Mot1 affects the magnitude of induction and rate of shut-off of HSP26. (A) Northern blot analysis of HSP26 message in wild-type and mot1 mutant cells following 35°C heat shock for the indicated times (min). ACT1 message levels were used as a loading control. The graph shows quantitation of the results. The increase in HSP26 message in mot1 cells was statistically significant: for example, at 40 min, message levels were elevated 2.8±0.5-fold in mot1-14 cells compared to wild type. See also Dasgupta et al (2002). (B) Levels of TBP associated with the HSP26 promoter were determined by ChIP. Wild-type or mot1-42 cells were harvested following 0, 5 or 45 min of heat shock. TBP levels were normalized to the input chromatin (labeled ‘total'), and assigned values relative to the TBP level in wild-type, non-heat-shocked cells. (C) Levels of TFIIB associated with the HSP26 promoter were determined by ChIP following heat shock as described in panel B. (D) Levels of RNA Pol II large subunit associated with the HSP26 promoter were determined by ChIP as described in panel B. ‘Mock', control in which no antibody was added. (E) ChIP for Mot1-TAP associated with the HSP26 promoter was performed as described in panel B. The untagged strain contained untagged Mot1, whereas the tagged strain contained Mot1-TAP. In panels B–D, the graphs indicate the mean relative ChIP signal (normalized to the value for wild-type cells at time zero), with standard deviations derived from at least three independent experiments. Western analysis (not shown) indicated that TBP and TFIIB levels were indistinguishable in extracts from wild-type and mot1 cells. Levels of Pol II large subunit were modestly reduced (∼3-fold) in mot1 extracts compared to wild-type extracts.

INO1 promoter is regulated by TBP recruitment. (A) TBP and Mot1 ChIPs were performed using chromatin from Mot1-myc cells by immunoprecipitation with myc antibody, rabbit polyclonal TBP antiserum or no added antibody (mock). TBP or Mot1 occupancy of the INO1 promoter was determined in cells grown in the indicated media. TBP and Mot1 levels were normalized to the input chromatin levels (total), and the levels of TBP and Mot1 associated with INO1 in YPD were arbitrarily assigned the value of 1.0. (B) ChIP was performed to determine the level of TBP bound to the INO1 promoter in wild-type (WT) or mot1-42 cells. Cells were grown in YPD and then heat-shocked at 35°C for 45 min to inactivate mot1-42 prior to harvest. (C) ChIP was performed as in panel B to determine the level of TFIIB associated with the INO1 promoter in wild-type or mot1-42 cells. (D) ChIP was performed as in panel B to determine relative Pol II association with the INO1 promoter in wild-type and mot1-42 cells. Pol II ChIP signal in mot1-42 cells was significantly greater than that in wild-type cells (1.4±0.1). (E) TBP, TFIIB and Pol II association with the ACT1 promoter, determined as in panel B. In all panels, the graphs show the mean relative ChIP signals (normalized to the wild-type value) with standard deviations determined from at least three independent experiments.

Mot1 catalyzes the formation of a transcriptionally active form of TBP. (A) Northern blot analysis of BNA1 and URA1 message levels in wild-type (WT) and mot1-42 cells. Blots were stripped and reprobed for ACT1 as a loading control. (B) Mot1-TAP binding to BNA1 and URA1 promoters in vivo was detected by ChIP using chromatin from wild-type cells with untagged or TAP-tagged Mot1, as indicated. (C) TBP binding to BNA and URA1 promoters in vivo was determined by ChIP using chromatin from wild-type (WT) or mot1-42 mutant cells. Mean relative ChIP signals are shown in the graph, with standard errors. (D) TFIIB binding to BNA1 and URA1 promoters in vivo was determined by ChIP using chromatin from wild-type (WT) or mot1-42 mutant cells. The bar graph shows the mean relative ChIP signal, normalized to the wild-type signal for each gene, with error bars representing standard deviations from multiple independent experiments. (E) RNA Pol II binding to BNA1 and URA1 promoters in vivo was determined by ChIP using chromatin from wild-type (WT) or mot1-42 mutant cells. The bar graph shows the mean relative ChIP signals, normalized to the wild-type value. Error bars are standard deviations. (F) ChIP data in panels C–E were used to calculate the ratio of TFIIB to TBP and RNA Pol II to TBP at the BNA1 and URA1 promoters; ratios are shown in the table.

Differential effects of Mot1 and SAGA. (A–C) Northern blot analysis of RNA from the strains indicated above each lane. ‘WT', wild-type cells. BNA1 and URA1 message levels were reduced ∼10- and ∼5-fold, respectively, in mot1-42 cells. Message levels for these genes were reduced 2- to 2.5-fold in spt3Δ cells; a similar intermediate effect was observed for the URA1 message level in gcn5Δ cells. BNA1 message was reduced ∼4-fold in ahc1Δ cells compared to wild type, and both BNA1 and URA1 message levels were reduced (∼3- and ∼10-fold, respectively) in spt7Δ cells. (D) Serial dilution spot assays of the indicated strains. Cells were grown in rich media at 30°C and then spotted in 10-fold dilutions on YPD and incubated at 30 or 35°C for 2–3 days.

Mot1-mediated derepression of INO1 requires Ino2 but not Ume6. (A) Northern blots of RNA from the indicated strains were probed for INO1 or ACT1 (loading control). Cells were grown in YPD and then heat-shocked for 45 min at 35°C to inactivate mot1-42. Quantitation of the results is shown in the bar graph below; error bars represent the standard deviation obtained from three independent experiments. (B) ChIP analysis of Ino2-myc or Ino4-myc binding to the INO1 promoter or the HSP26 promoter (which served as a negative control). Chromatin was prepared from cells grown in YPD. (C) Northern blot analysis of the INO1 message from cells grown in YPD or inositol starvation media with or without 100 μM inositol. ACT1 message levels were used as a loading control. (D) Serial dilution spot assay of the indicated strains. ‘WT' is wild type.

Mot1 accelerates the rate at which the INO1 promoter is inactivated. (A) Wild-type (WT) and mot1-42 cells were grown at 30°C in inositol starvation medium and then shifted to 35°C for 15 min. Following this, 100 μM inositol was added and cells were harvested at the indicated times. INO1 and ACT1 message levels at each time point were determined by Northern blotting. The results are shown in the graph, in which RNA levels were normalized to ACT1 and are plotted relative to the message level present in cells at time zero. (B) Wild-type and mot1-42 strains were grown at 30°C in synthetic media lacking methionine and cysteine. The cells were shifted to 35°C for 15 min and methionine and cysteine were then added and MET15 and ACT1 message levels were measured as in panel A. The results are quantified in the graph.

Taf1 and Taf4 chromatin distribution in wild-type and mot1-42 cells. (A) Wild-type and taf1 cells were grown in rich medium at 30°C and then heat-shocked at 35°C for 45 min. Cells were harvested and Northern analysis was carried out on total RNA isolated from the cells using radiolabeled probes to detect the indicated transcripts. (B) Wild-type (TAF1) and taf1 cells were grown and heat-shocked as in panel A. TBP binding to the BNA1 and URA1 promoters was then determined by ChIP. Quantitations of the results are shown by the bar graph with standard errors. (C) ChIP was performed to detect chromatin-bound Taf1-myc in wild-type (WT) or mot1-42 cells. Cells with untagged Taf1 were used as controls. Quantitative PCR was performed using the indicated promoter primers; band intensities were normalized to the input and untagged signals and values are shown relative to the ACT1 PCR signal in wild-type cells, which has been arbitrarily set to 10. Quantitations of the results with standard errors are represented in the bar graph. (D) ChIP was performed and quantified as in panel C to detect chromatin-bound Taf4 at the indicated promoters. Cells with untagged Taf4 were used as controls and relative ChIP signals were calculated as in panel C.

Chromatin structure of URA1 is similar in wild-type and mot1-42 cells. (A) Map of the URA1 locus, indicating the 5′ and 3′ probes used for indirect end labeling. (B) Naked genomic DNA (lanes 2–7) or chromatin from wild-type or mot1-42 cells (lanes 8–19) was digested with MNase and digestion products spanning the URA1 5′ probe shown in panel A were detected by Southern blotting as described in Materials and methods. Naked genomic DNA isolated from wild-type or mot1-42 cells was treated with 10, 17.5 or 35 U/ml MNase (increasing amounts symbolized by the ramps above the lanes). Chromatin from these cells was treated with doubling concentrations of MNase ranging from 1.875 to 75 U/ml. Markers were created by digestion with restriction endonucleases with sites at the indicated distances from the probe. The positions of the URA1 ORF and 5′ untranslated region are indicated and the arrow indicates the direction of transcription. Closed arrows denote sites in chromatin protected from MNase digestion compared to naked DNA controls. Open arrows denote chromatin sites with increased sensitivity to MNase compared to naked DNA. (C) Southern blot of MNase-digested naked DNA or chromatin as in panel B but probed with the 3′ probe depicted in panel A.