The epidermis is a highly regenerative barrier protecting organisms from environmental insults, including UV radiation, the main cause of skin cancer and skin aging. Here, we show that time-restricted feeding (RF) shifts the phase and alters the amplitude of the skin circadian clock and affects the expression of approximately 10% of the skin transcriptome. Furthermore, a large number of skin-expressed genes are acutely regulated by food intake. Although the circadian clock is required for daily rhythms in DNA synthesis in epidermal progenitor cells, RF-induced shifts in clock phase do not alter the phase of DNA synthesis. However, RF alters both diurnal sensitivity to UVB-induced DNA damage and expression of the key DNA repair gene, Xpa. Together, our findings indicate regulation of skin function by time of feeding and emphasize a link between circadian rhythm, food intake, and skin health.

Despite the circadian clock’s multiple roles in skin biology, other than the SCN, little is known of the factors that entrain the skin circadian clock. Restriction of food intake to defined time periods is known to change the phase of the circadian clock and gene expression programs, especially in primary metabolic organs such as the liver (). But not all peripheral tissues are entrained by RF (), and the effect of RF on the skin has not been investigated. Hence, we examined whether RF can entrain the circadian clock in skin and affect skin function. We discovered that RF can shift the circadian clock of skin but that the phase of the skin circadian clock is not as tightly coupled to feeding time (FT) as that of the liver. We found RF schedule-specific changes in the skin transcriptome, including changes in the expression of multiple metabolic genes and the nucleotide excision repair factor, Xpa. Although the phase of the cell cycle was insensitive to changes in circadian clock phase, RF decreased overall progenitor proliferation rates, and daytime RF reversed diurnal rhythm of epidermal sensitivity to UVB-induced DNA damage. This study points to unexpected influences of time of feeding on the biology of skin, suggesting that time of feeding may affect UVB-induced conditions such as skin cancer and premature aging.

Previous studies showed important roles for the circadian clock in skin biology (). The clock is highly active in the progenitor cells of the secondary hair germ, where it plays a role in the initiation of the hair growth cycle (). It also contributes to heterogeneity in hair follicle stem cells by regulating the sensitivity to activation signals (). In addition, in the matrix of growing hair follicles, the clock determines diurnal variation in cell division, which affects the sensitivity of hair follicles to external gamma radiation (). The circadian clock also gates the response to UVB radiation in the skin (), at least in part by controlling the expression of Xpa, a rate limiting enzyme involved in the repair of UVB-induced DNA damage (). In fact, the skin is most sensitive to UVB-induced tumor induction at night, when expression of Xpa is lowest (). Studies also showed a link between the circadian clock and skin aging as Bmal1-deleted mice exhibit accelerated skin aging (), perhaps related to excessive reactive oxygen species (ROS) generation. In the interfollicular epidermal progenitor cells, the clock is required for a prominent diurnal variation in DNA synthesis (). Although the function of these diurnal rhythms in epidermal progenitor cell DNA synthesis remains unknown, transcriptome studies from yeast to mammals have suggested that the circadian clock may coordinate the timing of different cellular processes (); in the case of epidermal progenitor cells, its role may be to synchronize intermediary metabolism and the cell cycle, thus minimizing cellular damage from oxidative phosphorylation-generated ROS (). The circadian clock, then, may be a mediator of the long-appreciated yet incompletely understood crosstalk between metabolism and the cell cycle ().

The hierarchically organized mammalian circadian clock comprises the central clock, located in the suprachiasmatic nucleus (SCN), and peripheral clocks, possessed by almost all cells (). Entrained by the day-night cycle, the central clock synchronizes the phases of peripheral clocks, thus coordinating the locomotor and metabolic activity of the animal with the Earth’s rotation. At a molecular level, the central and peripheral clocks are transcription-translation feedback loops wherein the heterodimeric CLOCK/BMAL1 transcription complex activates a large number of genes. These include PERs and CRYs that form heterodimers to inhibit CLOCK/BMAL1 activity, thus establishing an oscillating transcriptional output with 24 hr periodicity (). The direct and indirect targets of the circadian clock encode key regulators of many, if not most, biological processes, including metabolism (), cell proliferation (), and response to therapeutic treatment ().

Acting as a strong barrier to physical, chemical, and pathogenic insults from the exterior, and to water loss from the interior, the epidermis, the outermost layer of the skin, is a stratified epithelium. Its homeostasis is balanced by stem cell progeny production in the basal layer and loss of cells through terminal differentiation culminating in shedding of corneocytes at the skin’s surface (). Skin biology research focuses largely on the responses to various forms of external injury, including UV irradiation, a major cause of DNA damage, accelerated skin aging, and cancer (). Recent work, however, has unearthed an important role for the circadian clock in regulating skin function (). This raises the intriguing possibility that signals that influence circadian clocks, such as the time of feeding, could act as a regulator of skin function; the clocks in some peripheral tissues, especially metabolic organs, including the liver, can be entrained by time-restricted feeding (RF) ().

In sum, these results demonstrate that daytime RF affects the sensitivity to DNA damage in the skin of mice and dampens the expression of a key DNA repair factor.

Previous work in mice showed that sensitivity to UVB-induced DNA damage in the epidermis is diurnal with more damage when UVB is applied during the night than during the day (). This diurnal variation depends on the circadian clock as mutations in Bmal1 () and Cry1/Cry2 () obliterate the diurnal variation. To test whether daytime feeding, with its consequent shift in the phase of the clock, modulates the epidermal sensitivity to UVB-induced DNA damage, we applied UVB during the day (ZT9) and night (ZT21) to the shaved backs of AD, EN, ED, and MD mice, collecting the skin 15 min after UVB exposure. Consistent with previous studies (), mice that ate mainly (AD) or only (EN) at night formed more cyclobutane pyrimidine dimers (CPDs) when exposed to UVB during the night than during the day ( Figure 6 A). In contrast, mice fed during the day (ED and MD) exhibited a reverse pattern, forming more CPDs when exposed to UVB during the day than during the night ( Figure 6 A). Similar trends were observed in an earlier RF experiment with fewer mice in which we measured both CPDs and the second most common UVB-induced lesion, (6-4) photoproducts ([6-4]PP) ( Figure S5 ). Thus, while not altering the phase of S phase in epidermal progenitor cells, daytime RF reverses the diurnal rhythm of sensitivity to UVB-induced DNA damage. In addition, we found that the expression of Xpa, the gene encoding a rate-limiting protein necessary for nucleotide excision repair of UVB-induced DNA (), is dampened in EN, MD, and ED compared with AD. Furthermore, Xpa expression oscillates in a diurnal fashion in AD but not as robustly in the RF schedules ( Figure 6 B).

(B) Skin Xpa expression is dampened under RF, as detected by qPCR. Values represent mean ± SEM (n = 3–5). Two-way ANOVA shows significance for RF group (p < 0.0001), ZT time (p < 0.002), and RF group X ZT time (p < 0.02). Tukey’s post hoc test shows that AD has greater Xpa expression compared with EN (p < 0.0001) and MD (p < 0.0001), while AD is not significantly different from ED (p < 0.056).

(A) Quantification of CPD photoproducts after UVB exposure at ZT9 and ZT21. The shaved back skins of mice from AD, EN, ED, and MD feeding schedules were exposed to single dose of 500 J/m 2 UVB. Greater UVB-induced DNA damage is seen in AD and EN mice exposed to UVB at night versus the day, while mice fed during the day (ED and MD) have greater UVB-induced DNA damage when exposed to UVB during the day than during the night. Values represent mean ± SEM, n = 15. Statistical significance was determined using Welch’s t test, shown as ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

To determine whether the RF-induced phase shifts in the skin’s circadian clock alter the phase of the diurnal variation in epidermal progenitor cell proliferation, we measured the proportion of epidermal progenitor cells in S phase by BrdU staining over a 28 hr period. The proportion of cells in S phase is diurnal under each of the five RF schedules. Interestingly, despite the different phases in the circadian clock in different RF schedules, the phase of S phase was not shifted by RF ( Figure 5 A). We also evaluated whether RF affects the proliferation rate of interfollicular epidermal progenitor cells, finding that each of the RF schedules resulted in a similar and decreased peak and overall progenitor cell proliferation compared with AD ( Figure 5 B). Together with previous findings showing that the circadian clock is required for diurnal DNA synthesis rhythms in epidermal progenitor cells (), these results indicate that whereas the clock is critical for establishing diurnal variation in progenitor cell proliferation, it alone does not control the phase of the cell cycle.

(B) The average percentage of BrdU-positive epidermal cells in each RF schedule. Values represent mean ± SEM, n = 4. AD had higher proliferation rate than all RF feeding schedules. Statistical significance was determined using Welch’s t test, shown as ∗∗ p < 0.01 and ∗∗∗ p < 0.001.

(A) BrdU-positive cells were counted in the epidermis by immunohistochemistry. Values represent mean ± SEM, n = 2–5. The proportion of cells in S phase is diurnal under each of the five different RF schedules (one-way ANOVA p < 0.01). The dots above the curves indicate the peak time of BrdU incorporation. Values represent mean ± SEM, n = 4. Watson-Williams test was used to compare the peak times. The phase of diurnal rhythms in BrdU incorporation were unchanged among the four RF schedules.

Previous studies showed diurnal changes in epidermal progenitor cell proliferation with 3- to 4-fold greater number of cells in S phase during the night than during the day (). Deletion of Bmal1, either constitutionally or selectively in epidermal cells, obliterates the diurnal variation in cell proliferation, indicating that the circadian clock controls the diurnal variation in epidermal progenitor cell proliferation ().

We identified 2,026 exons differentially regulated by food intake ( Figures 4 B, S4 A, and S4B). Exons showing the most significant change in response to feeding were linked to metabolism ( Figure 4 C), including Pdk4, Ucp3, and Scd2. The food intake-affected exons fell into two groups: those that decreased (998) after food intake and those that increased (1,028) after food intake ( Figures 4 B–4E, S4 A, and S4B). Genes showing decreased expression after food intake were overrepresented in GO categories, including response to starvation, autophagy, response to oxidative stress, negative regulation of cell proliferation, and lipid oxidation ( Figure 4 D; Table S3 ), and those showing increased expression included lipid biosynthesis and protein synthesis ( Figure 4 E; Table S3 ). These results indicate that the metabolism of skin is oxidative before feeding and becomes anabolic after feeding. We also identified 1,890 introns and 662 antisense transcripts affected by food intake ( Figures S4 A and S4B). About half of the food intake-affected genes were identified as having diurnal expression in one or more of the three feeding schedules ( Figure S4 C), suggesting that food intake contributes to regulation of the diurnal transcriptome in the skin. In conclusion, these data indicate that expression of many genes in the skin, including those involved in oxidative-reductive metabolism and cell proliferation, respond acutely to food intake, and that the metabolic status of skin is determined by feeding.

We next considered the possibility that food intake could acutely affect skin gene expression. To search for such effects in the data, we rearranged the RNA-seq reads across all three feeding schedules according to the feeding time ( Figure 4 A), generating feeding time series (FT0–FT4–FT8), where FT0 represents the initiation of feeding and FT4 and FT8 represent times 4 and 8 hr after the start of feeding.

(D and E) Graphs of representative enriched GO categories for genes downregulated (D) or upregulated (E) after feeding. The numbers at the end of the bars refer to the number of genes affected in each category.

(B) A total of 2,026 exons were affected by feeding on the basis of the regrouping described in (A) (one-way ANOVA p < 0.01). Shown is the heatmap of food intake-affected genes at FT0, FT4, and FT8. The color key represents the Z score of expression value, with red being highly expressed genes and green being minimally expressed genes.

(A) A schematic showing how the RNA-seq reads from data presented in Figures 2 and 3 were regrouped and analyzed on the basis of the timing of food availability. Feeding time zero (FT0) indicates reads immediately before food availability, feeding time 4 (FT4) indicates reads 4 hr after the onset of food availability, and feeding time 8 (FT8) indicates reads 8 hr after the onset of food availability, as described in Experimental Procedures . Feeding groups are indicated below each column. The white-black bar indicates day and night, respectively.

Together, these data indicate that RF shifts the phase of most diurnal genes in skin by transcriptional mechanisms. The RF-affected genes, although participating in similar functions, are largely unique to each RF schedule. In addition, we identified two antisense transcripts that may have a regulatory relationship with the gene from which they are encoded.

We found only 37 antisense transcripts with diurnal expression in all three feeding schedules ( Figure S3 D). Some of these, including the clock regulator Dec2 (also known as Bhlhe41), show expression in phase with the exons ( Figure 3 C) while others, including Erc2, which is involved in regulation of neurotransmitter release, show antiphasic expression to the exons ( Figure 3 D).

We also identified diurnal intron ( Figure S3 C; Table S2 ) and antisense ( Figure S3 D; Table S2 ) transcripts. Because transcription affects both intron and exon components of transcripts, the comparison of phase shifts between introns and exons in a gene gives insights into the extent to which the phases of diurnal genes and biological processes under different RF schedules are controlled at a transcriptional or a post-transcriptional level. Overall, we found that the phase shift in peak expression in exons and introns of a gene was correlated (Pearson R = 0.68), suggesting that transcriptional regulation is an important mechanism underlying food entrainment in the skin ( Figures 3 A and S3 E–S3G; Table S2 ). This mode of regulation was reflected in our Gene Ontology (GO) analysis showing that the circadian clock genes are clearly regulated transcriptionally ( Figure 3 B). However, there were a number of genes that were exceptions to this general rule, as exemplified by cytokinesis genes, which appear to be regulated post-transcriptionally ( Figure 3 B).

(C and D) The expression of the exon and antisense transcripts for two diurnally expressed genes, Dec2 (C) and Erc2 (D), featuring diurnally expressed antisense transcripts in all three RF schedules. For Dec2, the exon and antisense oscillations are in phase with one another, while for Erc2, the exon and antisense have antiphasic oscillations.

(B) Enrichment of biological processes was determined for genes with diurnally expressed introns and exons by Fisher’s exact test. Rows are GO terms, and columns are RF schedules. Enrichment factor was determined as the ratio of diurnal genes in a term over all genes in a term; this is represented by circle size. The color of the circles indicates p value.

(A) Linear correlation of the time-shift of exon and intron expression under different RF schedules. Examples of genes for which the differential timing of intron and exon expression is greater than 4 hr are indicated with gene symbols.

(B–E) Heatmaps of the diurnal exons common under all (B) or two (C–E) of the RF schedules (corresponding to the B–E labels in [A]). Note that in (B), most genes in MD are phase advanced, while those in ED are phase delayed compared with EN. The white-black bars below indicate day and night, respectively. Colored bars under the heatmaps indicate the time of food availability. The color key for the heatmaps show the Z score of expression value, where red is highly expressed and green is minimally expressed. The colored lines in black bars to the right of the heatmaps indicate genes annotated to cell death (CD), reduction-oxidation (RO), cell cycle (CC), and circadian clock (CL) biological processes.

(A) Venn diagram depicting the overlap of diurnal exons expressed in skin of mice after time-restricted feeding. Of 7,317 total diurnally oscillating exon transcripts, 147 are common within all 3 RF schedules. Exons for core circadian clock genes (such as Clock, Npas2, and Arntl) maintained diurnal rhythm in all RF schedules (core clock gene examples listed in [B], to the right).

To define the diurnal transcriptome of skin under different RF schedules, we performed RNA sequencing (RNA-seq) on telogen skin collected every 4 hr for 28 hr under the three 4 hr RF schedules: ED, MD, and EN ( Figure 1 A; Table S1 ). We selected mice from these RF schedules for further study in order to minimize effects of differences in caloric intake (see Experimental Procedures ). EN mice were selected as the control because their circadian phase and time of feeding is similar to that of AD mice ( Figures 1 A and 1B); AD mice feed mainly during the early night (). Analysis of ED and MD mice allows us to observe the effect of maximum diurnal phase shifts as their circadian phases are nearly 9 hr apart in skin ( Figures 1 B and S2 A).

Collectively, these data demonstrate that time of feeding influences the phase and peak expression of the skin circadian clock in manner distinct from that of the liver. Although feeding appears to be a direct zeitgeber for the liver with Per2 expression having a constant relation to feeding time, there is a less direct relationship between the initiation of feeding and the phase of the skin circadian clock.

To determine if the shift in the phase of the circadian clock was consistent in the skin and liver, we compared the phase shift of Per2 in the RF groups relative to AD or EN, and found that although MD exhibited the same phase advance in skin and liver, the phase shifts for ED and LD were different in these organs ( Figure S2 C). Expression results for core clock genes Dbp ( Figures S2 D–S2F) and Per1 ( Figures S2 G–S2I) matched the Per2 results, indicating a true phase shift of the core clock machinery. We also studied the phase and peak expression of Per2 in isolated epidermis of EN and ED, finding that they are similar to that in whole skin ( Figures S2 J, S2K, 1 B, and 1C).

After implementing these RF schedules for 18–21 days, we harvested skin and liver from cohorts of mice every 4 hr for 28 hr starting at ZT0. We then determined the circadian clock phase by analyzing the peak time of skin mRNA expression of Per2, a commonly used indicator of circadian phase. The phase of Per2 in mice fed during the night (EN) was equivalent to that of AD ( Figure 1 B). Using AD as a reference, we found that MD induced a phase advance on average of 4.19 ± 0.43 hr; in contrast, ED caused a phase delay on average of 4.72 ± 0.38 hr ( Figures 1 C and S2 A). The phase of Per2, then, was almost 9 hr apart for MD and ED, the groups with the most widely separated phases ( Figure 1 B). The magnitude of phase advances was the same in LD and MD ( Figure S2 A). We also found that the amplitude of Per2 was significantly lower in day-fed mice compared with EN ( Figure 1 C). Using either AD or EN as a reference, the phase shift of Per2 in ED was significantly different compared with LD and MD ( Figure S2 A). By contrast, in the liver, the phase of Per2 expression in all feeding groups was tightly linked to the time of initiation of food intake ( Figures 1 D and S2 B), and the peak expression of neither ED nor LD was significantly different from EN ( Figure 1 E).

To evaluate the effect of RF schedules on body weight and food intake, we recorded food intake daily and body weight immediately prior to food availability every other day for 21–26 days for the AD, EN, MD, and ED groups ( Figures S1 A and S1B). Both MD and ED mice ate significantly less than the AD mice during the first 2 days of RF, but by the end of the experiment, there was no significant difference in food intake across the groups ( Figure S1 A). All groups weighed approximately the same prior to the beginning of the RF (data not shown), but throughout the RF experiment, body weight was significantly affected by RF schedule: AD weighed more than all RF groups, ED and EN had approximately equal body weight, and MD weighed consistently less than all other groups ( Figure S1 B). We performed two RF experiments as described in Experimental Procedures , with similar results for body weight and food intake in both experiments (data not shown). We also measured skin compartment width (epidermis; dermis, including dermal fat layer; and muscle layer) by histology and found no significant changes except that dermis width was decreased by about 16%–17% in EN and MD compared with AD ( Figures S1 C–S1F).

To determine whether RF can shift the phase of the skin circadian clock, we administered five different feeding schedules ( Figure 1 A). The ad libitum (AD) group of mice had unlimited access to food. The early daytime (ED) feeding group had access to food from zeitgeber time (ZT) 0 for 4 hr. The midday (MD) feeding group had access to food from ZT5 for 4 hr. The early nighttime (EN) feeding group had access to food starting at ZT12. Finally, the long daytime (LD) feeding group had access to food from ZT3 for 8 hr. These feeding lengths and times were modeled on previous RF studies ().

(B–E) Per2 gene expression in the skin (B and C) and liver (D and E) as measured by qPCR. (B and D) The values represent mean ± SEM, n = 3–5. The peak time, a proxy for circadian phase, is shown above the curves as mean ± SEM, n = 4. The Watson-Williams test was used to compare the peak times. (C and E) The peak expression values from (B) and (D) are shown as mean ± SEM, n = 4. Statistical significance comparing peak expression values was determined using Welch’s t test, shown as ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

(A) Schematic showing the RF schedules. Colored boxes indicate timing of food access: AD, ad libitum; ED, early day; MD, midday; EN, early night; and LD, long day. Day and night are indicated below with white and black bars. The sampling time points are indicated by ZT on the x axis.

Discussion

This work shows that time of feeding is an important regulator of skin function. As summarized in Figure 6 C, we found that (1) RF shifts the phase of the skin circadian clock, in a pattern distinct from that of the liver; (2) RF alters the expression of many diurnally expressed genes in the skin, including that of the key DNA repair factor Xpa; (3) feeding acutely causes large-scale gene expression changes in the skin, most prominently of metabolic genes; and (4) daytime RF reverses the time-of-day-dependent sensitivity to UVB-induced DNA damage in the skin. Together, our results indicate that timing of food intake has a more pronounced influence on skin biology than previously recognized, representing a modifiable regulator of skin health.

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Okamura H. Molecular clocks in mouse skin. Studies focusing on the liver, a key organ for organismal metabolism, showed that the phase of the circadian clock is entrained by RF () and that a significant portion of the liver transcriptome is affected by the time of feeding (). The effect of RF on clocks in peripheral organs not primarily involved in organismal metabolism is less well studied (); in particular, there are no such studies in the skin. Our findings indicate that both the phase and peak expression of the skin circadian clock are distinct from that of the liver. In the skin, day-fed mice (MD, ED, and LD) exhibit lower amplitude of the core clock gene Per2 than mice fed during the night (EN), while in the liver, Per2 amplitude does not seem to be sensitive to time of feeding. Furthermore, mice fed during the ED exhibited a 4.7 hr phase delay in Per2 expression compared with AD mice in the skin, while in the liver the phase of Per2 expression is advanced by 10.9 hr, corresponding to the initiation of food intake. These findings suggest that RF controls the phase of the skin circadian clock by different mechanisms than in the liver, where feeding appears to be a more direct and dominant cue ( Figure 1 ). These observations suggest that in addition to regulation of the skin clock by the SCN’s central clock (), the skin clock is independently modulated by the timing of food intake. The exact mechanism by which feeding time controls the skin clock likely involves many factors such as physical activity, sleep-wake cycles, metabolism, and circulating hormones.

Comparison of the telogen skin transcriptome under three different RF schedules (ED, MD, and EN) reveals that feeding schedule dictates the diurnal expression of approximately 10% of the skin transcriptome ( Figure 2 ). Specifically, RF schedules generate diurnal gene rhythms that are largely unique to each RF schedule while dampening diurnal expression of other genes that are diurnal in the other RF schedules ( Figure 2 ). Furthermore, while feeding time largely defines the identity of individual genes with oscillating expression, the functional categories annotated to these genes are similar among the three RF schedules. We also found that some diurnally expressed genes are conserved in all the three RF schedules; these genes are enriched for regulators of the circadian clock. The phase shift of the clock-related transcripts under the three RF schedules correlates well with the phase shift of the core circadian clock as indicated by the peak time of Per2 expression ( Figure 1 ). In addition, the shift of expression of exons is linearly correlated with that of introns, indicating that shift of transcriptional activity is an important mechanism underlying the shift of the whole transcriptome ( Figure 3 ). Genes annotated to regulation of circadian rhythm tend to be diurnally expressed in introns and exons, while some genes annotated to the cell cycle, especially cytokinesis, tend to be diurnally expressed in exons and not introns. These findings suggest that different diurnal processes may be regulated to varying extents by both transcriptional and/or post-transcriptional mechanisms.

In contrast to exons, there are very few diurnal antisense transcripts. Also, the expression of antisense transcripts is largely uncorrelated with the expression of either introns or exons of the corresponding diurnal genes. We have identified a few antisense genes, however, that exhibit unique diurnal expression in relation to their corresponding genes, indicating a potential regulatory role.

We observed striking changes in skin gene expression directly in response to food intake ( Figure 4 ). This suggests that at least part of the RF schedule-mediated changes in gene expression are a direct feeding effect, although we cannot rule out contributions by changes in rhythms of activity level and sleep caused by restricted food availability. An analysis of these gene expression changes indicates that after feeding, cellular metabolism becomes biosynthetic and reductive. Instead of oxidation of fatty acids, the skin transcriptome becomes more characteristic of the synthesis and import of lipids, especially steroids, which are involved in cellular membrane systems. In addition, in response to food intake, genes involved in transcription, translation, and protein folding and localization are upregulated and those involved in apoptosis are downregulated. This analysis indicates that components of the global diurnal gene expression program are acutely responsive to food intake.

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Kovacs C.J. Digestive tract cell proliferation and food consumption patterns of Ha/ICR mice. In previous studies, we showed that the circadian clock intrinsic to keratinocytes is required for the diurnal fluctuation in the proportion of epidermal progenitors undergoing S phase (). Interestingly, in the present study, we find that despite the shift in the phase of the circadian clock by RF, the rhythm of DNA synthesis in epidermal progenitors of the skin did not shift ( Figure 5 ). Together these findings indicate that while an intact clock is required for the diurnal variation in DNA synthesis, the phase of the clock is not the dominant regulator of the phase of the S phase oscillations in the mouse skin. These findings are in agreement with studies showing that the cell mitotic cycle can be uncoupled from the circadian clock in immortalized rat-1 fibroblasts () and Lewis lung carcinoma cells () and a recent study showing that cell division cycles could be gated by WNT-signaling (). Although a previous study showed that RF can shift the daily proliferative rhythm of the digestive tract (), in the gut, cell proliferation may be mechanically stimulated by food.

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Gratton E. In vivo single-cell detection of metabolic oscillations in stem cells. Given our finding that oxidative metabolism in the skin is affected by RF, it is likely that a deviation in the time of food intake may lead to asynchrony between oxidative metabolism and DNA replication, which normally is coordinated with cell cycle stages in epidermal progenitors (). We hypothesize that such asynchrony between the timing of peak oxidative phosphorylation metabolism and cell division due to unusual feeding times could contribute to increased ROS-mediated DNA damage in progenitor and stem cells, leading to aging and carcinogenesis.

In conclusion, our findings show that RF (EN, ED, and MD) decreases the proportion of cells in S phase and dampens expression of DNA repair factor Xpa. In addition to these changes, daytime RF (ED and MD) also shifts the phase of core clock genes and oxidative metabolism genes and reverses the rhythm of sensitivity to UVB-induced DNA damage. By disrupting the natural expression and diurnal variation of such important processes in the skin, abnormally timed food intake may contribute to the development of skin pathologies involving sun damage, skin aging, and skin cancer.