Transient thresholding: a mechanism enabling non-cooperative transcriptional circuitry to form a switch

ABSTRACTThreshold generation in fate-selection circuits is often achieved through deterministic bistability, which requires cooperativity (i.e., nonlinear activation) and associated hysteresis. However, the Tat positive-feedback loop that controls HIV’s fate decision between replication and proviral latency lacks self-cooperativity and deterministic bistability. Absent cooperativity, it is unclear how HIV can temporarily remain in an off state long enough for the kinetically slower epigenetic silencing mechanisms to act— expression fluctuations should rapidly trigger active positive feedback and replication, precluding establishment of latency. Here, using flow cytometry and single-cell imaging, we find that the Tat circuit exhibits a transient activation threshold. This threshold largely disappears after ∼40 hours—accounting for the lack of deterministic bistability—and promoter activation shortens the lifetime of this transient threshold. Continuous differential equation models do not recapitulate this phenomenon. However, chemical reaction (master equation) models where the transcriptional transactivator and promoter toggle between ‘inactive’ and ‘active’ states can recapitulate the phenomenon since they intrinsically create a single-molecule threshold transiently requiring excess molecules in the ‘inactive’ state to achieve at least one molecule (rather than a continuous fractional value) in the ‘active’ state. Given the widespread nature of promoter toggling and transcription factor modifications, transient thresholds may be a general feature of inducible promoters.