A fat-2(wa17) suppressor screen in C. elegans reveals genetic adaptations to polyunsaturated fatty acid deficiency

Reviewer #1 (Public review):

Summary:
This study addresses the roles of polyunsaturated fatty acids (PUFAs) in animal physiology and membrane function. A C. elegans strain carrying the fat-2(wa17) mutation possess a very limited ability to synthesize PUFAs and there is no dietary input because the E. coli diet consumed by lab grown C. elegans does not contain any PUFAs. The fat-2 mutant strain was characterized to confirm that the worms grow slowly, have rigid membranes, and have a constitutive mitochondrial stress response. The authors showed that chemical treatments or mutations known to increase membrane fluidity did not rescue growth defects. A thorough genetic screen was performed to identify genetic changes to compensate for the lack of PUFAs. The newly isolated suppressor mutations that compensated for FAT-2 growth defects included intergenic suppressors in the fat-2 gene, as well as constitutive mutations in the hypoxia sensing pathway components EGL-9 and HIF-1, and loss of function mutations in ftn-2, a gene encoding the iron storage protein ferritin. Taken together, these mutations lead to the model that increased intracellular iron, an essential cofactor for fatty acid desaturases, allows the minimally functional FAT-2(wa17) enzyme to be more active, resulting in increased desaturation and increased PUFA synthesis.

Strengths:
(1) This study provides new information further characterizing fat-2 mutants. The authors measured increased rigidity of membranes compared to wild type worms, however this rigidity is not able to be rescued with other fluidity treatments such as detergent or mutants. Rescue was only achieved with polyunsaturated fatty acid supplementation.
(2) A very thorough genetic suppressor screen was performed. In addition to some internal fat-2 compensatory mutations, the only changes in pathways identified that are capable of compensating for deficient PUFA synthesis was the hypoxia pathway and the iron storage protein ferritin. Suppressor mutations included an egl-9 mutation that constitutively activates HIF-1, and Gain of function mutations in hif-1 that are dominant. This increased activity of HIF conferred by specific egl-9 and hif-1 mutations lead to decreased expression of ftn-2. Indeed, loss of ftn-2 leads to higher intracellular iron. The increased iron apparently makes the FAT-2 fatty acid desaturase enzyme more active, allowing for the production of more PUFAs.
(3) The mutations isolated in the suppressor screen show that the only mutations able to compensate for lack of PUFAs were ones that increased PUFA synthesis by the defective FAT-2 desaturase, thus demonstrating the essential need for PUFAs that cannot be overcome by changes in other pathways. This is a very novel study, taking advantage of genetic analysis of C. elegans, and it confirms the observations in humans that certain essential PUFAs are required for growth and development.
(4) Overall, the paper is well written, and the experiments were carried out carefully and thoroughly. The conclusions are well supported by the results.

Weaknesses:
Overall, there are not many weaknesses. The main one I noticed is that the lipidomic analysis shown in Figs 3C, 7C, S1 and S3. Whie these data are an essential part of the analysis and provide strong evidence for the conclusions of the study, it is unfortunate that the methods used did not enable the distinction between two 18:1 isomers. These two isomers of 18:1 are important in C. elegans biology, because one is a substrate for FAT-2 (18:1n-9, oleic acid) and the other is not (18:1n-7, cis vaccenic acid). Although rarer in mammals, cis-vaccenic acid is the most abundant fatty acid in C. elegans and is likely the most important structural MUFA. The measurement of these two isomers is not essential for the conclusions of the study, but the manuscript should include a comment about the abundance of oleic vs vaccenic acid in C. elegans (authors can find this information, even in the fat-2 mutant, in other publications of C. elegans fatty acid composition). Otherwise, readers who are not familiar with C. elegans might assume the 18:1 that is reported is likely to be mainly oleic acid, as is common in mammals.

Other suggestions to authors to improve the paper:
(1) The title could be less specific; it might be confusing to readers to include the allele name in the title.
(2) There are two errors in the pathway depicted in Figure 1A. The16:0-16:1 desaturation can be performed by FAT-5, FAT-6, and FAT-7. The 18:0-18:1 desaturation can only be performed by FAT-6 and FAT-7

Reviewer #2 (Public review):

Summary:
The authors use a genetic screen in C. elegans to investigate the physiological roles of polyunsaturated fatty acids (PUFAs). They screen for mutations that rescue fat-2 mutants, which have strong reductions in PUFAs. As a result, either mutations in fat-2 itself, or mutations in genes involved in the HIF-1 pathway, were found to rescue fat-2 mutants.

Strengths:
As C. elegans can produce PUFAs de novo as essential lipids, the genetic model is well suited to study the fundamental roles of PUFAs, and the results are very interesting. The genetic screen finds mutations in convergent pathways, suggesting that it has reached near-saturation. The link between the HIF-1 pathway and lipid unsaturation is very interesting. As many of the mutations found to rescue fat-2 mutants are of gain-of-function, it is unlikely that similar discoveries could have been made with other approaches like genome-wide CRISPR screenings, making the current study distinctive.

Weaknesses:
The authors make very important statements, but some are not sufficiently supported by data. In page 5, they conclude that membrane rigidity is a minor cause of fat-2 mutant defects, which is a relevant observation regarding why PUFAs are important. However, they use treatments that have rescued fluidity in another mutant (paqr-2), but do not test if they have the same fluidifying effects in fat-2 mutants.

The screening results seem to converge into the HIF-1 pathway, which is hypothetically correct according to the literature. However, the authors do not validate this hypothesis, which is a critical limitation, especially because many of the mutations they obtained seem to be of gain-of-function. Therefore, without experimental testing, it cannot be concluded that the mutations have the expected effect on the HIF-1 pathway.

In some of the mutants, the rescues in lipid compositions seem to be weak, and it is arguable whether phenotypic rescues are really via a restoration in lipid compositions.

The hypothesis linking iron homeostasis and the rescue of fat-2 mutants is interesting, but the data of rescue by iron repletion seem to be against it. The results might be due to the inefficiency in iron repletion, as the authors suggest, but this has not been formally addressed.

Therefore, the authors propose multiple very interesting and important hypotheses, but experimental validations remain limited.

Author response:

We thank the editors at eLife and the reviewers for the care with which our mansucript has been reviewed and the constructive feedback that we have received. Both reviewers viewed the manuscript positively and in particular praised the merits of the forward genetic screen that led to the discovery of a new link between the HIF-1 pathway and fatty acid desaturation.

We agree with all points by Reviewer #1. We will modify our manuscript to clarify that two types of C18:1 fatty acids are present in our lipidomics, and that the majority is likely vaccenic acid that is not a FAT-2 substrate. The title will be modified and Fig. 1A corrected.

All points raised by Reviewer #2 are also valid and we will try to address most of them experimentally, though not always as suggested. In particular, we plan to use FRAP to verify that membrane-fluidizing treatments are effective in the fat-2 mutant. We also plan to use qPCR to test whether the novel egl-9(lof) and hif-1(gof) alleles lead to the expected downregulation of ftn-2. We note that the pathway connecting EGL-9, HIF-1 and FTN-2 is well supported by published work and that the alleles isolated in our screen are consistent with it, with the addition that FAT-2 is likely a regulated outcome of FTN-2 inhibition/mutation. We also plan to monitor FAT-2 protein levels using Western blots and thus provide more clarity about the mechanism of action of the novel fat-2(wa17) suppressors. The manuscript will be modified to tone down interpretations not directly supported by experiments.