Synthetic Transcription Factors Research Articles

Improving the Z3EV promoter system to create the strongest yeast promoter.

Promoters for artificial control of gene expression are central tools in genetic engineering. In the budding yeast S. cerevisiae, a variety of constitutive and controllable promoters with different strengths have been constructed using endogenous gene promoters, synthetic transcription factors and their binding sequences, and artificial sequences. However, there have been no attempts to construct the highest-strength promoter in yeast cells. In this study, by incrementally increasing the binding sequences of the synthetic transcription factor Z3EV, we were able to construct a promoter (P36) with approximately 1.4 times the strength of the TDH3 promoter. This is stronger than any previously reported promoter. Although the P36 promoter exhibits some leakage in the absence of induction, the expression induction by estradiol is maintained. When combined with a multicopy plasmid, it can express up to approximately 50% of total protein as a heterologous protein. This promoter system can be used to gain knowledge about the cell physiology resulting from the ultimate overexpression of excess proteins and is expected to be a useful tool for heterologous protein expression in yeast.

Hypoxia-responsive synthetic gene circuits to improve safety and potency of CAR T cell therapy for solid tumors.

37 Background: Engineered cell therapies expressing a chimeric antigen receptor (CAR) in human T cells have demonstrated great promise for the treatment of hematological malignancies. However, unique challenges remain in translating the success of CAR T cell therapies to solid tumors, namely: (1) specificity: there are few truly unique antigens present on solid tumors, leading to off-target effects that damage normal tissue and pose safety risks; and (2) suppression: solid tumors develop microenvironments that are immunosuppressive and decrease T cell survival and tumor-directed cytotoxicity. To address these challenges and improve the safety and potency of solid tumor cell therapy, we engineered cells with synthetic genetic circuits containing a previously validated hypoxia biosensor (HBS) that responds to upregulation of the native HIF1α and HIF2α transcription factors by hypoxia, enabling the cells to sense and respond to the hypoxic tumor microenvironment ubiquitous among solid tumors. Methods: We evaluated CAR expression from circuit architectures containing the HBS promoter driving expression of both CAR and native HIF1α, HIF2α, as well as from circuit architectures containing the HBS promoter and our toolkit of synthetic transcription factors and promoters. Circuits were integrated into HEK293 cells via PiggyBac transposon vector to generate stable cell lines. Cells were subsequently cultured in normoxia (21% O2) and hypoxia (1% O2), and harvested for flow cytometric analysis to evaluate CAR surface expression across a three day time frame. Results: We identified novel circuits providing fast reporter output with minimal background signal under hypoxic conditions, and elucidated design rules that enable or restrict amplification of transgene in native HIF1α and HIF2α-containing circuits. We observed differences in CAR transgene expression in hypoxia between circuits containing HIF1α and HIF2α-mediated feedback, suggesting mechanistic differences in regulation of HIF1α and HIF2α when overexpressed. Additionally, we observed greatly amplified transgene expression in circuit topologies containing feedback mediated by our synthetic transcription factor and promoter toolkit compared to native HIF1α and HIF2α-containing circuits and cells constitutively expressing CAR. Conclusions: We present a new therapeutic technology that senses hypoxic environments and enables high-output sense and respond behaviors with therapeutic outputs. We envision that this technology will enable development of safer, more effective cell therapies for solid tumors, as well as provide new biological and mechanistic insights that can inform the future development of hypoxia-responsive cell therapies.

Programmable promoter editing for precise control of transgene expression.

Subtle changes in gene expression direct cells to distinct cellular states. Identifying and controlling dose-dependent transgenes require tools for precisely titrating expression. To this end, we developed a highly modular, extensible framework called DIAL for building editable promoters that allow for fine-scale, heritable changes in transgene expression. Using DIAL, we increase expression by recombinase-mediated excision of spacers between the binding sites of a synthetic zinc finger transcription factor and the core promoter. By nesting varying numbers and lengths of spacers, DIAL generates a tunable range of unimodal setpoints from a single promoter. Through small-molecule control of transcription factors and recombinases, DIAL supports temporally defined, user-guided control of transgene expression that is extensible to additional transcription factors. Lentiviral delivery of DIAL generates multiple setpoints in primary cells and iPSCs. As promoter editing generates stable states, DIAL setpoints are heritable, facilitating mapping of transgene levels to phenotypes. The DIAL framework opens new opportunities for tailoring transgene expression and improving the predictability and performance of gene circuits across diverse applications.

Exploring plant-derived phytochrome chaperone proteins for light-switchable transcriptional regulation in mammals

Synthetic biology applications require finely tuned gene expression, often mediated by synthetic transcription factors (sTFs) compatible with the human genome and transcriptional regulation mechanisms. While various DNA-binding and activation domains have been developed for different applications, advanced artificially controllable sTFs with improved regulatory capabilities are required for increasingly sophisticated applications. Here, in mammalian cells and mice, we validate the transactivator function and homo-/heterodimerization activity of the plant-derived phytochrome chaperone proteins, FHY1 and FHL. Our results demonstrate that FHY1/FHL form a photosensing transcriptional regulation complex (PTRC) through interaction with the phytochrome, ΔPhyA, that can toggle between active and inactive states through exposure to red or far-red light, respectively. Exploiting this capability, we develop a light-switchable platform that allows for orthogonal, modular, and tunable control of gene transcription, and incorporate it into a PTRC-controlled CRISPRa system (PTRCdcas) to modulate endogenous gene expression. We then integrate the PTRC with small molecule- or blue light-inducible regulatory modules to construct a variety of highly tunable systems that allow rapid and reversible control of transcriptional regulation in vitro and in vivo. Validation and deployment of these plant-derived phytochrome chaperone proteins in a PTRC platform have produced a versatile, powerful tool for advanced research and biomedical engineering applications.

Engineering Material Properties of Transcription Factor Condensates to Control Gene Expression in Mammalian Cells and Mice.

Phase separation of biomolecules into condensates is a key mechanism in the spatiotemporal organization of biochemical processes in cells. However, the impact of the material properties of biomolecular condensates on important processes, such as the control of gene expression, remains largely elusive. Here, the material properties of optogenetically induced transcription factor condensates are systematically tuned, and probed for their impact on the activation of target promoters. It is demonstrated that transcription factors in rather liquid condensates correlate with increased gene expression levels, whereas stiffer transcription factor condensates correlate with the opposite effect, reduced activation of gene expression. The broad nature of these findings is demonstrated in mammalian cells and mice, as well as by using different synthetic and natural transcription factors. These effects are observed for both transgenic and cell-endogenous promoters. The findings provide a novel materials-based layer in the control of gene expression, which opens novel opportunities in optogenetic engineering and synthetic biology.

A zinc finger transcription factor enables social behaviors while controlling transposable elements and immune response in prefrontal cortex

The neurobiological origins of social behaviors are incompletely understood. Here we utilized synthetic biology approaches to reprogram the function of ZFP189, a transcription factor whose expression and function in rodent prefrontal cortex was previously demonstrated to be protective against stress-induced social deficits. We created novel synthetic ZFP189 transcription factors including ZFP189VPR, which activates the transcription of target genes and therefore exerts opposite functional control from the endogenous, transcriptionally repressive ZFP189WT. Following viral delivery of these synthetic ZFP189 transcription factors to mouse prefrontal cortex, we observe that ZFP189-mediated transcriptional control promotes mature dendritic spine morphology on transduced pyramidal neurons. Interestingly, inversion of ZFP189-mediated transcription in this brain area, achieved by viral delivery of synthetic ZFP189VPR, precipitates social behavioral deficits in terms of social interaction, motivation, and the cognition necessary for the maintenance of social hierarchy, without other observable behavioral deficits. RNA sequencing of virally manipulated prefrontal cortex tissues reveals that ZFP189 transcription factors of opposing regulatory function (ZFP189WT versus ZFP189VPR) have opposite influence on the expression of genetic transposable elements as well as genes that participate in adaptive immune functions. Collectively, this work reveals that ZFP189 function in the prefrontal cortex coordinates structural and transcriptional neuroadaptations necessary for complex social behaviors while regulating transposable element-rich regions of DNA and the expression of immune-related genes. Given the evidence for a co-evolution of social behavior and the brain immune response, we posit that ZFP189 may have evolved to augment brain transposon-associated immune function as a way of enhancing an animal’s capacity for functioning in social groups.

Development of new binary expression systems for plant synthetic biology.

A novel plant binary expression system was developed from the compactin biosynthetic pathway 27 of Penicillium citrinum ML-236B. The system achieved >fivefold activation of gene expression in 28 transgenic tobacco. A diverse and well-characterized genetic toolset is fundamental to achieve the overall goals of plant synthetic biology. To properly coordinate expression of a multigene pathway, this toolset should include binary systems that control gene expression at the level of transcription. In plants, few highly functional, orthogonal transcriptional regulators have been identified. Here, we describe the process of developing synthetic plant transcription factors using regulatory elements from the Penicillium citrinum ML-236B (compactin) pathway. This pathway contains several genes including mlcA and mlcC that are transcriptionally regulated in a dose-dependent manner by the activator mlcR. In Nicotiana benthamiana, we first expressed mlcR with several cognate synthetic promoters driving expression of GFP. Synthetic promoters contained operator sequences from the compactin gene cluster. Following identification of the most active synthetic promoter, the DNA-binding domain from mlcR was used to generate chimeric transcription factors containing variable activation domains, including QF from the Neurospora crassa Q-system. Activity was measured at both protein and RNA levels which correlated with an R2 value of 0.94. A synthetic transcription factor with a QF activation domain increased gene expression from its synthetic promoter up to sixfold in N. benthamiana. Two systems were characterized in transgenic tobacco plants. The QF-based plants maintained high expression in tobacco, increasing expression from the cognate synthetic promoter by fivefold. Transgenic plants and non-transgenic plants were morphologically indistinguishable. The framework of this study can easily be adopted for other putative transcription factors to continue improvement of the plant synthetic biology toolbox.

Reshaping the yeast galactose regulon via GPCR signaling cascade

Dynamically regulated systems are preferable to control metabolic pathways for an improved strain performance with better productivity. Here, we harnessed to the G protein-coupled receptor (GPCR) signaling pathway to reshape the yeast galactose regulon. The galactose-regulated (GAL) system was coupled with the GPCR signaling pathway for mating pheromone via a synthetic transcription factor. In this study, we refabricated the dynamic range, sensitivity, and response time of the GAL system to α factor by modulating the key components of the GPCR signaling cascade. A series of engineered yeasts with self-secretion of α factor were constructed to achieve quorum-sensing behaviors. In addition, we also repurposed the GAL system to make it responsive to heat shock. Taken together, our work showcases the great potential of synthetic biology in creating user-defined metabolic controls. We envision that the plasticity of our genetic design would be of significant interest for the future fabrication of novel gene expression systems.

High-throughput functional characterization of combinations of transcriptional activators and repressors.

Despite growing knowledge of the functions of individual human transcriptional effector domains, much less is understood about how multiple effector domains within the same protein combine to regulate gene expression. Here, we measure transcriptional activity for 8,400 effector domain combinations by recruiting them to reporter genes in human cells. In our assay, weak and moderate activation domains synergize to drive strong gene expression, whereas combining strong activators often results in weaker activation. In contrast, repressors combine linearly and produce full gene silencing, and repressor domains often overpower activation domains. We use this information to build a synthetic transcription factor whose function can be tuned between repression and activation independent of recruitment to target genes by using a small-molecule drug. Altogether, we outline the basic principles of how effector domains combine to regulate gene expression and demonstrate their value in building precise and flexible synthetic biology tools. A record of this paper's transparent peer review process is included in the supplemental information.

Major viral diseases in grain legumes: designing disease resistant legumes from plant breeding and OMICS integration.

Grain legumes play a crucial role in human nutrition and as a staple crop for low-income farmers in developing and underdeveloped nations, contributing to overall food security and agroecosystem services. Viral diseases are major biotic stresses that severely challenge global grain legume production. In this review, we discuss how exploring naturally resistant grain legume genotypes within germplasm, landraces, and crop wild relatives could be used as promising, economically viable, and eco-environmentally friendly solution to reduce yield losses. Studies based on Mendelian and classical genetics have enhanced our understanding of key genetic determinants that govern resistance to various viral diseases in grain legumes. Recent advances in molecular marker technology and genomic resources have enabled us to identify genomic regions controlling viral disease resistance in various grain legumes using techniques such as QTL mapping, genome-wide association studies, whole-genome resequencing, pangenome and 'omics' approaches. These comprehensive genomic resources have expedited the adoption of genomics-assisted breeding for developing virus-resistant grain legumes. Concurrently, progress in functional genomics, especially transcriptomics, has helped unravel underlying candidate gene(s) and their roles in viral disease resistance in legumes. This review also examines the progress in genetic engineering-based strategies, including RNA interference, and the potential of synthetic biology techniques, such as synthetic promoters and synthetic transcription factors, for creating viral-resistant grain legumes. It also elaborates on the prospects and limitations of cutting-edge breeding technologies and emerging biotechnological tools (e.g., genomic selection, rapid generation advances, and CRISPR/Cas9-based genome editing tool) in developing virus-disease-resistant grain legumes to ensure global food security.

Requirements for mammalian promoters to decode transcription factor dynamics.

In response to different stimuli many transcription factors (TFs) display different activation dynamics that trigger the expression of specific sets of target genes, suggesting that promoters have a way to decode dynamics. Here, we use optogenetics to directly manipulate the nuclear localization of a synthetic TF in mammalian cells without affecting other processes. We generate pulsatile or sustained TF dynamics and employ live cell microscopy and mathematical modelling to analyse the behaviour of a library of reporter constructs. We find decoding of TF dynamics occurs only when the coupling between TF binding and transcription pre-initiation complex formation is inefficient and that the ability of a promoter to decode TF dynamics gets amplified by inefficient translation initiation. Using the knowledge acquired, we build a synthetic circuit that allows obtaining two gene expression programs depending solely on TF dynamics. Finally, we show that some of the promoter features identified in our study can be used to distinguish natural promoters that have previously been experimentally characterized as responsive to either sustained or pulsatile p53 and NF-κB signals. These results help elucidate how gene expression is regulated in mammalian cells and open up the possibility to build complex synthetic circuits steered by TF dynamics.

Transcriptional kinetic synergy: A complex landscape revealed by integrating modeling and synthetic biology.

Transcription factors (TFs) control gene expression, often acting synergistically. Classical thermodynamic models offer a biophysical explanation for synergy based on binding cooperativity and regulated recruitment of RNA polymerase. Because transcription requires polymerase to transition through multiple states, recent work suggests that "kinetic synergy" can arise through TFs acting on distinct steps of the transcription cycle. These types of synergy are not mutually exclusive and are difficult to disentangle conceptually and experimentally. Here, we model and build a synthetic circuit in which TFs bind to a single shared site on DNA, such that TFs cannot synergize by simultaneous binding. We model mRNA production as a function of both TF binding and regulation of the transcription cycle, revealing a complex landscape dependent on TF concentration, DNA binding affinity, and regulatory activity. We use synthetic TFs to confirm that the transcription cycle must be integrated with recruitment for a quantitative understanding of gene regulation.

Substitution to hydrophobic linker and formation of host–guest complex enhanced the effect of synthetic transcription factor made of pyrrole−imidazole polyamide

GAA repeat expansion in the first intron of the frataxin (FXN) gene represses the transcription of FXN, and that induces Friedreich’s ataxia (FRDA). Pyrrole−imidazole polyamides (PIPs) are the class of oligopeptide that targets double-stranded DNA with sequence selectivity. Previously, bromodomain inhibitors such as JQ1 conjugated with PIPs were reported to selectively increase transcription. Here, we report the synthesis of a compound that increases the transcription of FXN in cells derived from an FRDA patient. The compound was effective in lower (one tenth) concentration than the compound that previously reported. High concentration of the compound is toxic, but toxicity was reduced with a host–guest complex.

Forced enhancer-promoter rewiring to alter gene expression in animal models

Transcriptional enhancers can be in physical proximity of their target genes via chromatin looping. The enhancer at the β-globin locus (locus control region [LCR]) contacts the fetal-type (HBG) and adult-type (HBB) β-globin genes during corresponding developmental stages. We have demonstrated previously that forcing proximity between the LCR and HBG genes in cultured adult-stage erythroid cells can activate HBG transcription. Activation of HBG expression in erythroid cells is of benefit to patients with sickle cell disease. Here, using the β-globin locus as a model, we provide proof of concept at the organismal level that forced enhancer rewiring might present a strategy to alter gene expression for therapeutic purposes. Hematopoietic stem and progenitor cells (HSPCs) from mice bearing human β-globin genes were transduced with lentiviral vectors expressing a synthetic transcription factor (ZF-Ldb1) that fosters LCR-HBG contacts. When engrafted into host animals, HSPCs gave rise to adult-type erythroid cells with elevated HBG expression. Vectors containing ZF-Ldb1 were optimized for activity in cultured human and rhesus macaque erythroid cells. Upon transplantation into rhesus macaques, erythroid cells from HSPCs expressing ZF-Ldb1 displayed elevated HBG production. These findings in two animal models suggest that forced redirection of gene-regulatory elements may be used to alter gene expression to treat disease.

Enhancement of Vivid-based photo-activatable Gal4 transcription factor in mammalian cells.

The Gal4/UAS system is a versatile tool to manipulate exogenous gene expression of cells spatially and temporally in many model organisms. Many variations of light-controllable Gal4/UAS system are now available, following the development of photo-activatable (PA) molecular switches and integration of these tools. However, many PA-Gal4 transcription factors have undesired background transcription activities even in dark conditions, and this severely attenuates reliable light-controlled gene expression. Therefore, it is important to develop reliable PA-Gal4 transcription factors with robust light-induced gene expression and limited background activity. By optimization of synthetic PA-Gal4 transcription factors, we have validated configurations of Gal4 DNA biding domain, transcription activation domain and blue light-dependent dimer formation molecule Vivid (VVD), and applied types of transcription activation domains to develop a new PA-Gal4 transcription factor we have named eGAV (enhanced Gal4-VVD transcription factor). Background activity of eGAV in dark conditions was significantly lower than that of hGAVPO, a commonly used PA-Gal4 transcription factor, and maximum light-induced gene expression levels were also improved. Light-controlled gene expression was verified in cultured HEK293T cells with plasmid-transient transfections, and in mouse EpH4 cells with lentivirus vector-mediated transduction. Furthermore, light-controlled eGAV-mediated transcription was confirmed in transfected neural stem cells and progenitors in developing and adult mouse brain and chick spinal cord, and in adult mouse hepatocytes, demonstrating that eGAV can be applied to a wide range of experimental systems and model organisms.Key words: optogenetics, Gal4/UAS system, transcription, gene expression, Vivid.

Inducible, Tissue-Specific Gene Expression in Arabidopsis Using GR-LhG4-Mediated Trans-Activation.

Inducible, tissue-specific gene expression is a potent tool to study gene regulatory networks as it allows spatially and temporally controlled genetic perturbations. To this end, we generated a toolkit that covers many cell types in the three main meristems: the root apical meristem, the shoot apical meristem, and the vascular cambium. The system is based on an extensive set of driver lines expressing a synthetic transcription factor under cell type-specific promoters. Induction leads to nuclear translocation of the transcription factor and expression of response elements under control of a cognate synthetic promoter. In addition, a fluorescent reporter incorporated in driver lines allows to monitor induction. All previously generated driver lines are available from the Nottingham Arabidopsis Stock Center. This protocol describes how users can create their own constructs compatible with the existing set of lines and as well as induction and imaging procedures.

Reduced Cytotoxicity by Repetitive mRNA Transfection in Differentiated Neurons.

mRNA-based protein expression technology has been used to express functional proteins. We have previously generated dopamine neurons from rat-embryo derived neural precursor cells (NPCs) through repeated transfection of synthetic transcription factor mRNA encoding dopamine-inducible genes. However, NPCs began to die approximately 10 d post-transfection. In this study, we examined a long-term transfection protocol that did not affect cell viability. Experiments were performed in eight groups sorted according to the start date of mRNA transfection. mRNA was transfected into NPCs daily for 21 d and live cell images of each group were recorded. NPCs which were differentiated for more than five days showed sustained gene expression and appreciable viability despite daily mRNA transfection for 21 d. Repeated mRNA transfection requires cells with a sufficient differentiation period.

Development of a synthetic transcription factor-based S-adenosylmethionine biosensor in Saccharomyces cerevisiae.

S-Adenosylmethionine (SAM) is a crucial small-molecule metabolite widely used in food and medicine. The development of high-throughput biosensors for SAM biosynthesis can significantly improve the titer of SAM. This paper constructed a synthetic transcription factor (TF)-based biosensor for SAM detecting in Saccharomyces cerevisiae. The synthetic TF, named MetJ-hER-VP16, consists of an Escherichia coli-derived DNA-binding domain MetJ, GS linker, the human estrogen receptor binding domain hER, and the viral activation domain VP16. The synthetic biosensor is capable of sensing SAM in a dose-dependent manner with fluorescence as the output. Additionally, it is tightly regulated by the inducer SAM and β-estradiol, which means that the fluorescence output is only available when both are present together. The synthetic SAM biosensor could potentially be applied for high-throughput metabolic engineering and is expected to improve SAM production.

Engineering a two-gene system to operate as a highly sensitive biosensor or a sharp switch upon induction with β-estradiol

The human estrogen receptor has been used for about thirty years, in the yeast S. cerevisiae, as a component of chimeric transcription factors. Its ligand, β-estradiol, permits to control the protein translocation into the nucleus and, as a consequence, the expression of the gene(s) targeted by the synthetic transcription factor. Activators that are orthogonal to the yeast genome have been realized by fusing the human estrogen receptor to an activation and a DNA-binding domain from bacteria, viruses, or higher eukaryotes. In this work, we optimized the working of a β-estradiol-sensing device—in terms of detection range and maximal output signal—where the human estrogen receptor is flanked by the bacterial protein LexA and either the strong VP64 (from herpes simplex virus) or the weaker B42 (from E. coli) activation domain. We enhanced the biosensor performance by thoroughly engineering both the chimeric activator and the reporter protein expression cassette. In particular, we constructed a synthetic promoter—where transcription is induced by the chimeric activators—based on the core sequence of the yeast CYC1 promoter, by tuning parameters such as the length of the 5′ UTR, the distance between adjacent LexA binding sites (operators), and the spacing between the whole operator region and the main promoter TATA box. We found a configuration that works both as a highly sensitive biosensor and a sharp switch depending on the concentration of the chimeric activator and the strength of its activation domain.

A Modified Yeast Two-Hybrid Platform Enables Dynamic Control of Expression Intensities to Unmask Properties of Protein-Protein Interactions.

The yeast two-hybrid (Y2H) assay is widely used for protein–protein interaction characterization due to its simplicity and accessibility. However, it may mask changes in affinity caused by mutations or ligand activation due to signal saturation. To overcome this drawback, we modified the Y2H system to have tunable protein expression by introducing a fluorescent reporter and a pair of synthetic inducible transcription factors to regulate the expression of interacting components. We found that the application of inducers allowed us to adjust the concentrations of interacting proteins to avoid saturation and observe interactions otherwise masked in the canonical Y2H assay, such as the abscisic acid-mediated increase in affinity of monomeric abscisic acid receptors to the coreceptor. When applied in future studies, our modified system may provide a more accurate characterization of protein–protein interactions.