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1.
Figure 6

Figure 6. A Schematic of the Circuitry of the UPR. From: IRE1-Independent Gain Control of the Unfolded Protein Response.

The model depicts the circuitry of the UPR (red) and the S-UPR (blue). Transcriptional control of HAC1 is indicated by an icon representing a rheostat affording gain control of the UPR; Ire1p-dependent HAC1u mRNA splicing is indicated by an icon representing an on/off switch. The I/T and UP signals in the S-UPR are integrated by an AND gate (semicircle, top right), i.e., both conditions must be met to propagate the S-UPR signal. The putative UMF may collaborate with Hac1p to control transcription of UPR target genes (shown) and also be involved in regulating HAC1 transcription (not shown); alternatively, different factors may be involved. The collaboration of Hac1p and UMF is indicated by the diamond-shaped icon, which integrates the information coming from both Hac1p and UMF concentration and activity.

Jess H Leber, et al. PLoS Biol. 2004 August;2(8):e235.
2.
Figure 3

Figure 3. Activation of the HAC1 Promoter Controls Increase in HAC1 mRNA Abundance. From: IRE1-Independent Gain Control of the Unfolded Protein Response.

(A) Analysis of HAC1 promoter activity during bipartite stress conditions. Δhac1 cells containing either a construct restoring HAC1 expression (lanes 1–4) or a construct expressing GFP driven by the HAC1 promoter (lanes 5–8) were grown at 23 °C and shifted to 37 °C concurrent with addition of DTT as indicated.
(B) Determination of mRNA half-life during HAC1-mRNA-inducing conditions. polIIts cells were grown at 23 °C and were shifted to 37 °C either in the absence (open symbols) or presence (filled symbols) of DTT. HAC1 mRNA abundance (squares) and ACT1 mRNA abundance (circles) are normalized to the abundance of the PolIII transcript SCR1.

Jess H Leber, et al. PLoS Biol. 2004 August;2(8):e235.
3.
Figure 1

Figure 1. ER-Distal Secretory Stress Boosts HAC1 mRNA Abundance. From: IRE1-Independent Gain Control of the Unfolded Protein Response.

(A) Determination of HAC1 mRNA abundance during the UPR. The UPR was induced in WT cells by addition of either 6 mM DTT (lanes 1–4) or 1 μg/ml tunicamycin (lanes 5–8) for the times indicated. Total RNA was harvested at the indicated intervals, and the relative abundance of HAC1 and ACT1 mRNAs was analyzed by Northern blot analysis (see Materials and Methods). Splicing was calculated at the ratio of spliced (HAC1i) to total (HAC1i + HAC1u) mRNA.
(B) Determination of HAC1 mRNA abundance during ER-distal secretory stress. WT, sec12–1, sec14–3, and sec1–1 strains were grown at 23 °C and shifted to 37 °C.
(C) Determination of HAC1 mRNA abundance during ER-proximal secretory stress. WT, sec14–3, sec61–101, sec62–101, and sec63–201 strains were grown at 23 °C and shifted to 37 °C.

Jess H Leber, et al. PLoS Biol. 2004 August;2(8):e235.
4.
Figure 2

Figure 2. HAC1 mRNA Induction Requires a Bipartite Signal and Is IRE1-Independent. From: IRE1-Independent Gain Control of the Unfolded Protein Response.

(A) Determination of HAC1 mRNA abundance during ER stress and temperature shift. WT cells were grown at 23 °C and shifted to 37 °C (lanes 1–4 and 9–12) or kept constant at 30 °C (lanes 5–8). DTT was added as indicated (lanes 5–8 and 9–12).
(B) Determination of HAC1 mRNA abundance during ER stress and inositol deprivation. WT cells were grown at 30 °C in synthetic medium supplemented with inositol and shifted to synthetic medium lacking inositol (lanes 1–4 and 9–12), or continuously grown in medium supplemented with inositol (lanes 5–8). Tunicamycin was added to a final concentration of 1 μg/ml as indicated (lanes 5–8 and 9–12).
(C) Distinction between heat shock response and HAC1-mRNA-inducing conditions. WT (lanes 1–4 and 9–12) and HSF1c (lanes 5–8) strains were grown at 23 °C and shifted to 37 °C (lanes 1–4 and 5–8) or continuously grown at 37 °C (lanes 9–12), and DTT added as indicated.
(D) Analysis of IRE1 pathway for a role in HAC1 mRNA induction. Δire1 cells were grown at 23 °C and shifted to 37 °C (lanes 1–4 and 9–12) or continuously grown at 30 °C (lanes 5–8), and DTT was added as indicated (lanes 5–8 and 9–12). Note that in Δire1 cells, HAC1 mRNA is modestly induced in response to DTT alone (lanes 5–8). This observation is indicative of feedback regulation, whereby a block in the UPR induces the I/T signal.

Jess H Leber, et al. PLoS Biol. 2004 August;2(8):e235.
5.
Figure 4

Figure 4. HAC1 Promoter Regulation Is Required to Survive Stress. From: IRE1-Independent Gain Control of the Unfolded Protein Response.

(A) Determination of Hac1p levels during either ER stress alone or during both ER stress and temperature shift. WT cells were either grown at 30 °C and treated with DTT (lanes 1–4) or grown at 23 °C and simultaneously shifted to 37 °C and treated with DTT (lanes 5–8). Protein lysates were prepared, and protein levels were analyzed by Western blot analysis. The relative Hac1p/Pgk1p ratio is normalized to the DTT-treated sample (lane 4).
(B) Characterization of HAC1 expression in strain used to approximate basal HAC1 expression. Cells expressing HAC1 from the endogenous promoter (lanes 1–4) or the ADH1 promoter (lanes 5–8) were grown at 30 °C in synthetic medium supplemented with inositol and shifted to synthetic medium lacking inositol simultaneous with the addition of tunicamycin.
(C) Reduced viability of strains unable to express HAC1 at elevated levels. The strains described in (B) were plated in serial dilutions (left to right) on synthetic medium lacking inositol (“−ino”) and synthetic medium lacking inositol and containing tunicamycin (“−ino +TM”).

Jess H Leber, et al. PLoS Biol. 2004 August;2(8):e235.
6.
Figure 5

Figure 5. Differential UPR Target Gene Induction by Elevated Hac1p Levels. From: IRE1-Independent Gain Control of the Unfolded Protein Response.

(A) Comparison of UPR target gene induction under either UPR or S-UPR conditions. Whole-genome mRNA expression analysis was carried out on WT cells harvested after 60 min of treatment, either grown at 30 °C and treated with 6 mM DTT (x-axis), or grown at 23 °C and simultaneously shifted to 37 °C and treated with 6 mM DTT (y-axis). Fold changes in gene expression are in reference to the untreated (t = 0) samples. Shown are only those genes designated as targets of the UPR (see Materials and Methods). The dashed diagonal line represents equal induction under both conditions.
(B) Comparison of UPR target gene induction under either UPR or S-UPR conditions (alternate display). The data from (A) were analyzed to generate a ratio (x-axis) for each gene, dividing the induction during S-UPR-inducing conditions by the induction during UPR-inducing conditions, with target genes of similar ratio grouped together (y-axis).
(C) Characterization of HAC1 expression in a strain constitutively expressing HAC1 at high levels. Cells expressing HAC1 from the endogenous promoter (WT; lanes 1 and 2), or a modified promoter constitutively expressing HAC1 at high levels (HAC1proHI; lanes 3 and 4) were treated with 6 mM DTT for 60 min. Although the basal transcription of HAC1proHI is elevated, the promoter is still capable of further induction during the S-UPR (unpublished data).
(D) Determination of Hac1p level in a strain constitutively expressing HAC1 at high levels. Protein lysates were prepared from the strains described in (C), and protein levels were analyzed by Western blot analysis. The relative Hac1p/Pgk1p ratio is normalized to the WT DTT-treated (t = 60) sample from Figure 4A.
(E) Transcriptional response of different classes of UPR targets to high levels of Hac1p. Whole-genome mRNA expression analysis was carried on HAC1proHI and WT cells treated with 6 mM DTT and harvested after 60 min. For the genes in each of the three classes of UPR targets defined in (B), a ratio (x-axis) is calculated by dividing the fold induction in DTT-treated HAC1proHI cells by the fold induction in DTT-treated WT cells. This ratio is plotted against the number of genes with a similar ratio (y-axis). The Class 2 target YFR026C (asterisk), which is DTT-induced approximately 10-fold more in HAC1proHI than in WT cells, is of unknown function. a, DER1; b, INO1; c, YOR289W; d, YHR087W.
(F) Transcriptional response of different classes of UPR targets to UMF. Whole-genome mRNA expression analysis was carried on ADH1pro-HAC1 cells grown at 23 °C and simultaneously shifted to 37 °C and treated with 6 mM DTT, and WT cells treated with 6 mM DTT, both harvested after 60 min. For the genes in each of the three classes of UPR targets defined in (B), a ratio (x-axis) is calculated by dividing the fold induction in ADH1pro-HAC1 cells under S-UPR-inducing conditions by the fold induction in WT cells under UPR-inducing conditions. This ratio is plotted against the number of genes with a similar ratio (y-axis). a, DER1; b, INO1; c, YOR289W; d, YHR087W.

Jess H Leber, et al. PLoS Biol. 2004 August;2(8):e235.

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