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

Reviewer #1 (Public Review):

Summary:

In this manuscript, Tung and colleagues identify Calreticulin as a repressor of ATF6 signaling using a CRISPR screen and characterize the functional interaction between ATF6 and CALR.

Strengths:

The manuscript is well written and interesting with an innovative experimental design that provides some new mechanistic insight into ATF6 regulation as well as crosstalk with the IRE1 pathway. The methods used were fit for purpose and reasonable conclusions were drawn from the data presented. Findings are novel and bring together glycoprotein quality control and activation of one sensor of the UPR. This is a novel perspective on how the integration of ER homeostasis signals could be sensed in the ER.

Weaknesses:

Several points remain to be documented to support the authors' model.

Reviewer #2 (Public Review):

Summary:

In this study, the authors set out to use an unbiased CRISPR/Cas9 screen in CHO cells to identify genes encoding proteins that either increase or repress ATF6 signaling in CHO cells.

Strengths:

The strengths of the paper include the thoroughness of the screens, the use of a novel, double ATF6/IRE1 UPR reporter cell line, and follow-up detailed experiments on two of the findings in the screens, i.e. FURIN and CRT, to test the validity of involvement of each as direct regulators of ATF6 signaling. Additional strengths are the control experiments that validate the ATF6 specificity of the screens, as well as, for CRT, the finding of focus, determining roles for the glycosylation and cysteines in ATF6 as mechanistically involved in how CRT represses ATF6, at least in CHO cells.

Weaknesses:

The weaknesses of the paper are that the authors did not describe why they focused only on the top 100 proteins in each list of ATF6 activators and repressors. Additionally, there were a few methodology items missing, such as the nature of where the insertion site in the CHO cell genome of the XBP1::mCherry reporter. Since the authors go to great lengths to insert the other reporter for ATF6 activation in a "safe harbor" location, it leads to questions about whether the XBP1::mCherry reporter insertion is truly innocuous. An additional weakness is that the evidence for the physical interaction between ATF6LD and CRT is not strong, being dependent mainly on a single IP/IB experiment in Figure 4C that comprises only 1 lane on the gel for each of the test cases. Moreover, while that figure suggests that the interaction between CRT and ATF6 is decreased by mutating out the glycosylation sites in the ATF6LD, the BLI experiment in the same figure, 4B, suggests that there are no differences in the affinities of CRT for ATF6LD WT, deltaGly and deltaCys. An additional detail is that I found Figure 6A to be difficult to interpret, and that 6B was required in order for me to best evaluate the points being made by the authors in this figure.

Overall, I believe that this work will positively impact the field as it provides a list of potential regulators of ATF6 activation and repression that others will be able to use as a launch point for discovering such interactions in cells and tissues or interest beyond CHO cells. However, I agree with the authors that these findings were in CHO cell lines and that it is possible, if not likely, that some of the interactions they found will be cell type/line specific.