Synthetic biologists would like to be able to make gene regulatory circuits that mimic key properties of eukaryotic gene regulation. Taking a cue from multimeric transcription factor complexes, Bashor et al. developed synthetic transcriptional circuits that produce nonlinear behavior from cooperativity (see the Perspective by Ng and El-Samad). Their system uses clamp proteins with multiple protein-interaction domains. Circuit behavior can be tuned by altering the number or affinities of the interactions according to a mathematical model. The authors created synthetic circuits with desired functions common in biology, for example, switch-like behavior or Boolean decision functions.

Science, this issue p. 593; see also p. 531


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Synthetic transcriptional synergy


The architecture of transcription factors (TFs) is surprisingly modular (1). Each factor broadly consists of a DNA binding domain and an activation domain that recruits the cell’s transcription apparatus. These domains can be combined in a plug-and-play manner to build synthetic TFs (synTFs). Such synTFs have been successfully used to program activation or repression of a gene of interest (24), a functionality that has proven essential for many studies in molecular biology and applications in biotechnology. On page 593 of this issue, Bashor et al. (5) present a successful approach for engineering cooperative synTF assemblies. These assemblies bring engineered control of gene expression closer to achieving the richness of behaviors exhibited by naturally cooperative TFs.


Engineered control

Synthetic transcription factors (synTFs) can bind to DNA in two ways.




When a synTF is designed to have one-to-one binding to its cognate DNA target (the promoter region of a particular gene), the dose response of gene expression is largely graded (until saturation), typically fitted by a Michaelis-Menten saturation curve. As a result, the types of gene expression regulation that can be programmed with synTFs in this manner occupy only a fraction of the capability of natural TFs.

By contrast, the dose-response relationship between many naturally occurring TFs and their promoters is sigmoidal, a result that has often been ascribed to the phenomenon of cooperativity. Cooperativity is a general process in which one molecule (e.g., a TF) can bind another (e.g., DNA) at multiple sites, with binding at one site affected (e.g., enhanced) by occupancy at other sites. It is broadly accepted based on studies of bacterial gene regulation (6) that cooperativity of TF binding can increase the “sharpness” of gene expression. Sharp (or switch-like) regulation of transcription allows a gene to switch decisively “on” in an all-or-none manner, at a precise and narrow concentration range of its inducer. Switch-like gene regulation is essential for many biological processes, most notably in developmental contexts (7).

Previous studies have reported (38) synTFs (those with a zinc finger structural motif) that interact with a single binding motif on a promoter to initiate transcription. To generate a cooperative response, Bashor et al. used a scaffold of covalently linked protein domains (called PDZ domains) that bind to a PDZ domain–interacting ligand fused to a particular synTF. In the presence of the PDZ scaffold, binding of a single synTF-PDZ ligand fusion to DNA increases the probability that another synTF-PDZ ligand fusion will bind to an adjacent DNA binding motif, forming a complex of synthetic transcriptional activators (see the figure). The formation of this cooperative assembly is highly tunable; varying the number of DNA binding motifs in the promoter and the number of PDZ domains, as well as the affinity of the PDZ ligand and the affinity of the DNA binding domain, enables programmable dose responses with customizable shape and sharpness characteristics. Using a thermodynamic model of their system, Bashor et al. identified the parameters necessary to implement OR and AND logic on a system with inputs consisting of two orthogonal synTFs. By testing several variants of assemblies of these two synTFs, the authors demonstrated experimentally that cooperative assembly of synTFs is required for Boolean-like behavior. Finding that cooperative synTF assembly also delays activation and hastens deactivation of transcription, the authors constructed circuits that could perform complex dynamic signal processing, such as a persistence filtering device, in which gene expression ensues only for long inputs but not short pulses, and a decoding device that responds preferentially to certain input frequencies. Overall, these rationally designed synthetic transcriptional cooperative assemblies will enable a large array of applications that rely on switch-like gene expression control.

Cooperativity is a highly nuanced phenomenon (9) that plays a complex role in transcriptional regulation. For example, in addition to configurational cooperativity, cells make use of allosteric cooperativity. In the context of transcription, allosteric cooperativity would stabilize a modified conformation of a transcriptional regulator or DNA after a binding event, therefore altering binding of additional molecules. Both types of cooperativity may be at play in the formation of large multisubunit transcriptional complexes that involve interactions between the DNA and its structural components, the general transcriptional machinery, and coactivator or co-repressor molecules. This is perhaps best exemplified by the interferon-β enhanceosome, an intricate protein-DNA complex that regulates gene expression in response to viral infection. High cooperativity in this structure transforms weak interactions between individual molecules into a tight and functional assembly (10). Further, recent theoretical studies suggest that pairwise cooperative binding of TFs is not sufficient to explain the sharpness observed in the regulation of certain eukaryotic genes such bicoid in Drosophila melanogaster. Higher-order cooperativity from pioneer factors (transcription factors that target DNA sites at silent genes) and chromatin remodeling can generate a sharper response than that expected from pairwise cooperativity, but the assumption of thermodynamic equilibrium still limits what is achievable. Greater sharpness can be achieved by expending energy to maintain the system away from equilibrium (11). Furthermore, formation of phase-separated superenhancers by cooperative interactions has been implicated in transcriptional regulation (12). The ordered assembly of these components and the contribution of cooperativity to gene expression characteristics remain unclear.

Cells harness cooperativity in a variety of contexts that extend beyond transcriptional control. One example is T cell activation, which depends on the cooperative clustering of the T cell receptor with costimulatory molecules in an ordered immunological synapse (13). T cells can also be synthetically activated with chimeric antigen receptors (CARs), which are fusions of motifs from the T cell receptor and costimulatory molecules. CARs have demonstrated therapeutic success, but they do not form an ordered immunological synapse (14). It is therefore possible that reengineering a CAR to include multisubunit cooperativity could enhance its function and further its application in engineered cell therapy (15). A modular framework for constructing synthetic cooperative responses could be a major advance for the field of synthetic biology, boosting the ability to dissect the requirements, constraints, principles, and properties of cooperative processes.



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Complex signal processing in synthetic gene circuits using cooperative regulatory assemblies




Eukaryotic genes are regulated by multivalent transcription factor complexes. Through cooperative self-assembly, these complexes perform nonlinear regulatory operations involved in cellular decision-making and signal processing. In this study, we apply this design principle to synthetic networks, testing whether engineered cooperative assemblies can program nonlinear gene circuit behavior in yeast. Using a model-guided approach, we show that specifying the strength and number of assembly subunits enables predictive tuning between linear and nonlinear regulatory responses for single- and multi-input circuits. We demonstrate that assemblies can be adjusted to control circuit dynamics. We harness this capability to engineer circuits that perform dynamic filtering, enabling frequency-dependent decoding in cell populations. Programmable cooperative assembly provides a versatile way to tune the nonlinearity of network connections, markedly expanding the engineerable behaviors available to synthetic circuits.




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