Small-molecule inducible transcriptional control in mammalian cells

Abstract

Tools for tuning transcription in mammalian cells have broad applications, from basic biological discovery to human gene therapy. While precise control over target gene transcription via dosing with small molecules (drugs) is highly sought, the design of such inducible systems that meets required performance metrics poses a great challenge in mammalian cell synthetic biology. Important characteristics include tight and tunable gene expression with a low background, minimal drug toxicity, and orthogonality. Here, we review small-molecule-inducible transcriptional control devices that have demonstrated success in mammalian cells and mouse models. Most of these systems employ natural or designed ligand-binding protein domains to directly or indirectly communicate with transcription machinery at a target sequence, via carefully constructed fusions. Example fusions include those to transcription activator-like effectors (TALEs), DNA-targeting proteins (e.g. dCas systems) fused to transactivating domains, and recombinases. Similar to the architecture of Type I nuclear receptors, many of the systems are designed such that the transcriptional controller is excluded from the nucleus in the absence of an inducer. Techniques that use ligand-induced proteolysis and antibody-based chemically induced dimerizers are also described. Collectively, these transcriptional control devices take advantage of a variety of recently developed molecular biology tools and cell biology insights and represent both proof of concept (e.g. targeting reporter gene expression) and disease-targeting studies.

Keywords: CAR T cells; TetR; CRISPRa/CRISPRi; TALEs; bacterial transcriptional factors; hormone receptors.

Fig. 1. Inducible gene expression using the HIT-TALE-SunTag system.

A. In the absence of the inducer (4-OHT), the fusion protein containing estrogen receptor (ER^T2) and the activator domains (VP64, p65, and HSF-1) remain sequestered in the cytoplasm by interactions with the HSP90 chaperone. The nuclear-tagged TALE-GCN4 complex is localized at the target locus but without the activator domains, TALE-GCN4 complex cannot turn ON transcription of the target gene. B. Upon addition of 4-OHT, the ER^T2 fusion complex dissociates from HSP90 and translocates in the nucleus where the single-chain antibody (scFv) interacts with the GCN4 peptide and recruits the multiple activator domains to the target site to activate gene transcription. The DNA binding TALE array determines the target site. The same design can be adapted to dCas9-sgRNA based systems wherein DNA-targeting specificity is determined by the sgRNA.

Fig. 2. Dual input transcriptional regulation.

A. Two layers of inducibility are achieved with the ChaCha system. In the first layer, doxycycline (Dox) induces the expression of ARRB2-dCas-activator fusion protein through a Tet-ON system (see text for details) while a second layer of control is achieved when a GPCR agonist leads to GPCR signal activation. Without the appropriate GPCR agonist, increasing dox concentration does not alter target gene expression. The binding of the GPCR agonist induces a conformational change in the V2 domain and allows the interaction between ARRB2 and V2. This ARRB2-V2 interaction further leads to the proteolytic release of the dCas9-activator. The nuclear-tagged dCas9-VPR localizes in the nucleus and upregulates target gene expression in the presence of constitutively expressed sgRNAs. B. Multilayered transcriptional activation is induced by VEGF and rapamycin inducers. VEGF binds to VEGFR and induces the proteolytic release of the membrane-tethered split-dCas9 fragments. Rapamycin induces heterodimerization of rapamycin-binding proteins (FKBP-FRB), leading to the recruitment of the FRB-fused activator to the target gene by dCas9-sgRNA complex. Once at the target gene, the dCas9-VP64 transactivator turns ON gene transcription.

Fig. 3. Small-molecule induced proteolytic release of synthetic transcriptional factor.

A. Modular Extracellular Sensor Architecture (MESA) platform induces receptor dimerization in the presence of small-molecule inducers leading to proteolytic release of a synthetic transcriptional factor (tet transactivator). The TF released upon proteolysis localizes to the nucleus to enable activation of the target gene expression. B. StaPL module for inducible transcriptional regulation. In the absence of an inducer, cis-cleaving NS3 protease releases the associated TF. The unlinked TF remains in the cytoplasm while the activator domains translocate to the nucleus but cannot activate the target gene without the associated TF. In presence of a small-molecule drug, the activity of the protease is inhibited thereby allowing nuclear localization of the linked TF and activator domains at the target site to turn ON gene expression.