Fus3-triggered Tec1 degradation modulates mating transcriptional output during the pheromone response

The yeast transcription factor Ste12 controls both mating and filamentation pathways. Upon pheromone induction, the mitogen-activated protein kinases, Fus3 and Kss1, activate Ste12 by relieving the repression of two functionally redundant Ste12 inhibitors, Dig1 and Dig2. Mating genes are controlled by the Ste12/Dig1/Dig2 complex through Ste12-binding sites, whereas filamentation genes are regulated by the Tec1/Ste12/Dig1 complex through Tec1-binding sites. The two Ste12 complexes are mutually exclusive. During pheromone response, Tec1 is degraded upon phosphorylation by Fus3, preventing cross-activation of the filamentation pathway. Here, we show that a stable Tec1 also impairs the induction of mating genes. A mathematical model is developed to capture the dynamic formation of the two Ste12 complexes and their interactions with pathway-specific promoters. By model simulations and experimentation, we show that excess Tec1 can impair the mating transcriptional output because of its ability to sequester Ste12, and because of a novel function of Dig2 for the transcription of mating genes. We suggest that Fus3-triggered Tec1 degradation is an important part of the transcriptional induction of mating genes during the pheromone response.


Model and equations
The model aims to study emergent properties arising from interactions and regulations among several transcription factors in the yeast mating pathway. The system consists of Tec1, Ste12, Dig1, Dig2, PRE, TCS, and their reaction products. Dig1 in the system is not explicitly modeled, and only the complex Ste12/Dig1 is included in the system. The system is activated by pheromone through active Fus3 and Kss1 that in turn inhibit Dig1 and Dig2 (Cook, 1996;Tedford, 1997), and positively regulate Tec1 degradations (Chou, 2004). And the TEC1 expression is also positively regulated by the PRE signal (Oehlen, 1998). For simplicity, the active Fus3 and Kss1 are assumed to be proportional to the pheromone level (Sabbagh, 2001) (posters and keynote presentation by Roger Brent, et al. at ICSB07). The biochemical reactions along with the rate constants are presented in Figure S1. The corresponding mass action equations take the following form:    The input active Fus3 (U) and active Kss1 (V) are chosen as two given functions in terms of time: where the variable a is the strength of a-factor (pheromone) with a unit nM. The expressions of Fus3 and Kss1 mimic experimentally observed temporal dynamics for Fus3 and Kss1's response to pheromone (Sabbagh, 2001). It is also consistent with previous simulations on the dynamics of active Fus3 (Wang, 2006). The level of active Fus3 and Kss1 are assumed to be proportional to the level of pheromone based on previous experimental observation (Sabbagh, 2001) (posters and keynote presentation by Roger Brent, et. al at ICSB0). The two functions are plotted in Figure S2.
The degradation of Tec1 is positively regulated by Fus3 (Bao, 2004;Chou, 2004). Tec1 degradation rates are modeled in terms of the Hill function of Fus3: As observed in the experiment, there exists a positive feedback on Tec1 expression (Bao, 2004) from the mating signal. So we also assume Tec1 is positively regulated by the mating signal.

Temporal dynamics of all complexes
The parameters of the systems in Figure 1 are chosen based on a biochemical analysis of various complexes in (Chou, 2006). The initial concentrations used in Fig. 1 of the paper are listed in Table 1, and the reaction rates and feedback parameters for Fig. 1 are listed in Table 2. Figure S2A plots the concentration of temporal dynamics of each complex in the system for the wild-type case shown in Fig. 1B of the paper. Figure S2B plots the total level of Dig2 and Ste12, Tec1, and PRE and TCS outputs. The total Dig2 and total Ste12 levels are constant in time, behaving like the experiment. The nonlinear response of total Tec1 is consistent with the experimental observation, and the specificity of PRE output agrees well with the experiments (Bao, 2004;Chou, 2004). Figure S3 shows how the level of degradation of Tec1 affects TCS outputs. We varied the mean strength of Tec1 degradation in the model. As expected, Tec1 degradation strength dictates the level of Tec1, which in turn controls the TCS-lacZ level. For the case of zero degradation of Tec1 in the model, which correlates to the stable Tec1 T273V , the Tec1 level monotonically increases from the start while the fold change of TCS-lacZ always increases in response to pheromone. On the other extreme, when Tec1 degradation is large, the TCS-lacZ level decreases after pheromone induction. In both cases, the simulated results are in agreement with the experimental data (Chou, 2004) (Bao, 2004).

Strategy one --Ste12/Dig1/Dig2 is more effective in activating mating gene transcription than Tec1/Ste12/Dig1
The ranges of randomly chosen parameters in Fig. 2 of the paper are listed in Table 2. Figure S4A shows that the qualitative pattern of PRE outputs under various mutations presented in Fig. 1C remains unchanged as long as w d is larger than v d. Figure S4B demonstrates that the strategy remains valid for higher levels of induced pheromone. Figure S4C shows that at this pheromone level, when w d and v d are changed to be the same, the system behaves similar to that at the low pheromone level. Figure S4D is the experimental result on the dig2 deletion at different levels of pheromone to show that the dig2 effect is independent of pheromone levels.
Strategy two --Tec1 can sequester Ste12 from forming the Ste12/Dig1/Dig2 complex to reduce the mating transcriptional output when the amount of Ste12 is limiting The parameters for Figure 1D are listed in Table 3. Figure S5 shows that the qualitative pattern on the PRE outputs under the conditions of stable Tec1 and tec1 deletion remain the same when the system is induced by different levels of pheromone. In Figure S6, we set the overall activation rate of Fus3 on Ste12 in Tec1/Ste12/Dig1 to be the same as the rate for Ste12 in Ste12/Dig1/Dig2 as in the case in Figure 3. A consistent pattern between panel (A) and panel (B) in Figure S6 suggests that 50 sample points are sufficient to identify the correlation. In both panels (A) and (B), there are few samples satisfying the condition in which both ratio of PRE output for the tec1 deletion over PRE output for WT (R 1 ) and ratio of PRE output for WT over PRE output for stable Tec1 (R 2 ) are greater than 1. As the initial amount of Ste12 decreases, there are more sample points satisfying the condition which resembles the experimental result shown in Figure 1C. Figure S7 is an example in which more sample points were added around the case which agrees well with the experimental result. The binding affinities are defined as Figure S8, we systematically varied the initial amount of Ste12 to study how it affects strategy two. In Figure S9, we systematically test the condition under which the correlation on affinities for strategy two remains true. Both figures show strategy two is a robust strategy as long as Ste12 is sequestered. In Figure S10, we find that, under the same conditions, the lower the initial amounts of PREs and TCSs were, the fewer the red dots were, however, with a clearer relationship between the two affinities: and . In Figure S11, as the ratio of w Dig2|PRE K Tec1|PRE K d over v d increases from 0.5 to 4, the output level of the PRE in wild type stain increases while PRE signals of other cases hardly change. Consequently, the difference in PRE level between the tec1 deletion and the wild type becomes smaller. Table 1. The initial concentrations used in Figure 1.

Figures and Tables --Supplement
name value unit Range of the random selection k on 10 -2 μM -1 sec -1 0.0010~0.1000 k off 10 -6 sec -1 l on 5x10 -3 μM -1 sec -1 0.0005~0.0500 l off 10 -6 sec -1 v on 5x10 -3 μM -1 sec -1 0.0005~0.0500 v off 10 -6 sec -1 w on 5x10 -3 μM -1 sec -1 0.0005~0.0500 w off 10 -6 sec -1 z on 5x10 -3 μM -1 sec -1 0.0005~0.0500  Table 2. Rate constants for Figure 1 and the range for randomly selected parameters in Figure 2 and Figure 3. Other parameters are the same as in Table 1 and Table 2. The other parameters are the same as in Table 1 and Table 2 Table 4. The range of randomly selected parameters used in Figure S7B.   Figure S3. TCS outputs at different Tec1 degradation rates. For each case, the top panel shows the total Tec1 level as a function of time, while the bottom panel shows the corresponding fold change of the TCS output. "xDeg" represents a case in which the mean value of Tec1 degradation is "x" fold of the Tec1 degradation strength used in Figure S2 with all other parameters being same as in Figure S2.  Figure S4 The effect of w d and the level of induced pheromone using on strategy one. A Red dot represents a case in which the ratio of PRE output for tec1 deletion over PRE output for WT at 120 min is larger than one and the ratio of PRE output for WT over PRE output for stable TEC1 at 120 min is larger than 1.1; a green dot represents a case not satisfying the above condition. (The definitions of red dots and green dots remain the same throughout the rest of the Supplement unless specified otherwise.) (A) The parameters and conditions are the same as Figure 2A Table  2; (B) After one set of parameter (see Table 4) in (A) is selected for the pattern resembling the experimental observation, another 100 sample points are randomly selected centering on this set of parameters.     Figure S9. The correlation between affinity rates using different criteria defining the red dots. All other conditions are the same as in Figure 3. A red dot represents a case in which the ratio of PRE output for the tec1 deletion over PRE output for WT at 120 min is larger than one and the ratio of PRE output for WT over PRE output for stable TEC1 at 120 min is larger than ( Figure S10. The correlation between affinity rates, with different initial amounts of PRE and TCSs. (A) The initial amounts of PREs and TCSs are given in the Table 1 but the initial Ste12 amount is 0.005 μM; 50 sample points; 200 nM induced pheromone; the same Fus3 activation rates; (B) the initial amount of PRE and TCS is 10% of (A) with other conditions being the same; (C) the initial amount of PRE and TCS is 1% of (A) with other conditions being the same. The other parameters used for these cases are the same as in Figure 1D.