Unraveling Transcriptional Dynamics in Mammalian Cells using a Synthetic Reporter

An outstanding challenge in mammalian synthetic biology is engineering genetic circuits with quantitatively precise behavior. One important reason for this challenge is that transcription occurs in stochastic bursts, the dynamics of which are regulated by the binding of transcription factors (TFs) and co-factors to gene promoters. Unfortunately, a lack of understanding of how promoter sequence elements contribute to the dynamics of transcriptional initiation and gene expression has prevented the design of circuits with predictable temporal and population-level behavior. To uncover these relationships, we have engineered a synthetic reporter locus in a mammalian cell line that permits simultaneous single-cell measurements of TF concentration, protein expression, mRNA levels, and kinetics of transcriptional initiation. Using the locus, we show that protein and mRNA distributions can be predicted directly from transcriptional initiation dynamics. We then demonstrate that alteration of promoter sequence elements and the recruitment of various TFs and co-factors to the locus, both individually and in combination, can be used to systematically alter transcriptional bursting dynamics, thereby allowing us to begin to establish an engineering grammar for quantitatively programming trans-regulatory network connections in mammalian cells. In future work, we aim to leverage the rules learned from our platform to advance our ability to engineer complex gene circuitry in mammalian cells.