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Stochastic signalling rewires the interaction map of a multiple feedback network during yeast evolution.

Hsu C, Scherrer S, Buetti-Dinh A, Ratna P, Pizzolato J, Jaquet V, Becskei A - Nat Commun (2012)

Bottom Line: The wiring strength in these feedback loops is set by the number of Gal4p binding sites.In this way, rapid adaptation to galactose can be triggered even by weakly expressed genes.Our results indicate that nonlinear stochastic transcriptional responses enable feedback loops to function autonomously, or contrary to what is dictated by the strength of interactions enclosing the circuit.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, Klingelbergstrasse 50/70, Basel 4056, Switzerland.

ABSTRACT
During evolution, genetic networks are rewired through strengthening or weakening their interactions to develop new regulatory schemes. In the galactose network, the GAL1/GAL3 paralogues and the GAL2 gene enhance their own expression mediated by the Gal4p transcriptional activator. The wiring strength in these feedback loops is set by the number of Gal4p binding sites. Here we show using synthetic circuits that multiplying the binding sites increases the expression of a gene under the direct control of an activator, but this enhancement is not fed back in the circuit. The feedback loops are rather activated by genes that have frequent stochastic bursts and fast RNA decay rates. In this way, rapid adaptation to galactose can be triggered even by weakly expressed genes. Our results indicate that nonlinear stochastic transcriptional responses enable feedback loops to function autonomously, or contrary to what is dictated by the strength of interactions enclosing the circuit.

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Retroactive effect of the GAL3 promoter in the presence of glucose.(a) The cells containing a one-copy PGAL3–rtTA direct regulatory circuit were grown in raffinose medium (green) or in raffinose medium with 0.32% glucose (red) for both the overnight preculturing and the subsequent 6-h growth period, following which RNA was isolated. Error bars indicate standard deviation, n=3. (b) Cells containing either the direct regulatory circuit PGAL3–rtTA (left panel) or the feedback circuit with P[tetO]3-in-GAL1–rtTA activated by PGAL3–rtTA (right panel) were grown overnight in raffinose medium with 0.32% glucose. Subsequently, the cells were induced by 5 μM doxycycline for 6 h in the same medium (dark red dashed lines) and GFP fluorescence was measured. The black lines denote the cellular autofluorescence. The kurtosis for the background distribution is 0.56 and for the direct regulatory circuit is −0.07.
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f6: Retroactive effect of the GAL3 promoter in the presence of glucose.(a) The cells containing a one-copy PGAL3–rtTA direct regulatory circuit were grown in raffinose medium (green) or in raffinose medium with 0.32% glucose (red) for both the overnight preculturing and the subsequent 6-h growth period, following which RNA was isolated. Error bars indicate standard deviation, n=3. (b) Cells containing either the direct regulatory circuit PGAL3–rtTA (left panel) or the feedback circuit with P[tetO]3-in-GAL1–rtTA activated by PGAL3–rtTA (right panel) were grown overnight in raffinose medium with 0.32% glucose. Subsequently, the cells were induced by 5 μM doxycycline for 6 h in the same medium (dark red dashed lines) and GFP fluorescence was measured. The black lines denote the cellular autofluorescence. The kurtosis for the background distribution is 0.56 and for the direct regulatory circuit is −0.07.

Mentions: Next, we studied the deactivation of the GAL network by glucose (Fig. 5b–h)31. The transcriptional inhibition of the GAL regulon by glucose is mediated by two proteins: Gal80p and the Mig1p repressor. As Mig1p represses GAL4, both these pathways converge on Gal4p (refs 32 and 33). Thus, the Gal4p-independent effects of glucose on GAL genes can be discerned by measuring their expression in Δgal80 cells, in which the Mig1p sites in PGAL4 are also mutated (PGAL4-with-MutatedMig1pBS–GAL4). Among the GAL genes, it was only GAL3 whose expression was significantly repressed by glucose in these cells (Fig. 1b). This suggests that repression of GAL3 by glucose is used to adjust the activity of the GAL signalling pathway. Interestingly, the percentage of ON cells in glucose containing media was higher for wild-type cells than for PGAL1–GAL3 cells, even though both of them drove similar GAL3 expression levels (Supplementary Fig. S3). The differential activation of the GAL3 loop in these cells may be due to the different bursting kinetics of the GAL1 and GAL3 promoters. Indeed, the amplification of GAL3 basal bursts in the direct regulatory circuit results in a distribution similar to that of GAL2 (Fig. 6). Thus, this bursting is likely to enable the GAL3 promoter to generate ON cells even when repressed by intermediate glucose concentrations.


Stochastic signalling rewires the interaction map of a multiple feedback network during yeast evolution.

Hsu C, Scherrer S, Buetti-Dinh A, Ratna P, Pizzolato J, Jaquet V, Becskei A - Nat Commun (2012)

Retroactive effect of the GAL3 promoter in the presence of glucose.(a) The cells containing a one-copy PGAL3–rtTA direct regulatory circuit were grown in raffinose medium (green) or in raffinose medium with 0.32% glucose (red) for both the overnight preculturing and the subsequent 6-h growth period, following which RNA was isolated. Error bars indicate standard deviation, n=3. (b) Cells containing either the direct regulatory circuit PGAL3–rtTA (left panel) or the feedback circuit with P[tetO]3-in-GAL1–rtTA activated by PGAL3–rtTA (right panel) were grown overnight in raffinose medium with 0.32% glucose. Subsequently, the cells were induced by 5 μM doxycycline for 6 h in the same medium (dark red dashed lines) and GFP fluorescence was measured. The black lines denote the cellular autofluorescence. The kurtosis for the background distribution is 0.56 and for the direct regulatory circuit is −0.07.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3293423&req=5

f6: Retroactive effect of the GAL3 promoter in the presence of glucose.(a) The cells containing a one-copy PGAL3–rtTA direct regulatory circuit were grown in raffinose medium (green) or in raffinose medium with 0.32% glucose (red) for both the overnight preculturing and the subsequent 6-h growth period, following which RNA was isolated. Error bars indicate standard deviation, n=3. (b) Cells containing either the direct regulatory circuit PGAL3–rtTA (left panel) or the feedback circuit with P[tetO]3-in-GAL1–rtTA activated by PGAL3–rtTA (right panel) were grown overnight in raffinose medium with 0.32% glucose. Subsequently, the cells were induced by 5 μM doxycycline for 6 h in the same medium (dark red dashed lines) and GFP fluorescence was measured. The black lines denote the cellular autofluorescence. The kurtosis for the background distribution is 0.56 and for the direct regulatory circuit is −0.07.
Mentions: Next, we studied the deactivation of the GAL network by glucose (Fig. 5b–h)31. The transcriptional inhibition of the GAL regulon by glucose is mediated by two proteins: Gal80p and the Mig1p repressor. As Mig1p represses GAL4, both these pathways converge on Gal4p (refs 32 and 33). Thus, the Gal4p-independent effects of glucose on GAL genes can be discerned by measuring their expression in Δgal80 cells, in which the Mig1p sites in PGAL4 are also mutated (PGAL4-with-MutatedMig1pBS–GAL4). Among the GAL genes, it was only GAL3 whose expression was significantly repressed by glucose in these cells (Fig. 1b). This suggests that repression of GAL3 by glucose is used to adjust the activity of the GAL signalling pathway. Interestingly, the percentage of ON cells in glucose containing media was higher for wild-type cells than for PGAL1–GAL3 cells, even though both of them drove similar GAL3 expression levels (Supplementary Fig. S3). The differential activation of the GAL3 loop in these cells may be due to the different bursting kinetics of the GAL1 and GAL3 promoters. Indeed, the amplification of GAL3 basal bursts in the direct regulatory circuit results in a distribution similar to that of GAL2 (Fig. 6). Thus, this bursting is likely to enable the GAL3 promoter to generate ON cells even when repressed by intermediate glucose concentrations.

Bottom Line: The wiring strength in these feedback loops is set by the number of Gal4p binding sites.In this way, rapid adaptation to galactose can be triggered even by weakly expressed genes.Our results indicate that nonlinear stochastic transcriptional responses enable feedback loops to function autonomously, or contrary to what is dictated by the strength of interactions enclosing the circuit.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, Klingelbergstrasse 50/70, Basel 4056, Switzerland.

ABSTRACT
During evolution, genetic networks are rewired through strengthening or weakening their interactions to develop new regulatory schemes. In the galactose network, the GAL1/GAL3 paralogues and the GAL2 gene enhance their own expression mediated by the Gal4p transcriptional activator. The wiring strength in these feedback loops is set by the number of Gal4p binding sites. Here we show using synthetic circuits that multiplying the binding sites increases the expression of a gene under the direct control of an activator, but this enhancement is not fed back in the circuit. The feedback loops are rather activated by genes that have frequent stochastic bursts and fast RNA decay rates. In this way, rapid adaptation to galactose can be triggered even by weakly expressed genes. Our results indicate that nonlinear stochastic transcriptional responses enable feedback loops to function autonomously, or contrary to what is dictated by the strength of interactions enclosing the circuit.

Show MeSH
Related in: MedlinePlus