Author response:
The following is the authors’ response to the original reviews
Correction: In the process of revising the preprint, we discovered that for the 15SY dataset that a single time point (ZT2) out of the 12 timepoint series was inadvertently combined with temporally adjacent time samples (ZT20, 22, 24). We corrected the accompanying GEO submission (Series GSE145509). With this update, we repeated the rhythm analysis with an updated RAIN algorithm as the original Boot-eJTK could not be run as it was outdated with dependent packages no longer maintained or supported, and some are no longer available through standard package managers. The analysis with the corrected ZT2 sample and did not find any significant changes in the major claims of the papers. One minor change is that we no longer observe significant DR-dependent increases in proteasome gene levels. Nonetheless, we still find DR-dependent cycling of proteasome genes and proteasome module network connectivity consistent with the DR-sensitivity of the proteasome pathway. Figure 4 has been updated to reflect this change. After correcting this issue, we revised the manuscript in response to the reviewer comments.
eLife Assessment
This study describes important findings on how a core component of the circadian clock impacts the effect of dietary restriction (DR) on longevity and fecundity in Drosophila, which lead the authors to postulate rhythmic control of proteostasis in the fat body as a critical aspect of DR effects. The evidence presented is still incomplete, not fully supporting the conclusions of the study, as alternative hypotheses/explanations have not yet been systematically explored. The work will nevertheless be of substantial interest to researchers working in circadian and cell biology, metabolism, and aging, with an interesting hypothesis to be explored further.
We sincerely thank eLife for considering our manuscript and express our gratitude to all three reviewers for their time and constructive comments. While acknowledging that there are alternative hypotheses for some of our findings, which make our evidence incomplete, we appreciate that our manuscript was recognized as important and of substantial interest. We have revised the manuscript to acknowledge that the major findings under light-dark conditions could be attributable to being driven by light rather than the circadian clock. We add new data demonstrating that the far majority of cycling genes in LD in control flies are disrupted in ClkJrk consistent with circadian regulation (see also below). Future investigations are needed to test the alternative hypotheses and explanations. Nevertheless, we believe that the manuscript still represents a meaningful advancement in understanding how molecular circadian clocks in peripheral tissues interact with diet to influence systemic lifespan and aging.
Public Reviews:
Reviewer #1 (Public Review):
The studies by Hwangbo et al. diligently attempt to account for many of the typically neglected dietary and non-dietary factors.
Strengths:
- Work addresses many potential artifacts of dietary (e.g., dehydration stress, macronutrient ratios, and protein source) and non-dietary (e.g., leaky expression of S106-GAL4) manipulations-important factors that are too often overlooked.
- Balanced and complementary behavioral, molecular, and bioinformatic experiments
- Show necessity of proteostatic subunits in the fat body for DR-mediated longevity. The findings in the current manuscript lay the ground for future studies that test sufficiency of fat body prosβ3 and rpn7, or necessity of other proteostatic genes in other tissues.
Weaknesses:
- Could the lack of DR response in clock mutants across dietary concentrations be simply because the clock mutants are better at compensatory feeding adjustments to dietary dilutions? If this were the case, there are two major implications to the authors' conclusions:
a) The Clk mutants are differently responding to dietary dilutions, not to dietary restriction, per se.
b) Nutritional intake was unaffected by the dietary manipulations. If the changes in fat body proteostasis and lifespan were due to nourishment, it would be expected that the physiology and lifespan do not change.
Accurate measurements of food consumption and the resulting protein intake could potentially clarify this critical question.
We thank the reviewer for their positive feedback and also appreciate their raising the important issue of whether Clk^Jrk mutants may be more effective at compensatory feeding. Xu et al. (2008) reported that overall food consumption in Clk^Jrk flies was indistinguishable from control flies.
We also directly assessed food intake in ~1 week old flies on three diets (1% SY, 5% SY, 15% SY) over two days (48 hours) using the Con-Ex method (Shell et al. 2018). We observed either no significant changes or relatively modest changes in food consumption between iso31 controls and Clk^Jrk flies that are limited compared to the large differences in caloric content between the diets. While we cannot rule out changes in feeding patterns throughout the lifespan, we believe that minimal differential compensatory feeding in the Clk^Jrk mutants are not sufficient to be the primary cause of the lifespan differences observed across diets. These data are added as new Figure 1-figure supplement 3.
Reviewer #1 (Recommendations For The Authors):
Hard to find information:
- Type of yeast used. Should be addressed in the Methods section at least, instead of having to dig through several paragraphs into the Results section.
Missing information:
- Agar type and concentration
- Mifepristone diets: pipetted on top or mixed into food?
The relevant information has been updated in the Materials and Methods
Wrong information:
- Line #159-160: "Fig. 1 and Fig. S3" should be "Fig. 1 and Fig. S2" and then "(Fig. S3)" added to the end of the sentence.
This information has been corrected in the revision
Presentation:
- Paragraphs are very long.
- Interactions should be denoted by ×, not * or x.
- Remove markers from mortality graphs. Having bulky markers reduces perceived differences between curves.
These changes have been updated in the revised version.
Reviewer #2 (Public Review):
Dietary restriction (DR) increases lifespan, an effect that has been consistently observed in several organisms, but we still lack a clear mechanism to explain this phenomenon. In this work, Hwangbo et al. revisited the role of the circadian clock in DR-mediated lifespan effects. They found that the increase in lifespan produced by DR is missing on a clock mutant, a clock dependency that is also observed at the level of nutrient-dependent egg laying. By conducting RNA-seq with an impressive temporal resolution, they showed that DR triggers an increment in the number of cycling genes expressed in the fat body, the fly functional analog of the mammalian liver. Interestingly, from these genes, a group of them are de novo daily expressed genes, meaning that their expression was not rhythmic under the control diet but appear rhythmically expressed under DR. Among those, genes encoding proteasome subunits are enriched. The authors finally showed that adult-specific knockdown of these genes in the fat body prevents the increase in lifespan under DR, further supporting a role of the proteasome in this process. Overall, the conclusions are mostly supported by the evidence presented, and the authors' discussion nicely frame their results with other research in the field.
Strengths:
- Many studies have limited their observations of DR on lifespan to a few dietary conditions which makes the reach of some previous conclusions somewhat limited. The dilution strategy that the authors used in this work provides a strong indication that the effect of DR on lifespan relies on clock expression regardless of the conditions used. Furthermore, the inclusion of the egg-laying assay is a good addition to support this hypothesis.
- Because the strength of the rhythmicity statistics relies heavily on the number of data points collected, the temporal resolution used for the RNA-seq experiments (every 2 hrs per 48hrs) is remarkable. This allows exquisite dissection of the phase of rhythmic genes in different conditions. The dataset produced in this work might be of use to other groups interested in weighting the role of other represented gene clusters in DR.
We are grateful for the reviewer’s positive feedback regarding the robust experimental design in the manuscript.
Weaknesses:
I see only minor flaws in this work, that if addressed, might strengthen the authors' conclusions, particularly:
- The results of the lifespan assays are quite variable and in some instances contradictory (Fig. S8) across trials, possibly because there are other unaccounted variables we still do not understand. The fecundity assay, in contrast, seems to be a better readout (Fig. 2). Confirming at least the two genes picked for the study (Fig. 5) would be good support for the claim that the proteasome mediates the effects of DR.
We appreciate the reviewer’s comment of seeing “only minor flaws”. We reiterate that we focused on those results which were replicated across trials, providing confidence in the overall conclusions. Nonetheless, we agree that exploring the proteasome role on the DR effect on fecundity would be intriguing and may complement the lifespan data. We now acknowledge this point in our discussion.
- According to the model, the acute effect of DR on gene expression is related to CLOCK protein function. However, I am not sure how this link was established. It is tempting to assume that CLOCK upstream is the reason for having an increase in rhythmic genes under DR, but the experiments did not test this. The tests conducted either assessed the role of clk or the effect of an impaired proteasome on DR-dependent extension of lifespan. Thus, it is difficult to assert the authors' claims on the link between CLK and the changes in cycling genes and to the proteasome upon DR.
We have updated our text and model figure suggesting a direct CLK role in the Discussion.
Reviewer #2 (Recommendations For The Authors):
As mentioned before, the experiments, in particular the RNA-seq datasets are excellent. Additionally, the discussion provides a good overview of other relevant papers on DR, and the conclusions are mostly supported by the data. Here I provide a couple of suggestions that I believe might improve this work:…
- Although the model is simple and understandable (Fig. 6), the inclusion of an overall summary or explanation in the figure legend would be appreciated, especially for readers that are not familiar with the terminology.
- It might be the formatting while parsing the files but some of the in-text citations are between curly brackets (e.g., lines 80, 92, 93).
- By definition, and unlike Canton-S or Oregon-R strains, w1118 flies are not wild-type but a genetic control. I believe that reference to this on the figures and text may need correction.
We updated and/or our corrected each of these in the revised version. For wild-type, we more explicitly define this as wild-type for the relevant genetic locus.
Reviewer #3 (Public Review):
In this study, Hwangbo and co-workers investigate the extent to which the well-established life extending effects of DR rely on the molecular circadian clock and how the landscape of clock-controlled gene expression changes in the face of DR within the fat body of the fly, a tissue that performs the functions associate with both the liver and adipose tissue of mammals. The authors evidence that DR extends lifespan in a manner that depends on only one of the two major limbs of the fly's molecular circadian clock, namely the positive limb, that DR produces major changes in the identities of cycling clock output genes, and that genes related to the proteosome represent a major component of DR-induced transcript cycling. Though interesting, these conclusions are not strongly supported by the data and there are two major reasons for this. First, the authors rely on only one loss of function genotype each for the loss of positive and negative limb clock gene function. Second, though they wish to address the "circadian transcriptome" under normal and DR conditions, the authors conduct all their work under strong Light/Dark cycles, making it impossible to address circadian phenomena. These shortcomings are problematic in the extreme, as they leave open obvious alternative explanations for the results and fail to directly determine if the rhythmic expression, they observe are clock controlled or merely driven by the light/dark cycles, which themselves produce major effects on activity, feeding, etc., that may be responsible for differentially driving rhythmic transcripts under normal and DR conditions in the fat bodies.
Major Weakness One: The use of only genotype each for the loss of positive (Clk^JRK) and negative (Per^01) limb of the circadian represents a major challenge for a central conclusion of the study. Phenotypes caused by the loss of a single clock gene may be due to the loss of circadian timekeeping, or they may represent a pleiotropic effect of the loss of function mutant being used. There are multiple precedents for pleiotropic (non-circadian) effects of clock gene mutants. It is, therefore, possible that the differences in the extent of DR mediated life extension between Clk^JRK and Per^01 may not represent a difference between breaking the positive and negative limbs of the clock but may simply reflect a pleiotropic effect of the dominant negative Clk^JRK. This possibility is acknowledged by the authors (lines 343-344). This could be addressed quite easily by extending the analysis to other loss of function mutants, for example, tim01 for the negative limb and cyc01 for the positive. Given the central focus here on the "circadian transcriptome," leaving open this alternative explanation for Clk's role in DR induced life extension represents a major weakness of the study. Furthermore, given the fact that Clk^JRK appears to be short lived on most of the media tested in the study, is it really surprising or informative that they would display lower life extension under DR?
We confirmed that the large majority of LD oscillating genes in wild-type controls are disrupted in ClkJrk consistent with circadian clock regulation (Figure 3-figure supplement 1). As noted, we formally acknowledged that the circadian clock mutant alleles used here, and in fact any circadian clock alleles, can have pleiotropic, i.e., non-circadian, clock effects. This would only be partially mitigated by adding more (but also potentially pleiotropic) clock mutant alleles. Very challenging circadian resonance experiments (see Xu et al, 2019) are the gold standard for resolving circadian clock v. non-clock effects which are beyond the scope of this study which we now add to our discussion.
We also note that foxo mutants are both short-lived and exhibit a robust lifespan extension to dietary restriction and thus the ClkJrk mutant is distinct in this regard. We have added this point to the Discussion.
Major Weakness Two: The authors have not established that any of cycling transcripts they have detected in the fat body under normal and DR conditions are driven by the circadian clock. This is because: 1.) they have conducted their transcriptomic analysis on cells taken from flies entrained to light dark cycles, which can themselves drive daily changes in expression levels and 2.) they have not shown that the cycling measured on normal diet or DR conditions depends on a functional circadian clock. The "significant reorganization of the circadian transcriptome" is presented as a major conclusion of this study, but the authors have not addressed circadian control of transcription at all here, either by an examination of transcription under free-running conditions and/or in loss of function clock mutants.
In addition, there is a logical gap in this study. The authors have shown that DR produces less life extension in Clk^JRK mutants than Per^01 or wild-type controls. They then show that DR produces changes in the rhythmic transcriptome when flies are place on DR. The central model presented in Fig. 6 shows/concludes that CLK drives increases in proteome-related transcript rhythms under DR. This conclusion could have been directly tested by asking if the changes in rhythmic gene expression induced by DR are gone the loss of function Clk mutants, or if the transcriptomic landscapes fail to differ between feeding conditions in these mutants.
In conclusion, the study falls far short of directly testing the ideas it puts forth, greatly limiting its impact and interest.
As noted above, we also examined the diurnal transcriptome in ClkJrk (at 4 hour resolution) and found that of the 290 genes that were detectably rhythmic in wild-type just 13 were rhythmic in ClkJrk consistent with the notion that oscillations depend on Clk (Figure 3-figure supplement 1). We now add this analysis to the manuscript. Nonetheless, we cannot exclude a role for light and thus have opted to use “diurnal” in place of “circadian” where appropriate for observed rhythms under LD conditions.
Reviewer #3 (Recommendations For The Authors):
Line 140 "showed an almost identical response" was a little hard to understand at first. Consider clarifying.
This has been rephrased for clarity
The authors claim that Clk mutants are "much longer lived" than wild-type controls on two of the relatively low calorie diets. Figure S3C certainly argues otherwise, and it's not clear how the data in 1C and S2C and warrant the use of "much" here.
This wording has been rephrased and corrected in the revised version. The low-calorie diet shown in Figure 1-figure supplement 3 contains a higher sucrose concentration (5%) than those used in Figure 1 and Figure 1-figure supplement 2 (1%). This observation suggests that sucrose may play an independent role in the survival of ClkJrk mutants under malnutrition conditions.
The authors should provide the rationale for the use of a dominant negative form of Clk for their experiments. Would the available amorphic allele be a better choice?
As ClkJrk is the first described Clk allele and it is probably the most well characterized. As a dominant negative version which is still capable of dimerizing and binding DNA it is less susceptible to compensation by redundant bHLH transcription factors as has been observed for between mouse Clock and NPAS2 (Debruyne et al, 2006).
It is not clear why the authors have chosen to examine transcriptomes so soon after transfer to DR. Why not wait longer. The authors provide context that changes are already taking place at the early time-point used, but would waiting a bit provide a more robust indication of how DR is changing the fat body?
We noted in the manuscript that the effects of DR on survival are evident relatively soon (~2d) after a diet shift. We were interested in identifying those changes in daily transcription that would be occurring during that early time span and potentially be a cause rather than an effect of survival changes.
