Single-particle imaging of stress-promoters induction reveals the interplay between mapk signaling, chromatin and transcription factors

ABSTRACT Precise regulation of gene expression in response to environmental changes is crucial for cell survival, adaptation and proliferation. In eukaryotic cells, extracellular signal

integration is often carried out by Mitogen-Activated Protein Kinases (MAPK). Despite a robust MAPK signaling activity, downstream gene expression can display a great variability between

single cells. Using a live mRNA reporter, here we monitor the dynamics of transcription in _Saccharomyces cerevisiae_ upon hyper-osmotic shock. We find that the transient activity of the

MAPK Hog1 opens a temporal window where stress-response genes can be activated. We show that the first minutes of Hog1 activity are essential to control the activation of a promoter.

Chromatin repression on a locus slows down this transition and contributes to the variability in gene expression, while binding of transcription factors increases the level of transcription.

However, soon after Hog1 activity peaks, negative regulators promote chromatin closure of the locus and transcription progressively stops. SIMILAR CONTENT BEING VIEWED BY OTHERS OSMOTIC

DISRUPTION OF CHROMATIN INDUCES TOPOISOMERASE 2 ACTIVITY AT SITES OF TRANSCRIPTIONAL STRESS Article Open access 05 December 2024 LIVE-CELL IMAGING REVEALS THE SPATIOTEMPORAL ORGANIZATION OF

ENDOGENOUS RNA POLYMERASE II PHOSPHORYLATION AT A SINGLE GENE Article Open access 26 May 2021 REVERSIBLE PHASE SEPARATION OF HSF1 IS REQUIRED FOR AN ACUTE TRANSCRIPTIONAL RESPONSE DURING

HEAT SHOCK Article 07 March 2022 INTRODUCTION A crucial function of all cellular life is the ability to sense its surroundings and adapt to its variations. These changes in the extracellular

environment will induce specific cellular responses orchestrated by signal transduction cascades, which receive cues from plasma membrane sensors. This information is turned into a

biological response by inducing complex transcriptional programs implicating hundreds of genes1,2,3. Tight regulation of signaling is thus crucial to ensure the correct temporal modulation

of gene expression, which can otherwise alter the cell physiology4,5,6. Interestingly, single-cell analyses have revealed that genes regulated by an identical signaling activity can display

a high variability in their transcriptional responses7,8,9,10. This noise in transcriptional output questions how signal transduction can faithfully induce different loci and which molecular

mechanisms contribute to the variability in gene expression. In eukaryotic cells, various environmental stimuli are transduced by the highly conserved Mitogen-Activated Protein Kinases

(MAPK) cascades11,12. They control a wide range of cellular responses such as cell proliferation, differentiation, or apoptosis. In _Saccharomyces cerevisiae_, a sudden increase in the

osmolarity of the medium is sensed by the High Osmolarity Glycerol (HOG) pathway, which leads to the activation of the MAPK Hog1, a homolog of p38 in mammals13,14. Upon hyper-osmotic stress,

the kinase activity of Hog1 promotes the adaptation of the cells to their new environment by driving an increase in the internal glycerol concentration, thereby allowing to balance the

internal and the external osmotic pressures. In parallel to its cytoplasmic activity, Hog1 also transiently accumulates into the nucleus to induce the expression of hundreds of

osmostress-responsive genes (Fig. 1a). The MAPK is recruited to promoter regions by Transcription Factors (TFs) and, in turn, Hog1 recruits chromatin remodeling complexes, the Pre-Initiation

Complex, and the RNA Polymerase II (PolII) to trigger gene expression15,16. Once cells have adapted, Hog1 is inactivated and exits the cell nucleus, transcription stops, and chromatin is

rapidly reassembled at HOG-induced gene loci. Biochemical analyses of this pathway have identified the key players implicated in gene expression and the central role played by the MAPK in

all these steps15. In parallel, single-cell measurements have uncovered the large variability present in their expression. In particular, translational reporters and RNA-FISH measurements

have identified that slow chromatin remodeling promoted by the MAPK at each individual locus is generating strong intrinsic noise in the activation of many stress-responsive genes9,17. In

order to get deeper insights into the regulation of osmostress-genes expression kinetics, we aimed at monitoring the dynamics of mRNA production in live single cells. Phage coat

protein-based assays, like the MS2 or PP7 systems, have been used to visualize mRNA in live single cells18,19,20. These experiments contributed to revealing the bursty nature of

transcription, whereby a set of polymerases simultaneously transcribing a gene generates a burst in mRNA production, which is followed by a pause in transcription21,22,23. In this study, we

dissect the kinetics of transcription of osmostress genes. The production of mRNA is monitored using the PP7 phage coat protein assay. This reporter allows us to measure with high temporal

resolution and in a fully automated manner, the fluctuations in transcription arising in hundreds of live single cells. This analysis enables to dissect the contribution of various players

to the overall transcriptional output. We show that the first few minutes of MAPK activity will determine if a gene is transcribed. We also demonstrate that the chromatin state of a promoter

will control the timing of activation and thus the variability in the transcription, while the TF binding will influence the level and duration of the mRNA production. RESULTS CELLULAR

RESPONSE TO HYPER-OSMOTIC STRESS High osmotic pressure is sensed and transduced in the budding yeast _Saccharomyces cerevisiae_ via the HOG signaling cascade, which culminates in the

activation of the MAPK Hog1 (Fig. 1a). Upon activation, this key regulator accumulates in the nucleus to trigger gene expression in a stress level-dependent manner (Supplementary Fig. 1a).

The activity of the kinase can be monitored by following its own nuclear enrichment24,25. In parallel to Hog1, the general stress-response pathway is induced by the hyper-osmotic shock and

the transcription factor Msn2 also relocates into the nucleus with dynamics highly similar to the ones observed for Hog1 (Supplementary Fig. 1b, c) 26,27. Nuclear Hog1 and Msn2 (together

with its paralog Msn4) induce osmostress-genes expression, with approximately 250 genes being up-regulated upon osmotic shock1,28,29. The activity of the pathway is limited to the cellular

adaptation time, which coincides with the nuclear exit of Hog1 and the recovery of the cell size (Supplementary Fig. 1a, d). The fast and transient activity of the osmostress response as

well as the homogenous activation of the MAPK within the population9,25 (Supplementary Fig. 1e), make this signaling pathway an excellent model to understand the induction of eukaryotic

stress-responsive genes, which are often accompanied by important chromatin remodeling. MONITORING THE DYNAMICS OF OSMOSTRESS-GENES TRANSCRIPTION In order to quantify the dynamics of

transcription in live single cells, we use the PP7 system to label the production of messenger RNAs (mRNA)30. Briefly, constitutively expressed and fluorescently labeled PP7 phage coat

proteins strongly associate to a binding partner: an array of twenty-four PP7 mRNA stem-loops (PP7sl). In our settings, this PP7 reporter construct is placed under the control of a promoter

of interest and integrated in the genome at the _GLT1_ locus (Fig. 1b)30 in a strain bearing a nuclear tag (Hta2-mCherry) and expressing a fluorescently-tagged PP7 protein

(PP7ΔFG-GFPenvy31,32, abbreviated PP7-GFP, Methods). Upon activation of the promoter, local accumulation of newly synthesized transcripts at the Transcription Site (TS) leads to the

formation of a bright fluorescent focus due to the enrichment in PP7-GFP fluorescence above the background signal (Fig. 1c and Supplementary Movie 1). The fluorescence intensity at the TS is

proportional to the number of mRNA being transcribed and thus to the instantaneous load of RNA polymerases. After termination, single mRNAs are exported out of the nucleus and their fast

diffusion in the cytoplasm prevents their detection under the selected illumination conditions. Typically, time-lapses with fifteen-second intervals for twenty-five minutes with six Z-planes

for the PP7-GFP channel on four fields of view were performed. Image segmentation and quantification were performed automatically, allowing to extract up to four hundred single-cell traces

for each experiment33. The mean intensity of the 20 brightest pixels in the nucleus, from which the median cell fluorescence was subtracted, was used as a measurement of TS intensity and

thus as proxy for transcriptional activity (Fig. 1d, Methods). Figure 1d displays the average TS fluorescence from more than 200 cells bearing the p_STL1_-PP7sl reporter following the

activation of the HOG pathway by various NaCl concentrations. The HOG-induced _STL1_ promoter has been extensively studied at the population and single-cell level9,17,34,35. As expected,

increasing the salt concentration leads to a proportionally increasing transcriptional output from the cell population, whereas no change in TS fluorescence is detected in the control

medium. The hundreds of dynamic measurements acquired with the PP7 reporter form a rich dataset where multiple features can be extracted from each single-cell trace (Fig. 1e, Methods). Our

automated image segmentation and analysis allow to reliably quantify the appearance (Start Time) and disappearance (End Time) of the TS (Supplementary Fig. 2 and Methods). The maximum

intensity of the trace and the integral under the curve provide estimates of the transcriptional output from each promoter (Fig. 1e). In addition, transcriptional bursts can be identified by

monitoring strong fluctuations in the TS intensity. VALIDATING THE LIVE MRNA REPORTER ASSAY The mRNA dynamics measured with the PP7 assay are in close agreement with previously reported

data sets34,36. Nonetheless, we also verified with a dynamic protein expression reporter that comparable results can be obtained (Supplementary Fig. 3a). The dynamic Protein Synthesis

Translocation Reporter (dPSTR) enables to monitor the kinetics of gene expression from a promoter of interest. It by-passes the slow maturation time of Fluorescent Proteins (FP) by

monitoring the relocation of the fluorescent signal in the nucleus of the cell37. For the PP7 assay, as well as the dPSTR and many other expression reporters, an additional copy of the

promoter of interest is inserted in a non-native locus. In order to address if this modified genomic environment alters the dynamics of gene expression, we used CRISPR-Cas9 to integrate the

PP7sl downstream of the endogenous _STL1_ promoter (Supplementary Fig. 4). Interestingly, we observe only minor differences between the p_STL1_ at its endogenous location and at the _GLT1_

locus. This observation strongly suggests that the −0.8 kb to TSS of the _STL1_ promoter sequence placed at a non-endogenous locus replicates many of the properties of the endogenous

promoter. INTRINSIC NOISE IN OSMOSTRESS-GENE ACTIVATION The microscopy images presented in Fig. 1c illustrate the noise that can be observed in the activation of the p_STL1_ promoter upon

osmotic stress, which has been previously reported9,17. In order to verify that this noise is not due to a lack of activation of the MAPK Hog1 in the non-responding cells, we combined the

p_STL1_-PP7sl reporter and the Hog1-mCherry relocation assay in the same strain. As expected, we observe an absence of correlation between the two measurements (Supplementary Fig. 5).

Indeed, cells with similar Hog1 relocation behaviors can display highly variable transcriptional outputs. An additional manner to observe this heterogeneity is to monitor the activation of

two _STL1_ promoters within the same cell. Using a diploid strain where both _GLT1_ loci were modified with either a p_STL1_-_24xPP7sl_ or a p_STL1_-_24xMS2sl_ and expressing PP7-mCherry and

MS2-GFP proteins, we observe an uncorrelated activation of both loci within each single cell (Supplementary Fig. 6 and Supplementary Movie 2). This observation confirms the high intrinsic

noise generated by the _STL1_ promoter upon osmotic stress9,37. The highly dynamic measurements provided by the PP7 reporter allows us to decipher some of the parameters that contribute to

this large variability. HIGH VARIABILITY IN OSMOSTRESS-GENES TRANSCRIPTION DYNAMICS In addition to p_STL1_, five other stress-responsive promoters often used in the literature to report on

the HOG pathway transcriptional activity were selected for this study34,38. Each reporter strain differs only by the one thousand base pairs of the promoter present in front of the PP7sl

(800 bp for p_STL1_, 660 for p_ALD3_9,39); however, each strain displays a different transcriptional response following a 0.2 M NaCl stimulus (Fig. 2a). Because the level of accumulation of

the PP7 signal at the transcription site and the timing of the appearance and disappearance of the TS is different for each tested promoter, it implies that the promoter sequence dictates

multiple properties of the transcription dynamics. These dynamic measurements are in general agreement with control experiments performed with the dPSTR assay (Supplementary Fig. 3b) and

previously published population-averaged data34,37. Importantly, expressing three times more phage coat proteins did not alter substantially the parameters extracted from the PP7

measurements for the two strongest promoters, denoting the absence of titration of PP7-GFP reporter proteins in our experimental settings (Supplementary Fig. 7). The automated analysis

allows to identify the presence or absence of a transcription site in each single cell and thus the fraction of cells that induce the promoter of interest (Fig. 2b). Interestingly, even in

absence of stimulus, some promoters display a basal level of transcription. In the p_GRE2_, p_HSP12_ and p_GPD1_ reporter strains, an active transcription site can be detected in 5–20% of

the cells in the few time points before the stimulus (Fig. 2c, Supplementary Movie 3). If the period of observation is extended to a 25-min time lapse without stimulus, this fraction

increases twofold to threefold (Supplementary Fig. 8). Upon activation by 0.2 M NaCl, the fraction of responding cells for the three promoters that display basal expression overcomes 85%,

while it remains below 65% for the three promoters without basal induction. CHROMATIN STATE SETS THE TIMING OF TRANSCRIPTION INITIATION A key parameter controlled by the promoter sequence is

the timing of induction. In Fig. 2d, the time when cells become transcriptionally active (Start Time) is plotted as a Cumulative Distribution Function (CDF) only for the cells where a TS is

detected after the stimulus, thereby excluding basal expressing cells and non-responding cells. Treatment with 0.2 M NaCl results in a sudden activation of transcription (Fig. 2d). This

contrasts with non-induced samples, where the CDF of the promoters displaying basal activity rises almost linearly due to stochastic activation during the recording window (Supplementary

Fig. 8c). Upon stress, the promoters displaying basal activity are induced faster than the promoters that are repressed under log-phase growth, with p_GPD1_ being activated the fastest (~1 

min), while p_ALD3_ and p_STL1_ require more than 4 min for activation (Fig. 2e). However, there is a great variability in transcription initiation between cells of the same population,

since we generally observe 3–4 min delay between the 10th and 90th percentiles, with the exception of p_GPD1_ where the induction is more uniform and <2 min delay is observed (Fig. 2e).

Comparison between individual replicates demonstrates the reliability of our measurement strategy. Interestingly, we observe a positive correlation between faster transcriptional activation

from p_GPD1_, p_HSP12,_ and p_GRE2_ and the presence of basal expression. These promoters also display the highest numbers of responding cells upon a 0.2 M NaCl shock. These results suggest

that basal expression is associated with a more permissive chromatin state, which enables a faster activation and higher probability of transcription among the cell population. To test this

hypothesis, we disrupted the function of the SAGA chromatin remodeling complex by deleting _GCN5_40. As expected, we observe fewer transcribing cells and a slower induction of the p_STL1_

promoter in this background (Fig. 2f). Less remarkably, abolishing histone H2AZ variants exchange at +1 and −1 nucleosomes by deleting _HTZ1_41 only results in a reduced percentage of

transcribing cells. Conversely, chromatin state at the _STL1_ promoter can be loosened by relieving the glucose repression using raffinose as a C-source42. Interestingly, a fraction of the

cells grown in these conditions displays basal expression from the p_STL1_-PP7sl reporter and the Start Times measured for cells grown in raffinose are accelerated by 1 min compared with the

glucose control experiment (Fig. 2g). The link between the chromatin state under log-phase growth and the ability to induce stress-responsive genes is confirmed by these results. A promoter

that is tightly repressed will need more Hog1 activity and thus more time to become transcriptionally active, therefore displaying a lower fraction of responding cells. EARLY HOG1 ACTIVITY

DICTATES TRANSCRIPTIONAL COMPETENCE The period of Hog1 activity provides a temporal window, where transcription can potentially be initiated. However, the switch to a transcriptionally

active state takes place almost exclusively within the first few minutes after the stimulus. When comparing the characteristic timing of Hog1 nuclear enrichment to the CDF of Start Times for

cells bearing the p_STL1_ reporter (Fig. 3a and Supplementary Fig. 9a), we observe that 90% of the transcribing cells initiate transcription during the first few minutes of the stress

response, while Hog1 nuclear accumulation rises and before it drops below 80% of its maximum (decay time). A similar behavior is observed for all the promoters tested, independently of the

presence of basal levels (Fig. 3a, b). For p_ALD3_, which is the slowest promoter tested, 87% of the Start Times are detected before the decay of Hog1 activity (7 min) while the full

adaptation time takes 14 min at 0.2 M NaCl. Interestingly, promoter output also decreases with the time after stimulus. Cells that start transcribing p_STL1_ earlier display a larger

integral over the PP7 signal and a higher maximum intensity compared with cells that initiate transcription later (Fig. 3c and Supplementary Fig. 9b). A similar behavior is quantified for

all tested promoters (Supplementary Fig. 9c, d). These measurements demonstrate that the high Hog1 activity present in the first minutes of the response is key to determine both the

transcriptional state and overall output of the promoters. TFS CONTROL THE DYNAMICS AND LEVEL OF MRNA PRODUCTION Promoters dictate the timing of transcriptional activation of the ORF and the

level at which the mRNA is produced. To extract the transcriptional level of each promoter, we used as a proxy the maximum of the PP7 trace of each single cell where a transcription event

could be detected (Fig. 4a). This value represents the maximal loading of polymerases on the locus during the period of transcription. Similar results are obtained when comparing the

integral below the PP7 trace, which represents the total transcriptional output from a promoter (Supplementary Fig. 10a). As shown in Fig. 4a, each promoter has an intrinsic capability to

induce a given level of transcription, which is independent from the presence of basal transcription or the locus activation time. Indeed, p_GRE2_ displays the lowest level of induction

among all tested promoters, despite the presence of basal transcription and being the second-fastest promoter activated. As expected, the recruitment of the RNA polymerases is stimulated by

the stress; the three promoters with basal activities display a higher transcriptional level upon a 0.2 M NaCl stress than in normal growth conditions (Fig. 4b and Supplementary Fig. 10b).

Both the general stress transcription factors Msn2 and Msn4 and the TFs activated by the MAPK Hog1 (Hot1, Sko1, Smp1) contribute to the transcriptional up-regulation29,38,43. Based on

studies on synthetic promoters, it has been established that TF binding site number and distance from the Transcription Start Site (TSS) influence the promoter output44. Unfortunately,

osmostress promoters display a wide diversity in numbers and affinities of TF binding sites and no obvious prediction of the transcriptional activity can be drawn (Supplementary Fig. 11).

While multiple Msn2/4 binding sites can be found on the _GPD1_ and _STL1_ promoter sequences, their activations are only mildly affected by deletions of these two TFs (Supplementary Fig. 

12). Both _GPD1_ and _STL1_ are primarily Hog1 targets28,29,38. However, their requirements for Hog1 activity is strikingly different. In strains where the MAPK has been anchored to the

plasma membrane to limit its nuclear enrichment45, p_STL1_ induction is virtually abolished (only 1.5% transcribing cells), while p_GPD1_ activity is barely affected (Supplementary Fig. 13).

Similarly, deletion of either TF Sko1 or Hot1 profoundly alter the capacity of p_STL1_ to be induced (Fig. 4c–f) while these same deletions have weaker effects on the _GPD1_ promoter.

Because the induction of the _STL1_ promoter requires an efficient chromatin remodeling, every defect (TF deletion or absence of Hog1 in the nucleus) strongly alters its capability to induce

transcription. In comparison, the p_GPD1_ is less perturbed by these same defects. These results suggest that TFs act in a cooperative manner on p_STL1_, while they act independently of

each other on p_GPD1_. BURSTS OF POLII TRANSCRIPTION IN OSMOSTRESS-GENES ACTIVATION The PP7 and MS2 systems have allowed to directly visualize transcriptional bursting. In order to identify

bursts arising from osmostress promoters, we sought to detect strong fluctuations in each single-cell trace. Fluctuations in TS intensities were filtered to retain only peaks separated by

pronounced troughs (Methods). In 20–30% of the traces, two or more peaks are identified (Fig. 5a, b). The total length of the transcript downstream of the promoter is 8 kb (1.5 kb for the

stem loops +6.5 kb for _GLT1_). Based on a transcription speed of 20 bp/s30, the expected lifetime of a transcript at the TS is 6.6 min. This corresponds well to the mean duration observed

for the p_ALD3_, p_CTT1_, p_STL1,_ and p_GRE2_ reporters (Fig. 5c). However, it is unlikely that the strong TS intensities recorded are generated by a single transcript, but rather by a

group of RNA PolII that simultaneously transcribe the locus, probably forming convoys of polymerases46. Indeed, single mRNA FISH experiments have shown that following a 0.2 M NaCl stress,

the endogenous _STL1_ locus produces on average 20 mRNAs per cell, with some cells producing up to 10036. For p_HSP12_ and p_GPD1_, the average peak duration is longer than 11 min (Fig. 5c),

suggesting that multiple convoys of polymerases are traveling consecutively through the ORF. Unfortunately, the long half-lives of the transcripts on the locus prevent a separation of

individual groups of polymerases. However, when we isolate individual peaks in the single-cell traces, their durations become closer to the expected value of 6.6 min (Fig. 5d). In addition,

the output of the transcription estimated by the maximum intensity of the trace, or the integral under the whole curve, is equal or lower for traces with multiple pulses compared with traces

where only a single peak is present, indicating a pause in transcription (Fig. 5e and Supplementary Fig. 14). Together these data strengthen the notion that these stress-responsive

promoters are highly processive, displaying an elevated rate of transcription once activated. Only brief pauses in the transcription can be observed in a small fraction of the responding

cells. MAPK ACTIVITY OPENS AN OPPORTUNITY WINDOW FOR TRANSCRIPTION We have shown that transcription initiation is dictated by early Hog1 activity. Next, we want to assess what the

determinants of transcription shutoff are and by extension, how the duration of transcriptional activity is controlled. In the HOG pathway, the duration of transcription has been reported to

be limited by the cellular adaptation time34,38. Therefore, the duration of transcription is shorter after a 0.1 M NaCl stress and longer after a 0.3 M stress, compared with a 0.2 M stress

(Fig. 6a). For the _STL1_ promoter, the last time point where a PP7 signal is detected at the TS matches the timing of nuclear exit of the MAPK at all concentrations tested (Fig. 6b). In

order to challenge this link between Hog1 activity and transcriptional arrest, we sought to modulate the MAPK activity pattern by controlling the cellular environment in a dynamic manner.

Using a flow channel set-up, we generated a step, a pulse, or a ramp in NaCl concentrations (Fig. 6c, Methods). These experiments were performed in a strain carrying the p_STL1_-PP7sl

reporter in conjunction with Hog1-mCherry, allowing to monitor kinase activity and the downstream transcriptional response in the same cell. The step stimulus at 0.2 M NaCl mimics the

experiments performed in wells, where the concentration of the osmolyte is suddenly increased at time zero and remains constant throughout the experiments (Supplementary Movie 4). The mean

responses at the population level (Fig. 6c) confirm this relationship between Hog1 adaptation time and transcription shutoff time. However, at the single-cell level, no direct correlation is

observed between these two measurements due to important single-cell variability (Fig. 6f). In the pulse assay, 7 min after the initial 0.2 M step, the NaCl concentration is set back to 0 M

(Supplementary Movie 5). This shortens the MAPK activity period, as Hog1 leaves the nucleus immediately when cells are brought back in normal growth medium. Removing the kinase from the

nucleus has a direct impact on the transcriptional process. First, fewer cells become transcriptionally active. Second, the active TS sites disappear within a few minutes after the end of

the pulse (Fig. 6d, e). Therefore in this context, we observe a direct correlation between Hog1 activity and transcription, which is in line with the known role played by MAPKs, and Hog1 in

particular, on multiple steps of the transcriptional process47,48. The ramp experiment starts with a pulse at 0.2 M NaCl followed by a slow increase of the NaCl concentration up to 0.6 M

over the next 20 min (Supplementary Movie 6). This constant rise in external osmolarity extends the Hog1 activity window by preventing the adaptation of the cells. More cells can become

transcriptionally active and the transcription shut off is delayed (Fig. 6d, e). However, in these conditions, there is a clear lack of correlation between Hog1 activity, which is sustained

in many cells over the 30 min of the time-lapse, and the transcription output of the p_STL1_ that stops much earlier (Fig. 6f). Taken together, these experiments demonstrate that the MAPK

activity is required but not sufficient to sustain the transcriptional process. In the ramp experiment, transcription cannot be sustained throughout the whole Hog1 activity window,

demonstrating that other factors contribute to limiting the duration of the transcription. PROMOTER IDENTITY INFLUENCES THE TRANSCRIPTION SHUTOFF TIME In order to test whether the promoter

identity plays a role in the process of transcriptional shutoff, we quantified the duration of the transcriptional period for the six promoters and plotted the cumulative distributions of

End Times following a 0.2 M NaCl stress (Fig. 7a, b). Interestingly, despite similar cell volume adaptation times for all the experiments, the promoters display substantially different

kinetics of inactivation. Promoters transcribing at a lower level (p_CTT1_ and p_GRE2_) terminate transcription earlier. This shorter transcriptional window may reflect an inferior

recruitment of transcriptional activators to the promoter, enabling an earlier inhibition of transcription due to chromatin closure. In addition, promoters with basal activity display an

extended period of transcription after adaptation (Fig. 7a, b). For p_GPD1_ and p_GRE2_, this results in a biphasic decay, where the first phase corresponds to the arrest of Hog1-induced

transcription and the second phase can be associated to the basal transcription arising from these promoters (Fig. 7b). Note that basal transcription may even be increased due to a higher

basal Hog1 signaling activity post high osmolarity conditions49. Remarkably, p_HSP12_ transcription persists beyond the adaptation time, with nearly 30% of the cells displaying an active TS

at the end of the experiment. This suggests that basal expression from this promoter is strongly increased post-stimulus. In contrast to p_GPD1_ and p_GRE2_, p_HSP12_ possesses numerous

Msn2/4 binding sites. Although the relocation dynamics of Hog1 and Msn2 are highly similar during the adaptation phase, Msn2 displays some stochastic secondary pulses27 that are not

correlated to Hog1 relocation events. This could explain the stronger basal expression arising from this promoter post-adaptation (Supplementary Fig. 1e, f). To summarize, these measurements

demonstrate that the pattern of MAPK activity provides a temporal window where transcription can take place. When the signaling cascade is shut off, transcription ceases soon afterward.

However, the promoter identity, and probably its propensity to recruit positive activators, will determine for how long it can sustain an open chromatin environment favorable to

transcription before Hog1 activity decreases due to cellular adaptation. DISCUSSION In this study, we have constructed PP7 reporter strains to monitor the transcription dynamics of

osmostress promoters. The second exogenous copy of the promoter is integrated at the _GLT1_ locus. This strategy provides a similar genomic environment for all the promoters, in order to

compare their specific characteristics. Interestingly, we saw only minor differences in the CDF of Start Times of the p_STL1_ when integrated at its endogenous locus compared with the _GLT1_

locus. This observation provides a strong evidence that TF binding and chromatin state of the duplicated promoter sequences mimic closely the ones at the native environment of the gene.

Note that the signal at the TS is expected to be proportional to the length of the transcribed mRNA. The _GLT1_ locus with its 6.5 kB length was expected to provide a signal four times

stronger than the endogenous _STL1_ ORF (1.7 kB). The unexpectedly high signal obtained from the PP7 reporter at the endogenous locus may be indicative of global difference in transcription

rates between the _GLT1_ and _STL1_ ORFs alternatively, the smaller _STL1_ ORF might enhance transcription efficiency via gene-looping50,51. Our data illustrate the complex balance that

exists between positive and negative regulators taking place at the stress-induced loci. At each locus, positive and negative regulators will control the level and duration of transcription.

We have shown that the first few minutes of Hog1 activity are essential to initiate the transcription. Transcription factors, chromatin remodelers such as the SAGA and RSC complexes,

together with Hog1 will contribute to open and maintain an accessible chromatin environment at the stress-response loci35,40. Once initiated, transcription seems highly processive and only

in a small fraction of traces, we are able to detect a pause in transcription. However, it has been shown that PolII recruits additional chromatin remodelers, including the Ino80 complex and

Asf1 that will redeposit nucleosomes after acute transcription52. These conflicting activities will determine the overall duration of transcription at a locus. Indeed, promoters with lower

transcriptional activity, such as p_GRE2_ and p_CTT1_, recruit fewer positive activators and will be repressed faster by the negative regulators. The repression level of a promoter during

log-phase growth will determine the speed and the noise of the transcription activation process. Thus, for each promoter, a trade-off has to be found between these two contradictory

requirements. For instance, _GPD1_, which is essential for survival to osmotic stress, has an important basal expression level and can thus be rapidly induced upon stress. Interestingly, the

chromatin state, encoded in part by the promoter sequence, can be tuned by external growth conditions. Thus, the noise generated by a promoter is not rigidly set by its DNA sequence but

fluctuates based on the environment. In higher eukaryotes, the stress response MAPKs p38 and JNK relocate to the nucleus upon activation53,54. Early genes such as c-Fos or c-Jun, are induced

within minutes after activation of signaling3,55. Interestingly, these loci display basal expression and require minimal chromatin modification for their induction56,57. Conversely, delayed

primary response and secondary response genes require more chromatin remodeling to induce their activation55,58. These similarities with the regulation of Hog1-dependent genes induction

suggest a high conservation in the mechanisms used by MAPK in eukaryotes to regulate the dynamics of gene expression. METHODS PLASMIDS AND YEAST STRAINS Plasmids, yeast strains and primers

used in this study are listed in Supplementary Tables 1–3. All strains were constructed in the _W303_ background. Transformations were performed with a standard lithium-acetate protocol.

Gene deletions and gene taggings were performed with either pFA6a cassettes59,60 or pGT cassettes61. Transformants were selected with auxotrophy markers (Uracil, Histidine, Leucine,

Tryptophan, Adenine) and gene deletions were performed with antibiotic resistance to Nourseothricin (NAT) or Kanamycin (KAN). In order to generate the membrane-anchored Hog1, the

pGTT-mCherry vector was modified by inserting annealed oligos (oSP1648/9) encoding the last 30 bp of the Ras2 sequence to obtain the pGTT-mCherry-CaaX plasmid. A strain possessing the

Hog1-mCherry:_LEU2_ modification was transformed with the pGTT-mCherry-CaaX plasmid linearized with XbaI and SacI to induce a marker switch and introduce the membrane anchoring motif. PP7

AND MS2 STRAINS CONSTRUCTION The PP7-GFP plasmids are based on the bright and photostable GFPenvy fluorescent protein32. The PP7 protein was derived from Larson et al.30 (Addgene# 35194)

with an additional truncation in the capsid assembly domain (PP7∆FG residues 67–75: CSTSVCGE31). The expression of the PP7 construct is controlled by an _ADH1_ promoter and a _CYC1_

terminator. The final construct pVW284 was cloned in a Single Integration Vector _URA3_ (pSIVu61). The PP7-mCherry was cloned by replacing the GFP by the mCherry sequence. The MS2-GFP was

generated by using the original _MS2_ sequence from Betrand et al.18, which also lacks the capsid assembly domain (Addgene# 27117), inserted into the pVW284. The PP7 stem-loops plasmids are

based on the previously published p_POL1 24xPP7sl_ integrative plasmid30 (Addgene #35196). The stress-responsive promoters replace the _POL1_ promoter in the original construct using 1 kb

(0.8 kb for p_STL1_, 0.66 kb for p_ALD3_) upstream of the start codon. The p_STL1_-_24xMS2sl_ was generated by replacing the PP7 stem-loops with the MS2 stem-loops obtained from Betrand et

al.18 (Addgene# 31865). A strain bearing the Hta2-mCherry nuclear marker and expressing the PP7-GFP was transformed with plasmids containing the different osmostress promoters driving the

PP7sl production, linearized with a NotI digestion and integrated upstream of the _GLT1_ ORF, as previously published30. Correct integration into the _GLT1_ locus was screened by colony PCR

with primers in the _GLT1_ ORF (oSP061, +600 bp) and in the _TEF_ terminator (oSP062) of the selection marker on genomic DNA extractions. The integrity of the PP7 stem-loops array was

assessed with primers within the _TEF_ terminator (oSP062) and in _GLT1_ ORF (oVW447, +250 bp) for all the promoters and deletions, after each transformation performed. For all the strains

used in the study, at least two clones with correct genotypes were isolated and tested during a salt challenge time-lapse experiment. From the data analysis, the most frequent phenotype was

isolated and the strain selected. To tag the endogenous locus of _STL1_ with the _24xPP7sl_, the plasmid pSP264 with the _STL1_ promoter was modified by replacing the _GLT1_ ORF sequence by

a 500 bp sequence starting 100 bp after the start codon of _STL1_. The plasmid was digested SacI-NotI and purified over a gel to isolate a fragment that contains the

p_STL1_-_24xPP7sl_-_STL1_100-600. A double-strand break was generated in the _STL1_ ORF using Cas9 and a sgRNA targeting the PAM motif GGG 62 bp upstream of the start codon. The Cas9 and

sgRNA are expressed from a 2 µ plasmid (Addgene #3546462) slightly modified from the work from Laughery et al. (Addgene# 6763963). The purified DNA fragment containing the stem-loops was

used as repair DNA to promote homologous recombination at the _STL1_ locus (Supplementary Fig. 4a). The correct size of the inserted fragment was verified by colony PCR around the _PP7sl_

insert. Multiple positive clones were screened by microscopy. The results from two transformants are presented in this work to ensure that potentially undesired DNA alterations by Cas9 do

not affect the response in the HOG pathway. In order to generate the diploid reporter strain, a MATa strain containing the _PP7-mCherry::URA3_, p_STL1 24xPP7sl_:_GLT1_ and

_Hta2-tdiRFP_:_TRP1_ was crossed to a MATα strain bearing the _MS2-GFP_::_URA3_, p_STL1 24xMS2sl_:_GLT1_ and _Hta2-tdiRFP_:_NAT_. Haploid cells were mixed on a YPD plate for a few hours

before cells were resuspended in water and spread with beads on a selection plate (SD-TRP + NAT). The plasmids generated for this study are available on Addgene. YEAST CULTURE Yeast cells

were grown in YPD medium (YEP Broth: CCM0405, ForMedium) for transformation or in Synthetic Defined (SD) medium (YNB:CYN3801/CSM: DCS0521, ForMedium) for microscopy experiments. Before

time-lapse experiments, cells were grown at least 24 h in log-phase. A saturated overnight culture in SD medium was diluted into fresh SD-full medium to OD600 0.025 in the morning and grown

for roughly 8 h to reach OD600 0.3–0.5. In the evening, cultures were diluted by adding (0.5/OD600)µl of cultures in 5 ml SD-full for an overnight growth that kept cells in log-phase

conditions. Cultures reached an OD600 of 0.1–0.3 in the morning of the second day and were further diluted when necessary to remain below an OD600 of 0.4 during the day. To prepare the

samples, these log-phase cultures were further diluted to an OD600 0.05 and sonicated twice 1 min (diploids were not sonicated) before placing 200 µl of culture into the well of a 96-well

glass-bottom plate (MGB096-1-2LG, Matrical Bioscience) previously coated with a filtered solution of Concanavalin A diluted to 0.5 mg/ml in water (C2010, Sigma-Aldrich)64. Cells were let to

settle for 30–45 min before imaging. Osmotic shock was performed under the microscope, by adding 100 µl of a three times concentrated SD-full+NaCl stock solutions to the 200 µl of medium

already in the well, to reach the final desired salt concentration. MICROSCOPY Images were acquired on a fully automated inverted epi-fluorescence microscope (Ti2-Eclipse, Nikon) placed in

an incubation chamber set at 30 °C. Excitation was provided by a solid-state light source (SpectraX, Lumencor) and dedicated filter sets were used to excite and detect the proper

fluorescence wavelengths with a sCMOS camera (Flash 4.0, Hamamatsu). A motorized XY-stage was used to acquire multiple fields of views in parallel and a piezo Z-stage (Nano-Z200, Mad City

Labs) allowed fast Z-dimension scanning. Micro-manager was used to control the multidimensional acquisitions65. Experiments with PP7 stem-loops were acquired with a 60X oil objective. For

strains with PP7-GFP and Hta2-mCherry, GFP (40 ms, 3% LED power) and RFP (20 ms), along with two bright field images were recorded every 15 s for the GFP and every minute for the other

channels, for a total duration of 25 min. Six z-stacks were performed on the GFP channels covering ±1.2 µm from the central plane with 0.4 µm steps. An average bleaching of 32% for the GFP

and 26% for the RFP for the whole time-lapse was quantified in a strain without the PP7 stem-loops, to avoid artifacts from the appearance of bright fluorescent foci. For all time-lapse

experiments, media addition was performed before time point 4, defined as time zero. All microscopy experiments were performed in duplicate for non-induced control experiments and at least

triplicate for the NaCl induced experiments. FLOW CHAMBER EXPERIMENTS The flow experiments were performed in Ibidi chambers (µ-Slide VI 0.4, Ibidi). Two 50 ml Falcon tube reservoirs

containing SD-full +0.5 µg/ml fluorescein-dextran (D3305, ThermoFischer) and SD-full +0.6 M NaCl were put under a pressure of 30 mbar (FlowEZ, Fluigent). The media coming from each reservoir

were connected using FEP tubing (1/16″ OD × 0.020″ ID, Fluigent) to a 3-way valve (2-switch, Fluigent). The concentration of NaCl in the medium was controlled using a Pulse-Width Modulation

strategy66,67. Periods of 4 s were used and within this time, the valve controlled the fraction of time when SD-full versus SD-full + NaCl was flowing. TTL signals generated by an Arduino

Uno board and dedicated scripts were used to control precisely the switching of the valve. The fluorescein present in the SD-full medium quantified outside the Cell object provided an

estimate of the NaCl concentration in the medium. Some strong fluctuations in this signal were probably generated by dust particles in the imaging oil or FLSN-dextran aggregates in the flow

chamber. Following 24 h log-phase growth, cells bearing the p_STL1_-PP7sl reporter, Hog1-mCherry, and Hta2-tdiRFP tags were diluted to OD 0.2, briefly sonicated and loaded in the ibidi

channel previously coated by Concanavalin A. Cells were left to settle in the channel for 10 min before SD-full flow was started. RAFFINOSE EXPERIMENT For the experiments comparing

p_STL1_-PP7sl induction in glucose versus raffinose, cells were grown overnight to saturation in SD-full medium. The cultures were diluted to OD 0.025 (Glucose) or 0.05 (Raffinose) and grown

at 30 °C for at least four hours. In the raffinose medium, the expression level of the PP7-GFP was twofold lower than in glucose. Because of this low fluorescence intensity, cells were

imaged with a 40X objective, and a single Z-plane was acquired. Manual curation of the images was performed to define the Start Time in more than 250 cells. This experiment was performed in

duplicate. DATA ANALYSIS Time-lapse movies were analyzed in an automated way: cell segmentation, tracking, and feature measurements were performed by the YeastQuant platform33 based on

Matlab. Summary of the dataset, strains, and cell numbers are provided in Supplementary Table 4. All PP7 experiments were realized in at least two or three fully independent replicate

experiments. A representative experiment was selected for each strain and inducing conditions, based on cell size and cell adaptation dynamics. The replicates which did not pass one of these

controls were discarded from the replicate analyses. Individual single-cell traces were filtered based on cell shape and GFP intensity to remove segmentation errors or other experimental

artifacts. In addition, cells in mitosis were removed from the analysis with a 0.95 filter on the nuclei eccentricity, to remove artifacts from locus and PP7 signal duplication. The Hta2

signal combined with the two bright field images allowed to define the nucleus and cell borders. The GFP z-stacks were converted by a maximum intensity projection in a single image that was

used for quantification. In order to avoid improper quantification of transcription sites at the nuclear periphery, the Nucleus object defined by the histone fluorescence was expanded by 5

pixels within the Cell object to define the ExpNucl object. Further analysis was performed by dedicated Matlab scripts. The transcription site intensity was quantified by the difference

between the mean intensity of the 20 brightest pixels (HiPix) in the ExpNucl and the median intensity from the whole cell. This provides a continuous trace that is close to zero in absence

of TS and increases by up to few hundred counts when a TS is present. To identify the presence of a transcription site, a second feature named ConnectedHiPix was used (Supplementary Fig. 

2a). Starting from the 20 HiPix, a morphological opening of the image was performed to remove isolated pixels and retaining only the ones that clustered together which correspond to the

transcription site. The ConnectedHiPix value was set to the mean intensity of the pixel present in the largest object remaining after the morphological operation. If no pixel remained after

the morphological operation, the ConnectedHiPix was set to “NaN”. In each single-cell trace, ConnectedHiPix values only detected for a single time point were removed. After this filtering,

the first and last time points where a ConnectedHiPix was measured were defined as transcription initiation (Start Time) and shutoff (End Time), respectively. Manual curation of Start and

End Times from raw microscopy images was performed to validate this transcription site detection strategy (Supplementary Fig. 2b, c). In order to detect individual transcriptional bursts in

the HiPix traces, the _findpeak_ algorithm was used to identify in the trace all the peaks larger than a threshold of seven counts within the Start and End Times. Following this first

process, a set of conditions were defined to retain only the more reliable fluctuations: the drop following the peak has to be larger the fourth of the peak intensity; the intensity of the

following peak has to rise by more than a third of the value at the trough. In addition, the value of the peak has to be at least one-fifth of the maximum intensity of the trace in order to

remove small intensity fluctuations being considered as peaks. REPORTING SUMMARY Further information on research design is available in the Nature Research Reporting Summary linked to this

article. DATA AVAILABILITY The raw images and additional features measurements that support the findings of this study are available from the corresponding author upon reasonable request.

All other relevant data supporting the key findings of this study are available within the article and its Supplementary Information files or from the corresponding author upon reasonable

request. The source data for Figs. 1d, 2a, f, g, 3a, 4b, c, and 6c and Supplementary Figs. 1, 4–6, 8, 12, and 13 are provided as Source Data file. A reporting summary for this article is

available as a Supplementary Information file. CODE AVAILABILITY A general description of the image analysis platform has been published previously33. A more recent version of the code has

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Cell Experiments_. (ETH Zurich, 2014). https://doi.org/10.3929/ethz-a-010350761. Download references ACKNOWLEDGEMENTS We thank the members of the Pelet lab and Martin lab and for helpful

discussions. Marta Schmitt, Yves Dusserre, Gaëlle Spack, Joan Jordan, and Clémence Varidel for technical assistance. David Shore and his lab for helpful discussions and reagents.

Marie-Pierre Peli-Gulli and Claudio de Virgilio for plasmids, Tineke Lenstra for suggesting the PP7ΔFG allele. Agathe Pelet for manual curation of microscopy images. Eulalia de Nadal,

Mariona Nadal-Ribelles, and Veneta Gerganova for critically reading the manuscript. Work in the Pelet lab is supported by SystemsX.ch (IPhD 51PHP0_157354), the Swiss National Science

Foundation (SNSF, PP00P3_172900 and 31003A_182431), and the University of Lausanne. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Fundamental Microbiology, University of

Lausanne, 1015, Lausanne, Switzerland Victoria Wosika & Serge Pelet Authors * Victoria Wosika View author publications You can also search for this author inPubMed Google Scholar * Serge

Pelet View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS VW and SP designed the experiments, analyzed the data, and wrote the manuscript. VW

established the conditions for the PP7 imaging. VW and SP performed the experiments. CORRESPONDING AUTHOR Correspondence to Serge Pelet. ETHICS DECLARATIONS COMPETING INTERESTS The authors

declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature Communications_ thanks Gustav Ammerer, Stefan Hohmann and the other, anonymous, reviewer(s) for their

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Single-particle imaging of stress-promoters induction reveals the interplay between MAPK signaling, chromatin and transcription factors. _Nat Commun_ 11, 3171 (2020).

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