Understanding Transcriptional Regulation by Characterizing Gene Regulatory Functions of Synthetic Loci in Mammalian Cells*

Gene expression is dynamically regulated by transcription factors (TFs) during development and in response to environmental signals. The relationship between TF concentration and gene expression is referred to as gene regulatory function (GRF). Mammalian GRFs integrate information from TF occupancy, epigenetic modifications and chromatin structure. A quantitative understanding of the underlying mechanisms regulating GRF is essential for understanding fundamental biological processes and achieving predictive control over gene expression. However, endogenous mammalian gene loci exhibit complex pleiotropic regulatory interactions that limit our ability to decouple underlying mechanisms governing their GRFs. To overcome this limit, we constructed a fully-composable synthetic reporter locus that enables single-cell fluorescent measurement of steady-state and dynamic transcriptional activities. By expressing fluorescently labeled synthetic TFs—individually or in combination—that regulate the locus, we can precisely quantify changes in transcription rate and noise.  In addition, we identified combinations of regulatory mechanisms that synergistically regulate GRF. To understand the GRFs, we developed non-equilibrium theoretical models that allow us to make experimentally testable predictions. In the future, we envision using these experimental and theoretical approaches in tandem to establish quantitative rules for engineering complex gene networks in mammalian cells.