A genome-wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6α

Activating transcription factor 6 (ATF6) is one of three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its crucial role in long-term ER stress adaptation, regulation of ATF6 alpha (alpha) signalling remains poorly understood, possibly because its activation involves ER-to-Golgi and nuclear trafficking. Here, we generated an ATF6 alpha/Inositol-requiring kinase 1 (IRE1) dual UPR reporter CHO-K1 cell line and performed an unbiased genome-wide CRISPR/Cas9 mutagenesis screen to systematically profile genetic factors that specifically contribute to ATF6 alpha signalling in the presence and absence of ER stress. The screen identified both anticipated and new candidate genes that regulate ATF6 alpha activation. Among these, calreticulin (CRT), a key ER luminal chaperone, selectively repressed ATF6 alpha signalling: Cells lacking CRT constitutively activated a BiP::sfGFP ATF6 alpha-dependent reporter, had higher BiP levels and an increased rate of trafficking and processing of ATF6 alpha. Purified CRT interacted with the luminal domain of ATF6 alpha in vitro and the two proteins co-immunoprecipitated from cell lysates. CRT depletion exposed a negative feedback loop implicating ATF6 alpha in repressing IRE1 activity basally and overexpression of CRT reversed this repression. Our findings indicate that CRT, beyond its known role as a chaperone, also serves as an ER repressor of ATF6 alpha to selectively regulate one arm of the UPR.