Functional roles of pulsing in genetic circuits

Abstract

A fundamental problem in biology is to understand how genetic circuits implement core cellular functions. Time-lapse microscopy techniques are beginning to provide a direct view of circuit dynamics in individual living cells. Unexpectedly, we are discovering that key transcription and regulatory factors pulse on and off repeatedly, and often stochastically, even when cells are maintained in constant conditions. This type of spontaneous dynamic behavior is pervasive, appearing in diverse cell types from microbes to mammalian cells. Here, we review recent work showing how pulsing is generated and controlled by underlying regulatory circuits and how it provides critical capabilities to cells in stress response, signaling, and development. A major theme is the ability of pulsing to enable time-based regulation analogous to strategies used in engineered systems. Thus, pulsatile dynamics is emerging as a central, and still largely unexplored, layer of temporal organization in the cell.

Fig. 1. Pulsing is ubiquitous in cellular regulation

(A)Pulsatile dynamics involve the transient, simultaneous activation of many molecules of a given type (circles), even under constant environmental conditions. Cells pulse asynchronously, making pulsing difficult to detect with static snapshots and necessitating tracking of cell lineages over time (right, schematic). (B) Pulsing occurs in a diverse array of pathways, molecular types, organisms, and time scales (–6). For each example, a schematic of the type of regulation is shown at left, a typical filmstrip is shown at center, and a qualitative schematic plot of typical dynamics is shown at right.

Fig. 2. Pulsing enables diverse cellular functions

(A) Cells modulate pulse characteristics, including amplitude, frequency, and duration, to implement diverse regulatory functions. (B) A transcription factor (green) may activate different target promoters at different thresholds or with different affinities (light and dark arrows). Concentration-based regulation (amplitude modulation, AM) would therefore lead to different, nonproportional, response profiles (bottom left). In contrast, frequency-modulated (FM) pulsing, by effectively controlling the fraction of time that all target genes are expressed, leads to expression of targets in fixed proportions (bottom right), indicated by overlap of expression curves (each is normalized to its own maximum) (11). (C) Pulsed regulation functions in a developmental timer. B. subtilis respond to sudden nutrient limitation by proliferating for multiple cell cycles before sporulating (schematic). A model of the underlying circuit (inset) is based on a positive-feedback loop (arrows) with a hypothesized time delay (Δt). This circuit can generate progressive growth in pulses of phosphorylation of the sporulation master regulator Spo0A (green trace), via steplike growth in the kinase concentration (blue trace). The timer terminates when a threshold level of Spo0A is reached (dashed line) (10). (D) Examples in which dynamic multiplexing enables a single pathway to transmit multiple signals (2, 13). In each case, distinct types and levels of inputs generate distinct dynamic activation patterns for the indicated regulatory protein.

Fig. 3. Circuit mechanisms of pulse generation and modulation. (A)

The B. subtilis competence circuit generates stereotyped pulses of ComK activation (9). Green and red arrows represent positive and negative feedbacks, respectively. Pulses are stereotyped, as indicated by three identical traces (bottom left) and unimodal pulse size distribution (bottom right). (B) The B. subtilis general stress response (σ^B) circuit combines transcriptional feedback and an ultrasensitive phosphoswitch, and produces a nonstereotyped distribution of pulse amplitudes (bottom right) (1). (C and D) Mammalian cell pulse-generation mechanisms use multiple negative feedbacks. (C) In the p53 circuit, doublestranded DNA damage activates p53 pulses through ATM kinase. Pulses may lead to subsequent DNA repair (12). (D) NF-κB pulse mechanism. This circuit displays digital activation behavior, with the fraction of cells that pulse depending on the level of stimulus (5).

Fig. 4. Other potential regulatory functions of pulsing

(A) Regulation could occur through modulation of the relative pulse dynamics of two factors that co-regulate a common target. Inputs could modulate the relative level of synchronization of the regulator pulses. Here, we assume a target promoter activated only when both factors are present simultaneously. (B) Pulsing may reduce conflicts between incompatible pathways by temporally alternating between states in which only one or the other pathway is active (middle), rather than by simultaneously expressing conflicting programs (bottom). (C) Stochastic pulsing systems enable random switching between cellular states. In contrast to sequential switching, stochastic state switching allows cells to diversify cellular states on the time scale of about one cell cycle when the pulse duration is similar to the cell cycle time.