Distinct transcriptional responses elicited by unfolded nuclear or cytoplasmic protein in mammalian cells
- Stanford University, United States
Figures
Figure 1 with 1 supplement
Unfolded DD induces transcriptional response.
(A) Schematic representation of the strategy used to create a unfolded protein in cells using the destabilizing domain (DD). (B–D) Changes in transcript levels measured by mRNA-seq are shown for …
Figure 1—figure supplement 1
Unfolded DD responsive genes from mRNA-seq.
Changes in transcript levels measured by mRNA-seq are shown for genes that respond strongly to HS (red), tunicamycin (green), and the appearance of the unfolded DD (black). Transcript levels are …
Figure 2 with 1 supplement
Cellular transcriptional response is dose-dependent.
Unfolded DD was created in two cell lines, which express DD-GFP levels that differ by a factor of three (Figure 2B). Cells were harvested at the indicated times following withdrawal of Shield-1 …
Figure 2—figure supplement 1
Lower levels of unfolded DD do not induce the stress response, but a different unfolded protein does.
(A) Unfolded DD was created in two NIH3T3 cell lines, which express DD-GFP levels that differ by a factor of three (Figure 2B). Cells were harvested 45, 135, and 405 min following withdrawal of S1, …
Figure 3
Transcriptome profiles of unfolded DD compared to HS and ER stress.
(A) NIH3T3 cells expressing DD-GFP in the presence of S1 were perturbed by HS (42°C), treatment with 5 μM tunicamycin, or by withdrawal of S1 to create unfolded DD. Cells were harvested 45, 135, and …
Figure 4 with 1 supplement
Comparison of unfolded DD stress with known stress responses.
(A) Venn diagram showing pairwise comparisons of the genes induced by unfolded DD (gray) with the genes induced by the HS response (HSR) (red), the erUPR (green), and the p53-mediated response to …
Figure 4—figure supplement 1
Comparison of the unfolded DD stress with proteasome inhibition.
Venn diagram showing pairwise comparisons of the genes induced by unfolded DD (gray) with the genes induced by proteasome inhibition using bortezomib (brown) The p-values are calculated by …
Figure 5 with 2 supplements
p53 protein accumulates upon appearance of the unfolded DD.
(A) S1 was withdrawn from NIH3T3 cells expressing the DD-GFP fusion protein for the indicated times, and equal amounts of total protein were immunoblotted with antibodies against p53 or GAPDH. (B) …
Figure 5—figure supplement 1
Investigating potential sources of p53 activation.
(A) S1 was withdrawn from cultures of NIH3T3 cells stably transduced with the DD-GFP fusion protein, and the rate of DD-GFP degradation was quantified using analytical flow cytometry. (B) Unfolded …
Figure 5—figure supplement 2
Involvement of other transcription factors.
(A) Cells expressing DD-GFP stabilized by S1 were treated with siRNA pools against HSF1, cultured for 2 days, then RNA was harvested for RT-qPCR analysis 135 min following withdrawal of S1. …
Figure 6 with 1 supplement
Nuclear or cytosolic unfolded proteins elicit distinct responses.
(A) Images of live NIH3T3 cells stably expressing nuclear DD-GFP or cytosolic DD-GFP. Hoechst 33,342 was used to image nuclei. (B, C) Unfolded DD was induced by withdrawal of S1, and transcript …
Figure 6—figure supplement 1
Additional comparisons of nuclear vs cytosolic unfolded protein stress.
(A) Comparison of fluorescent level between nuclear DD cells and cytosolic DD cells by analytical flow cytometry. (B) Flow cytometry data of the kinetics of nuclear DD-GFP and cytosolic DD-GFP …
Figure 7 with 1 supplement
The cUPR provides protection against subsequent proteotoxic stress.
(A) NIH3T3 cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in the presence of 1 μM S1. The stabilizing S1 was withdrawn for 45, 135, or 405 min and then re-administered to the …
Figure 7—figure supplement 1
The effect of transcription factors on cell growth.
(A) NIH3T3 cells expressing DD-GFP were co-cultured with unmodified 3T3 cells in the presence of 1 μM S1. The stabilizing S1 was withdrawn for 45, 135, or 405 min and then re-administered to the …
Additional files
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Supplementary file 1
List of genes significantly up- or down-regulated by the three stress conditions (false discovery rate [FDR] <0.05).
- https://doi.org/10.7554/eLife.07687.017
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Supplementary file 2
mRNA-seq data from all three perturbations.
- https://doi.org/10.7554/eLife.07687.018
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Supplementary file 3
Gene ontology analysis for genes responding to unfolded destabilizing domain (DD) stress.
- https://doi.org/10.7554/eLife.07687.019
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Supplementary file 4
Gene ontology analysis for genes responding to unfolded destabilizing domain (DD) stress in different timepoints.
- https://doi.org/10.7554/eLife.07687.020
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Supplementary file 5
mRNA-seq data from bortezomib perturbations.
- https://doi.org/10.7554/eLife.07687.021
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Supplementary file 6
List of primers used in RT-qPCR experiments in this paper.
- https://doi.org/10.7554/eLife.07687.022
