Dietary Restriction Impacts Peripheral Circadian Clock Output Important for Longevity in Drosophila

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.

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.

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.
- 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.

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?

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.