Variable gene dosage is a major source of fluctuations in gene expression in both endogenous and synthetic circuits. Synthetic incoherent feedforward regulatory motifs using RNA interference are shown to robustly adapt to changes in DNA template amounts in mammalian cells.
Variable gene dosage is a major source of fluctuations in gene product levels in both endogenous and synthetic circuits.To mitigate gene expression variability, we designed, simulated, constructed, and tested regulatory circuits, implementing an incoherent feedforward motif.A number of control mechanisms including transcription and post-transcriptional regulation were tested in mammalian cells.Feedforward regulation displayed better adaptation than negative feedback, and circuits based on RNA interference were the most robust to variation in DNA template amounts.
Natural and synthetic biological networks must function reliably in the face of fluctuating stoichiometry of their molecular components. These fluctuations are caused in part by changes in relative expression efficiency and the DNA template amount of the network-coding genes. Indeed, changes in gene dosage are clearly a major source of variation in cells, and yet those changes are very common in both normal processes (sex determination, ploidy change) and disease (gene amplification in cancer). In synthetic networks, the problem is exacerbated due to commonly used transient delivery methods that result in very large cell-to-cell variability in gene dosage. The basic question on gene dosage compensation in nature (Veitia et al, 2008; Acar et al, 2010) and a practical challenge of overcoming sensitivity to DNA copy number in synthetic circuits prompted us to investigate mechanisms to reduce this variability using sophisticated internal regulatory mechanisms. Indeed, the baseline expression unit in many synthetic circuits is an open-loop promoter-ORF combination. We hypothesized that some sort of internal regulation will result in ‘expression units' whose gene product (i.e. protein) output will depend only mildly on the intracellular concentration of its DNA template. In other words, we searched for architecture that would lead to ‘adaptation' of the gene product to the amount of DNA template.
By examining large body of published work, we found frequent reference to a so-called ‘incoherent feedforward' network motif (Mangan and Alon, 2003). The canonical three-node incoherent loop contains input, auxiliary regulator, and output nodes. The output is controlled directly by the input and the auxiliary regulator. The latter is also controlled by the input, introducing an additional indirect effect of the input on the output. In incoherent loops, the input controls the auxiliary regulator node in such a way that input's overall indirect action on the output via this node counteracts its direct effect. In a motif named ‘type I incoherent feedforward loop' (I1-FFL), the input's direct effect is activating, as is its control of the auxiliary node, while the auxiliary node controls the output through repression. One of the most studied properties of such motifs is their transient response to persistent stimulus, that is visually characterized as a ‘bump' or ‘pulse' (hence the name ‘pulse generator') that then goes back to the original steady state of the system (Basu et al, 2004). We hypothesized that changing DNA amount could serve as an input to an incoherent circuit if the auxiliary regulator and the output nodes are coexpressed from this DNA; in other words DNA can be viewed as an ‘activator' of both the regulator and the output. We conjectured that this might lead to adaptation to changes in DNA template.
We designed and simulated in silico a number of network architectures that all exhibit incoherent feedforward connectivity. We also compared them with the well-studied feedback loop circuit that in theory weakens but does not eliminate gene product dependency on the DNA template amount. The schematics of the circuits are shown in Figure 1.
Experimental measurement of input–output response of these circuits, or their transfer function, indeed uncovered adaptation of the output to DNA template abundance. Such adaptation has not been observed with feedback loop, as expected. Among various architectures, the post-transcriptional circuits showed faster adaptation, higher absolute expression levels and lower ‘noise' (Figure 4).
We also simulated and measured stochastic variability in the circuits by collecting all the cells with similar input values and statistically analyzing output values in those cells. We found that substantial noise component could not be accounted for by known noise sources, and concluded that the very step of negative regulation, both by a repressor LacI and by a microRNA, significantly increases cell-to-cell variability. This needs to be addressed in further studies. We also found that the negative feedback loop did not result in reduced noise as we expected, yet it did not result in noise increase as in the incoherent motif. This means that there may be effective noise reduction but it is not sufficient to produce narrow distributions of outputs for a given input.
We conclude that expression units that incorporate incoherent feedforward control of the gene product provide adaptation to the amount of DNA template and can be superior to simple combinations of constitutive promoters with ORFs. We also emphasize the relevance of our findings to the long-standing question of gene dosage compensation in cells, and note that similar incoherent architectures with microRNA negative regulators have been found in cells, suggesting that their physiological role is to curb variability in gene dosage and/or promoter strength.
Natural and synthetic biological networks must function reliably in the face of fluctuating stoichiometry of their molecular components. These fluctuations are caused in part by changes in relative expression efficiency and the DNA template amount of the network-coding genes. Gene product levels could potentially be decoupled from these changes via built-in adaptation mechanisms, thereby boosting network reliability. Here, we show that a mechanism based on an incoherent feedforward motif enables adaptive gene expression in mammalian cells. We modeled, synthesized, and tested transcriptional and post-transcriptional incoherent loops and found that in all cases the gene product adapts to changes in DNA template abundance. We also observed that the post-transcriptional form results in superior adaptation behavior, higher absolute expression levels, and lower intrinsic fluctuations. Our results support a previously hypothesized endogenous role in gene dosage compensation for such motifs and suggest that their incorporation in synthetic networks will improve their robustness and reliability.