Attenuated SIRT1 Activity Leads to PER2 Cytoplasmic Localization and Dampens the Amplitude of Bmal1 Promoter-Driven Circadian Oscillation

The circadian clock possesses robust systems to maintain the rhythm approximately 24 h, from cellular to organismal levels, whereas aging is known to be one of the risk factors linked to the alternation of circadian physiology and behavior. The amount of many metabolites in the cells/body is altered with the aging process, and the most prominent metabolite among them is the oxidized form of nicotinamide adenine dinucleotide (NAD+), which is associated with posttranslational modifications of acetylation and poly-ADP-ribosylation status of circadian clock proteins and decreases with aging. However, how low NAD+ condition in cells, which mimics aged or pathophysiological conditions, affects the circadian clock is largely unknown. Here, we show that low NAD+ in cultured cells promotes PER2 to be retained in the cytoplasm through the NAD+/SIRT1 axis, which leads to the attenuated amplitude of Bmal1 promoter-driven luciferase oscillation. We found that, among the core clock proteins, PER2 is mainly affected in its subcellular localization by NAD+ amount, and a higher cytoplasmic PER2 localization was observed under low NAD+ condition. We further found that NAD+-dependent deacetylase SIRT1 is the regulator of PER2 subcellular localization. Thus, we anticipate that the altered PER2 subcellular localization by low NAD+ is one of the complex changes that occurs in the aged circadian clock.


INTRODUCTION
The oxidized form of nicotinamide adenine dinucleotide (NAD + ) found in all living cells plays critical roles in a wide range of physiological processes. NAD + acts as a coenzyme for enzymes which are involved in energy metabolism and homeostasis pathways such as glycolysis, TCA cycle, and oxidative phosphorylation (Campbell, 1995). NAD + also acts as a co-substrate for enzymes such as sirtuins (SIRTs) and poly(ADP-ribose) polymerases (PARPs) to regulate a wide array of cellular processes such as survival/cell death, circadian clock, and aging (Imai and Guarente, 2014;Verdin, 2015). In mammals, cellular NAD + is primarily biosynthesized by the NAD + salvage pathway, in which nicotinamide phosphoribosyltransferase (NAMPT) is the ratelimiting enzyme and converts nicotinamide into nicotinamide mononucleotide (NMN), the NAD + precursor (Revollo et al., 2007;Imai and Guarente, 2014;Nakahata and Bessho, 2016).
NAD + amount declines during the aging process, causing defects in nuclear and mitochondrial functions and resulting in many age-associated diseases (Gomes et al., 2013;Mouchiroud et al., 2013). Intriguingly, administration of NAD + precursors such as NMN and nicotinamide riboside (NR) can ameliorate many age-associated pathologies, thereby leading to healthy longevity (Yoshino et al., 2018;Custodero et al., 2020;Hong et al., 2020). Similar to organismal aging, NAD + amount is also reported to decline with cellular aging, i.e., cellular senescence (Khaidizar et al., 2017). Overexpression of NAMPT in primary fibroblast cells delays the onset of replicative senescence (van der Veer et al., 2007;Khaidizar et al., 2017) and confers a protective effect against stress-induced premature senescence (Nuriliani et al., 2020). These reports demonstrate that a decline in NAD + is one of the causes to induce cellular senescence as well as organismal aging.
The aforementioned findings prompted us to reveal the molecular mechanisms of how NAD + amount, especially low NAD + , affects the circadian clock system, which might give some hints to unravel molecular links between aging and circadian clock. In this study, we reduced intracellular NAD + amount by inhibiting NAMPT enzymatic activity and found that it attenuates the amplitude of Bmal1 promoter-driven luciferase oscillation and promotes cytoplasmic localization of PER2, leading to attenuated CRY/PER-dependent repression, thereby enhancing E-box-regulated circadian gene expressions. At the molecular level, we revealed that the translocation of PER2 with low NAD + is dependent upon SIRT1 activity, but not PARP1 activity.

Cell Culture
NIH3T3 cells were cultured in DMEM-4.5 g/L glucose (Nacalai Tesque, Japan) supplemented with 10% FBS (Sigma) and antibiotics (100 units/ml penicillin, 100 µg/ml streptomycin, Nacalai Tesque, Japan) at 37 • C and 5% CO 2 in a humidified incubator. Cells were treated with FK866/GMX1778 with or without 1 mM nicotinamide mononucleotide for 24 h and harvested for measuring NAD + amount, performing qPCR, immunofluorescence, and western blotting. For experiments analyzing circadian oscillations of clock genes, luciferase, and PER2 protein, cells were pretreated with FK866 for 24 h before Dex synchronization and also treated with FK866 after synchronization.

SDS-PAGE, Western Blotting Analysis, RNA Extraction, and qPCR
Protocols for SDS-PAGE, western blotting analysis, RNA extraction, and qPCR were followed as described previously (Khaidizar et al., 2017

Real-Time Luciferase Monitoring Assay
The method was described previously (Ahmed et al., 2019).

Immunofluorescence
NIH3T3 cells were seeded on a sterilized cover glass (Matsunami; 24 × 24 mm; thickness no. 1) placed on a six-well plate so as to have 4 × 10 5 cells/well as a single experiment. Cells were co-transfected with indicated expression vectors using FuGENE HD (Promega) and cultured for 48 h. The cells after washing twice with phosphate buffered saline (PBS) were fixed with 1 ml of 4% paraformaldehyde for 15 min with shaking at room temperature. Then, cells were washed three times with PBS and permeabilized with 1 ml of 0.5% Triton X-100 for 10 min with shaking at room temperature. After blocking the cells with 1% bovine serum albumin (BSA) for 30 min at room temperature, the cells were washed three times with PBS and incubated with Myc antibody diluted with 1% BSA/PBS for 1 h at room temperature. Then, cells were incubated with secondary antibody conjugated with Alexa Fluor Plus 488 diluted with 1% BSA/PBS for 1 h at room temperature. Nuclei were also stained with 1 µg/ml Hoechst 33342 (Nacalai Tesque, Japan) to justify subcellular localizations of clock proteins. Fluorescent images were acquired with a confocal laser scanning microscope (ZEISS; LSM 700), and subcellular localizations of clock proteins were counted at least 100 cells. We interpreted the result for nuclear localization of PER2/CRY1 (N) as positive when fluorescence intensity of PER2/CRY1 signal that overlapped with Hoechst 33342 signal was higher than the fluorescence intensity of PER2/CRY1 in the cytoplasm (Supplementary Figure 1).
Meanwhile, we interpreted the result for cytoplasmic localization of PER2/CRY1 (C) as positive when PER2/CRY1 signal that did not overlap with Hoechst 33342 signal exhibited higher fluorescence intensity than PER2/CRY1 signal intensity that overlapped with Hoechst 33342 signal. Finally, we interpreted the result for nuclear and cytoplasm localization of PER2/CRY1 (N + C) as positive when PER2/CRY1 fluorescence signal at both nucleus and cytoplasm had the same intensities. We performed the experiments independently for at least three times and showed the data as mean ± SEM.
Nuclear Extract From NIH3T3 Cells NIH3T3 cells treated with or without 7.5 nM FK866 for 24 h in a 10-cm dish were synchronized with 100 µM dexamethasone for 1 h. Synchronized cells after 24-44 h were collected every 4 h. Cells were washed twice with PBS and harvested by cell scrapers, centrifuged at 300 × g for 5 min at 4 • C, and resuspended in 50 µl lysis buffer [10 mM HEPES (pH 7.9), 1.5 mM MgCl 2 , 10 mM KCl]. Cells were vortexed for 10 s and allowed to stand on ice for 10 min, and the same step was repeated again to extract cell components other than the nuclear fraction. The nuclear fraction was washed three times with lysis buffer, added with 50 µl RIPA buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl 2 , 1 mM EDTA, 0.2% SDS, 1% NP-40], vortexed for 30 s, and then allowed to stand on ice for 20 min. The nuclear fraction was centrifuged at 15,000 × g for 5 min at 4 • C, and the supernatant was collected as a nuclear extract. Total cell extract was obtained by extracting cells with RIPA buffer.

Dual-Luciferase Promoter Assay
NIH3T3 cells seeded in a 24-well plate were co-transfected with 500 ng of Per1 firefly reporter, 50 ng of EF1a Renilla reporter, and indicated expression vectors using FuGENE HD (Roche). Samples were prepared using a Dual-Luciferase promoter assay kit (Promega) according to their instructions and analyzed by a multiwell luminescence measurement plate reader (Mithras, LB940). Firefly luciferase activity of the reporter was normalized by the activity of Renilla luciferase under control of EF1α promoter.

Establishment of Sirt1 Knockdown Cell
To establish Sirt1 knockdown cells, lentivirus vector pLLX-shRNA (Zhou et al., 2006), which expresses GFP and a puromycin resistant gene, was used. After virus infection to Bmal1-luc/NIH3T3 (Yoshitane et al., 2012), cells were selected by puromycin for 1 week and confirmed by GFP fluorescence (all cells were ∼100% GFP positive). Three different target sequences for mouse Sirt1 and scramble sequence are shown in Supplementary Table 2.

Statistics
Values are reported as mean ± SEM. Statistical differences were determined by a Student's two-tailed t-test. Statistical significance is displayed as * p < 0.05, * * p < 0.01, or * * * p < 0.001.

RESULTS
To investigate how the circadian clock system is affected by low NAD + in cells, we first evaluated how much NAD + was decreased by the inhibition of the rate-limiting enzyme, NAMPT, in the NAD + salvage pathway ( Figure 1A). We used FK866 (Hasmann and Schemainda, 2003) to inhibit the NAMPT activity and found that 2.0, 5.0, and 7.5 nM FK866 treatment decreased NAD + to approximately 50, 33, and 25%, respectively ( Figure 1B). In addition, the acetylated form of BMAL1, which has been reported to be increased by FK866 treatment (Nakahata et al., 2009), was increased by FK866 treatment in a dosedependent manner (Supplementary Figure 2A). We further confirmed that the effect of FK866 treatment at 7.5 nM on NAD + amount was abrogated by the co-treatment with the NAD + precursor, NMN ( Figure 1C). A similar tendency was observed by another NAMPT inhibitor, GMX1778 (Watson et al., 2009) (Supplementary Figures 3A,B). We then performed a real-time luciferase assay to address whether circadian clock properties are affected by low NAD + . To address that, we used NIH3T3 cells stably expressing Bmal1 promoter-driven luciferase (Yoshitane et al., 2012). The amplitude, which was defined in this study as the height from the first peak to the first trough, of Bmal1 promoter-driven luciferase oscillation was attenuated in FK866-treated cells, compared with that in control cells (Figures 1D,E). A delayed phase was also observed in FK866-treated Bmal1-luc oscillation. Intriguingly, the attenuated amplitude and delayed phase were abrogated in FK866 and NMN co-treated cells, indicating that the amplitude and phase are regulated by NAD + amount. The reason why these alterations were abrogated after 60 h Dex treatment is presumably due to the recovery of intracellular NAD + amount (Supplementary Figure 4). We further checked endogenous circadian gene expressions in unsynchronized cells with low NAD + . Among core circadian genes, E-box-regulated genes, such as Rev-erbα, Rev-erbβ, Per1, and Per2, were upregulated; however, the RORE-regulated gene, Bmal1, was downregulated ( Figure 1F). Moreover, these alterations were abrogated in FK866 and NMN co-treated cells, indicating that the alterations of these genes are regulated by NAD + amount. Furthermore, we demonstrated that circadian profiles of endogenous Bmal1 and Rev-erbα transcripts were also affected, lower Bmal1 and higher Rev-erbα, in synchronized cells with low NAD + ( Figure 1G). These results demonstrate that low NAD + alters transcript levels of not only E-box-regulated genes as we have reported previously (Nakahata et al., 2008), but also RORE-regulated genes, which presumably regulate the amplitude of circadian clocks.
As our previous studies have demonstrated that the circadian gene expressions are epigenetically regulated by NAD +dependent deacetylases, SIRT1 and SIRT6 (Nakahata et al., 2008;Masri et al., 2014), in this study, we sought to find out whether the properties of circadian clock proteins, such as protein stability and subcellular localization, are regulated by NAD + amount. First, whether low NAD + affects protein stability of clock proteins was investigated using cycloheximide, which is an inhibitor of protein biosynthesis due to its prevention in translational elongation. However, the stabilities of CLOCK, NAD + amount was normalized by protein amount. (C) NAD + amount after 24 h treatment of 7.5 nM FK866 with or without 1 mM NMN was measured by HPLC. NAD + amount was normalized by protein amount. (D) Bmal1-luc oscillation patterns in NIH3T3 cells treated with FK866 with or without NMN were monitored by using a real-time luciferase monitoring system. One representative result is shown for each condition. (E) Relative amplitudes were analyzed. In this study, the amplitude was defined as the height from the first peak to the first trough. The value of control (ctrl) was set to 1. (F) Circadian gene expression levels after treatment with 7.5 nM FK866 alone or 7.5 nM FK866 and 1 mM NMN were quantified by qPCR. Each sample was normalized by the amount of 18S rRNA. Each gene expression level in untreated cells (ctrl) was set to 1. (G) Circadian profiles of Bmal1 and Rev-erbα synchronized by DEX were quantified. Each sample was normalized by 18S rRNA. Time 12 of control cells was set to 1 for each gene. All data represented here, except (E) (n = 4), are the mean ± SEM of three independent samples. * p < 0.05, * * p < 0.01, and * * * p < 0.001, compared to each of the untreated control by Student's two-tailed t-test.
BMAL1, PER2, and CRY1 were not significantly affected by low NAD + (Supplementary Figure 5). Next, whether low NAD + affects subcellular localization of clock proteins was investigated [(A-D), bottom] Subcellular localizations were counted and quantified; N, nucleus; C, cytoplasm; N + C, both nucleus and cytoplasm. The data represented are the mean ± SEM of at least three independent samples. * p < 0.05, * * p < 0.01, compared to each subcellular localization in control cells by Student's two-tailed t-test.
(E) PER2 protein levels in nuclear extract and total cell lysate were detected under the 7.5 nM FK866-treated condition (top). PER2 protein levels were quantified using ImageJ software (bottom). Each sample was normalized by LAMIN B1 protein level. TUBULIN was used to confirm fractionation. The data represented as filled circle and triangle are individual data of two independent samples and bars are the average of two samples. (Figures 2A,B and Supplementary Figure 6). Among clock proteins tested in this study, subcellular localization of PER2 was clearly affected by FK866 treatment (Figure 2A). Cytoplasmic and nuclear PER2 under control condition were 18.8 ± 3.4 and 49.3 ± 4.2%, respectively, whereas cytoplasmic and nuclear PER2 under FK866-treated condition were 32.0 ± 7.9% (p = 5.1 × 10 −3 , compared with cytoplasmic PER2 under control condition) and 34.5 ± 9.2% (p = 3.8 × 10 −3 , compared with nuclear PER2 under control condition), respectively. Furthermore, GMX1778 treatment also changed the ratio of PER2 subcellular localization from 22.2 ± 0.5 to 26.3 ± 0.1% in the cytoplasm (p = 1.0 × 10 −4 ) and from 62.0 ± 1.9 to 50.0 ± 0.6% in the nucleus (p = 2.0 × 10 −4 ) (Supplementary Figure 7). Importantly, the effect of FK866 or GMX1778 on PER2 subcellular localization was abrogated when cells were treated with both FK866 and NMN or GMX1778 and NMN (Figure 2A and Supplementary Figure 7). These results indicate that PER2 subcellular localizations are controlled by intracellular NAD + amount; lower NAD + promotes the translocation of PER2 from the nucleus to the cytoplasm. Similarly, cytoplasmic and nuclear CRY1 under control condition were 6.3 ± 0.9 and 75.6 ± 3.9%, respectively, while cytoplasmic and nuclear CRY1 under FK866treated condition were 10.6 ± 2.8% (p = 0.026, compared with cytoplasmic CRY1 under control condition) and 67.1 ± 8.5% (p = 0.12, compared with nuclear CRY1 under control condition), respectively ( Figure 2B), indicating the similarity to the change in PER2 by FK866 treatment. These results prompted us to investigate whether the increase in PER2 cytoplasmic localization triggered by FK866 induces the increase in CRY1 cytoplasmic localization, because it is well known that PER2 determines CRY1 localization (Albrecht et al., 2007). Cytoplasmic localizations of CRY1 co-expressing PER2 with or without FK866 were 15.4 ± 0.7 and 10.8 ± 0.8% (p = 4.8 × 10 −3 ) and nuclear localizations were 71.8 ± 2.2 and 81.7 ± 1.3% (p = 8.6 × 10 −3 ), respectively ( Figure 2C). The alteration of CRY1 subcellular localization by FK866 treatment was more when PER2 was co-expressed, suggesting that the alteration of PER2 subcellular localization leads to that of CRY1.
Since PER2 protein amount and subcellular localization show time-of-day variations, whether circadian subcellular localization of PER2 is affected by NAD + amount was next investigated ( Figure 2E). Although nuclear localization of PER2 clearly showed the time-of-day variation under the control condition as reported previously (Lee et al., 2001;Yagita et al., 2001), the nuclear accumulation of PER2 was reduced, but still showed circadian oscillation, under the FK866-treated condition ( Figure 2E, left panel). Intriguingly, PER2 in total cell lysate under the FK866-treated condition was comparable with that under the control condition ( Figure 2E, right panels), suggesting that the reduction of PER2 nuclear localization under the FK866treated condition is not due to the reduction of PER2, but the decrease in PER2 nuclear entry and/or the increase in PER2 nuclear export.
CRY acts as a transcriptional repressor with PER against CLOCK/BMAL1-dependent, namely E-box-regulated, transcriptions. As the decrease in nuclear localization of PER2 by FK866 suggested the attenuation of PER/CRY-dependent repression against E-box-regulated genes, we next addressed that possibility. To address that, luciferase reporter assay using Per1 promoter that possesses several E-boxes was assessed using PER2-NES, which mimics PER2 subcellular localizations under the FK866-treated condition. As reported previously, Per1 promoter activity was activated by CLOCK/BMAL1 and this activation was repressed by CRY1 (Figure 3). When PER2-NES was co-expressed, the repression by CRY1 against CLOCK/BMAL1-dependent Per1 expression was attenuated; namely, the luciferase intensities were increased in a PER2-NES dose-dependent manner (Figure 3). On the other hand, when PER2-WT or PER2-NLS was co-expressed, the repression by CRY1 was not affected. The results from Figures 2E, 3 indicate that PER2 subcellular localization determines PER/CRYdependent repression potential, and support that the mechanism shown; 100 ng of CLOCK, 100 ng of BMAL1, 10 ng of CRY1, 500 ng of Per1 firefly reporter, and 50 ng of EF1a Renilla reporter were co-transfected in NIH3T3 cells. Indicated amounts of PER2-WT, PER2-NLS, or PER2-NES were also co-transfected in NIH3T3 cells. Firefly luciferase intensity was normalized by Renilla luciferase intensity. Basal per1 promoter activity (far left bar) was set to 100. The data represented are the mean ± SEM of three independent samples. * p < 0.05, * * p < 0.01, and * * * p < 0.001, compared with the sample with CLOCK/BMAL1 and CRY1, the third bar from left, by Student's two-tailed t-test. of upregulation of E-box-regulated genes under the FK866treated condition (Figure 1C) is due to the preferential cytoplasmic localization of PER2 with CRY1.
To further investigate whether the acetylation status of lysine 680 residue of PER2, where PER2 is acetylated and deacetylated by SIRT1 (Levine et al., 2020), affects its subcellular localization, we analyzed subcellular localizations of lysine 680 residue mutants of PER2 (Figure 5). Non-acetyl mimetic PER2 K 680R mutant only slightly increased in the cytoplasmic population of PER2, compared with the cytoplasmic population of PER2 WT (p = 0.010). Moreover, acetyl mimetic PER2 K 680Q had no effect on PER2 subcellular localization. These mutant PER2 experiments suggest that K680 of PER2 is not the responsible lysine to regulate subcellular localization by SIRT1, although we cannot deny the possibility that acetyl mimetic mutant of PER2 K 680Q does not completely substitute the functions of acetyl-K680 of PER2. The data represented are the mean ± SEM of independent three samples. * * * p < 0.001, compared to each subcellular localization in control (ctrl) cells by Student's two-tailed t test. (B,C) Bmal1-luc oscillation patterns in NIH3T3 cells treated with either NAM or EX-527 were monitored by using a real-time luciferase monitoring system. One representative result is shown for each condition. (D) Bmal1-luc oscillation patterns in WT (ctrl) or Sirt1 knockdown (shSirt1 #3) NIH3T3 cells were monitored by using a real-time luciferase monitoring system. One representative result is shown for each condition. (E) Relative amplitudes were analyzed. The value of control (ctrl) was set to 1. The data represented are the mean ± SEM of independent 8, 5, 3 or 3 samples for the control, NAM, EX-627, or shSirt1 #3, respectively. * p < 0.05, * * p < 0.01, compared to the control (ctrl) by Student's two-tailed t-test.

DISCUSSION
In this study, we found that low intracellular NAD + , which was induced by the pharmacological inhibition of NAMPT, promoted PER2 subcellular localization from the nucleus to the cytoplasm and increased and decreased the expressions of E-boxand RORE-regulated circadian genes, respectively, leading to the attenuated Bmal1-luc circadian oscillation. Furthermore, we demonstrated that SIRT1 was responsible for NAD + -dependent PER2 subcellular regulation, and suggested that lysine 680 residue on PER2 is not responsible for SIRT1-regulated PER2 subcellular localization.
Since several lines of evidence indicate that a decline in NAD + is a hallmark of senescence/aging (Braidy et al., 2011;Yoshino et al., 2011;Canto et al., 2012;Massudi et al., 2012;Camacho-Pereira et al., 2016;Zhang et al., 2016;Khaidizar et al., 2017), in this study, we used NAMPT inhibitors, FK866 and GMX1778, to reduce intracellular NAD + , which, we expect, mimics cellular senescence. However, many other physiological events and metabolites such as AMP/ATP ratio and polyamines are known to be changed with senescence/aging (James et al., 2015;Zwighaft et al., 2015). An increase in the AMP/ATP ratio promotes AMP-activated protein kinase (AMPK), which acts as a sensor of the reduced energetic state and further activates catabolic pathways while inhibiting anabolic ones (Hardie, 2003;Garcia and Shaw, 2017). Meanwhile, it has been reported that mTOR, which is an intracellular nutrient sensor for high cellular energy state and associated with autophagy, is also upregulated during senescence (Herranz et al., 2015;Laberge et al., 2015;Nacarelli and Sell, 2017). These signaling molecules have been reported to be involved in the circadian clock. AMPK is a rhythmically expressed kinase and phosphorylates CK1ε, resulting in enhanced phosphorylation and degradation of PER2 (Um et al., 2007;Sahar and Sassone-Corsi, 2012) and CRY1 (Lamia et al., 2011;Sahar and Sassone-Corsi, 2012;Jordan and Lamia, 2013). mTOR perturbation, such as RNAi knockdown or mTOR inhibitors, alters circadian rhythms in fibroblast, SCN, and animal behaviors (Zhang et al., 2009;Ramanathan et al., 2018). Several lines of evidence mentioned here imply that the altered aforementioned signaling pathways during senescence may affect circadian clock properties, although it is largely unknown whether these pathways alter circadian clock properties in senescent cells. We recently demonstrated that altered circadian properties such as period extension and phase delay occur in senescent human cells (Ahmed et al., 2019(Ahmed et al., , 2021. Therefore, we assume that these circadian alterations in senescent cells will be useful indexes to evaluate whether low NAD + or other conditions are sufficient for the circadian clock to mimic senescent condition.
Real-time luciferase monitoring assay revealed that the attenuated amplitude of Bmal1-luc oscillations was observed by either NAMPT inhibitors, FK866 and GMX1778; Sirtuins and PARPs broad inhibitor, NAM; SIRT1-specific inhibitor, EX-527; or Sirt1 knockdown. However, the delayed phase of Bmal1luc oscillations was not observed by SIRT1-specific inhibitor, EX-527, or Sirt1 knockdown. These results indicate that SIRT1 deacetylation activity is involved in amplitude regulation, but not in phase regulation. This is supported by the report that circadian profiles of Dbp and Per2 in Sirt1 −/− cells showed higher amplitude and no delayed phase (Nakahata et al., 2008). In contrast, delayed phases were observed with FK866, GMX1778, or NAM treatment, suggesting that NAD + -dependent enzymes other than SIRT1 are involved in the phase regulation. The investigation to address which enzyme is associated with phase regulation will be needed to understand the mechanisms of how circadian clocks work in senescent cells. Actually, we have recently shown that senescent cells possess altered circadian clocks with a prolonged period and delayed phase (Ahmed et al., 2019(Ahmed et al., , 2021. In this study, we investigated whether acetylation of lysine 680 residue on PER2 is responsible for PER2 subcellular localization; however, neither acetyl mimetic mutant (K680Q) nor nonacetyl mimetic mutant (K680R) of PER2 changed subcellular localization such as low NAD + or SIRT1 inhibitor-treated conditions. Levine and coworkers recently reported that PER2 is acetylated on K680, which is deacetylated by SRIT1; furthermore, FK866 treatment increases PER2 phosphorylation at numerous sites (Levine et al., 2020). This suggests that acetylation of K680 or other lysine residues of PER2 might be a trigger for subsequent phosphorylation(s), which might determine PER2 subcellular localization. Our current results cannot deny the possibility that acetyl mimetic mutant PER2 K 680Q may not trigger subsequent phosphorylation(s) because of differences of three-dimensional structures triggered by the acetylated form of lysine residue and substituted glutamine residue. Further experiments including structural biological experiments will be needed to elucidate which lysine residue is responsible for subcellular localization of PER2 regulated by SIRT1.
Our current results suggest that PER/CRY-dependent repression against E-box-regulated genes might be dependent on the NAD + /SIRT1 axis. Intriguingly, we have reported that SIRT1 deacetylates lysine 9/14 residues of histone H3 on the promoters of E-box-regulated genes to repress their expressions epigenetically (Nakahata et al., 2008). These results imply that the NAD + /SIRT1 axis coordinates the transcriptional repression of E-box-regulated genes by different ways. In addition to the coordinated mechanisms for E-box-regulated genes, RORE-regulated genes might also be coordinated by the NAD + /SIRT1 axis. It has been reported that NAD + activates Bmal1 transcription by SIRT1 through the deacetylation of PGC1α (Chang and Guarente, 2014), while we demonstrated in this study that the low NAD + condition increased Reberbα/β which in turn might repress Bmal1 gene expression. These coordinated Bmal1 gene regulations by the NAD + /SIRT1 axis presumably regulate the amplitude of Bmal1 circadian oscillation, namely the low NAD + condition or decreased SIRT1 activity attenuates the amplitude of Bmal1 circadian oscillation. Therefore, these coordinated transcriptional regulations of E-box-and RORE-regulated circadian genes by the NAD + /SIRT1 axis might interlock circadian negative feedback loops.
In addition to our finding that low NAD + retains PER2 in the cytoplasm, some studies have demonstrated that the NAD + /SIRT1 axis regulates PER2 properties; PER2 in Sirt1 −/− cells is more stable (Asher et al., 2008) and localizes predominantly in the nucleus (Levine et al., 2020), while PER2 in nicotinamide-treated cells localizes predominantly in the cytoplasm (Miki et al., 2012). Our pharmacologically conducted results were consistent with the results pharmacologically performed by Miki and colleagues, but not with the results performed genetically. These conflicting results by pharmacological (acute) and genetical (chronic) approaches suggest the possibility that responses of molecular clock are different at early and chronic senescence/aging phases. This possibility is worth verifying so further experiments using different stages of senescent cells/aging animals will be needed to reveal this possibility.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
AA performed and analyzed the experiments. YN designed the research and wrote the manuscript. TS, YuF, HY, and YoF provided the reagents. KS assisted with the experiments and reviewed the manuscript. TM and YB designed the research and reviewed the manuscript. All authors contributed to the article and approved the submitted version.

ACKNOWLEDGMENTS
We thank Dr. R. Ahmed for her critical comments on the manuscript.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnins. 2021.647589/full#supplementary-material Supplementary Figure 1 | Representative Immunofluorescence images. N, nucleus; C, cytoplasm; N + C, nuclear and cytoplasm. We interpreted a result for nuclear localization of PER2/CRY1 (N) as positive when fluorescence intensity (Alexa Fluor Plus 488) of PER2/CRY1 signal that overlapped with Hoechst 33342 signal was higher than the fluorescence intensity of PER2/CRY1 in the cytoplasm Meanwhile, we interpreted a result for cytoplasmic localization of PER2/CRY1 (C) as positive when PER2/CRY1 signal that did not overlap with Hoechst 33342 signal exhibited higher fluorescence intensity than PER2/CRY1 signal intensity that overlapped with Hoechst 33342 signal. Finally, we interpreted a result for nuclear and cytoplasm localization of PER2/CRY1 (N + C) as positive when PER2/CRY1 fluorescence signal at both nucleus and cytoplasm had the same intensities. Bmal1-luc oscillation patterns treated with GMX1778 with or without NMN were monitored by using a real-time luciferase monitoring system. (D) Relative amplitudes were analyzed. The value of control (ctrl) was set to 1. All data represented in Supplementary Figure 3 are the mean ± SEM of independent three samples. * * p < 0.01 and * * * p < 0.001, compared to each of the untreated control by Student's two-tailed t-test, n.s., means not significant.
Supplementary Figure 4 | Effects of FK866 or GMX1778 on intracellular NAD + amount. NIH3T3 were treated with NAMPT inhibitors, FK866 or GMX1778, throughout this experiment.
Supplementary Figure 5 | FK866 has no effect on stabilities of clock proteins. NIH3T3 cells expressing Myc-CLOCK (A), Myc-BMAL1 (B), Myc-PER2 (C), or Myc-CRY1 (D) were treated with cycloheximide (CHX) at 100 µg/ml. At indicated times, cells were lysed and protein extracts were immunoblotted with antibody to Myc (top) and antibody to Tubulin (middle) as loading control. Cells were either untreated (ctrl) or treated with 7.5 nM FK866. The results are representative of three independent experiments. (Bottom) The immunoblots were quantified by densitometric analysis. The graph shows the percentage of protein amount relative to time 0 (100%). Myc-tagged proteins were normalized by each Tubulin amount.
Supplementary Figure 9 | Sirt1 knockdown attenuates the amplitude of Bmal1-luc oscillation. (A) Knockdown efficiencies against Sirt1 were analyzed by qPCR. Sirt1 amount in control (scramble) was set to 1. The data represented are the mean ± SEM of independent three samples. (B) Bmal1-luc oscillation patterns in Sirt1 knockdown (shSirt1 #1 or #2) NIH3T3 cells were monitored by using a real-time luciferase monitoring system. (C) Relative amplitudes were analyzed. The value of control (ctrl) was set to 1. The data represented are the mean ± SEM of independent 8, 3, or 3 samples for control, shSirt1 #1 or #2, respectively. * * p < 0.01, compared to control cells by Student's two-tailed t-test, n.s., means not significant.