Peet, D. J. | bioRxiv Science

bioRxiv2023

dFLASH; dual FLuorescent transcription factor Activity Sensor for Histone integrated live-cell reporting and high-content screening

Live-cell reporting of regulated transcription factor (TF) activity has a wide variety of applications in synthetic biology, drug discovery, and functional genomics. As a result, there is high value in the generation of versatile, sensitive, robust systems that can function across a range of cell types and be adapted toward diverse TF classes. Here we present the dual FLuorescent transcription factor Activity Sensor for Histone integrated live-cell reporting (dFLASH), a modular sensor for TF activity that can be readily integrated into cellular genomes. We demonstrate readily modified dFLASH platforms that homogenously, robustly, and specifically sense regulation of endogenous Hypoxia Inducible Factor (HIF) and Progesterone receptor (PGR) activities, as well as regulated coactivator recruitment to a synthetic DNA-Binding Domain-Activator Domain fusion proteins. The dual-colour nuclear fluorescence produced normalised dynamic live-cell TF activity sensing with facile generation of high-content screening lines, strong signal:noise ratios and reproducible screening capabilities (Z = 0.68-0.74). Finally, we demonstrate the utility of this platform for functional genomics applications by using CRISPRoff to modulate the HIF regulatory pathway, and for drug screening by using high content imaging in a bimodal design to isolate activators and inhibitors of the HIF pathway from a [~]1600 natural product library.

synthetic biology↗

bioRxiv2022

Core and Flanking bHLH-PAS:DNA interactions mediate specificity and drive obesity

The basic-Helix-Loop-Helix Per-Arnt-Sim (PAS) homology domain (bHLH-PAS) transcription factor (TF) family comprises critical biological sensors of physiological (hypoxia, tryptophan metabolites, neuronal activity, and appetite) and environmental (diet derived metabolites and environmental pollutants) stimuli to regulate genes involved in signal adaptation and homeostasis1. bHLH TFs bind DNA as homo or heterodimers via E-box (CANNTG) response elements, however the DNA binding specificity of the PAS domain-containing bHLH subfamily remains unresolved1. We systematically analysed cognate DNA binding hierarchies of prototypical bHLH-PAS family members (ARNT, ARNT2, HIF1, HIF2, AhR, NPAS4, SIM1) and demonstrate distinct core (NNCGTG) specificities for different heterodimer classes. The results also show that bHLH-PAS TFs bind over a large footprint 12-15bp and recognise preferential DNA sequences flanking the core. For example, specificity beyond otherwise identical core binding by SIM1 and the HIFs is mediated through N-terminal HIF-DNA interactions. We also reveal an intimate relationship between DNA shape and both core and flanking TF binding allowing motif sequence flexibility and underpinning TF binding specificity. Furthermore, DNA-shape affinity relationships revealed that novel downstream PAS-A-loop DNA interactions are associated with AT-rich sequences that lead to high-affinity binding, and that loss of this function underpins a monogenic cause of human hyperphagic obesity in a recapitulated SIM1.R171H knock-in mouse model. Importantly, models of protein-DNA binding accurately predict in vivo occupancy, while response element methylation blocks DNA binding and predicts cell type specific chromatin occupancy. These data provide a definitive and accurate map of bHLH-PAS TF specificity and target selectivity through novel flanking protein-DNA interactions that are crucial for in vivo biological function.

molecular biology↗