Synthetic incoherent feedforward circuits show adaptation to the amount of their genetic template

Bleris, Leonidas; Xie, Zhen; Glass, David S.; Adadey, Asa; Sontag, Eduardo D.; Benenson, Yaakov

doi:10.1038/msb.2011.49

Cited by 160 publications

(233 citation statements)

References 45 publications

(56 reference statements)

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“…S5). Strikingly, the exact same topology was shown experimentally to confer robustness of protein concentration to variation in transcription levels in synthetic genetic circuits (Bleris et al 2011). These observations suggest that the incoherently regulated targets we detected in Nematostella are genes whose product amounts should be constant and resistant to fluctuation in transcription levels.…”

Section: Discussionmentioning

confidence: 98%

Cnidarian microRNAs frequently regulate targets by cleavage

et al. 2014

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In bilaterians, which comprise most of extant animals, microRNAs (miRNAs) regulate the majority of messenger RNAs (mRNAs) via base-pairing of a short sequence (the miRNA ''seed'') to the target, subsequently promoting translational inhibition and transcript instability. In plants, many miRNAs guide endonucleolytic cleavage of highly complementary targets. Because little is known about miRNA function in nonbilaterian animals, we investigated the repertoire and biological activity of miRNAs in the sea anemone Nematostella vectensis, a representative of Cnidaria, the sister phylum of Bilateria. Our work uncovers scores of novel miRNAs in Nematostella, increasing the total miRNA gene count to 87. Yet only a handful are conserved in corals and hydras, suggesting that microRNA gene turnover in Cnidaria greatly exceeds that of other metazoan groups. We further show that Nematostella miRNAs frequently direct the cleavage of their mRNA targets via nearly perfect complementarity. This mode of action resembles that of small interfering RNAs (siRNAs) and plant miRNAs. It appears to be common in Cnidaria, as several of the miRNA target sites are conserved among distantly related anemone species, and we also detected miRNA-directed cleavage in Hydra. Unlike in bilaterians, Nematostella miRNAs are commonly coexpressed with their target transcripts. In light of these findings, we propose that post-transcriptional regulation by miRNAs functions differently in Cnidaria and Bilateria. The similar, siRNA-like mode of action of miRNAs in Cnidaria and plants suggests that this may be an ancestral state.

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Section: Discussionmentioning

confidence: 98%

Cnidarian microRNAs frequently regulate targets by cleavage

et al. 2014

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“…In fact, attempts to fundamentally address the issue by recognizing and filtering out the effects of indirect interactions at a global scale have begun to surface (11). Meanwhile, parallel developments in synthetic biology (23) have endowed researchers with new tools that allow precise emulation of naturally occurring topologies (21,22). Networks orthogonal to the cellular milieu can serve as a biomolecular topological "ground truth" (20,24).…”

Section: Discussionmentioning

confidence: 99%

“…S1 and S2). The kinetic parameters were selected from literature (21,30). We performed sensitivity analysis of the output node Z protein concentration against the mRNA species of nodes X and Y (thereby emulating RNAi perturbation).…”

Section: Significancementioning

confidence: 99%

“…On the other hand, engineered synthetic gene circuits are orthogonal to the endogenous pathways yet operate within the natural cellular context using the available resources. Thus, synthetic networks are a versatile platform for investigating specific connectivities and topological properties and can ultimately guide us to deriving fundamental insights about biological systems and pathways (20)(21)(22)(23).…”

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confidence: 99%

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Discriminating direct and indirect connectivities in biological networks

Kang

Moore

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Reverse engineering of biological pathways involves an iterative process between experiments, data processing, and theoretical analysis. Despite concurrent advances in quality and quantity of data as well as computing resources and algorithms, difficulties in deciphering direct and indirect network connections are prevalent. Here, we adopt the notions of abstraction, emulation, benchmarking, and validation in the context of discovering features specific to this family of connectivities. After subjecting benchmark synthetic circuits to perturbations, we inferred the network connections using a combination of nonparametric single-cell data resampling and modular response analysis. Intriguingly, we discovered that recovered weights of specific network edges undergo divergent shifts under differential perturbations, and that the particular behavior is markedly different between topologies. Our results point to a conceptual advance for reverse engineering beyond weight inference. Investigating topological changes under differential perturbations may address the longstanding problem of discriminating direct and indirect connectivities in biological networks. A focal point of systems biology is the reverse engineering of gene regulatory networks (1-5). The methods have shifted from intuitive inference of local connectivities to comprehensive analysis of large networks, involving heterogeneous data sets from high-throughput experiments and complex theoretical tools (6-10). Despite significant advances, a fundamental reverse engineering bottleneck is the ability to discriminate between direct and indirect connections. In a simple case, assuming three nodes in a cascade formulation, where an input node is activating an intermediary node which in turn is activating an output node, a reverse engineering algorithm may infer an activating edge from the input node to the output, even though there is no direct biological interaction.Unfortunately, the limitation in correctly distinguishing the effects stemming from indirect connectivities is pervasive (11-13) and justifies the urgent need for new and reliable methods to eliminate spurious edges. Importantly, remedies to address this problem should not further muddle the interpretation by removing true network edges (14). A number of theoretical approaches have been proposed to overcome this hurdle (4, 15-18), but the ability to experimentally verify the conclusions drawn by reverse engineering tools remains paramount.The majority of efforts to address the verification issue adopt in silico benchmark suites that are based on biological pathway approximations (19). Although these models do include a number of commonly observed topologies and have provided significant insights, they do not fully capture the complexity of the biological realm and the associated heterogeneity and intrinsic variability. On the other hand, engineered synthetic gene circuits are orthogonal to the endogenous pathways yet operate within the natural cellular context using the available resources. Thus, sy...

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“…Synthetic biology provides a complementary approach to investigate miRNA-mediated regulations in a controlled and largely isolated biological setting by using engineered genetic circuits (11). Synthetic circuits have been constructed to help elucidate the underlying design principles of network motifs that combine transcriptional and miRNA-mediated regulations (12)(13)(14). It has also been demonstrated that miRNAs can generate a threshold in target gene expression by using a bidirectional reporter assay in mammalian cells (15).…”

mentioning

confidence: 99%

Model-guided quantitative analysis of microRNA-mediated regulation on competing endogenous RNAs using a synthetic gene circuit

Yuan

Liu

Xie

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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108

104

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Competing endogenous RNAs (ceRNAs) cross-regulate each other at the posttranscriptional level by titrating shared microRNAs (miRNAs). Here, we established a computational model to quantitatively describe a minimum ceRNA network and experimentally validated our model predictions in cultured human cells by using synthetic gene circuits. We demonstrated that the range and strength of ceRNA regulation are largely determined by the relative abundance and the binding strength of miRNA and ceRNAs. We found that a nonreciprocal competing effect between partially and perfectly complementary targets is mainly due to different miRNA loss rates in these two types of regulations. Furthermore, we showed that miRNA-like off targets with high expression levels and strong binding sites significantly diminish the RNA interference efficiency, but the effect caused by high expression levels could be compensated by introducing more small interference RNAs (siRNAs). Thus, our results provided a quantitative understanding of ceRNA cross-regulation via shared miRNA and implied an siRNA design strategy to reduce the siRNA off-target effect in mammalian cells. RNAs that are loaded onto RNA-induced silencing complexes (RISC) and subsequently bind to their target RNAs. In mammalian cells, the perfect pairing of miRNA to target RNAs causes RNA cleavage through the RNA interference (RNAi) pathway, whereas partial pairing results in translational repression and RNA destabilization (1, 2). miRNA-mediated regulation can be triggered by only 6-nt complementarity of the miRNA 5′-end "seed region" to the target RNA, which confers each miRNA species the capacity to interact with multiple RNA species, including gene-coding mRNAs (3, 4), long noncoding RNAs (5), and circular RNAs (6). Similarly, each RNA species can interact with multiple miRNA species through various miRNA response elements (MREs) (7).The complex interaction network of miRNAs and their target RNAs has been shown to allow indirect cross-regulation between different competing endogenous RNAs (ceRNAs) by sequestering shared miRNAs, which is essential for regulating many biological functions (7). The strength of ceRNA regulation is largely determined by the relative abundance and binding strength of ceRNAs and miRNAs and whether the miRNA-bound ceRNA decays through a stoichiometric mechanism or a catalytic mechanism (8-10). The threshold-like behavior of the ceRNA regulation has been experimentally observed by measuring the abundance of two ceRNAs, phosphatase and tensin homolog (PTEN) and vesicle-associated membrane protein (VAMP)-associated protein A (VAPA) across various cell lines (8). Nevertheless, many quantitative predictions deduced from miRNA-ceRNA computational models have not been experimentally validated. Another intriguing question is whether the miRNA-mediated catalytic mechanism can be affected by the miRNA-mediated stoichiometric mechanism through a ceRNA effect or vice versa.Currently, the ability to systematically elucidate features of the ceRNA effect is impeded by th...

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Synthetic incoherent feedforward circuits show adaptation to the amount of their genetic template

Abstract: 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.

Cited by 160 publications

References 45 publications

Cnidarian microRNAs frequently regulate targets by cleavage

Cnidarian microRNAs frequently regulate targets by cleavage

Discriminating direct and indirect connectivities in biological networks

Model-guided quantitative analysis of microRNA-mediated regulation on competing endogenous RNAs using a synthetic gene circuit

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