Genetic and Epigenetic Strategies Potentiate Gal4 Activation to Enhance Fitness in Recently Diverged Yeast Species

Abstract

Certain genes show more rapid reactivation for several generations following repression, a conserved phenomenon called epigenetic transcriptional memory. Following previous growth in galactose, GAL gene transcriptional memory confers a strong fitness benefit in Saccharomyces cerevisiae adapting to growth in galactose for up to 8 generations. A genetic screen for mutants defective for GAL gene memory revealed new insights into the molecular mechanism, adaptive consequences, and evolutionary history of memory. A point mutation in the Gal1 co-activator that disrupts the interaction with the Gal80 inhibitor specifically and completely disrupted memory. This mutation confirms that cytoplasmically inherited Gal1 produced during previous growth in galactose directly interferes with Gal80 repression to promote faster induction of GAL genes. This mitotically heritable mode of regulation is recently evolved; in a diverged Saccharomyces species, GAL genes show constitutively faster activation due to genetically encoded basal expression of Gal1. Thus, recently diverged species utilize either epigenetic or genetic strategies to regulate the same molecular mechanism. The screen also revealed that the central domain of the Gal4 transcription factor both regulates the stochasticity of GAL gene expression and potentiates stronger GAL gene activation in the presence of Gal1. The central domain is critical for GAL gene transcriptional memory; Gal4 lacking the central domain fails to potentiate GAL gene expression and is unresponsive to previous Gal1 expression.

Keywords: GAL genes; adaptive fitness; epigenetics; evolution; stochastic expression; trade-offs; transcriptional activation; transcriptional memory; transcriptional potentiation.

Figure 1. Expression changes during GAL memory and their fitness effect

A. Model for GAL1 regulation and memory. Top panel: during activation, Gal3 sequesters the Gal80 repressor from the Gal4 activator, leading to GAL gene expression. Bottom panel: during reactivation, residual Gal1 augments Gal3 co-activation, leading to faster expression kinetics. B – G. Naïve cells (ACT), naïve cells expressing ectopic Gal1 (ACT + eGAL1), or cells that were grown in galactose overnight and shifted to glucose for 12 hours (REACT) were shifted to media containing galactose and either GAL1-mCherry fluorescence (B–D & F) or OD₆₀₀ (E and G) were measured. GAL1-mCherry fluorescence was measured relative to constitutively expressed CFP using flow cytometry. C & D. Effect of Gal80 inhibition on Gal1-mCherry expression. D. Overlay of histograms for Gal1-mCherry from corresponding strains at the indicated times in B and C. E. At time = 0, all cultures were diluted to an OD₆₀₀ of 0.1 in galactose and OD₆₀₀ was measured every 20 minutes using 96-well plate reader. Open circles represent the ratio of OD₆₀₀ between REACT and ACT. F. Gal1-mCherry levels relative to CFP control after 7 hours in different concentration of galactose, plotted as fraction of expression in 1% galactose. G. Growth and Gal1-mCherry expression (inset) in 0.2% glucose + 1.8% galactose. * = p ≤ 0.05, Student’s t test. Error bars for Gal1mCherry fluorescence represent SEM from ≥ 3 biological replicates. The line and the bounding envelope for the OD₆₀₀ measurements is the mean and SEM, respectively, from ≥ 5 biological replicates. The yeast strains and the number of biological replicates for all experiments are listed in Table S1 and S2, respectively.

Figure 2. Genetic screen for mutants defective for GAL memory identifies gal1-D117V

A. Schematic of the 2-step FACS based screen (see Methods for details). B. Gal1-mCherry intensity relative to CFP internal control in wild-type and gal1-D117V mutant, measured by flow-cytometry. Cells were shifted from glucose to galactose for activation (ACT) or grown in galactose overnight, shifted to glucose for 12 hours and then shifted to galactose for reactivation (REACT). Error bar represents SEM from ≥ 4 biological replicates. C. Growth of wild-type and gal1-D117V mutant cells assayed by measuring OD₆₀₀ every 20 minutes during continuous growth in galactose (gal → gal), during activation (ACT) or reactivation (REACT) after 12hours of repression. The line represents the mean and the envelope represent the SEM from ≥ 4 biological replicates. D. Co-crystal structure between Gal3 (pink) and Gal80 (blue), highlighting the salt bridge between the Gal3-Asp111 and Gal80-Arg367 (inset). E. Lysates from strains expressing Gal80-13xmyc and Gal1-mCherry were subjected to co-immunoprecipitation using anti-myc antibody. The immunoprecipitated fractions (IP; top), the input fractions (middle) and the supernatant fraction after immunodepletion (bottom) were resolved by SDS PAGE and immunoblotted against either mCherry (top two panels) or the myc epitope tag (bottom panels). F. Overlay of histograms for ACT and REACT of gal1D117V in B. The yeast strains and the number of biological replicates for all experiments are listed in Table S1 and S2, respectively. Additional characterization of gal1-D117V mutant, Figure S1.

Figure 3. Recently diverged Saccharomyces species utilize genetic and epigenetic switches to adapt to growth in galactose

A–I. Cells were shifted from glucose to galactose for activation (ACT) or grown in galactose overnight, repressed for 12h (S. cerevisiae) or 18h (S. uvarum) in glucose and then shifted to galactose for reactivation (REACT). A. Gal1-mCherry fluorescence during activation and reactivation in S. cerevisiae and S. uvarum, normalized to expression at 10 h. B. Ratio of reactivation to activation from the data in B. C. OD₆₀₀ of S. uvarum during activation and reactivation. D–F. The GAL1 promoter from S. uvarum was introduced in place of the endogenous GAL1 promoter in S. cerevisiae. Gal1-mCherry (D) and Gal7-Venus (E) fluorescence relative to CFP and OD₆₀₀ (F) were measured during activation (ACT) and reactivation (REACT). Inset: Basal GAL1 mRNA, relative to ACT1, transcribed from the P_GAL1 from S. cerevisiae and S. uvarum in glucose media. G–I. The GAL1 promoter from S. uvarum driving expression of GAL1 or gal1-D117V was introduced in place of the endogenous GAL1 gene in S. cerevisiae. Gal1-mCherry (G) and Gal7-Venus (H) fluorescence relative to CFP and OD₆₀₀ (I) was measured during activation (ACT) and reactivation (REACT). Error bars represent SEM from ≥ 3 biological replicates for Gal1-mCherry fluorescence, ≥5 biological replicates for OD₆₀₀ and 12 replicates for RNA estimation. The yeast strains and the number of biological replicates for all experiments are listed in Table S1 and S2, respectively. GAL1 expression from PGAL1 from other Saccharomyces species, Figure S2; Growth trade-off for basal GAL1 expression, Figure S3.

Figure 4. The Gal4 central domain is required for GAL memory

A. Schematic of the putative domain organization with a large central domain of Gal4 (based on a structural prediction), between the N-terminal DNA binding domain and unstructured C-terminal activation domain. B–F. Naïve cells (ACT), naïve cells expressing ectopic GAL1 (ACT+eGAL1), or cells that were grown in galactose overnight and shifted to glucose for 12 hours, were shifted to galactose (REACT) to assay the Gal1-mCherry fluorescence relative to constitutively expressed CFP. B. Wild-type and gal4Δcd mutant. Inset: immunoblot of Gal4-myc immunoprecipitated from wild-type and gal4Δcd mutant cells; arrows: Gal4, * = non-specific bands. C. and D. Central domain of Gal4 was replaced with either 5-tandem repeats of β-spectrin domain (C) or the central domain from Leu3 (D). E. Wild-type, gal3Δ, gal4Δcd and gal4Δcd gal3Δ strains with or without eGAL1. Only the 0h and 10h time points are plotted for gal3Δ and gal4Δcd gal3Δ mutants. F. gal4Δcd strains with and without gal80Δ and gal4V864E mutation. G. Overlay of histograms of biological replicates from the indicated strains and time points in B and F. Error bars represent SEM from ≥ 3 biological replicates. The yeast strains and the number of biological replicates for all experiments are listed in Table S1 and S2, respectively. Additional characterization of gal4Δcd mutant, Figure S4.

Figure 5. An inter-domain interaction that is regulated by Gal80 potentiates Gal4 activation

A. Confocal micrograph showing nuclear localization of the Gal4 central domain-GFP expressed from P_ADH1 (eCD-GFP) in cells having the nuclear envelope/endoplasmic reticulum marked with a RFP tagged protein. B. Schematic for the experimental setup in C–F. Gal80 dimer binds to the activation domain of Gal4Δcd and to eCD. C, D & F. Gal1-mCherry fluorescence relative to CFP in presence of eCD and eCDmut (L282P). C. gal4Δcd mutant with or without eGAL1. Inset: immunoblot of eCD-GFP and eCDmut-GFP. D. gal4Δcd or gal4Δcd gal80Δ mutants. E. ChIP against eCD-GFP or eCDmut-GFP in the indicated strains. Recovery of the GAL1 promoter and a control locus, PRM1, were quantified relative to input by real time quantitative PCR. * p ≤ 0.05 (Student’s t-test) relative to the ChIP enrichment of PRM1. F. Wild-type cells with and without eGAL1. Error bars represent SEM from ≥ 3 biological replicates for Gal1-mCherry fluorescence and ≥ 3 replicated for ChIP experiments. The yeast strains and the number of biological replicates for all experiments are listed in Table S1 and S2, respectively. Further characterization of eCD, Figure S5. See also Figure S4.

Figure 6. Model for epigenetic potentiation of Gal4 activation through interdomain potentiation

A. In wild-type cells during early activation, Gal80 repression is relieved in subset of population, leading to lower-level expression. Inter-domain interaction between central domain and activation domain potentiates higher activation levels in cells relieved of Gal80. B. During memory (or in the presence of basal Gal1 expression), Gal80 repression is relieved early in whole population leading to unimodal, fully potentiated GAL gene expression. C. gal4Δcd cells show uniform, low-level activation.