Costa, E. J. | bioRxiv Science

bioRxiv2026

Stable epigenetic states set single-cell activation thresholds in mammalian expression systems

Quantitatively relating transcription factor (TF) input to gene expression output is central to understanding mammalian gene regulation and essential for designing predictable synthetic expression systems. However, even minimal synthetic systems often exhibit unexplained behaviors. In a widely used inducible mammalian expression system, we show that transcriptional responses appear graded and sigmoidal at the population level but are largely all-or-none at the single-cell level. By combining single-cell sorting and single-molecule footprinting with mathematical modeling of transcriptional regulation, we found that this behavior is not caused by bursty transcription or bistability, but by long-lived, chromatin-encoded variability in TF occupancy and activation strength. This variability produced a range of activation thresholds in switch-like single-cell responses that were stable over time, resulting in bimodal gene expression across the population. These results advance our basic understanding of how TFs interact with chromatin to modulate quantitative features of single-cell and population level transcriptional responses.

systems biology↗

bioRxiv2025

Bifunctional transcriptional effector domains control gene expression pulses in an occupancy-dependent manner

Dynamic gene expression pulses enable adaptive response to stimuli and can be generated in natural and synthetic systems. Controlling these dynamics typically involves circuits consisting of multiple genes and transcription factors (TFs). Here, we discover a new class of bifunctional transcriptional effector domains that can first activate and subsequently repress the same gene, producing dynamic gene expression pulses from a single input. These pulse dynamics arise from distinct, temporally separated chromatin states defined by active and repressive chromatin modifications. The balance between active and repressed states is determined by the DNA occupancy of the bifunctional TF. Bifunctional domains activate at low occupancy but switch to repression at high occupancy, resulting in a non-monotonic TF input-gene expression output relationship tunable by TF concentration and number of DNA binding sites. We develop a kinetic model that links TF occupancy to gene expression transitions, allowing for the programming of eight distinct cell "states" - combinations of On/Off states of 3 reporter genes - using a single bifunctional effector. This work establishes the theoretical framework and molecular mechanisms of pulse-generating gene regulation by bifunctional domains and creates a foundation for engineering complex multi-gene circuits.

systems biology↗