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| Status |
Public on Apr 23, 2013 |
| Title |
Stress-Independent Activation of XBP1s and/or ATF6 Reveals Three Functionally Distinct ER Proteostasis Environments [HEK293DYG] |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
The unfolded protein response (UPR) maintains endoplasmic reticulum (ER) proteostasis through the activation of transcription factors such as XBP1s and ATF6. The functional consequences of these transcription factors for ER proteostasis remain poorly defined. Here, we describe methodology that enables orthogonal, small molecule-mediated activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the same cell independent of stress. We employ transcriptomics and quantitative proteomics to evaluate ER proteostasis network remodeling owing to the XBP1s and/or ATF6 transcriptional programs. Furthermore, we demonstrate that the three ER proteostasis environments accessible by activating XBP1s and/or ATF6 differentially influence the folding, trafficking, and degradation of destabilized ER client proteins without globally affecting the endogenous proteome. Our data reveal how the ER proteostasis network is remodeled by the XBP1s and/or ATF6 transcriptional programs at the molecular level and demonstrate the potential for selectively restoring aberrant ER proteostasis of pathologic, destabilized proteins through arm-selective UPR-activation.
The unfolded protein response adapts endoplasmic reticulum (ER) proteostasis via stress-responsive transcription factors including XBP1s and ATF6. Here, R. Luke Wiseman and colleagues implement technology for the orthogonal, ligand-dependent activation of XBP1s and/or ATF6 in a single cell. They characterize how XBP1s and/or ATF6 activation impacts ER proteostasis pathway composition and function. Adapted ER environments influence the proteostasis of destabilized protein variants without affecting the endogenous proteome. The work informs the development of proteostasis environment-adapting therapeutics for protein misfolding-related diseases. In order to activate both XBP1s and ATF6 in the same cell, we incorporated DHFR.ATF6 and tet-inducible XBP1s into a HEK293T-REx cell line stably expressing the tet-repressor. The HEK293DYG control cell line expresses tet-inducible eGFP and DHFR.YFP and is used as a control to demonstrate that the addition of doxycycline (dox) and trimethoprim (TMP) do not induce UPR genes.
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| Overall design |
HEK293DYG cells were treated for 12 h with vehicle or 1 μg/mL dox and 10 μM TMP in biological triplicate. Cells were harvested and RNA was extracted using the RNeasy Mini Kit (Qiagen). Genomic DNA was removed by on-column digestion using the RNase-free DNase Set (Qiagen). Data from HEK293DYG cells showed no significant overlap in the ligand-treated transcriptomes obtained from HEK293DAX cells.
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| Contributor(s) |
Shoulders MD, Wiseman RL |
| Citation(s) |
23583182 |
| |
| Submission date |
Mar 07, 2013 |
| Last update date |
Jul 26, 2018 |
| Contact name |
R. Luke Wiseman |
| E-mail(s) |
wiseman@scripps.edu
|
| Organization name |
The Scripps Research Institute
|
| Department |
Molecular & Experimental Medicine
|
| Lab |
MEM220
|
| Street address |
10550 N. Torrey Pines Rd. MEM 220
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platforms (1) |
| GPL6244 |
[HuGene-1_0-st] Affymetrix Human Gene 1.0 ST Array [transcript (gene) version] |
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| Samples (6)
|
| GSM1094311 |
HEK293DYG cell line treated for 12 h with DMSO Replicate #1 |
| GSM1094312 |
HEK293DYG cell line treated for 12 h with DMSO Replicate #2 |
| GSM1094313 |
HEK293DYG cell line treated for 12 h with DMSO Replicate #3 |
| GSM1094314 |
HEK293DYG cell line treated for 12 h with 1 µg/mL doxycycline and 10µM trimethoprim Replicate #1 |
| GSM1094315 |
HEK293DYG cell line treated for 12 h with 1 µg/mL doxycycline and 10µM trimethoprim Replicate #2 |
| GSM1094316 |
HEK293DYG cell line treated for 12 h with 1 µg/mL doxycycline and 10µM trimethoprim Replicate #3 |
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| This SubSeries is part of SuperSeries: |
| GSE44951 |
Stress-Independent Activation of XBP1s and/or ATF6 Reveals Three Functionally Distinct ER Proteostasis Environments |
|
| Relations |
| BioProject |
PRJNA192602 |
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