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

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 (⍺) signalling remains poorly understood, possibly because its activation involves ER-to-Golgi and nuclear trafficking. Here, we generated an ATF6⍺/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⍺ signalling in the presence and absence of ER stress. The screen identified both anticipated and new candidate genes that regulate ATF6⍺ activation. Among these, calreticulin (CRT), a key ER luminal chaperone, selectively repressed 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 interacted 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 this repression. Our findings indicate that CRT, beyond its known role as a chaperone, also serves as an ER repressor of ATF6⍺ to selectively regulate one arm of the UPR.