Transient Thresholding: A Mechanism Enabling Noncooperative Transcriptional Circuitry to Form a Switch
Threshold 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 h—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 because 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.
© 2017 Biophysical Society. Received 9 December 2016, Accepted 1 May 2017, Available online 11 June 2017. We thank Brandon Razooky for key initial observations and reagents, and Marielle Cavrois of the Gladstone Institutes Flow Cytometry Core and Kurt Thorn of the University of California San Francisco Nikon Imaging Center for technical help and advice. The Gladstone Institutes Flow Cytometry Core is supported by National Institutes of Health (NIH) grant Nos. P30 AI027763 and S10 RR028962. K.H.A. was supported by a National Science Foundation (NSF) Graduate Research Fellowship. M.T. acknowledges support from the National Institutes of Health (NIH) Office of the Director, the National Cancer Institute, and the National Institute of Dental and Craniofacial Research under NIH grant No. DP5 OD012194. L.S.W. acknowledges support from the NIH Director's New Innovator Award Program, grant No. OD006677, and NIH grant No. R01 AI109593.
Submitted - 134858.full.pdf
Supplemental Material - mmc1.pdf