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. 2019 May 24;8:e45002. doi: 10.7554/eLife.45002

Figure 5. Cycloheximide treatment induces a rapid RNAPII transcriptional response opposite and epistatic to that of RASTR.

(A) Schematic related to experiments in subsequent panels describing the effect of cycloheximide treatment on de novo RP production and auxin treatment on Top1-AID (or Top1/2-AID) degradation. (B) Northern blots of pre-rRNA after 0, 5, 10, and 20 min of cycloheximide (CHX) treatment. (C) Scatter plot comparing RNAPII (Rpb1) ChIP-seq after 20 min of cycloheximide treatment (y-axis, CHX) to that of non-treated cells (x-axis, vehicle) at the indicated groups of target genes. (D) Scatter plots comparing RNAPII ChIP-seq in auxin-treated to untreated cells (left panel) and in auxin + cycloheximide (CHX)-treated to untreated cells (right panel). In both cases cells express Top1-AID. (E) Scatter plots comparing RNAPII ChIP-seq as in (D), but for Top1/2-AID cells. (F) Box plots showing RNAPII ChIP-seq fold-change for Hsf1 target genes after cycloheximide (CHX) and/or auxin (Aux) treatment of Top1-AID or Top1/2-AID cells, as indicated (data taken from experiments shown in D and E). p-Values are shown above the indicated comparisons together with significance according to student’s t-test (***: p<0.001, ns: not significant). (G–H) Top1/2-AID strains expressing Rpl25-eGFP (G) or Ifh1-eGFP (H) and Nhp6-mCherry were grown exponentially and samples were used for fluorescence microscopy analysis after 20 min of auxin (top) or vehicle treatment (bottom), in the absence (left) or presence (right) of cycloheximide (CHX).

Figure 5.

Figure 5—figure supplement 1. Characterization of Ifh1-FRB Top1-AID Top2-AID and Ifh1-FRB Top1-AID strains; binding of Ifh1 at Hsf1 target and RP gene promoters.

Figure 5—figure supplement 1.

(A) Box plots showing RNAPII ChIP-seq fold-change for Hsf1 target genes after auxin (Aux) and/or cycloheximide (CHX) treatment of Top1-AID or Top1/2-AID cells, as indicated. (B) A tom1-Δ strain expressing Ifh1-eGFP and Nhp6-mCherry was grown to mid-exponential phase and cell samples were used for fluorescence microscopy analysis following 20 min of vehicle (top panels) or cycloheximide (CHX; bottom panels) treatment. (C) Ifh1 occupancy at the RPL30 promoter 20 min following cycloheximide (CHX, red) or vehicle (Veh, blue) treatment in wild type (TOM1) or tom1-Δ cells. (D) Ifh1 occupancy at the RPL30 and RPL37A promoters 0 or 20 min following mock or Top1/2-AID depletion (- or Aux) in the presence (CHX) or absence (-) of cycloheximide. (E, F) Numbers of cells where Rpl25-eGFP (E) or Ifh1-eGFP (F) nuclear foci were identified, presented as a percentage. In each experiment 75–100 cells were used for the quantification. Data are reported as averages from two experiments, with standard deviations indicated.