FIGURE 2.

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.