Studies of individual living cells have revealed that many transcription factors activate in dynamic, and often stochastic, pulses within the same cell. occur inside a pulsatile fashion in response to numerous inputs10,14,17. Mig1 is known to quickly localize to the nucleus in response to an increase in glucose levels36, and may also show pulsatile activation38. Two phases of dynamic pulsing To analyze Msn2 and Mig1 dynamics in the same cell, we constructed strains expressing fusions of Msn2 and Mig1 proteins to the distinguishable fluorescent proteins39 mKO2 and mCherry, respectively (Fig. 1a). To simplify the analysis, we knocked out their paralogs Msn4 and Mig2 (Methods). We attached solitary cells to the glass surface of a microfluidic channel, keeping buy 503468-95-9 a constant flow of press, while acquiring time-lapse movies. By analyzing individual cells in these movies, we could track the nuclear localization dynamics of both proteins over time (Methods). Number 1 Temporally organized pulsing of transcription factors Msn2 and Mig1 in response to glucose reduction We 1st analyzed the effects of glucose reduction, which is known to induce changes in nuclear localization for both transcription factors35,36. In response to a sudden step from 0.2% to 0.1% blood sugar, both protein exhibited pulses of nuclear localization, but did so with different timing (Fig. 1b). Msn2 localized towards the nucleus instantly, while Mig1 exited the nucleus. Subsequently, in many cells (75%), Msn2 exited the nucleus followed by the re-entry of Mig1 (Fig. 1b; Supplementary Video 1). This transient response terminated within 30 min (Fig. 1b, bottom). We describe events like this in which Msn2 and Mig1 pulses are temporally separated, as non-overlapping (observe Fig. 1b, top and Methods). After this event, Msn2 and Mig1 exhibited sporadic pulsing that was unsynchronized between cells (Supplementary Video 1). During this steady-state period, we observed both overlapping (i.e., coincident) events, in which Msn2 and Mig1 pulses overlap, as well as nonoverlapping events in which Msn2, but not Mig1 localized to the nucleus (Fig. 1b, top and Methods). These data provoke two interrelated questions about whether and how relative pulse timing could function in combinatorial rules (Fig. 1c): 1st, do inputs modulate the relative timing of transcription element pulses, either during the transient response to a change in conditions, or during the subsequent period of repeated pulsing? Second, if so, how does such pulse timing modulation impact downstream combinatorial gene rules? To address these questions, we constructed strains containing synthetic target promoters incorporating binding sites for either or both transcription factors (Fig. 1a). These promoters drove manifestation of a transcriptional reporter consisting of 24 binding sites for any separately indicated PP7 RNA binding protein fused to GFP40 (Fig. 1a). These strains enabled us to simultaneously adhere to localization dynamics of Msn2 and Mig1 and downstream target manifestation in the same cell. Relative pulse timing in the transient response We 1st analyzed transient reactions to changes in various input conditions (i.e., different Msn2 stressors) other than the known common input glucose (Fig. 2a). Addition of 100mM NaCl produced transient non-overlapping pulses of Msn2 and Mig1 in solitary cells and in populace averages (Fig. 2b, Extended Data Fig. 1aCc, Supplementary Video 2) that were much like those observed buy 503468-95-9 in the transient response to glucose reduction (Fig. 1b). Addition of 2.5% ethanol also activated both transcription factors. But in contrast to NaCl, it did so with overlapping, rather than non-overlapping, pulses (Fig. 2c, Extended Data Fig. 1dCf, Supplementary Video 3). The difference in relative timing between NaCl and ethanol was also buy 503468-95-9 apparent in cross-correlation analysis (Extended Data Fig. 1g). Collectively, these results indicate that unique inputs can generate reverse relative timing in the transient reactions of Msn2 and Mig1. Number 2 Different inputs create unique transient gene manifestation reactions by modulating relative pulse timing We hypothesized that control of temporal overlap could provide a mechanism for combinatorial gene rules. Non-overlapping pulse dynamics, in which the activator Msn2 is definitely active, but the repressor Mig1 is not, could activate combinatorial target genes more efficiently than overlapping pulses, Akap7 in which the two proteins are simultaneously bound to the same target promoter. Indeed, while both NaCl and ethanol led to.