,i } (see the “Results” section).
Biologically, Eqs. (7) and (8) suggest that correlations between mRNA levels may not directly arise from correlations between promoters states (which in fact are weak because of (k _{on,i }+k _{off,i })≫d _{1,i }), but rather originate from correlations between promoter parameters k _{on,i } and k _{off,i }, which themselves depend on the protein joint distribution.
Successive dynamical models introduced in this article. We recall for each step the main feature and the form of the mRNA stationary distribution. The full network model (step 3) is used for simulations, while the simplified one (step 4) is used to derive the approximate statistical likelihood
1 | Single-gene, discrete [29] | ||
◇ All molecules are discrete | |||
◇mRNA distribution: Beta-Poisson | ↓ | Abundant species treated continuously | |
2 | Single-gene, PDMP (1) | ||
◇ Only the promoter is discrete | |||
◇ mRNA distribution: Beta | ↓ | Introduction of interactions via k _{on}, k _{off} | |
3 | |||
◇ Both accurate and fast to simulate | |||
◇mRNA distribution: unknown | ↓ | Timescale separation of Protein/mRNA (d _{0}≫d _{1}) | |
4 | Simplified network (7) | ||
◇ mRNA is removed from the network | |||
◇ Conditional mRNA distribution: Beta (8) |
Hartree approximation
In this section, we present the Hartree approximation principle and provide an explicit formula in the particular case of no self-interaction. The simplified model (7) is still not analytically tractable, but it is now appropriate for employing the self-consistent proteomic field approximation introduced in [51, 52] and successfully applied in [53, 54]. More precisely, we will use its natural PDMP counterpart, which will be referred to as “Hartree approximation” since the main idea is similar to the Hartree approximation in physics [51]. It consists in assuming that genes behave as if they were independent from each other, but submitted to a common “proteomic field” created by all other genes. In other words, we transform the original problem of dimension 2^{ n } into n independent problems of dimension 2 that are much easier to solve (see section 2 of Additional file 1 for details).
where y=(y _{1},…,y _{ n })=(P _{1},…,P _{ n })=P, a _{ i }(y)=k _{on,i }(y)/d _{1,i }, b _{ i }(y)=k _{off,i }(y)/d _{1,i } and B is the standard Beta function. Note that promoter states have been integrated out since they are not required by Eq. (8).
The function u is a heuristic approximation of a probability density function. It is only valid when interactions are not too strong, that is, when k _{on,i } and k _{off,i } are close enough to constants, and it becomes exact when they are true constants. Besides, it does not integrate to 1 in general. However, this approximation turns out to be very robust in practice and it has the great advantage to be fully explicit (and significantly simpler than in the non-PDMP case), thus providing a promising base for a statistical model.
When k _{on,i } and k _{off,i } depend on P _{ i }, one can still explicitly compute the Hartree approximation in many cases: we will give an example in the next section. Alternatively, it is always possible to use formula (9) even with self-interactions, giving a correct approximation when the feedback is not too strong, as for other proteins.
An explicit form for interactions
We now propose an explicit definition of functions k _{on,i } and k _{off,i }. Recent work [36, 55, 56] showed that apparent increased transcription actually reflects an increase in burst frequency rather than amplitude. We therefore decided to model only k _{on,i } as an actual function and to keep k _{off,i } constant. In this view, the activation frequency of a gene can be influenced by ambiant proteins, whereas the active periods have a random duration that is dictated only by an intrinsic stability constant of the transcription machinery.
Our approach uses a description of the molecular activity around the promoter in a very similar way as Coulon et al. [42]. Accordingly, we make a quasi-steady state assumption to obtain k _{on,i }. This idea based on thermodynamics was also used in the DREAM3 in-Silico Challenge [57] to simulate gene networks. However, only mean transcription rate was described (instead of promoter activity in our work), which is inappropriate to model bursty mRNA dynamics at the single-cell level.
We herein derive k _{on,i } from an underlying stochastic model for chromatin dynamics. We first introduce a set of abstract chromatin states, each state being associated with one of two possible rates of promoter activation, either a low rate k _{0,i } or a high rate k _{1,i }≫k _{0,i }. More specifically, such chromatin states may be envisioned as a coarse-grained description of the chromatin-associated parameters that are critical for transcription of gene i. Second, we assume a separation of timescales between the abstract chromatin model and the promoter activity, so that the promoter activation reaction depends only on the quasi-steady state of chromatin. In other words, the effective k _{on,i } is a combination of k _{0,i } and k _{1,i } which integrates all the chromatin states: its value depends on the probability of each state and a fortiori on the transitions between them. We propose a transition scheme which leads to an explicit form for k _{on,i }, based on the idea that proteins can alter chromatin by hit-and-run reactions and potentially introduce a memory component. Some proteins thereby tend to stabilize it either in a “permissive” configuration (with rate k _{1,i }) or in a “non-permissive” configuration (with rate k _{0,i }), providing notions of activation and inhibition. A more precise definition and details of the derivation are provided in section 3 of Additional file 1.
The n×n matrix θ=(θ _{ i,j }) therefore plays the same role as the interaction matrix in traditional network inference frameworks [8]. For i≠j, θ _{ i,j } quantifies the regulation of gene i by gene j (activation if θ _{ i,j }>0, inhibition if θ _{ i,j }<0, no influence if θ _{ i,j }=0), and the diagonal term θ _{ i,i } aggregates the “basal input” and the “self-activation strength” of gene i. Note that self-inhibition could be considered instead, but the choice has to be made before the inference since the self-interaction form is notoriously difficult to identify, especially in the stationary regime. In the remainder of this article, we assume that parameters k _{0,i }, k _{1,i }, m _{ i,j } and s _{ i,j } are known and we focus on inferring the matrix θ.
In other words, the Hartree approximation (11) is a product of gene-specific distributions which are themselves mixtures of Beta distributions: for gene i, the a _{ i,r } correspond to “frequency modes” ranging from k _{0,i } to k _{1,i }, weighted by the probabilities p _{ i,r }(y). It is straightforward to check that inhibitors tend to select the low burst frequencies of their target (a _{ i,r }≈k _{0,i }) while activators select the high frequencies (a _{ i,r }≈k _{1,i }). If m _{ i,i }=0 for some i, then k _{on,i } does not depend on P _{ i } so one just has to replace the i-th term in the product (11) with the single Beta form as in Eq. (9), which is equivalent to taking the limit c _{ i }→+∞. Finally, when m _{ i,i }>0 but c _{ i } is not an integer, using ⌈c _{ i }⌉ instead keeps a satisfying accuracy.
The statistical model in practice
where we used conditional factorization and independence of the cells.
The basic statistical inference problem would be to maximize the marginal likelihood of mRNA with respect to θ. Since this likelihood has no simple form, a typical way to perform inference is to use an Expectation-Maximization (EM) algorithm on the complete likelihood (13). However, the algorithm may be slow in practice because of the computation of expectations over proteins. A faster procedure consists in simplifying these expectations using the distribution modes: the resulting algorithm is often called “hard EM” or “classification EM” and is used in the “Results” section. Moreover, it is possible to encode some potential knowledge or constraints on the network by introducing a prior distribution w(θ). In this case, from Baye’s rule, one can perform maximum a posteriori (MAP) estimation of θ by using the same EM algorithm but adding the penalization term log(w(θ)) to ℓ during the Maximization step (see section 4 of Additional file 1 and the “Results” section). Alternatively, a full bayesian approach, i.e. sampling from the posterior distribution of θ conditionally to (M _{1},…,M _{ m }), may also be considered using standard MCMC methods.
Taking advantage of the latent structure of proteins, we can also deal with missing data in a natural way: if the mRNA measurement of gene i is invalid in a cell k owing to technical problems, it is possible to ignore it by removing the i-th term in the conditional distribution of mRNAs (12). This only modifies the definition of v for cell k in Eq. (13), ensuring that all valid data is effectively used for each cell.
Results
In this part, we first compare the distribution of the mechanistic model (4) to the mRNA quasi-steady state combined with Hartree approximation for proteins, on a simple toggle-switch example. Then, we show that the single-gene model with auto-activation can fit marginal mRNA distributions from real data better than the constant- k _{on} model. Finally, we successfully apply the inference procedure to various two-gene networks simulated from the mechanistic model.
Relevance of the approximate likelihood
Fitting marginal mRNA distributions from real data
and thus Beta(a,b)≈γ(a,b). This way, formulas (11) and (12) can be easily transformed into Gamma-based distributions. Parameters s _{0} and k _{off} then aggregate in k _{off}/s _{0} because of the scaling property of the Gamma distribution, so only this ratio has to be inferred: from an applied perspective, it simply represents a scale parameter for each gene. This remark leads to a possible preprocessing phase that can be used for estimating the crucial basal parameters of the network, without requiring the knowledge of such scale parameters (see section 5 of Additional file 1).
Application of the inference procedure
By construction of the mechanistic model, the interaction matrix θ can describe any oriented graph by explicitly defining causal quantitative links between genes, which is difficult to do within traditional statistical frameworks (e.g. bayesian networks or undirected Markov random fields). The logical downside is that identifiability issues seem inevitable. In a first attempt to assess this aspect, we implemented the inference method presented above and tested it on various two-gene networks, assuming auto-activation for each gene (i.e. m _{ i,i }>0) with Eq. (10) to maximize variability without considering perturbations of the system (parameter list in section 6 of Additional file 1).
We decided to investigate the worst case scenario in terms of cell numbers. We are fully aware of the existence of technologies allowing to interrogate thousands of cells simultaneously, but most of the recent studies still rely upon a much smaller number of cells. For each network, we therefore simulated mRNA snapshot data for 100 cells using the full PDMP model (4). We then inferred the matrix θ using a “hard EM” algorithm based on the likelihood (13), that is, alternatively maximizing the likelihood with respect to θ and with respect to the (unknown) protein levels of each cell. A lasso-like penalization term, corresponding to a prior distribution, was added to the θ _{ i,j } for i≠j to obtain true zeros – so that the inferred network topology is clear – and to prevent keeping both θ _{ i,j } and θ _{ j,i } when one is significantly weaker (see section 4 of Additional file 1 for details of the penalization and the whole procedure).
Finally, we assessed the inference behavior in the presence of dropouts, i.e. genes expressed at a low level in a cell that give rise to zeros after measurement [4]. Our first tests tend to indicate that our approach is robust regarding dropouts, in the sense that up to 30% of simulated dropouts does not drastically affect the estimation of θ once the other parameters have been estimated correctly (see Additional file 1: Table S4 for an example).
Discussion
In this paper, we introduce a general stochastic model for gene regulatory networks, which can describe bursty gene expression as observed in individual cells. Instead of using ordinary differential equations, for which cells would structurally all behave the same way, we adopt a more detailed point of view including stochasticity as a fundamental component through the two-state promoter model. This model is but a simplification of the complexity of the real molecular processes [42]. Modifications have been proposed, from the existence of a refractory period [23] to its attenuation by nuclear buffering [62]. In bacteria, the two states originate from the accumulation of positive supercoiling on DNA which stops transcription [63]. In eukaryotes, although its molecular basis is not quite understood, the two-state model is a remarkable compromise between simplicity and the ability to capture real-life data [18, 22, 36–38]. Our PDMP framework appears to be conceptually very similar to the random dynamical system proposed in [64] but it has two major advantages: time does not have to be discretized, and the mathematical analysis is significantly easier. We also note that a similar framework appears in [65, 66] and that a closely related PDMP – which can be seen as the limit of our model for infinitely short bursts – has recently been described in [67].
We then derive an explicit approximation of the stationary distribution and propose to use it as a statistical likelihood to infer networks from single-cell data. The main ingredient is the separation of three physical timescales – chromatin, promoter/RNA, and proteins – and the core idea is to use the self consistent proteomic field approximation from [51, 52] in a slightly simpler mathematical framework, providing fully explicit formulas that make possible the massive computations usually needed for parameter inference. From this viewpoint, it is a rather simple approach and we hope it can be adapted or improved in more specific contexts, for example in the study of lineage commitment [68]. Besides, the main framework does not necessarily has to include an underlying chromatin model and thus it can in principle also be used to describe gene networks in procaryotes.
Mechanistic modelling and statistical inference
An important quality of the PDMP network model is that the simulation algorithm is comparable in speed with classic ODE and diffusion systems, while providing an effective approximation of the “perfect”, fully discrete, molecular counterpart [33, 35]. It is worth noticing that the PDMP – at least the promoter-mRNA system – naturally appears as an example of Poisson representation [28, 69], that is, not a simple approximation but rather the core component of the exact distribution of the discrete molecular model. Furthermore, such a simulation speed allowed us to compare our approximate likelihood with the true likelihood for a simple two-gene toggle switch, giving excellent results (Fig. 6). This obviously does not constitute a proof of robustness for every network: a proper quantitative (theoretical or numeric) comparison is beyond the scope of this article but would be extremely valuable. Intuitively, it should work for any number of genes, provided that interactions are not too strong.
Besides, some widely used ODE frameworks [8, 17, 57] can be seen as the fast-promoter limit of the PDMP model: this limit may not always hold in practice, especially in the bursty regime. In particular, Fig. 5 highlights the risk of using mRNA levels as a proxy for protein levels. It also explains why ordering single-cell mRNA measurements by pseudo-time may not always be relevant, as found in [38]. In [70], the authors use a hybrid model of gene expression to infer regulatory networks: it is very close to the diffusion limit of our reduced model (7) with the difference that the discrete component, called “promoter” by the authors, would correspond to the “frequency mode” in the present article, as visible for proteins in Fig. 5. From such a perspective, our approach adds a description of bursty mRNA dynamics that allows for fitting single-cell data such as in Fig. 7.
Finally, our method performed well for simple two-gene networks (Fig. 8), showing that part of the causal information remains present in the stationary distribution: this suggests that it is indeed possible to retrieve network structures with a mechanistic interpretation, even from bursty mRNA data.
Perspectives
We focused here on presenting the key ideas behind the general network model and the inference method: the logical next step is to apply it to real data and with a larger number of genes, which is the subject of work in progress in our group. In particular, we propose a functional preprocessing phase, detailed in section 5 of Additional file 1, that only requires the knowledge of the ratio d _{0,i }/d _{1,i } to estimate all the relevant parameters before inferring θ. The ratio between protein and mRNA degradation rates (or half-lives) hence appears to be the minimum required for such a mechanistic approach to be relevant. Depending upon the species, mRNA and protein half-lives values can be found in the literature (see e.g. [31] for human proteins half-lives), or should be estimated from ad hoc experiments.
From a computational point of view, the main challenge is the algorithmic complexity induced by the fact that proteins are not observed and have to be treated as latent variables. There is a priori no possibility of reducing this without loosing too much accuracy, and therefore some finely optimized algorithms may be required to make the method scalable. Furthermore, the identifiability properties of the interaction matrix θ seem difficult to derive theoretically. In this paper we focused on the stationary distribution for simplicity: importantly, several aspects such as time dependence (computing the Hartree approximation in transitory regime) or perturbations (changing the cell’s medium or performing knockouts [71], which can be naturally embedded in our framework) could greatly improve the practical identifiability.
From a biological point of view, our model does not really describe individual cells but rather a concatenation of trajectories obtained by following cells throughout divisions. Experiments suggest that it should be a relevant approximation, providing one considers mRNA and proteins levels in terms of concentrations instead of molecule numbers [72], which is made possible by the PDMP framework. In this view, the cell cycle results in increasing the apparent degradation rates – because of the increase in cell volume followed by division – and thus plays a crucial role for very stable proteins. However, at such a description level, many aspects of possible compensation mechanisms [73] and chromatin dynamics [74] remain to be elucidated. Regarding the latter aspect, our abstract chromatin states were not modeled from real-life data – chromatin composition for instance – but our approach is relevant in that partitioning into dual-type chromatin states as we did is now known as a pervasive feature of all eukaryotic genomes [75–78].
Conclusions
Protein and mRNA measurements in individual cells have revealed the importance of stochasticity in gene expression, which may potentially affect many aspects of gene regulation within cells. The traditional paradigm of gene network dynamics consisting in a deterministic structure plus an external noise – historically based on population-averaged data – should therefore be questioned, as such a noise appears to be itself part of the network structure and far from a small perturbation.
By modelling gene networks using piecewise-deterministic Markov processes, which are a simple way to introduce the minimum amount of mechanistic, non-diffusive stochasticity (corresponding to low molecule numbers), we derived a likelihood-based statistical model with interpretable parameters that successfully describes single-cell expression data. Our first results show that oriented interactions can indeed be inferred using such a method. Hence, this type of approach may take gene network inference to the next level by optimally exploiting single-cell data and improving the physical interpretability of inferred networks.
Declarations
Acknowledgements
We thank Geneviève Fourel (LBMC/ENSL) for critical reading of the manuscript and Anne-Laure Fougères (ICJ) for her constant support. We also thank all members of the SBDM and Dracula teams for enlightening discussions, and BioSyL Federation and Ecofect Labex for inspiring scientific events.
Funding
This work was supported by funding from the Institut Rhônalpin des Systèmes Complexes (IXXI) and from the French agency ANR (ICEBERG; ANR-IABI-3096).
Authors’ contributions
UH, AB, TE and OG designed the study. UH performed the theoretical derivations, implemented the algorithms and conceived/analyzed the examples. UH, AB, TE and OG interpreted the results and wrote the paper. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Authors’ Affiliations
References
- Hecker M, Lambeck S, Toepfer S, van Someren E, Guthke R. Gene regulatory network inference: data integration in dynamic models-a review. BioSystems. 2009; 96(1):86–103.PubMedView ArticleGoogle Scholar
- Kanter I, Kalisky T. Single cell transcriptomics: methods and applications. Front Oncol. 2015; 5:53.PubMedPubMed CentralView ArticleGoogle Scholar
- Tang F, Barbacioru C, Wang Y, Nordman E, Lee C, Xu N, Wang X, Bodeau J, Tuch BB, Siddiqui A, Lao K, Surani MA. mRNA-Seq whole-transcriptome analysis of a single cell. Nat Methods. 2009; 6(5):377–82.PubMedView ArticleGoogle Scholar
- Wagner A, Regev A, Yosef N. Revealing the vectors of cellular identity with single-cell genomics. Nat Biotechnol. 2016; 34(11):1145–1160.PubMedPubMed CentralView ArticleGoogle Scholar
- Huang S. Non-genetic heterogeneity of cells in development: more than just noise. Development. 2009; 136(23):3853–3862.PubMedPubMed CentralView ArticleGoogle Scholar
- Eldar A, Elowitz MB. Functional roles for noise in genetic circuits. Nature. 2010; 467(7312):167–173.PubMedPubMed CentralView ArticleGoogle Scholar
- Dueck H, Eberwine J, Kim J. Variation is function: Are single cell differences functionally important?Bioessays. 2015; 38:172–180.PubMedPubMed CentralView ArticleGoogle Scholar
- Mizeranschi A, Zheng H, Thompson P, Dubitzky W. Evaluating a common semi-mechanistic mathematical model of gene-regulatory networks. BMC Syst Biol. 2015; 9(5):1–12.Google Scholar
- Matsumoto H, Kiryu H, Furusawa C, Ko MSH, Ko SBH, Gouda N, Hayashi T, Nikaido I. Scode: An efficient regulatory network inference algorithm from single-cell rna-seq during differentiation. Bioinformatics. 2017; 33(15):2314–2321.PubMedView ArticleGoogle Scholar
- Symmons O, Raj A. What’s luck got to do with it: Single cells, multiple fates, and biological nondeterminism. Mol Cell. 2016; 62(5):788–802.PubMedPubMed CentralView ArticleGoogle Scholar
- Munsky B, Trinh B, Khammash M. Listening to the noise: random fluctuations reveal gene network parameters. Mol Syst Biol. 2009; 5(1):1–7.Google Scholar
- Zimmer C, Sahle S, Pahle J. Exploiting intrinsic fluctuations to identify model parameters. IET Syst Biol. 2015; 9(2):64–73.PubMedView ArticleGoogle Scholar
- Cai X, Bazerque JA, Giannakis GB. Inference of gene regulatory networks with sparse structural equation models exploiting genetic perturbations. PLoS Comput Biol. 2013; 9(5):1–13.View ArticleGoogle Scholar
- Djordjevic D, Yang A, Zadoorian A, Rungrugeecharoen K, Ho JWK. How difficult is inference of mammalian causal gene regulatory networks?PLoS One. 2014; 9(11):1–10.View ArticleGoogle Scholar
- Angulo MT, Moreno JA, Lippner G, Barabási A-L, Liu Y-Y. Fundamental limitations of network reconstruction from temporal data. J R Soc Interface. 2017; 14(127):1–6.View ArticleGoogle Scholar
- Moignard V, Woodhouse S, Haghverdi L, Lilly AJ, Tanaka Y, Wilkinson AC, Buettner F, Macaulay IC, Jawaid W, Diamanti E, Nishikawa S-I, Piterman N, Kouskoff V, Theis FJ, Fisher J, Göttgens B. Decoding the regulatory network of early blood development from single-cell gene expression measurements. Nat Biotechnol. 2015; 33(3):1–8.View ArticleGoogle Scholar
- Ocone A, Haghverdi L, Mueller NS, Theis FJ. Reconstructing gene regulatory dynamics from high-dimensional single-cell snapshot data. Bioinformatics. 2015; 31(12):89–86.View ArticleGoogle Scholar
- Kim JK, Marioni JC. Inferring the kinetics of stochastic gene expression from single-cell RNA-sequencing data. Genome Biol. 2013; 14:7.View ArticleGoogle Scholar
- Marbach D, Costello JC, Küffner R, Vega NM, Prill RJ, Camacho DM, Allison KR, DREAM5 Consortium, Kellis M, Collins JJ, Stolovitzky G. Wisdom of crowds for robust gene network inference. Nat Methods. 2012; 9(8):796–804.PubMedPubMed CentralView ArticleGoogle Scholar
- Raser JM, O’Shea EK. Control of stochasticity in eukaryotic gene expression. Science. 2004; 304(5678):1811–1814.PubMedPubMed CentralView ArticleGoogle Scholar
- Becskei A, Kaufmann BB, van Oudenaarden A. Contributions of low molecule number and chromosomal positioning to stochastic gene expression. Nat Genet. 2005; 37(9):937–944.PubMedView ArticleGoogle Scholar
- Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S. Stochastic mRNA Synthesis in Mammalian Cells. PLoS Biology. 2006; 4(10):1707–1719.View ArticleGoogle Scholar
- Suter DM, Molina N, Gatfield D, Schneider K, Schibler U, Naef F. Mammalian genes are transcribed with widely different bursting kinetics. Science. 2011; 332(6028):472–474.PubMedView ArticleGoogle Scholar
- Ko MSH. A stochastic model for gene induction. J Theor Biol. 1991; 153:181–194.PubMedView ArticleGoogle Scholar
- Ko MSH, Nakauchi H, Takahashi N. The dose dependence of glucocorticoid-inducible gene expression results from changes in the number of transcriptionally active templates. EMBO J. 1990; 9(9):2835–2842.PubMedPubMed CentralGoogle Scholar
- Larson DR. What do expression dynamics tell us about the mechanism of transcription?Curr Opin Genet Dev. 2011; 21(5):591–599.PubMedPubMed CentralView ArticleGoogle Scholar
- Gillespie DT. Exact stochastic simulation of coupled chemical reactions. J Phys Chem. 1977; 81(25):2340–2361.View ArticleGoogle Scholar
- Dattani J, Barahona M. Stochastic models of gene transcription with upstream drives: exact solution and sample path characterization. J R Soc Interface. 2017; 14(126):1–20.View ArticleGoogle Scholar
- Shahrezaei V, Swain PS. Analytical distributions for stochastic gene expression. PNAS. 2008; 105(45):17256–17261.PubMedPubMed CentralView ArticleGoogle Scholar
- Iyer-Biswas S, Hayot F, Jayaprakash C. Stochasticity of gene products from transcriptional pulsing. Phys Rev E Stat Nonlin Soft Matter Phys. 2009; 79:1–9.View ArticleGoogle Scholar
- Schwanhäusser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, Chen W, Selbach M. Global quantification of mammalian gene expression control. Nature. 2011; 495:337–342.View ArticleGoogle Scholar
- Davis MHA. Piecewise-deterministic markov processes: A general class of non-diffusion stochastic models. J R Stat Soc. 1984; 46(3):353–388.Google Scholar
- Crudu A, Debussche A, Radulescu O. Hybrid stochastic simplifications for multiscale gene networks. BMC Syst Biol. 2009; 3(1):89.PubMedPubMed CentralView ArticleGoogle Scholar
- Crudu A, Debussche A, Muller A, Radulescu O. Convergence of stochastic gene networks to hybrid piecewise deterministic processes. Ann Appl Probab. 2012; 22(5):1822–1859.View ArticleGoogle Scholar
- Lin YT, Galla T. Bursting noise in gene expression dynamics: linking microscopic and mesoscopic models. J R Soc Interface. 2016; 13:1–11.Google Scholar
- Viñuelas J, Kaneko G, Coulon A, Vallin E, Morin V, Mejia-Pous C, Kupiec J-J, Beslon G, Gandrillon O. Quantifying the contribution of chromatin dynamics to stochastic gene expression reveals long, locus-dependent periods between transcriptional bursts. BMC Biol. 2013; 11(1):15.PubMedPubMed CentralView ArticleGoogle Scholar
- Albayrak C, Jordi CA, Zechner C, Lin J, Bichsel CA, Khammash M, Tay S. Digital quantification of proteins and mrna in single mammalian cells. Mol Cell. 2016; 61:914–924.PubMedView ArticleGoogle Scholar
- Richard A, Boullu L, Herbach U, Bonnafoux A, Morin V, Vallin E, Guillemin A, Papili Gao N, Gunawan R, Cosette J, Arnaud O, Kupiec J-J, Espinasse T, Gonin-Giraud S, Gandrillon O. Single-cell-based analysis highlights a surge in cell-to-cell molecular variability preceding irreversible commitment in a differentiation process. PLoS Biol. 2016; 14(12):1–35.View ArticleGoogle Scholar
- Boxma O, Kaspi H, Kella O, Perry D. On/Off Storage Systems with State-Dependent Input, Output, and Switching Rates. Probab Eng Inf Sci. 2005; 19:1–14.View ArticleGoogle Scholar
- Benaïm M, Le Borgne S, Malrieu F, Zitt P-A. Quantitative ergodicity for some switched dynamical systems. Electron Commun Probab. 2012; 17(56):1–14.Google Scholar
- Ong KM, Blackford, JA Jr, Kagan BL, Simons, SS Jr, Chow CC. A theoretical framework for gene induction and experimental comparisons. PNAS. 2010; 107(15):7107–7112.PubMedPubMed CentralView ArticleGoogle Scholar
- Coulon A, Gandrillon O, Beslon G. On the spontaneous stochastic dynamics of a single gene: complexity of the molecular interplay at the promoter. BMC Syst Biol. 2010; 4:2.PubMedPubMed CentralView ArticleGoogle Scholar
- Coulon A, Chow CC, Singer RH, Larson DR. Eukaryotic transcriptional dynamics: from single molecules to cell populations. Nat Rev Genet. 2013; 14(8):1–13.View ArticleGoogle Scholar
- Friedman N, Rando OJ. Epigenomics and the structure of the living genome. Genome Res. 2015; 25(10):1482–1490.PubMedPubMed CentralView ArticleGoogle Scholar
- Bintu L, Yong J, Antebi YE, McCue K, Kazuki Y, Uno N, Oshimura M, Elowitz MB. Dynamics of epigenetic regulation at the single-cell level. Science. 2016; 351(6274):720–724.PubMedPubMed CentralView ArticleGoogle Scholar
- Benaïm M, Le Borgne S, Malrieu F, Zitt P-A. Qualitative properties of certain piecewise deterministic Markov processes. Annales de l’Institut Henri Poincaré - Probabilités et Statistiques. 2015; 51(3):1040–1075.View ArticleGoogle Scholar
- Faggionato A, Gabrielli D, Crivellari MR. Non-equilibrium thermodynamics of piecewise deterministic markov processes. J Stat Phys. 2009; 137:259–304.View ArticleGoogle Scholar
- Pakdaman K, Thieullen M, Wainrib G. Asymptotic expansion and central limit theorem for multiscale piecewise-deterministic Markov processes. Stoch Process Appl. 2012; 122:2292–2318.View ArticleGoogle Scholar
- Peccoud J, Ycart B. Markovian Modelling of Gene Product Synthesis. Theor Popul Biol. 1995; 48:222–234.View ArticleGoogle Scholar
- Li G-W, Xie XS. Central dogma at the single-molecule level in living cells. Nature. 2011; 475(7356):308–315.PubMedPubMed CentralView ArticleGoogle Scholar
- Sasai M, Wolynes PG. Stochastic gene expression as a many-body problem. PNAS. 2003; 100(5):2374–2379.PubMedPubMed CentralView ArticleGoogle Scholar
- Walczak AM, Sasai M, Wolynes PG. Self-consistent proteomic field theory of stochastic gene switches. Biophys J. 2005; 88:828–850.PubMedView ArticleGoogle Scholar
- Kim K-Y, Wang J. Potential energy landscape and robustness of a gene regulatory network: toggle switch. PLoS Comput Biol. 2007; 3(3):565–577.View ArticleGoogle Scholar
- Zhang B, Wolynes PG. Stem cell differentiation as a many-body problem. PNAS. 2014; 111(28):10185–10190.PubMedPubMed CentralView ArticleGoogle Scholar
- Senecal A, Munsky B, Proux F, Ly N, Braye FE, Zimmer C, Mueller F, Darzacq X. Transcription Factors Modulate c-Fos Transcriptional Bursts. Cell Rep. 2014; 8:75–83.PubMedPubMed CentralView ArticleGoogle Scholar
- Fukaya T, Lim B, Levine M. Enhancer control of transcriptional bursting. Cell. 2016; 166(2):358–368.PubMedPubMed CentralView ArticleGoogle Scholar
- Marbach D, Prill RJ, Schaffter T, Mattiussi C, Floreano D, Stolovitzky G. Revealing strengths and weaknesses of methods for gene network inference. PNAS. 2010; 107(14):6286–6291.PubMedPubMed CentralView ArticleGoogle Scholar
- Gu J, Gu Q, Wang X, Yu P, Lin W. Sphinx: modeling transcriptional heterogeneity in single-cell RNA-Seq. bioRxiv preprint. 2015.Google Scholar
- Ghazanfar S, Bisogni AJ, Ormerod JT, Lin DM, Yang JYH. Integrated single cell data analysis reveals cell specific networks and novel coactivation markers. BMC Syst Biol. 2016; 10:127.PubMedPubMed CentralView ArticleGoogle Scholar
- Mojtahedi M, Skupin A, Zhou J, Castano IG, Leong-Quong RYY, Chang HH, Giuliani A, Huang S. Cell fate decision as high-dimensional critical state transition. PLOS Biol. 2016; 14(12):1–28.View ArticleGoogle Scholar
- Sokolik C, Liu Y, Bauer D, McPherson J, Broeker M, Heimberg G, Qi LS, Sivak DA, Thomson M. Transcription factor competition allows embryonic stem cells to distinguish authentic signals from noise. Cell Syst. 2015; 1:117–129.PubMedPubMed CentralView ArticleGoogle Scholar
- Battich N, Stoeger T, Pelkmans L. Control of transcript variability in single mammalian cells. Cell. 2015; 163(7):1596–1610.PubMedView ArticleGoogle Scholar
- Chong S, Chen C, Ge H, Xie XS. Mechanism of transcriptional bursting in bacteria. Cell. 2014; 158(2):314–326.PubMedPubMed CentralView ArticleGoogle Scholar
- Antoneli F, Ferreira RC, Briones MRS. A model of gene expression based on random dynamical systems reveals modularity properties of gene regulatory networks. Math Biosci. 2016; 276:82–100.PubMedView ArticleGoogle Scholar
- Potoyan DA, Wolynes PG. Dichotomous noise models of gene switches. J Chem Phys. 2015; 143(19):195101.PubMedPubMed CentralView ArticleGoogle Scholar
- Hufton PG, Lin YT, Galla T, McKane AJ. Intrinsic noise in systems with switching environments. Phys Rev E. 2016; 93(5):052119.PubMedView ArticleGoogle Scholar
- Pájaro M, Alonso AA, Otero-Muras I, Vázquez C. Stochastic modeling and numerical simulation of gene regulatory networks with protein bursting. J Theor Biol. 2017; 421:51–70.PubMedView ArticleGoogle Scholar
- Teles J, Pina C, Edén P, Ohlsson M, Enver T, Peterson C. Transcriptional Regulation of Lineage Commitment - A Stochastic Model of Cell Fate Decisions. PLoS Comput Biol. 2013; 9(8):1–13.View ArticleGoogle Scholar
- Schnoerr D, Grima R, Sanguinetti G. Cox process representation and inference for stochastic reaction-diffusion processes. Nat Commun. 2016; 7:1–11.View ArticleGoogle Scholar
- Ocone A, Millar AJ, Sanguinetti G. Hybrid regulatory models: a statistically tractable approach to model regulatory network dynamics. Bioinformatics. 2013; 29(7):910–916.PubMedView ArticleGoogle Scholar
- Pinna A, Soranzo N, de la Fuente A. From knockouts to networks: establishing direct cause-effect relationships through graph analysis. PLoS One. 2010; 5(10):1–8.View ArticleGoogle Scholar
- Corre G, Stockholm D, Arnaud O, Kaneko G, Viñuelas J, Yamagata Y, Neildez-Nguyen TMA, Kupiec J-J, Beslon G, Gandrillon O, Paldi A. Stochastic Fluctuations and Distributed Control of Gene Expression Impact Cellular Memory. PLoS ONE. 2014; 9(12):115574.View ArticleGoogle Scholar
- Padovan-Merhar O, Nair GP, Biaesch AG, Mayer A, Scarfone S, Foley SW, Wu AR, Churchman LS, Singh A, Raj A. Single mammalian cells compensate for differences in cellular volume and dna copy number through independent global transcriptional mechanisms. Mol Cell. 2015; 58(2):339–352.PubMedPubMed CentralView ArticleGoogle Scholar
- Hathaway NA, Bell O, Hodges C, Miller EL, Neel DS, Crabtree GR. Dynamics and memory of heterochromatin in living cells. Cell. 2012; 149(7):1447–1460.PubMedPubMed CentralView ArticleGoogle Scholar
- Fourel G, Magdinier F, Gilson E. Insulator dynamics and the setting of chromatin domains. BioEssays. 2004; 26(5):523–532.PubMedView ArticleGoogle Scholar
- Kueng S, Oppikofer M, Gasser SM. Sir proteins and the assembly of silent chromatin in budding yeast. Annu Rev Genet. 2013; 47:275–306.PubMedView ArticleGoogle Scholar
- Rao SSP, Huntley MH, Durand NC, Stamenova EK, Bochkov ID, Robinson JT, Sanborn AL, Machol I, Omer AD, Lander ES, Aiden EL. A 3d map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell. 2014; 159(7):1665–1680.PubMedPubMed CentralView ArticleGoogle Scholar
- Obersriebnig MJ, Pallesen EMH, Sneppen K, Trusina A, Thon G. Nucleation and spreading of a heterochromatic domain in fission yeast. Nat Commun. 2016; 7:1–11.View ArticleGoogle Scholar