HSF-1 activates the ubiquitin proteasome system to promote non-apoptotic developmental cell death in C. elegans

Essential revisions: 1) While the implication of a number of interesting genes in this pathway was appreciated, there was a substantial concern about the interpretation of the genetic interaction data. Throughout the manuscript, in a number of cases, genetic interaction arguments were based on examining the enhancement of phenotypes based on the combination of non-null alleles (or RNAi). Observed enhancements, even if synergistic in nature, do not prove that genes act in the same pathway; they are equally consistent with genes acting in separate pathways. The interpretation of all these genetic interaction tests needs to be more correctly, and carefully, phrased.

We completely agree with this comment. Although none of our final conclusions depend on the data of synergy between non-null alleles, we have, nonetheless changed the text where such results may have appeared to be overreaching. The following specific changes were made:

Results section:

“as would be predicted if HSF-1 functions” changed to “as might be predicted if HSF-1 functions”.

“indicating that these genes likely function together to promote LCD” changed to “indicating that these genes likely function together, in sequence or in parallel, to promote LCD”.

“We conclude that SIAH-1, CUL-3, and RBX-1 likely function together to promote LCD and likely do so downstream of LET-70” changed to “We conclude that SIAH-1, CUL-3, and RBX-1 all function to promote LCD and likely do so downstream of LET-70”.

“consistent with the two genes acting in the same pathway” was removed.

2) The evidence that hsf-1 controls let-70 expression is very preliminary and this should be made explicitly clear and conclusions be toned down accordingly.

Our data demonstrate that (1) both hsf1 and let-70 are required for linker cell death, (2) that let-70::GFP expression is induced upon cell death onset, (3) that hsf-1 is required for this transcriptional induction, (4) that a conserved HSF-1 binding site is present upstream of let-70 coding sequences, (5) that deletion of these upstream sequences inhibits induction of let-70::GFP expression, and (6) that unlike all previously described LCD genes, let70 lesions cannot be rescued by an hsf-1 gain of function allele. We therefore feel that the conclusion that HSF-1 controls let-70 expression is reasonably well substantiated.

Nonetheless, we agree with the reviewer comment in the sense that we have not demonstrated that HSF-1 directly binds regulatory sequences upstream of the let-70 gene in vivo. However, this is a very tall order, as the experiment would require performing chromatin IP from single linker cells, which is currently a major technical challenge.

To avoid confusion about our claims, we have introduced the following changes:

Results section: “Taken together, our results strongly suggest that LET-70 functions downstream of a linker-cell-specific non-canonical function of HSF-1 to promote LCD” changed to “Taken together, our results suggest that LET-70 functions downstream of a linker-cell-‐specific non-canonical function of HSF-1 to promote LCD. Our data also suggest that other HSF-1 targets are likely relevant, and that let- 70 may be under the control of additional regulators.”

3) The authors try to describe a molecular-genetic framework governing the initiation and execution of LCD. The initiation and execution process should be more clearly defined and genes be more clearly categorized.

We thank the reviewer for this comment, as we were indeed not consistent in our use of these terms, and, in addition, the figure legend for our model had an error in color assignment. We have now made several changes to the text, and have reserved use of the terms “initiation” and “execution” only as they refer to the upstream or downstream pathway components, respectively. The following specific changes have been made:

Figure 7 legend: Pathway color designations changed to reflect the actual colors.

Introduction:

“Here we describe a molecular-genetic framework governing the initiation and execution of LCD in C. elegans” changed to “Here we describe a molecular-genetic framework governing LCD in C. elegans”.

“We demonstrate that LCD initiation is controlled” changed to “We demonstrate that LCD is controlled”.

“increases just before LCD initiation” changed to “increases just before LCD onset”.

Results:

“dictate cell death initiation” changed to “dictate cell death onset”.

“in which organelle changes accompanying cell death initiation are evident” changed to “in which organelle changes accompanying cell death are evident”.

“the parallel pathways controlling LCD initiation” changed to “the parallel pathways controlling LCD onset”.

“let-70 likely acts in the execution of LCD” changed to “let70 likely acts in promoting LCD”.

Discussion:

“the molecular identification of a LCD execution machinery” changed to “the molecular identification of LCD components and their interactions”.

“may lead to a block in cell death execution” changed to “may lead to a block in cell death”.

4) In the Introduction, the authors claimed that "our studies reveal intriguing design similarities between LCD and apoptosis" This is overstated. How similar are these two pathways? Depending on transcriptional factors and protein degradation? The hsf-1/transcriptional evidence is weak and the involvement of protein degradation relies on the implication of components of the proteasome pathway but those can play roles in protein-degradation independent processes. While it is interesting to speculate on difference, the conclusions need to be significantly toned down.

We agree with the reviewer that we may have been too exuberant about the similarities in our use of language. Nonetheless, the similarities we point out are apparent, and worth noting, in our opinion. We have made the following changes in the text to tone down the conclusions:

Introduction: “Our studies reveal intriguing design similarities” changed to “Our studies reveal design similarities”.

Discussion: “The logic of the LCD pathway is strikingly similar” changed to “The logic of the LCD pathway may be similar”.