Inhibition of gene expression by the Snf1^as inhibitor 2NM-PP1. Cultures of strains TYY923, TYY924, and TYY925 (all SNF1^as) were grown in 200 ml of YPD medium to an A₆₀₀ = 0.8 at 30 °C. Duplicate samples of 10 ml were removed for RNA isolation, and the cells in the remainder of the culture were pelleted and resuspended in 200 ml of YP medium containing 0.05% glucose and subsequently treated as described below. Each culture was divided into six equal portions after resuspension in low glucose and agitated vigorously at 30 °C. One portion received DMSO, the solvent for 2NM-PP1 as a control. The remaining five cultures received an amount of 2NM-PP1 to make the final concentration shown in the figure. Triplicate samples of 10 ml were collected for RNA isolation 4 h after resuspending the cells in low glucose medium (derepressing conditions). A, differential effects of Snf1^as inhibition on gene expression. mRNA levels were measured by RT-qPCR and normalized against ACT1 mRNA, which was unaffected by the inhibitor (supplemental Fig. 2B), and are expressed as the percentage of the mRNA amount measured in the absence of inhibitor. The control culture is represented in the figure as having 0.01 μm 2NM-PP1 to display the data on a log scale. The error bars represent the standard deviation from three biological replicates. The experiment was repeated twice with similar results. B, Snf1^as inhibition is correlated with promoter strength. The concentration of 2NM-PP1 that caused a 50% reduction in mRNA levels at 4 h was plotted against the relative level of mRNA (normalized to ACT1) at 4 h in the absence of inhibitor. The data are from supplemental Fig. 3 and similar data.

Chromatin immunoprecipitation analysis of Adr1 and RNA pol II binding and histone H3-K9,14 acetylation. Triplicate cultures of Snf1^as strain TYY1077 were grown in YPD containing 3% glucose. At A₆₀₀ ∼1.0, the cells were pelleted and resuspended in YP plus 0.05% glucose with the indicated amounts of 2NM-PP1 or with DMSO (final concentration 0.05%) in the control culture lacking 2NM-PP1. After 4 h of derepression, samples were collected for ChIP and RNA analysis. A, Adr1-Myc binding. Adr1-Myc binding was measured by qPCR after ChIP was performed as described under “Experimental Procedures.” The binding value represents the ratio of (ChIP(geneX)/input(geneX))/(ChIP(tel)/input(tel)). The control sample lacking 2NM-PP1 was set to 100%. The error bars represent the standard deviation of three biological replicates. B, RNA pol II binding. ChIP was performed using the extracts described above. ChIP grade anti-pol II antisera 8WG16 was used, and binding was determined as described under “Experimental Procedures.” C, histone H3-K9,14 acetylation. Strain TYY1077 was grown in YPD and derepressed in the presence of the indicated concentrations (in μm) of the Snf1^as inhibitor 2NM-PP1 or 0.05% DMSO (0 inhibitor) for 4 h. Another culture was derepressed for 3.5 h in the absence of inhibitor, and then inhibitor was added for 30 min before a sample was prepared for ChIP. Levels of histone H3-K9,14 acetylation were measured by ChIP as described under “Experimental Procedures.” H3-K9,14 acetylation at the indicated genes (transcription start sites) is expressed relative to acetylation at the ACT1 transcription start sites. D, comparison of Adr1 and RNA pol II binding, histone H3-K9,14 acetylation, and transcript levels of ADH2. The data are from A–C. The transcript levels for ADH2 were determined using aliquots from the same cultures that were sampled for ChIP analysis.

Snf1 activity is needed continuously during derepression to maintain high transcript levels for ADH2, FBP1, and POX1. A, mRNA accumulation stops abruptly when Snf1as is inhibited. Strain TYY925 (SNF1^as) was grown in 200 ml of YPD with 3% glucose. After removing 2–10-ml aliquots for RNA isolation representing repressing growth conditions, the remaining cells were collected by centrifugation and resuspended in 200 ml of YP with 0.05% glucose. After a subsequent 4-h incubation in derepressing conditions with vigorous shaking, 2NM-PP1 was added to a final concentration of 10 μm to a 100-ml portion of the culture. Another 100-ml portion of the culture received DMSO, the 2NM-PP1 solvent, at a final concentration of 0.05% to serve as the “no inhibitor” control. Samples of 10 ml were pipetted into 50-ml Falcon centrifuge tubes containing 10 g of ice at 0, 20, 30, 45, and 60 min after inhibitor (or DMSO) addition, and the cells were collected for RNA isolation and analysis. Triplicate samples were collected at 4 h, and the average of these values was used to calculate the relative mRNA level for each gene (compared with ACT1), expressed as the percentage of the transcript level present at 4 h. B, glucose addition and Snf1^as inhibition affects ADH2 mRNA levels similarly. Strain TYY1077 was grown and derepressed as described above. After 4 h of derepression, cultures received 2NM-PP1 (I, final 10 μm), glucose (D, final 3%), or 2NM-PP1 and glucose (10 μm and 3% final concentrations, respectively) or DMSO (0.05% final concentration). Samples of 10 ml were removed at the indicated times, and RNA was isolated and analyzed by RT-qPCR. Duplicate samples were prepared after 4 h of derepression, and the average of these values was used to calculate the relative mRNA level (compared with ACT1) after inhibitor addition, expressed as the percentage of the transcript level present at 4 h.

Continuous Snf1 activity is not needed for Adr1 and RNA pol II binding, histone H3-K9,14 acetylation, or RNA pol II CTD phosphorylation. A, Adr1-Myc binding after inhibiting Snf1^as and adding glucose. Adr1-Myc binding in strain TYY1077 was measured by ChIP after derepression as described in the legend to , but the cultures were derepressed for only 2 h in low glucose medium. The final concentration of 2NM-PP1 was 10 μm. Duplicate 60 samples were removed at the times indicated. A 10-ml portion of this sample was used for RNA preparation and analysis. The remaining 50 ml was used for ChIP analysis for Adr1-Myc13 as described under “Experimental Procedures.” ChIP-grade monoclonal antibody 9E10 from Santa Cruz Biotechnology (anti-Myc) was used. The data are expressed as binding (ChIP/input) for ADH2prm relative to ChIP/input at the TEL region used as a reference. B, histone H3-K9,14 acetylation after inhibiting Snf1^as or adding glucose. Strain TYY1077 was grown and treated as described in the legend. ChIP for K9,14-acetylated histone H3 was performed using anti-histone H3-K9,14 antisera from Santa Cruz Biotechnology as described under “Experimental Procedures.” The data are expressed as the ratio of ChIP/input without normalizing to the level of acetylation at the TEL region that changed less than 2-fold with any of the treatments. C and D, RNA pol II remains bound to the ADH2 transcription start sites (tss) and ORFs after inhibiting Snf1^as. Growth and treatment of the cells is described in the legend. RNA pol II binding was measured by ChIP as described under “Experimental Procedures” using ChIP-grade Abcam anti-pol II antibody 8WG16. The data are expressed as binding (ChIP/input) for ADH2tss and POX1tss relative to ChIP/input at the TEL region used as a reference. E and F, RNA pol II CTD associated with ADH2 is phosphorylated on Ser-2 and Ser-5 after Snf1^as inhibition. The cultures for ChIP analysis were derepressed for 6 h (none), and 2NM-PP1 (+I) or glucose (+D) was added after 2 h of derepression. Abcam anti-pSer-5 and anti-pSer-2 polyclonal antisera were used as described under “Experimental Procedures.” The data are expressed as binding (ChIP/input) for ADH2tss and ORF relative to ChIP/input at the TEL region used as a reference.

Stability of selected glucose-repressible mRNAs in derepressing growth conditions after inhibiting Snf1^as, addition of glucose, or inactivating RNA pol II (rpb1-1) at 36. 5 °C. Strain TYY1077 (SNF1^as) was grown in YPD medium and derepressed in YP containing 0.05% glucose as described under “Experimental Procedures.” Either glucose (D, final concentration 3%) and 2NM-PP1 (I, final concentration 10 μm) was added 4 h after initial glucose depletion, and samples were removed 5 and 15 min after glucose addition or 7.5 and 22 min after 2NM-PP1 addition. For inactivation of RNA pol II, strain KBY108 (rpb1-1 SNF1^as) was grown a 25 °C, and the temperature was rapidly raised to 36.5 °C as described under “Experimental Procedures.” Aliquots were removed for mRNA analysis 20, 40, and 60 min later. RT-qPCR was performed as described under “Experimental Procedures.” The RNA was converted to cDNA using random hexamers and quantified by qPCR. The mRNA values were normalized to the amount of 18 S ribosomal RNA (measured as cDNA) and are expressed relative to the amount of mRNA present in the absence of 2NM-PP1 or glucose for TYY1077 or relative to the amount of mRNA present immediately before heat inactivation of RNA pol II for strain KBY108 (rpb1-1 ts).

Active Snf1^as protects glucose-repressible mRNAs from glucose-induced rapid decay. Strain TYY1085 (reg1Δ SNF1^as) was grown in YPD medium and derepressed in YP containing 0.05% glucose as described under “Experimental Procedures.” Additions were made 4 h after initial glucose depletion, and samples were removed for RNA preparation 7.5, 30, and 60 min later. The additions were 2NM-PP1 (I, 10 μm final concentration), glucose (D, 3% final concentration), 2NM-PP1 plus glucose (10 μm and 3%, final concentrations, respectively), 1,10-o-phenanthroline (100 μg/ml final concentration), or DMSO (0.05% final concentration). Messenger RNAs were quantified by RT-qPCR as described under “Experimental Procedures.” Triplicate 0-min samples were prepared, and their average was used as the initial time point. The decay of ADH2 mRNA in a REG1 SNF1^as strain after depletion and subsequent re-addition of glucose at 4 h in a separate experiment is shown for reference. The error bars represent the standard deviation from five separate experiments for the REG1 SNF1^as strain. The data are expressed relative to ACT1 mRNA levels.

Model of Snf1 regulation of Adr1-dependent gene expression. Activation of and by Snf1 is indicated by green arrows; red arrows indicate pathways or functions that inactivate Snf1 and Snf1-dependent functions. In brief, activation of Snf1 occurs through phosphorylation of Snf1 on Thr-210, a process that is reversed in the presence of glucose through the action of two protein phosphatases, Glc7 and Sit4. The model depicts active Snf1 stimulating acetylation of nucleosomal histones, a process that could occur by directly stimulating a histone acetyltransferase (as shown), by inactivating a histone deacetylase, or by phosphorylation-dependent acetylation of histones. Once bound, Adr1 and Cat8 stimulate promoter nucleosome remodeling and PIC recruitment. A subsequent step in PIC activation is Snf1-dependent as is stabilization of cytoplasmic mRNA stability. The latter process could occur through protection of the 5′ end of glucose-repressed mRNAs, by altering cytoplasmic mRNA decay processes, or through some other unknown mechanism. The protection is represented by a phosphorylated diamond protecting the 5′ cap of a mRNA. Snf1 may also affect transcription elongation because RNA pol II is engaged in the ORF, but transcript accumulation is not observed after Snf1 is inhibited. The green arrow showing Snf1 affecting this process has a question mark (?) because we have not directly demonstrated an effect on transcript elongation.