Engineered signaling networks can impart cells with new functionalities useful for directing differentiation and actuating cellular therapies. For such applications, the engineered networks must be tunable, precisely regulate target gene expression, and be robust to perturbations within the complex context of mammalian cells. Here, we use bacterial two-component signaling proteins to develop synthetic phosphoregulation devices that exhibit these properties in mammalian cells. First, we engineer a synthetic covalent modification cycle based on kinase and phosphatase proteins derived from the bifunctional histidine kinase EnvZ, enabling analog tuning of gene expression via its response regulator OmpR. By regulating phosphatase expression with endogenous miRNAs, we demonstrate cell-type specific signaling responses and a new strategy for accurate cell type classification. Finally, we implement a tunable negative feedback controller via a small molecule-stabilized phosphatase, reducing output expression variance and mitigating the context-dependent effects of off-target regulation and resource competition. Our work lays the foundation for establishing tunable, precise, and robust control over cell behavior with synthetic signaling networks.
Synthetic biology has the potential to bring forth advanced genetic devices for applications in healthcare and biotechnology. However, accurately predicting the behavior of engineered genetic devices remains difficult due to lack of modularity, wherein a device’s output does not depend only on its intended inputs but also on its context. One contributor to lack of modularity is loading of transcriptional and translational resources, which can induce coupling among otherwise independently-regulated genes. Here, we quantify the effects of resource loading in engineered mammalian genetic systems and develop an endoribonuclease-based feedforward controller that can adapt the expression level of a gene of interest to significant resource loading in mammalian cells. Near-perfect adaptation to resource loads is facilitated by high production and catalytic rates of the endoribonuclease. Our design is portable across cell lines and enables predictable tuning of controller function. Ultimately, our controller is a general-purpose device for predictable, robust, and context-independent control of gene expression.
more » « less- Award ID(s):
- 1840257
- NSF-PAR ID:
- 10201201
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 11
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract -
INTRODUCTION Neurons are by far the most diverse of all cell types in animals, to the extent that “cell types” in mammalian brains are still mostly heterogeneous groups, and there is no consensus definition of the term. The Drosophila optic lobes, with approximately 200 well-defined cell types, provides a tractable system with which to address the genetic basis of neuronal type diversity. We previously characterized the distinct developmental gene expression program of each of these types using single-cell RNA sequencing (scRNA-seq), with one-to-one correspondence to the known morphological types. RATIONALE The identity of fly neurons is determined by temporal and spatial patterning mechanisms in stem cell progenitors, but it remained unclear how these cell fate decisions are implemented and maintained in postmitotic neurons. It was proposed in Caenorhabditis elegans that unique combinations of terminal selector transcription factors (TFs) that are continuously expressed in each neuron control nearly all of its type-specific gene expression. This model implies that it should be possible to engineer predictable and complete switches of identity between different neurons just by modifying these sustained TFs. We aimed to test this prediction in the Drosophila visual system. RESULTS Here, we used our developmental scRNA-seq atlases to identify the potential terminal selector genes in all optic lobe neurons. We found unique combinations of, on average, 10 differentially expressed and stably maintained (across all stages of development) TFs in each neuron. Through genetic gain- and loss-of-function experiments in postmitotic neurons, we showed that modifications of these selector codes are sufficient to induce predictable switches of identity between various cell types. Combinations of terminal selectors jointly control both developmental (e.g., morphology) and functional (e.g., neurotransmitters and their receptors) features of neurons. The closely related Transmedullary 1 (Tm1), Tm2, Tm4, and Tm6 neurons (see the figure) share a similar code of terminal selectors, but can be distinguished from each other by three TFs that are continuously and specifically expressed in one of these cell types: Drgx in Tm1, Pdm3 in Tm2, and SoxN in Tm6. We showed that the removal of each of these selectors in these cell types reprograms them to the default Tm4 fate. We validated these conversions using both morphological features and molecular markers. In addition, we performed scRNA-seq to show that ectopic expression of pdm3 in Tm4 and Tm6 neurons converts them to neurons with transcriptomes that are nearly indistinguishable from that of wild-type Tm2 neurons. We also show that Drgx expression in Tm1 neurons is regulated by Klumpfuss, a TF expressed in stem cells that instructs this fate in progenitors, establishing a link between the regulatory programs that specify neuronal fates and those that implement them. We identified an intronic enhancer in the Drgx locus whose chromatin is specifically accessible in Tm1 neurons and in which Klu motifs are enriched. Genomic deletion of this region knocked down Drgx expression specifically in Tm1 neurons, leaving it intact in the other cell types that normally express it. We further validated this concept by demonstrating that ectopic expression of Vsx (visual system homeobox) genes in Mi15 neurons not only converts them morphologically to Dm2 neurons, but also leads to the loss of their aminergic identity. Our results suggest that selector combinations can be further sculpted by receptor tyrosine kinase signaling after neurogenesis, providing a potential mechanism for postmitotic plasticity of neuronal fates. Finally, we combined our transcriptomic datasets with previously generated chromatin accessibility datasets to understand the mechanisms that control brain wiring downstream of terminal selectors. We built predictive computational models of gene regulatory networks using the Inferelator framework. Experimental validations of these networks revealed how selectors interact with ecdysone-responsive TFs to activate a large and specific repertoire of cell surface proteins and other effectors in each neuron at the onset of synapse formation. We showed that these network models can be used to identify downstream effectors that mediate specific cellular decisions during circuit formation. For instance, reduced levels of cut expression in Tm2 neurons, because of its negative regulation by pdm3 , controls the synaptic layer targeting of their axons. Knockdown of cut in Tm1 neurons is sufficient to redirect their axons to the Tm2 layer in the lobula neuropil without affecting other morphological features. CONCLUSION Our results support a model in which neuronal type identity is primarily determined by a relatively simple code of continuously expressed terminal selector TFs in each cell type throughout development. Our results provide a unified framework of how specific fates are initiated and maintained in postmitotic neurons and open new avenues to understanding synaptic specificity through gene regulatory networks. The conservation of this regulatory logic in both C. elegans and Drosophila makes it likely that the terminal selector concept will also be useful in understanding and manipulating the neuronal diversity of mammalian brains. Terminal selectors enable predictive cell fate reprogramming. Tm1, Tm2, Tm4, and Tm6 neurons of the Drosophila visual system share a core set of TFs continuously expressed by each cell type (simplified). The default Tm4 fate is overridden by the expression of a single additional terminal selector to generate Tm1 ( Drgx ), Tm2 ( pdm3 ), or Tm6 ( SoxN ) fates.more » « less
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Cells respond to biochemical and physical internal as well as external signals. These signals can be broadly classified into two categories: (a) ``actionable'' or ``reference'' inputs that should elicit appropriate biological or physical responses such as gene expression or motility, and (b) ``disturbances'' or ``perturbations'' that should be ignored or actively filtered-out. These disturbances might be exogenous, such as binding of nonspecific ligands, or endogenous, such as variations in enzyme concentrations or gene copy numbers. In this context, the term robustness describes the capability to produce appropriate responses to reference inputs while at the same time being insensitive to disturbances. These two objectives often conflict with each other and require delicate design trade-offs. Indeed, natural biological systems use complicated and still poorly understood control strategies in order to finely balance the goals of responsiveness and robustness. A better understanding of such natural strategies remains an important scientific goal in itself and will play a role in the construction of synthetic circuits for therapeutic and biosensing applications. A prototype problem in robustly responding to inputs is that of ``robust tracking'', defined by the requirement that some designated internal quantity (for example, the level of expression of a reporter protein) should faithfully follow an input signal while being insensitive to an appropriate class of perturbations. Control theory predicts that a certain type of motif, called integral feedback, will help achieve this goal, and this motif is, in fact, a necessary feature of any system that exhibits robust tracking. Indeed, integral feedback has always been a key component of electrical and mechanical control systems, at least since the 18th century when James Watt employed the centrifugal governor to regulate steam engines. Motivated by this knowledge, biological engineers have proposed various designs for biomolecular integral feedback control mechanisms. However, practical and quantitatively predictable implementations have proved challenging, in part due to the difficulty in obtaining accurate models of transcription, translation, and resource competition in living cells, and the stochasticity inherent in cellular reactions. These challenges prevent first-principles rational design and parameter optimization. In this work, we exploit the versatility of an Escherichia coli cell-free transcription-translation (TXTL) to accurately design, model and then build, a synthetic biomolecular integral controller that precisely controls the expression of a target gene. To our knowledge, this is the first design of a functioning gene network that achieves the goal of making gene expression track an externally imposed reference level, achieves this goal even in the presence of disturbances, and whose performance quantitatively agrees with mathematical predictions.more » « less
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Abstract Understanding the genetic influences of traits of nonmodel organisms is crucial to understanding how novel traits arise. Do new traits require new genes or are old genes repurposed? How predictable is this process? Here, we examine this question for gene expression influencing parenting behavior in a beetle,
Nicrophorus vespilloides . Parental care, produced from many individual behaviors, should be influenced by changes of expression of multiple genes, and one suggestion is that the genes can be predicted based on knowledge of behavior expected to be precursors to parental care, such as aggression, resource defense, and mating on a resource. Thus, testing gene expression during parental care allows us to test expectations of this “precursor hypothesis” for multiple genes and traits. We tested for changes of the expression of serotonin, octopamine/tyramine, and dopamine receptors, as well as one glutamate receptor, predicting that these gene families would be differentially expressed during social interactions with offspring and associated resource defense. We found that serotonin receptors were strongly associated with social and aggression behavioral transitions. Octopamine receptors produced a complex picture of gene expression over a reproductive cycle. Dopamine was not associated with the behavioral transitions sampled here, while the glutamate receptor was most consistent with a behavioral change of resource defense/aggression. Our results generate new hypotheses, refine candidate lists for further studies, and inform the genetic mechanisms that are co‐opted during the evolution of parent–offspring interactions, a likely evolutionary path for many lineages that become fully social. The precursor hypothesis, while not perfect, does provide a starting point for identifying candidate genes. -
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Abstract Expanding the genetic toolbox for prokaryotic synthetic biology is a promising strategy for enhancing the dynamic range of gene expression and enabling new engineered applications for research and biomedicine. Here, we reverse the current trend of moving genetic parts from prokaryotes to eukaryotes and demonstrate that the activating eukaryotic transcription factor QF and its corresponding DNA-binding sequence can be moved to
E. coli to introduce transcriptional activation, in addition to tight off states. We further demonstrate that the QF transcription factor can be used in genetic devices that respond to low input levels with robust and sustained output signals. Collectively, we show that eukaryotic gene regulator elements are functional in prokaryotes and establish a versatile and broadly applicable approach for constructing genetic circuits with complex functions. These genetic tools hold the potential to improve biotechnology applications for medical science and research.
