Cell signaling can direct either binary or graded transcriptional responses

Abstract

Transcriptional control is generally thought to operate as a binary switch, a behavior that might explain observations such as monoallelic gene expression, stochastic phenotypic changes and bimodal gene activation kinetics. By measuring the activity of the single-copy GAL1 promoter in single cells, we found that changes in the activities of either the transcriptional activator, Gal4 (by simple recruitment with synthetic ligands), or the transcriptional repressor, Mig1, generated graded (non-binary) changes in gene expression that were proportional to signal intensity. However, in the context of the endogenous glucose-responsive signaling pathway, these transcription factors formed part of a binary transcriptional response. Genetic studies demonstrated that this binary response resulted from regulation of a second repressor, Gal80, whereas regulation of Mig1 by a distinct signaling pathway generated graded changes in GAL1 promoter activity. Surprisingly, isogenetic cells can respond to glucose with either binary or graded changes in gene expression, depending on growth conditions. Our studies demonstrate that a given promoter can adapt either binary or graded behavior, and identify the Mig1 and Gal80 genes as necessary for binary versus graded behavior of the Gal1 promoter.

Fig. 1. Synthetic and natural modes of transcriptional activation of the integrated Gal1–GFP promoter. Regulation of a single-copy, integrated GAL1–GFP gene by FK506 and by glucose. (A) Activation with the synthetic ligand FK506. In cells expressing the Gal4BD–FKBP and Gal4AD–CN fusion proteins, FK506 generates an interaction between FKBP and calcineurin at the promoter, and thereby recruits the activation domain and stimulates transcription. The GAL1–GFP reporter, which consists of the full-length GAL1 promoter fused to the coding sequence of GFP, is shown schematically, with the binding sites for Gal4 and Mig1 indicated. (B) Activation by relief of glucose-induced repression. Glucose regulates GAL1 promoter activity by three mechanisms in wild-type cells (see text). The GAL1–GFP reporter, which consists of the full-length GAL1 promoter fused to the coding sequence of GFP, is shown schematically, with the binding sites for Gal4 and Mig1 indicated.

Fig. 2. Single-cell GAL1–GFP expression in response to varying the activity of the transcriptional activator Gal4. (A) Single-cell analysis of the response to directly increasing activity of the Gal4 activator with FK506. Yeast (YSB30) carrying integrated GAL1–GFP and GAL1–LacZ reporter genes and expressing Gal4BD–FKBP and Gal4AD–CN were treated with 0, 10, 50 and 250 ng/ml FK506. Cells were then analyzed for GFP expression by FACS and for (B) β-galactosidase expression in the entire population of cells. (C) Single-cell expression in response to different galactose concentrations and endogenous regulation. Yeast (YSB31) carrying the integrated GAL1–GFP reporter were grown in glucose media, washed, and then exposed to the indicated concentrations of galactose in 2% raffinose media. Cells were harvested at mid-log phase to avoid nutrient depletion and then analyzed for GFP expression by FACS.

Fig. 3. Single-cell GAL1–GFP expression in response to varying the activity of the transcriptional repressor Mig1. (A) Glucose represses at all levels of activation by FK506. Cells carrying an integrated GAL1–GFP reporter (YSB30) and expressing Gal4BD–FKBP and Gal4AD–CN were grown in either 2% glucose or 2% raffinose media and treated with the indicated concentrations of FK506. Cells were then analyzed for GFP expression by FACS. (B) Glucose repression produces graded transcriptional repression of Gal1 transcription. Cells carrying an integrated GAL1–GFP reporter (YSB30) and expressing Gal4BD–FKBP and Gal4AD–CN were treated with 150 ng/ml FK506 (to induce Gal1–GFP maximally) in media containing the indicated concentrations of glucose and 2% raffinose. Cells were harvested at mid-log phase and then analyzed for GFP expression by FACS.

Fig. 4. Single-cell GAL1–GFP expression in response to varying glucose levels in cells carrying the entire GAL1 regulatory network. Cells carrying the integrated GAL1–GFP reporter (YSB31) were grown in 2% glucose media, washed, and transferred to media containing the indicated concentrations of glucose and 2% galactose for 14 h. Cells were harvested at mid-log phase and then analyzed for GFP expression by FACS.

Fig. 5. Single-cell GAL1–GFP expression in response to glucose in cells lacking MIG1 or GAL80. (A) Binary responses to glucose in mig1Δ mutants. mig1Δ cells carrying the integrated GAL1–GFP reporter (YSB32) were grown in 2% glucose media, washed, and transferred to media containing the indicated concentrations of glucose and 2% galactose for 14 h. Cells were harvested at mid-log phase and then analyzed for GFP expression by FACS. (B) Graded responses to glucose in gal80Δ mutants. gal80Δ cells carrying the integrated GAL1–GFP reporter (YSB35) were grown in 2% glucose media, washed, and transferred to media containing the indicated concentrations of glucose and 2% galactose for 14 h. Cells were then analyzed for GFP expression by FACS.

Fig. 6. Single-cell GAL1–GFP expression in response to glucose in cells expanded in non-glucose media. Cells carrying the integrated GAL1–GFP reporter (YSB31) were grown overnight in 2% raffinose media, washed, and transferred to media containing the indicated concentrations of glucose and 2% galactose for 14 h. Cells were harvested at mid-log phase and then analyzed for GFP expression by FACS.