Single-cell analysis showed that changes in the intracellular distribution of transcription factors can regulate the expression of target genes under environmental stimuli.

Most transcription factors function continuously, but some transcription factors achieve pulsatile regulation by rapidly shuttling from and to the nucleus. Compared to the continuous action mode, pulsed regulation has some advantages. For example, information can also be encoded according to the frequency or amplitude of the pulses, increasing the amount of information transmitted. However, the study of this phenomenon is difficult because of the different patterns of transcription factor regulation pulses in different cells. Lin et al. overcame this hurdle, demonstrating that multiple transcription factor pulses can modulate gene expression in response to environmental stimuli.

The Msn2 protein expressed in budding yeast is the first transcription factor identified to regulate genes in a pulsed manner. When cells are exposed to light, Msn2 enters the nucleus, thereby activating the transcription of target genes. Such pulsing signals can be beneficial or harmful to cells because the information sent changes over time. In electronic devices, this problem is generally addressed by ensuring that multiple components can perform the same task. Theoretically, this principle also applies to cellular signaling pathways, where signal pulses are transmitted through multiple integrated pathways, thereby increasing the reliability of signal transmission. In fact, cell signaling pathways do employ this strategy, such as Msn2 antagonizing the transcriptional repressor protein Mig1 to finely control gene expression under various conditions.

Lin et al. used different fluorescent proteins to label Msn2 and Mig1 to study the dynamics of the pulsing signals of these two transcription factors. They connected the cells to a microfluidic device through which cell culture fluids flowed, which was able to monitor the movement of transcription molecules and subsequent gene expression regulated by two transcription factors.

Depletion of glucose in cell culture media triggers nuclear export of Mig1 and nuclear import of Msn2, thereby increasing the expression of target genes. If Msn2 and Mig1 regulate gene expression in a simple continuous pattern, or if they regulate in a pulse pattern of random duration, then glucose consumption gradually changes the average level of nuclear transcription factors across the cell population, and target gene expression gradually increases to a new stable state. In contrast, Lin et al. observed a "transient phase" following glucose depletion, during which the mean intranuclear levels of Msn2 and Mig1 increased and decreased, respectively, compared with the levels after reaching steady state (Figure 1A). ). This overshoot occurs when the system changes - shortening the time it takes for the expression level of the gene of interest to reach a new steady state. In fact, when the glucose concentration increases, a similar phenomenon occurs: the nuclear level of Mig1, which can inhibit the expression of target genes, increases, reduces the transcription level of the corresponding RNA of the target gene, and rapidly reduces the level of the corresponding protein.

Pulse gene regulation (Figure 1)

Figure 1 Interpretation of transcription factor pulses. Transcription factors Msn2 and Mig1 enter the nucleus in a pulsed manner and are able to activate and repress the transcription of the same target genes, respectively. a, Lin et al. found pulsed changes in transcription factor levels under environmental stimuli. Decreased glucose concentration in the cell culture medium resulted in a large decrease in nuclear Mig1 levels for a short period of time, while a rapid increase in Msn2. This "overshoot" allows cells to rapidly increase gene expression and adapt to changes. After this transient phase, the nuclear levels and gene expression of both remain stable from the perspective of the cell population. b, Lin et al. also found that the pulsing signals of all cells were synchronized only during the transient phase. After this phase, the levels of these two transcription factors changed randomly in single cells. Gene expression decreased when the pulses of Msn2 and Mig1 overlapped. Target gene expression increased when the pulses of Msn2 did not overlap with Mig1.

Lin et al. speculate that the overshoot observed in the experiment may not be a transient event, and may still exist under steady-state conditions, but cannot be observed at the cell population level because the effects at the population level cancel each other out. However, when analyzing single cells, Lin et al. found that under steady-state environmental conditions, the pulses of each transcription factor are sporadic and irregular, making it impossible to define a single pulse. In principle, many criteria could be used to define such pulses, but most are moot. Based on a neuroscience technique called spike-triggered averaging, Lin et al. developed an interesting and practical method to detect individual pulses. Lin et al. measured pulses by measuring the average levels of Mig1 and Msn2 near the peak over a set time period.

To confirm the effectiveness of this method, Lin et al. studied the expression of target genes under pulsed changes of Msn2 and Mig1. For example, if the Msn2 pulse did not overlap the Mig1 pulse (ie, the nuclear level of Mig1 did not increase), the expression of the target gene increased. Conversely, if the Msn2 pulse overlapped with the Mig1 pulse, the effect of Msn2 was counteracted and target gene expression was reduced. These observations suggest that transcription factor pulses also exist under steady-state conditions.

In the steady state following an increase in glucose concentration, the proportion of overlapping pulses increases, thereby enhancing the repression efficiency of target gene expression. In contrast, during the transient phase, Lin et al. could only detect non-overlapping pulses, suggesting that the duration of the pulses during the transient period is regulated. In this case, the pulses of the individual cells are synchronized, resulting in population-level overshoot.

The spike-triggered averaging technique is very powerful in analyzing the oscillating signal of cellular regularity. To date, most studies have focused on average cell behavior, but cellular responses such as cell cycle checkpoints have very large cell-to-cell differences. Spike-triggered averaging may help unravel the mechanisms regulating such responses.

Furthermore, the method used by Lin et al. can be used to analyze gene regulation without knowing all system parameters. For example, the current study shows that a perfectly overlapping inhibitory pulse can neutralize the activating pulse, and that this counteracting effect persists even when the inhibitory pulse is several minutes apart from the activating pulse. It is important to note that not all Msn2 pulses promote target gene expression even in the presence of low Mig1 activity. This suggests that large-scale action dynamics, stochastic modeling, and identification of response mechanisms must be included in future studies of complete models of gene regulation. Research in these areas has grown tremendously in recent years and may be integrated to pave the way for a detailed understanding of the dynamics of signaling pathways.

(Jilin Province Qijian Biotechnology Co., Ltd. www.qjbio.com.cn)