Control of transcriptional variability by overlapping feed-forward regulatory motifs

Abstract

In yeast, beta-oxidation of fatty acids (FAs) takes place in the peroxisome, an organelle whose size and number are controlled in response to environmental cues. The expression of genes required for peroxisome assembly and function is controlled by a transcriptional regulatory network that is induced by FAs such as oleate. The core FA-responsive transcriptional network consists of carbon source-sensing transcription factors that regulate key target genes through an overlapping feed-forward network motif (OFFNM). However, a systems-level understanding of the function of this network architecture in regulating dynamic FA-induced gene expression is lacking. The specific role of the OFFNM in regulating the dynamic and cell-population transcriptional response to oleate was investigated using a kinetic model comprised of four core transcription factor genes (ADR1, OAF1, PIP2, and OAF3) and two reporter genes (CTA1 and POT1) that are indicative of peroxisome induction. Simulations of the model suggest that 1), the intrinsic Adr1p-driven feed-forward loop reduces the steady-state expression variability of target genes; 2), the parallel Oaf3p-driven inhibitory feed-forward loop modulates the dynamic response of target genes to a transiently varying oleate concentration; and 3), heterodimerization of Oaf1p and Pip2p does not appear to have a noise-reducing function in the context of oleate-dependent expression of target genes. The OFFNM is highly overrepresented in the yeast regulome, suggesting that the specific functions described for the OFFNM, or other properties of this motif, provide a selective advantage.

FIGURE 1

The yeast oleate-responsive transcriptional network contains an OFFNM. Lines terminating in open arrows and blunted lines represent transcriptional up- and down-regulation, respectively. Lines terminating in solid arrows indicate molecular processes such as transport, transcription/translation, and dimerization. Dotted black arrows indicate indirect carbon-source-dependent activation. Red and blue arrows and blunted lines represent the Adr1p- and Oaf3p-driven coherent feed-forward motifs, respectively. The alternating red/blue dashed line represents the overlapping region. The inset panel demonstrates schematically the OFFNM. Intracellular FA (oleate) binds Oaf1p, activating the TF. Active Oaf1p forms a heterodimer with Pip2p, and this heterodimer targets the ORE on DNA as a transcriptional activator. The promoter of the gene PIP2 contains an ORE, and thus PIP2 is transcriptionally autoregulated in the presence of oleate. Adr1p is rapidly activated in the presence of nonfermentable carbon sources and targets UAS1 elements in the promoters of the target genes PIP2 and CTA1. Adr1p therefore drives a coherent feed-forward network motif targeting PIP2 and CTA1 (thick red lines). Oaf3p is a transcriptional inhibitor whose target footprint (in terms of number of genes) is strongly increased under oleate growth conditions (17). It drives a coherent inhibitory feed-forward network motif targeting PIP2 and CTA1 (thick blue lines).

FIGURE 2

The model recapitulates the measured relative dose response for POT1 expression. Data points indicate the activity of a luciferase reporter gene with the POT1 promoter in yeast cells grown overnight in media with oleate at the indicated initial concentration (13). The predicted POT1 expression levels from the model (line plot) have been normalized relative to the luciferase activity in 8 mM oleate.

FIGURE 3

An in silico model of a mutant strain in which CTA1 is solely ORE-activated (AOPY Mutant Model I) is predicted to have greater variability of CTA1 expression than the WT model. The histogram shows the simulated population heterogeneity of reporter expression (CTA1 mRNA level) in WT (black bars) and mutant strain (in which Adr1p does not regulate CTA1) (white bars) in oleate growth conditions. The abscissa is the CTA1 mRNA concentration after 100 min of stochastic simulation with initial conditions given by species concentrations obtained from the steady-state solution to the ODE kinetic model with constant 0.12% (w/v) oleate. Stochastic simulations were carried out for an ensemble of 1000 realizations of the stochastic process. CV_mut represents the steady-state CV of reporter expression levels for the AOPY Mutant Model I, and CV_WT represents the CV in the WT model.

FIGURE 4

An in silico model of a mutant strain in which both PIP2 and CTA1 are solely ORE-activated (AOPY Mutant Model II, corresponding to an adr1Δ strain with the ability to fully induce ORE-driven expression) is predicted to have greater variability of CTA1, but not PIP2 expression, than the WT model. The histograms show the simulated population heterogeneity of (A) CTA1 and (B) PIP2 mRNA levels in WT (black bars) and a mutant adr1Δ strain in which PIP2 and CTA1 have increased ORE-driven transcriptional activity (white bars) in oleate growth conditions. The abscissas represent the distribution of the (A) CTA1 and (B) PIP2 mRNA concentrations after 100 min of stochastic simulation with initial conditions given by the steady-state solution to the ODE kinetic model with constant 0.12% oleate. Stochastic simulations were carried out for an ensemble of 1000 realizations of the stochastic process. CV_mut represents the steady-state CV of expression levels of the indicated reporter in AOPY Mutant Model II, and CV_WT represents the CV of the indicated reporter in the WT model.

FIGURE 5

Deletion of Oaf3p in the model makes POT1 transcriptional activity undergo larger-amplitude oscillations in response to a temporally varying concentration of intracellular FA. WT and oaf3Δ models of POT1 transcription were solved for the case of a temporally oscillating concentration of oleate. (A) POT1 undergoes higher-amplitude oscillations in the oaf3Δ model than in the WT model. (B) The difference between POT1 mRNA variation amplitudes in the oaf3Δ model and WT increases with increasing period of oleate pulsing. When the period exceeds 10 h, the difference between the oaf3Δ model and WT model amplitudes starts to decrease. (C) The difference between the POT1 mRNA variation amplitudes in the oaf3Δ (A_oaf3Δ) and WT (A_WT) models, for different values of the period and amplitude of oleate concentration oscillation. Color indicates the POT1 amplitude difference between the oaf3Δ model and the WT model. Overall, the difference between the amplitude of POT1 variation in the two models is stronger at higher values of the oleate oscillation amplitude. Furthermore, as the oleate oscillation amplitude increases, the maximum POT1 amplitude difference (dark red) is observed at slightly increasing values of the oleate oscillation period (black circles).

FIGURE 6

OFFNMs are enriched in yeast regulome. Frequency of OFFNMs in the yeast regulome (extracted from the Yeast Proteome Database (33), see Fig. S1) and in random networks.