A genome wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6?
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. 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.