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| Status |
Public on Jul 26, 2024 |
| Title |
A genome wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6⍺ |
| Organism |
Cricetulus griseus |
| Experiment type |
Other
|
| Summary |
Activating transcription factor 6 alpha (ATF6⍺) is one of the three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its significant involvement in long-term ER stress adaption, regulation of ATF6⍺ signalling is still poorly understood, possibly because its activation involves Golgi and nucleus trafficking. Here, we have generated a dual CHO-K1 ATF6⍺/IRE1⍺ reporter cell line to perform an unbiased genome-wide CRISPR/Cas9 mutagenesis screen, in the presence and absence of ER stress, to systematically profile genetic factors that specifically contribute to ATF6⍺ signalling. Anticipated and new candidate genes that regulate ATF6⍺ activation were discovered. Among these, calreticulin (CRT), a key ER luminal chaperone, emerged as a selective repressor molecule of ATF6⍺ signalling. Cells lacking CRT constitutively activated a BiP::sfGFP ATF6⍺-dependent reporter, had higher BiP levels and an increased rate of trafficking and processing of ATF6⍺. Purified CRT interacts with the luminal domain of ATF6⍺ in vitro and the two proteins co-immunoprecipitated from cell lysates. CRT depletion exposed a negative feedback loop implicating ATF6⍺ in repressing IRE1⍺ activity basally and overexpression of CRT reversed phenotype. Our data indicate that CRT, in addition to its known role as a chaperone, also serves as an ER repressor of ATF6⍺ to maintain selective regulation of the UPR.
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| Overall design |
Examination of genomic DNA, pooled from sorted cells in different bins (based on ATF6::sfGFP and XBP1s::mCherry reporter signals) at different stages of the phenotypic enrichment process and from unsorted control cells, was subjected to high-throughput sequencing and MAGeCK bioinformatics analysis. In total 16 samples were examinated.
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| Contributor(s) |
Ordonez A, Harding HP, Ron D |
| Citation(s) |
39073063 |
| |
| Submission date |
Jan 31, 2024 |
| Last update date |
Aug 23, 2024 |
| Contact name |
Adriana Ordonez |
| E-mail(s) |
aog23@cam.ac.uk
|
| Organization name |
Cambridge Institute for medical Research
|
| Department |
Biochemistry
|
| Lab |
David Ron
|
| Street address |
Keith Peters Building, Biomedical Campus, Hills Rd
|
| City |
Cambridge |
| State/province |
Cambridgeshare |
| ZIP/Postal code |
CB2 0XY |
| Country |
United Kingdom |
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| Platforms (1) |
| GPL27425 |
Illumina NovaSeq 6000 (Cricetulus griseus) |
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| Samples (16)
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| Relations |
| BioProject |
PRJNA1071609 |
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