Timing of gene expression in a cell‐fate decision system

Abstract During development, morphogens provide extracellular cues allowing cells to select a specific fate by inducing complex transcriptional programs. The mating pathway in budding yeast offers simplified settings to understand this process. Pheromone secreted by the mating partner triggers the activity of a MAPK pathway, which results in the expression of hundreds of genes. Using a dynamic expression reporter, we quantified the kinetics of gene expression in single cells upon exogenous pheromone stimulation and in the physiological context of mating. In both conditions, we observed striking differences in the timing of induction of mating‐responsive promoters. Biochemical analyses and generation of synthetic promoter variants demonstrated how the interplay between transcription factor binding and nucleosomes contributes to determine the kinetics of transcription in a simplified cell‐fate decision system.

NLSs (cyan) fused to the second SynZip (dark blue), driven by the promoter of interest.
dPSTR complex formed by interaction between the two SynZips, causing nuclear accumulation of the fluorescent protein.

B
OFF ON SKARS module formed of a docking site (dark blue), an NLS (cyan), and a fluorescent protein (green).
Specific interaction with the active MAPK (light green) will lead to the inactivation of the NLS through phosphorylation. Principle of the relocation sensors used for the study.
A. Principle of the Synthetic Kinase Activity Relocation Sensor (SKARS, Durandau et al., BMC Biology, 2015). The SKARS is formed by a docking site that can specifically interact with a kinase of interest, a phosphorylatable NLS, and a fluorescent protein for visualization. When the kinase of interest is inactive, the NLS is functional and the fluorescent signal is nuclear. Phosphorylation of the NLS by the active kinase will lower its efficiency, inducing the relocation of the fluorescent signal throughout the cell.
B. Schematic representation of the dynamic Protein Synthesis Translocation Reporter (dPSTR, Aymoz et al., Nat Commun, 2016). The dPSTR converts protein expressoin arising from a promoter of interest into a relocation of a constitutive fluorescent signal from the cytoplasm into the nucleus of cells. It is based on two transcriptional units carried on a sole uniquely integrated vector. The fluorescent protein in constitutively expressed and present all over the cell in the dPSTR OFF state. A second peptide driven by the promoter of interest carries two Nuclear Localization Signals (NLS), allowing the nuclear recruitment of any protein. The physical interaction between the fluorescent protein and the induced peptide is taking place through the interaction of small synthetic peptides called SynZips, providing strong and specific interaction. The whole complex is then recruited in the nucleus, changing the fluorescent signal arising from the cytoplasm and the nucleus.

Metrics calculation
A. Population average nuclear enrichment of the pAGA1-dPSTR Y in course of time. Nuclear enrichment is the difference between nuclear and cytoplasmic fluorescence. All cells of the sample that passed quality control are taken in account here (low variability on nuclear and cell area and in nuclear CFP fluorescence Appendix Figure S5 Consistent differential kinetics between promoters after inverting the dPSTRs FP and SynZip pairs A. Histograms of the response time of pFIG1-dPSTR Y and pAGA1-dPSTR R in the same strain (Total number of cells: 193. Number of expressing cells considered in this histograms: 157 for pFIG1-dPSTR Y and 189 for pAGA1-dPSTR R ). Note the similarity with Figure 1F.
B. Instant correlation of nuclear enrichment of the two dPSTRs at time 0, 10, 25 and 60min after stimulation by pheromone. Note that pAGA1-dPSTR R is induced before pFIG1-dPSTR Y .
C. Correlation of the expression output of the two dPSTRs for all the cells of the experiment.   Figure S6 Temporal evolution of noise in pFIG1 and pAGA1 induction A. and B. Nuclear enrichment of pFIG1-dPSTR Y and pFIG1-dPSTR R (A) pAGA1-dPSTR Y and pAGA1-dPSTR R (B) as function of time. dPSTR Y is plotted in yellow, right axis, and dPSTR R in red, left axis.
C. and D. Correlation of the normalized dPSTR nuclear enrichment from all single cells at diferent time points after stimulation in strains carrying two dPSTRs measuring either pFIG1 (C) or pAGA1 (D).

E.
Intrinsic noise as percent of total noise calculated for pFIG1 and pAGA1 at all time points of the time-lapse movie, using the formula from Elowitz et al. 2002 (see Methods). The error bars represent the standard deviations calculated from 3 biological replicates.   A to D. Histograms of the basal nuclear enrichment of the dPSTR R are plotted for all cells from the representative experiments chosen for each promoter of the study. The orange dotted line on all plots is the nuclear enrichment observed in a strain carrying only the constitutive part of the dPSTR R (mCherry-SZ2), without the second half of the construct (MCS2, where the inducible promoter is). From this strain, the threshold for basal enrichment, due to the fluorescent protein itself, was determined (black doted line, N-C=350, N C =2888), with 5% of false positive cells. Promoters were classified in 4 categories: no basal level (A), low basal level (B), mid basal level (C) and high basal level (D). The percentages in brackets represent the proportion of cells from the entire population of the experiment that express the promoters in vegetative growth (expressing cells, EC). Colors of promoters in the legends indicate the promoter class: early: red; intermediate: green; late: blue.

E.
Correlation between the mean basal expression level and the median response time for all promoters in one representative experiment. The lines represent the 25th-75th percentiles. Note that there is no correlation between the basal expression level and the response time.  Appendix Figure S9 Dose response various mating-induced promoters.
A. Mean expression output for the indicated promoters in response to different pheromone concentrations. The expression output is defined as the maximal dPSTR nuclear enrichment following stimulation, for all cells of the experiment. Error bars represent the standard deviation of 3 replicates.

B.
Percentage of cells expressing the indicated promoter, according to various pheromone concentrations. The error bars represent the standard deviation of three experiments.
Correlation of normalized dPSTRs nuclear enrichments from all single cells at different time points after stimulation in the strains used in Figure 2A. Promoters in red were categorized as early, in green as intermediate, and in blue as late induced.
All Ste12 binding sites were mapped on the 14 promoters of the study, which are defined as nucleotides from the stop codon of the upstream ORF to the ATG of the gene of interest. Consensus PREs, meaning nTGAAACn, are represented here by red arrows indicating their positions and orientations, with their corresponding sequences above (green lower cases are mutations from the full consensus ATGAAACA  Appendix Figure S13 Mutants of the Group I induce pFIG1 and pAGA1 as the WT. A and B. Nuclear enrichment of the pAGA1-dPSTR Y (A) and pFIG1-dPSTR R (B) after stimulation by 1µM of pheromone in the indicated mutant. Lines represent the median of either the mutant (red) or the WT strain (black) for one representative experiment, with the shaded area representing the 25th-75th percentile.
C. Correlation of the expression output (maximal dPSTR nuclear enrichment following stimulation) of pAGA1-dPSTR Y and pFIG1-dPSTR R for all single cells of the experiment, for the indicated strain. Dotted lines represent the threshold of expression (defined as the 20% of the WT mean expression output for each dPSTR). The Venn diagram represents the proportion of cells expressing pAGA1 (red circle) or pFIG1 (blue circle) or none of them (black circle). 1∆ ∆ Appendix Figure S14 Mutants of the Group II have an impaired induction of both pFIG1 and pAGA1 in a similar manner.
A and B. Nuclear enrichment of the pAGA1-dPSTR Y (A) and pFIG1-dPSTR R (B) after stimulation by 1µM of pheromone in the indicated mutant. Lines represent the median of either the mutant (red) or the WT strain (black) for one representative experiment, with the shaded area representing the 25th-75th percentile.
C. Correlation of the expression output (maximal dPSTR nuclear enrichment following stimulation) of pAGA1-dPSTR Y and pFIG1-dPSTR R for all single cells of the experiment, for the indicated strain. Dotted lines represent the threshold of expression (defined as the 20% of the WT mean expression output for each dPSTR). The Venn diagram represents the proportion of cells expressing pAGA1 (red circle) or pFIG1 (blue circle) or none of them (black circle). Appendix Figure S15 Biochemistry experiments A and B. Northern blot detection of mRNAs from AGA1 and FIG1 in the reference strain used with the dPSTR (ySP643, left) or the strain carring the tags for biochemistry experiments (yCS418, right). lot is shown in A, and quantifications normalized on the maximal level of each promoter are shown in . This is the same image as the one presented in Figure 1 .

C and D.
MNase protection assay performed on the endogenous AGA1 (C) and FIG1 ( ) loci. The intensity of the -1 nucleosome (insert) is plotted in Figure 3C and . E. asal occupancy of Ste12 and ar4 on the A A1 and F 1 promoter relative to a no tag strain. The bars represents the mean of three replicates, and the error bars the standard deviations. Note that ar4 enrichment at the promoters is dependent on the presence of Ste12 (Student s t-test p al 6.10 -7 for pAGA1 and p al 3. .10 -3 for pFIG1).
F. estern blot quantifying ar4 amount in the strain carrying the two tags or in a 1 ∆ background.  F and G. Nuclear enrichment of the dPSTR R for the indicated constructs (top legend) in a WT background (F) or a ar ∆ background (G), from the same experiments used to plot Figure 3I. Constructs from top to bottom: pAGA1; pFIG1; chimera pFIG1 with the last 150bp from pAGA1 replacing those from pFIG1; pFIG1 with mutation of a non-consensus binding site into a 4th consensus site, chimera pFIG1 with 4 consensus binding sites and the last 150bp from pAGA1.  Microscopy images of a mating mixture containing the MATa strain (Hta2-CFP, pFIG1-dPSTR R and pAGA1-dPSTR Y ) and a MATα (cytoplasmic tdiRFP) recorded every 5 minutes. The MATa cells were segmented based on the Hta2-CFP tag and two bright field images to identify the nucleus (white circle in the tdiRFP images) and the cell boundaries (black contour in BF image and white contour in YFP and RFP images). Fusion events are detected by a sudden increase in tdiRFP fluorescence in the nucleus (magenta trace in the left graphs) and define the reference time for the synchronization of the single cell traces. The corresponding dPSTR nuclear enrichments of pFIG1-dPSTR R (red) and pAGA1-dPSTR Y (green) for these four fusion events are also shown.     D to F. Nuclear enrichment of pAGA1-dPSTR Y quantified in the same cells.
G. Cumulative probability of the response time of the pAGA1-dPSTR Y relative to fusion.
H to J. Correlation of the expression output of the dPSTR R relative to the pAGA1-dPSTR Y , one hour after the start of the imaging, in cells that do not undergo fusion. Note the lack of expression of the late promoters even in cells inducing strongly the pAGA1 promoter.    Tables  Appendix Table S1: List of yeast strains used in this study