SIRT1 Regulates Circadian Clock GeneExpression through PER2 Deacetylation Gad Asher,David Gatfield,Markus Hans Reinke,Charna Florian Kreppel,Raul Mostoslavsky,Frederick W. Alt,and Ueli 1Department of Molecular Biology, Sciences III, University of Geneva, 30, Quai Ernest Ansermet, CH-1211 Geneva-4, Switzerland2Division of Gene Therapy, University of Ulm, Helmholtzstrasse 8/1, D-89081 Ulm, Germany3Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA 02114, USA4Howard Hughes Medical Institute, Children's Hospital, Center for Blood Research, and Department of Genetics,Harvard University Medical School, Boston, MA 02115, USA*Correspondence: DOI 10.1016/j.cell.2008.06.050 The molecular oscillator in both master and subsidiary clocks (is thought to rely on The mammalian circadian timing system is com- a negative transcriptional feedback loop posed of a central pacemaker in the suprachiasmatic nucleus of the brain that synchronizes countless PAS domain helix-loop-helix proteins BMAL1 and CLOCK (or subsidiary oscillators in peripheral tissues. The its paralog NPAS2 []) rhythm-generating mechanism is thought to rely on bind as heterodimers to regulatory elements of Cry and Per genesand stimulate the transcription of these genes. Once the repres- a feedback loop involving positively and negatively sor proteins CRY and PER have reached a critical concentration, acting transcription factors. BMAL1 and CLOCK they attenuate the activity of BMAL1-CLOCK heterodimers and activate the expression of Period (Per) and Crypto- thereby repress the transcription of their own genes. In addition, chrome (Cry) genes, and once PER and CRY proteins an interconnecting feedback loop involving orphan nuclear re- accumulate to a critical level they form complexes ceptors of the REV-ERB and ROR families regulates the expres- with BMAL1-CLOCK heterodimers and thereby sion of Bmal1 ).
repress the transcription of their own genes. Here, Several lines of evidence suggest a strong interplay between we show that SIRT1, an NAD+-dependent protein metabolism and the circadian clock ( deacetylase, is required for high-magnitude circa- dian transcription of several core clock genes, in- feeding cycles as a Zeitgeber for peripheral clocks implies that cluding Bmal1, Rorg, Per2, and Cry1. SIRT1 binds the circadian clock plays an important role in nutrient process-ing and energy homeostasis. Indeed, transcriptome profiling CLOCK-BMAL1 in a circadian manner and promotes studies revealed that many genes involved in metabolism the deacetylation and degradation of PER2. Given are rhythmically expressed ( the NAD+ dependence of SIRT1 deacetylase activity, it is likely that SIRT1 connects cellular metabolism to Furthermore, at least the circadian core clockwork circuitry.
in vitro, the DNA-binding activity of BMAL1-CLOCK is stronglyinfluenced by the ratio of reduced to oxidized NAD cofactors, which are often considered as a readout of the cellular metabolicstate ).
The physiology and behavior of mammals are subject to daily SIRT1 is the mammalian homolog of yeast Sir2, an NAD+- oscillations driven by an endogenous circadian clock ( dependent deacetylase involved in transcriptional silencing, genome stability, and longevity ( the circadian timing system is composed of a central pacemaker ). The SIRT1 catalytic reaction involves in the brain's suprachiasmatic nucleus (SCN) and subsidiary the breakdown of one NAD+ molecule for each deacetylated oscillators in most peripheral tissues. While light-dark cycles acetyl lysine and the generation of nicotinamide and O-acetyl- are the predominant Zeitgebers (timing cues) for the SCN pace- ADP-ribose. SIRT1 was found to deacetylate not only histones maker, cyclic feeding behavior is a strong Zeitgeber for clocks but also several transcriptional regulatory proteins involved in operating in many peripheral tissues the control of metabolism, including members of the FOXO ). It is therefore likely that the SCN synchronizes peroxisome proliferator-activated receptor gamma peripheral oscillators by imposing rest-activity rhythms and (PPARg) coactivator 1a (PGC1a), and the nuclear receptor LXR thus feeding-fasting cycles.
Cell 134, 317–328, July 25, 2008 ª2008 Elsevier Inc. 317

Figure 1. Circadian Expression of SIRT1 ProteinMice were sacrificed at 4 hr intervals; liver nuclear protein extracts and total RNA were prepared.
(A) Protein extracts were analyzed by immunoblotting.
(B) RNA was analyzed by quantitative TaqMan real-time PCR using specific TaqMan probes.
MEFs were synchronized by a dexamethasone shock and samples were collected at 4 hr intervals, starting 24 hr after the shock.
(C) Protein extracts were analyzed by immunoblotting.
(D) RNA was analyzed by quantitative TaqMan real-time PCR using specific TaqMan probes.
Plotted values are the mean values ± standard deviation (SD) from three independent experiments.
Here we show that SIRT1 is expressed in a circadian manner expression as well as changes in BMAL1 phosphorylation were and that it is required for high-magnitude circadian expression of several core clock genes. Moreover, we present evidence that SIRT1 binds to CLOCK-BMAL1 heterodimers and promotes whether the daily changes in SIRT1 protein expression were the deacetylation and degradation of PER2.
due to corresponding changes in Sirt1 mRNA accumulation,we analyzed mouse liver RNA harvested around the clock by quantitative TaqMan real-time PCR. In contrast to severalwell-established circadian transcripts such as Per2, Rorg, and Circadian Expression of SIRT1 Protein in Mouse Liver Rev-Erba mRNAs, the levels of Sirt1 mRNA were nearly constant and in Cultured Fibroblasts throughout the day (B). Hence, posttranscriptional regu- In order to investigate whether SIRT1 might be involved in circa- latory mechanisms must have accounted for the observed circa- dian rhythm, we first examined the temporal expression of SIRT1 dian changes in SIRT1 protein levels.
in mouse liver. Mice were sacrificed at 4 hr intervals around the SIRT1 protein was also expressed in a circadian manner in clock, and liver nuclear proteins were prepared and analyzed.
dexamethasone-synchronized cultured mouse embryonic fibro- The results showed that SIRT1 accumulated in a circadian man- blasts (MEFs) (and NIH 3T3 cells A available ner with maximal and minimal levels reached at around Zeitgeber online). Again, no significant changes in Sirt1 mRNA accumula- time (ZT) 16 and ZT4, respectively (In parallel, we also tion were observed As expected Per2 and followed the temporal expression of known core clock proteins.
Cry1 mRNA levels were clearly rhythmic in these cells As reported previously, circadian changes in PER2 protein (Finally, we examined temporal SIRT1 accumulation 318 Cell 134, 317–328, July 25, 2008 ª2008 Elsevier Inc.

Figure 2. SIRT1 Deacetylase Activity Is Required for the High-Magnitude Oscillation of the Bmal1-Luciferase Reporter(A) NIH 3T3 cells were transfected with a 200 ng/plate Bmal1-luciferase reporter gene either alone (blue) or together with an HA-Flag-SIRT1 expression vector(green). The small insert shows the cells transfected with the Bmal1-luciferase reporter gene alone at a higher magnification.
(B) NIH 3T3 cells were transfected with a 200 ng/plate Bmal1-luciferase reporter gene either alone (blue) or together with an HA-Flag-SIRT1 H363Y expressionvector (green).
(C) NIH 3T3 cells were transfected with a 1 mg/plate Bmal1-luciferase reporter gene either with pU6 empty vector (blue) or together with pU6-Sirt1 siRNA expres-sion vector (red).
(D) Immunoblot analysis of protein extracts obtained from NIH 3T3 cells transfected with pU6 empty vector or pU6-Sirt1 siRNA expression vector.
(E) Two WT MEF cell lines (dark and light blue) and two Sirt1 KO MEF cell lines (dark and light orange), all obtained from different embryos harvested from the samepregnant female, were transduced with Bmal1-luciferase adenovirus.
Cells were synchronized by a dexamethasone shock and bioluminescence was recorded using photomultiplier tubes.
in synchronized NIH 3T3 fibroblasts by immunohistochemistry luciferase reporter plasmid resulted in a dramatic increase in experiments. SIRT1 staining was mostly nuclear and more in- the magnitude of the bioluminescence oscillations ( tense at 36 hr than at 24 hr following the dexamethasone shock The deacetylase activity of SIRT1 was required for this effect (C and S1D). Interestingly, even in knockout (KO) since cotransfection of SIRT1 H363Y, a catalytically inactive, MEFs stably expressing human SIRT1 from an expression vector dominant-negative SIRT1 mutant version containing the SV40 promoter and unrelated 50- and 30-untrans- virtually abolished the circadian bioluminescence oscillations lated regions, human SIRT1 protein was expressed differentially generated by the Bmal1-luciferase reporter gene ( at two examined time points (This experiment fa- We also tested the effect of SIRT1 expression on reporter vors a mechanism involving changes in protein stability rather genes driven by other promoters, in particular Per2-luciferase, than translation rates in circadian SIRT1 protein accumulation.
Whereas SIRT1 did not significantly affect CMV-luciferase or SIRT1 Deacetylase Activity Is Required Rev-Erba-luciferase expression, it increased the magnitude of for High-Magnitude Bmal1 Expression the bioluminescence oscillations of Per2-luciferase and Dbp- In order to examine whether SIRT1 might influence circadian gene expression, we followed real-time bioluminescence Nicotinamide (NAM), a product of the SIRT1 deacetylation recordings of a Bmal1-luciferase reporter whose expression is reaction, was reported to inhibit SIRT1 activity ( driven by the Bmal1 promoter Cotransfec- Treatment of NIH 3T3 cells stably expressing the tions of a SIRT1 expression vector together with the Bmal1- Bmal1-luciferase reporter gene with a moderate concentration Cell 134, 317–328, July 25, 2008 ª2008 Elsevier Inc. 319 of NAM (10 mM) had no significant effect on the daily biolumines- proteins for ubiquitination and degradation), and Cki3 (casein cence oscillations A higher concentration of NAM kinase I3) were only slightly affected by the absence of SIRT1 (50 mM) resulted in the dampening of circadian Bmal1-luciferase (The strong repression of Rorg mRNA expression in expression A). However, the most conspicuous effect the Sirt1 KO MEFs incited us to examine the levels of the other of NAM treatment was a dramatic period lengthening two Ror paralogs, Rora and Rorb . However, no significant a phenotype that was not observed in genetic loss- differences were observed for Rora mRNA levels, and Rorb, a of-function experiments (see below) and, therefore, was unlikely neuron-specific Ror isoform ), was unde- to involve SIRT1. In contrast, Sirtinol, a more specific and potent tectable in both WT and Sirt1 KO MEFs ( inhibitor of SIRT1 deacetylation activity (), We also examined the accumulation of various clock proteins strongly dampened the circadian Bmal1-luciferase reporter from nonsynchronized WT and Sirt1 KO MEFs. In keeping with gene expression (B) and thereby closely phenocopied the changes observed for their mRNA levels, both BMAL1 and the phenotypes observed in genetic Sirt1 loss-of-function CLOCK protein levels were significantly downregulated in the Sirt1 KO MEFs However, although the Per2 mRNA To further scrutinize possible roles of SIRT1 on the clock func- level was strongly decreased in Sirt1 KO MEFs ( tion we performed loss-of-function experiments. Cotransfection PER2 protein accumulation was actually higher in these cells of a Sirt1 siRNA expression vector with the Bmal1-luciferase (C). CRY1 protein accumulation was also slightly ele- reporter gene attenuated the circadian oscillations ( vated in the absence of SIRT1 C), in contrast to the in a dose-dependent manner (The effect of Sirt1 downregulation of its mRNA level ( siRNA expression on endogenous SIRT1 protein accumulation To verify whether the described changes in clock gene expres- was verified by analysis of protein extracts from the cells that sion were indeed due to the absence of SIRT1 in the KO MEFs, were used for the Bmal1-luciferase recordings (D). Since we generated rescue cell lines from Sirt1 KO MEFs that stably not all cells contributing to the protein extract were transfected, expressed a human SIRT1 cDNA. The comparison of WT, Sirt1 the downregulation of SIRT1 accumulation in transfected cells KO, and two human SIRT1-rescued KO MEFs cell lines showed must have been very efficient. The expression of Sirt1 siRNA that the mRNA levels encoding the different clock transcription also resulted in a decrease in the bioluminescence oscillations factors and human SIRT1 were restored to nearly normal levels of Per2-luciferase and to a lesser extent of Dbp-luciferase oscil- in both rescued cell lines (D). Similarly, the magnitude lations and S4C) but did not affect the biolumines- of the bioluminescence oscillations of Bmal1-luciferase re- cence of CMV-luciferase or Rev-Erba-luciferase (D porter were reestablished in the human SIRT-rescued KO and S4E). We also examined the effect of SIRT1 knockdown on Bmal1-luciferase reporter at a single-cell resolution. As can The observed changes in clock gene mRNA and protein levels be concluded from a comparison of (control) and were obtained in nonsynchronized cells and thus reflected (Sirt1 siRNA) only a small proportion of cells displayed strong average expression levels. Thus, we also wished to monitor bioluminescence cycles when Sirt1 expression was diminished.
the circadian expression of these genes in synchronized MEFs.
Experiments with MEFs from wild-type (WT) and Sirt1 KO em- In accordance with the observations made in the experiments bryos, transduced with an adenoviral vector harboring the with the Bmal1-luciferase reporter (E), endogenous Bmal1-luciferase reporter gene, substantiated the observations Bmal1 mRNA was expressed at low and nearly invariable levels made with Sirt1 siRNA-expressing cells. Thus, the magnitude throughout the day A). Similarly, Rorg mRNA accumula- of bioluminescence cycles was considerably higher in WT tion was strongly repressed in the absence of SIRT1 MEFs than in Sirt1 KO MEFs (E). A closer inspection of Per2 and Cry1 mRNA were still expressed in a circadian manner our loss-of-function data also revealed a modest phase advance in Sirt1 KO MEFs, but with a significantly reduced magnitude for the temporal expression of Bmal1-luciferase, Per2-luciferase, (Again, no significant changes in Rev-Erba and Dbp and Dbp-luciferase in the absence of SIRT1 (E and ).
mRNA accumulation were noticed These results However, in none of these experiments were significant differ- were thus in keeping with the changes observed in our analysis ences in period lengths noticed.
of nonsynchronized MEFs ( Similarly to the changes observed for Bmal1 mRNA expres- SIRT1 Influences the Expression of Endogenous sion A), BMAL1 protein levels were significantly downre- gulated in the Sirt1 KO MEFs B). In contrast, both PER2 To examine whether SIRT1 also affected the expression of en- and CRY1 protein levels were elevated and relatively constant in dogenous circadian genes, we analyzed the levels of various the absence of SIRT1 (B), in spite of their diminished transcripts in WT and Sirt1 KO MEFs by quantitative TaqMan mRNA levels Again, SIRT1 accumulated in a circa- real-time PCR. The levels of endogenous Bmal1 mRNA were dian manner with maximal expression between 32 to 36 hr reduced to around 40% in nonsynchronized Sirt1 KO MEFs following the dexamethasone shock B).
compared to WT MEFs Clock, Per1, and Cry1mRNA accumulation was attenuated to a similar extent in Sirt1 SIRT1 Binds to CLOCK-BMAL1 and PER2 KO MEFs (A), while Per2 and Rorg transcript levels in in a Circadian Fashion Sirt1 KO cells only amounted to 20% and 10%, respectively, of SIRT1 was previously reported to deacetylate several tran- those observed in WT cells (In contrast, the mRNA scriptional regulatory proteins levels of Rev-Erba, Dbp, bTrcp (an F box protein targeting PER including the basic helix-loop-helix 320 Cell 134, 317–328, July 25, 2008 ª2008 Elsevier Inc.

Figure 3. Analysis of mRNA and Protein Levels of Core Clock Proteins in Nonsynchronized WT, Sirt1 KO, and Sirt1 Rescue MEFs(A) RNA extracts from nonsynchronized WT and Sirt1 KO MEFs were analyzed by quantitative TaqMan real-time PCR using specific TaqMan probes.
(B) RNA extracts from nonsynchronized WT and Sirt1 KO MEFs were analyzed for the mRNA expression of different ROR isoforms by quantitative TaqMan real-time PCR.
(C) Protein extracts from nonsynchronized WT and Sirt1 KO MEFs were examined by immunoblotting.
(D) RNA extracts from nonsynchronized WT, Sirt1 KO, and two different monoclonal Sirt1 rescue MEF lines expressing human SIRT1 were analyzed by quan-titative TaqMan real-time PCR using specific TaqMan probes.
Plotted values are the mean values ± SD from three independent experiments.
repressors HES1 and HEY2 Since, observed around ZT0 After a longer exposure similarly to HES1 and HEY2, both CLOCK and BMAL1 contain weak binding of PER2 to CLOCK was also detected around helix-loop-helix domains, we first examined whether SIRT1 ZT4 (data not shown). Importantly, an immunoprecipitation ex- might interact with these transcription factors. Both endogenous periment with SIRT1 antibody with the same protein extracts CLOCK and BMAL1 coimmunoprecipitated with SIRT1 in ex- showed that SIRT1 bound to CLOCK in a circadian manner tracts obtained from mouse liver nuclei (and from cul- with maximal binding around ZT4 F). A similar experi- tured NIH 3T3 fibroblasts In addition, coimmunos- ment was conducted with whole-cell extracts from NIH 3T3 taining experiments for SIRT1 and CLOCK in NIH 3T3 cells cells synchronized by a dexamethasone shock. The binding of showed that at least a fraction of these proteins colocalized in CLOCK-BMAL1 to SIRT1 was circadian with maximal binding between 36 and 42 hr after the dexamethasone treatment The binding of SIRT1 to CLOCK and BMAL1 prompted us to (Interestingly, PER2 could also be detected in examine whether SIRT1 interacted with additional core clock SIRT1-associated complexes around 42 hr after the dexameth- proteins in a circadian manner. We thus analyzed the different asone shock.
binding partners of CLOCK around the clock in mouse liver nu-clear extracts (As expected, immunoprecipitaiton of SIRT1 Deacetylates PER2 CLOCK resulted in the coimmunoprecipitation of BMAL1, with To examine whether CLOCK, BMAL1, or PER2 are acetylated maximal binding around ZT8 E). In contrast, maximal in- and thereby potential substrates for SIRT1 deacetylation teractions of the repressors PER2 and CRY1 with CLOCK were activity, we performed immunoprecpitation experiments with Cell 134, 317–328, July 25, 2008 ª2008 Elsevier Inc. 321

Figure 4. Analysis of Circadian mRNA and Protein Levels of Core Clock Proteins in WT and Sirt1 KO MEFsWT and Sirt1 KO MEFs were synchronized by a dexamethasone shock, and protein and RNA were extracted at 4 hr intervals starting 24 hr after the shock.
(A) mRNA analysis was done by quantitative TaqMan real-time PCR using specific TaqMan probes. Plotted values are the mean values ± SD from three inde-pendent experiments.
(B) Protein extracts were analyzed by immunoblotting.
nonsynchronized WT and Sirt1 KO MEFs, using a pan acetyl ly- itation experiments were performed with a pan acetyl lysine an- sine antibody. The immunoprecipitated proteins were analyzed tibody, and the immunoprecipitated proteins were analyzed by by immunoblotting using antibodies for various clock proteins.
immunoblotting. In agreement with the previous experiments, These experiments failed to reveal acetylated forms of CLOCK, no acetylation of BMAL1 or CLOCK was detected BMAL1, CRY1, or SIRT1 but suggested that a frac- In WT MEFs, PER2 acetylation was maximal at around 32 hr after tion of PER2 was acetylated in Sirt1 KO MEFs Low the dexamethasone shock (C). The extent of PER2 acet- levels of acetylated PER2 were also detected in WT MEFs after ylation was significantly higher in KO MEFs than in WT MEFs, a long exposure (data not shown). To corroborate PER2 acety- and maximal acetylation in KO MEFs occurred between 24 and lation we transfected NIH 3T3 cells with expression vectors for 28 hr after the dexamethasone shock. Our findings thus sug- a tandem affinity purification (TAP) tagged PER2 (PER2-TAP) gested that PER2 was acetylated in a circadian manner, and or a TAP-tagged luciferase (luciferase-TAP) (as a negative that SIRT1 deacetylated PER2 in vivo. While PER2 protein levels control). Immunoblot analysis of purified TAP-tagged proteins were elevated and relatively constant in the Sirt1 KO MEFs confirmed that PER2 was acetylated ) and that the (PER2 acetylation and/or deacetylation was cyclic.
acetyl groups were removed in vitro by recombinant SIRT1 in This is in line with the circadian binding of SIRT1 to PER2 an NAD+-dependent manner B).
(To examine whether PER2 was also acetylated in Next, we monitored the acetylation of PER2 in synchronized mice, we immunoprecipitated PER2 from mouse liver nuclear WT and Sirt1 KO MEFs around the clock. Again, immunoprecip- extracts prepared around the clock. Immunoblot analysis of 322 Cell 134, 317–328, July 25, 2008 ª2008 Elsevier Inc.

Figure 5. SIRT1 Binds to CLOCK, BMAL1, and PER2 in a Circadian MannerSIRT1 was immunoprecipitated from mouse liver nuclear extracts (A) and from NIH 3T3 cells (B). The immunoprecipitated proteins were analyzed by immuno-blotting. Rabbit yeast RAP1 antibody was used as a negative control.
(C) Immunostaining of SIRT1 (red) and CLOCK (green) in NIH 3T3 cells was performed with rabbit SIRT1 and CLOCK antibodies. In blue: DAPI staining. In yellow:merge of SIRT1 and CLOCK staining.
(D) Mice were sacrificed at 4 hr intervals, and liver nuclear extracts were analyzed by immunoblotting.
(E) CLOCK was immunoprecipitated from mouse liver nuclear extracts, and the immunoprecipitated proteins were analyzed by immunoblotting. Rabbit yeastRAP1 antibody was used as a negative control.
(F) SIRT1 was immunoprecipitated from mouse liver nuclear extracts, and the immunoprecipitated proteins were analyzed by immunoblotting.
(G) NIH 3T3 cells were synchronized by a dexamethasone shock, and protein extracts were prepared at 6 hr intervals, starting 24 hr after the shock. SIRT1 wasimmunoprecipitated from NIH 3T3 cells, and the immunoprecipitated proteins were analyzed by immunoblotting.
precipitated proteins with the pan acetyl lysine antibody showed during 4 hr. The results indicated that the protein half-life of that PER2 was acetylated also in mouse liver D).
PER2 was significantly prolonged in the absence of SIRT1(and 7B). In keeping with these observations, the SIRT1-Dependent Deacetylation of PER2 Determines coexpression of SIRT1 together with PER2-TAP significantly PER2 Protein Stability reduced the accumulation C) and acetylation of PER2- The elevated PER2 acetylation and accumulation in Sirt1 KO TAP (whereas knockdown of SIRT1 expression with MEFs on one hand and the reduced Per2 mRNA levels on the Sirt1 siRNA resulted in a significant increase in PER2 accumula- other hand raised the possibility that acetylation of PER2 stabi- tion and acetylation E).
lized the protein. We thus compared the decay of PER2 protein To further address the dependency of PER2 degradation upon in the presence and absence of SIRT1. To this end, WT and Sirt1 deacetylation by SIRT1 we performed an in vitro assay with KO MEFs were incubated with or without cycloheximide 24 hr purified PER2-TAP and extracts obtained from WT and Sirt1 KO after synchronization and PER2 protein levels were recorded MEFs. PER2 was deacetylated only in the presence of extracts Cell 134, 317–328, July 25, 2008 ª2008 Elsevier Inc. 323

Figure 6. SIRT1 Deacetylates PER2(A) Protein extracts from nonsynchronized WT and Sirt1 KO MEFs were subjected to immunoprecipitation with rabbit pan acetyl lysine antibody, and the immu-noprecipitated proteins were analyzed by immunoblotting.
(B) Purified PER2-TAP was incubated in the absence or presence of recombinant SIRT1 and NAD+ for 3 hr at 30, and samples were analyzed by immunoblotting.
(C) WT and Sirt1 KO MEFs were synchronized by a dexamethasone shock, and protein extracts were prepared at 4 hr intervals, starting 24 hr after the shock.
Immunoprecipitation experiments were performed with rabbit pan acetyl lysine antibody, and the immunoprecipitated proteins were analyzed by immunoblotting.
(D) Mice were sacrificed at 4 hr intervals and liver nuclear extracts were prepared. PER2 was immunoprecipitated, and the immunoprecipitated proteins wereanalyzed by immunoblotting. Rabbit yeast RAP1 antibody was used as a negative control.
from WT MEFs together with NAD+. Deacetylation of PER2 re- tained from wild-type but not from Sirt1 KO MEFs, and recombi- sulted in PER2 degradation, which was blocked in the presence nant SIRT1 deacetylates purified PER2 in vitro in a NAD+-depen- of the proteasome inhibitor MG132 dent manner. The latter result should, however, be interpretedwith caution since the in vitro substrate specificity of recombi- nant SIRT1 is rather promiscuous ().
Other posttranslational modifications such as phosphoryla- Modulation of Circadian Oscillator Function by Protein tion, sumoylation, histone acetylation, and methylation have Acetylation and Deacetylation already been shown to play a key role in circadian gene expres- We identified SIRT1 as a regulator of circadian gene expression.
sion ). For example, sumoylation of SIRT1 accumulates in a circadian manner in mouse hepatocytes BMAL1 has been shown to play an important role in BMAL1 ac- and cultured fibroblasts and is required for high-magnitude cir- cumulation and clock rhythmicity (Like- cadian transcription of several core clock genes, including wise, phosphorylation of BMAL1 either by Casein Kinase I (CKI) Bmal1, Rorg, Per2, and Cry1. SIRT1 binds to CLOCK-BMAL1 (or by mitogen-activated protein kinases and PER2 in a circadian manner and supports the deacetylation (MAPK) () modulates BMAL1-CLOCK-depen- and degradation of PER2. In the absence of SIRT1, constitutively dent transcription. Recently, CLOCK was reported to acetylate high protein levels of PER2 may lead to the repression of Per1, BMAL1, thereby facilitating repression of BMAL1-CLOCK-de- Per2, Cry1, and Rorg mRNA expression. Repression of RORg, pendent transcription We suppose that an activator of Bmal1 transcription, is likely to account for the due to the sensitivity of our immunoblot experiments, the acety- dampening of Bmal1 mRNA and protein expression in Sirt1 KO lated fraction of endogenous BMAL1 in liver and fibroblast ex- tracts was not revealed. CKI has been reported to phosphorylate The enzyme(s) responsible for PER2 acetylation remain(s) to PER2 protein, thereby regulating PER2 protein stability ( be identified, but the acetyltransferase activity of CLOCK ). Likewise, CRY degradation mediated by the F box or p300, a coactivator associated with CLOCK- protein SCFFbxl3 has been demonstrated to be required for BMAL1 heterodimer (are attractive can- normal oscillator function didates. PGC1a, a recently identified key player in circadian oscillator function ), may also affect PER2 acety-lation via stimulating the acetyl transferase activity of p300 SIRT1 Affects Circadian Transcription in a Gene-Specific Manner Although our results cannot rigorously exclude a more compli- The extent to which SIRT1 affects circadian transcription ap- cated scenario, they suggest that SIRT1 deacetylates PER2 pears to be target gene specific. For example, in the absence directly. Thus, SIRT1 is associated with CLOCK-BMAL1-PER2 of SIRT1, Rorg mRNA levels are strongly repressed and Per2 complexes, purified PER2 is deacetylated in vitro by extracts ob- mRNA levels are significantly downregulated, whereas Rev-Erba 324 Cell 134, 317–328, July 25, 2008 ª2008 Elsevier Inc.

Figure 7. SIRT1-Dependent PER2 Deacetylation Determines PER2 Protein Stability(A) WT and Sirt1 KO MEFs were synchronized by a dexamethasone shock, and 24 hr after the shock cells were untreated or treated with cycloheximide. Cellswere harvested 1, 2, 3, and 4 hr following the treatment, and protein extracts were analyzed by immunoblotting.
(B) The graph illustrates the quantification of PER2 by densitometry of triplicate experiments (mean ± standard error).
(C) NIH 3T3 cells were transfected with PER2-TAP expression vector either alone or together with HA-FLAG-human SIRT1 expression vector. Protein extractswere analyzed by immunoblotting.
(D) PER2-TAP was purified from NIH 3T3 cells transfected with PER2-TAP expression vector either alone or together with HA-FLAG-human SIRT1 expressionvector and analyzed by immunoblotting.
(E) NIH 3T3 cells were transfected with the V5-PER2 expression vector either alone or together with the Sirt1 siRNA expression vector. Protein extracts wereprepared, and immunoprecipitation experiments were performed with mouse V5 antibody. The immunoprecipitated proteins were analyzed by immunoblotting.
(F) Purified PER2-TAP was incubated for 3 hr at 30 with protein extract obtained from WT or Sirt1 KO MEFs in the absence or presence of 100 mM NAD+ or 25 mMMG132, and samples were analyzed by immunoblotting.
(G) Hypothetical model showing the possible role of SIRT1 in circadian oscillator function. BMAL1-CLOCK heterodimers bind and activate transcription of the Per,Cry, Rre-Erba, and Rorg genes. Once the PER and CRY proteins accumulate to a critical level, they form complexes with BMAL1-CLOCK and thereby represstheir own transcription. In addition, there is an interconnecting feedback loop in which REV-ERBa represses and RORg activates Bmal1 transcription. SIRT1binds CLOCK-BMAL1 complexes and promotes PER2 deacetylation and degradation.
and Dbp mRNA levels are only slightly affected Con- SIRT1 is caused by a combination of diminished RORg ceivably, BMAL1 and CLOCK bind their DNA cognate sites in expression and impaired PGC1a coactivation. Indeed, Pgc1a Rev-Erba and Dbp with a higher affinity than those present in KO mice exhibit abnormal diurnal rhythms of activity, body Per2 and Rorg. The reduced BMAL1-CLOCK levels in Sirt1 KO temperature, and metabolic rate (Unfortu- cells might then still support high-amplitude/magnitude Rev- nately, such studies cannot be performed with Sirt1-deficient Erba and Dbp transcription.
mice because their postnatal survival rates are very poor and PGC1a was found to be expressed in a circadian manner the few surviving mice exhibit many developmental defects and to stimulate Bmal1 transcription as a coactivator of the ROR family of nuclear orphan receptors ).
SIRT1 deacetylates PGC1a and thereby modulates its coacti- Regulation of PER2 Protein Degradation vator activity (). Thus, it is possible that In mammals, the stability of PER proteins is regulated by the downregulation of Bmal1 expression in the absence of the F-box-containing E3 ubiquitin ligase bTrCP ( Cell 134, 317–328, July 25, 2008 ª2008 Elsevier Inc. 325 ). PER phosphorylation by CKI3 promotes the re- Plasmids and Transfections cruitment of bTrCP complexes, which in turn mediates the ubiq- The following plasmids were used: Bmal1-luciferase ), uitination and proteasomal degradation of PER Per2(E-BOX2)-luciferase (), Dbp-luciferase (), Rev-Erba-luciferase, and CMV-luciferase; pCDNA HA-Flag-SIRT1 en- Our results that both PER2 and acety- coding WT human SIRT1, pCDNA HA-Flag-SIRT1 H363Y encoding inactive lated PER2 levels are elevated in the absence of SIRT1 suggest deacetylase mutant of SIRT1, pBabe human SIRT1, pU6-siRNA-Sirt1, and that SIRT1-mediated deacetylation enhances the rate of PER2 pU6-empty vector (and pEF5/FRT7V5-PER2, pCMV degradation. Since ubiquitination and acetylation occur on lysine PER2-TAP, and pCMV luciferase-TAP. Transient transfections of NIH 3T3 cells residues, it is conceivable that the same lysine residues can be were carried out with FuGENE Transfection Reagent (Roche) according to the either acetylated or ubiquitinated. If true, acetylated PER2 could no longer be ubiquitinated and degraded by the proteasome, Generation and Transduction of Bmal1-Luciferase which would explain the augmented PER2 levels in Sirt1 KO Expressing Adenovirus cells. Interestingly, CRY1 protein levels are also elevated in the Bmal1-luciferase (cassettes was cloned into pCV100 absence of SIRT1, in spite of reduced Cry1 mRNA levels plasmid, first-generation adenoviral vector was amplified in N52.E6-producer ). It has been reported that PER2 inhibits the ubiquitination cells, and viruses were generated and purified as previously described ( and degradation of CRY proteins ). Thus, the MEFs were incubated with Bmal1-luciferase expressing adenovirus at an higher accumulation of PER2 in Sirt1 KO cells may account for multiplicity of infection (moi) of 5000 for 6 hr, cells were thoroughly washed the elevated CRY1 levels in these cells.
with PBS, and the medium was replaced. Forty-eight hours after transductioncells were shocked with dexamethasone and real-time bioluminescence was The Circadian Accumulation and Activity Circadian SIRT1 protein accumulation appears to be con- Cells were fixed with 4% paraformaldehyde for 10 min at room temperature.
trolled by posttranscriptional mechanisms as no significant Fixed cells were permeabilized with 0.5% Triton X-100 in TBS and washed changes in Sirt1 mRNA were observed ). Surprisingly, with TBS containing 0.1% Triton X-100 (TBS-T). Samples were blocked with temporal SIRT1 accumulation does not correlate with the cir- 2% BSA in TBS-T followed by incubation with rabbit anti-SIRT1 and rat anti- cadian interaction of SIRT1 with its identified core clock bind- CLOCK antibodies. Samples were washed with TBS-T and incubated withalexa 594-conjugated anti-rabbit and with FITC-conjugated anti-rat secondary ing partners. For example, in mouse liver nuclei, maximal antibodies. Nuclei were stained with DAPI. Microscopic images were obtained SIRT1 protein expression is observed at around ZT16, using a Leica SP2 confocal microscope.
whereas its maximal binding to CLOCK occurs at aroundZT4, when SIRT1 levels are minimal. Similarly, in NIH 3T3 RNA Analysis by Real-Time Quantitative PCR cells, maximal SIRT1 protein expression occurs between 32 RNA extraction and transcript quantification by TaqMan real-time PCR tech- and 36 hr after the dexamethasone shock, while its maximal nology was performed as previously described ), usingan ABI PRISM 7700 Sequence Detection System from PE-Applied Biosys- binding to the core clock components is observed around tems. The real-time PCR data were normalized to 45S pre-mRNA. Primers 42 hr. Therefore, SIRT1 might also regulate the expression and probes are listed in of circadian output genes expressed with a different phase,possibly through activation of coactivators and transcription Protein Extraction and Immunoblot Analysis factors such as PGC1a, FOXO, or LXR Proteins from mouse liver nuclei and cultured fibroblasts were prepared ) or through the circadian deacetylation according to the NUN procedure (Trichostatin A of histones in nucleosomes associated with clock-controlled was added during the extraction. SDS-PAGE and immunoblot analysis wereperformed according to standard protocols. Antibodies used were rabbit CRY1, PER2, BMAL1, and CLOCK (kindly provided by S. Brown and J. Rip- perger) and rabbit SIRT1 (Upstate), human-SIRT1 (Santa Cruz), pan acetylated Future experiments with Sirt1-deficient and -proficient lysine (Cell Signaling), TAP (OPEN BIOSYSTEMS), mouse V5 (Invitrogen), and cells should shed light on the role of SIRT1 in these additional U2AF65 (Sigma).
mechanisms involved in the regulation of circadian geneexpression.
Coimmunoprecipitation experiments were carried out with mouse liver nuclearextracts or with whole-cell NUN extracts. Extracts were incubated for 12 hr EXPERIMENTAL PROCEDURES with the indicated antibodies at 4C and further incubated with protein A beads(Roche) for an additional 2 hr at 4C. The beads were collected by centrifuga- Cells and Cell Culture tion and washed with NP40 buffer (100 mM Tris-HCL pH 7.5, 150 mM NaCl, NIH 3T3 cells and NIH 3T3-Bmal1-luciferase cells stably expressing Bmal1-lu- 2 mM EDTA, and 1% NP40). Laemmli sample buffer was added and samples ciferase reporter were grown as previously described ( were heated at 95C for 5 min and loaded on a polyacrylamide-SDS PAGE.
WT and Sirt1 KO MEFs were grown in Dulbecco's modified Eagle's medium Purification of the C-terminal TAP-tagged PER2 and TAP-tagged luciferase (DMEM) supplemented with 15% FBS, 100 units/ml penicillin, 100 mg/ml proteins were performed according to standard protocol as previously streptomycin, 2 mM glutamine, 8 mM nonessential amino acids (Sigma), described ().
1 mM Na-Pyruvate, 0.006 mM b-mercaptoethanol, and 18 mM HEPES(pH 7.0) and cultured at 37C in a humidified incubator with 5.6% CO2. Cells In Vitro Deacetylation Assay were synchronized with 100 nM dexamethasone and real-time biolumines- Purified PER2-TAP protein was incubated in deacetylation buffer (50 mM Tris- cence was recorded ). Nicotinamide and dexamethasone HCL pH 8, 50 mM NaCl, 4 mM MgCl2) in the presence of purified recombinant were prepared in H2O and ethanol, respectively. Trichostatin a, cycloheximide, human SIRT1 (BioMol, 5U) or in the presence of protein extracts from WT or and sirtinol (Sigma) were dissolved in DMSO.
Sirt1 KO MEFs lysed in RIPA lysis buffer (150 mM NaCl, 1% NP-40 [vol/vol], 326 Cell 134, 317–328, July 25, 2008 ª2008 Elsevier Inc.
0.5% Na-deoxycholate [DOC vol/vol], 0.1% SDS [vol/vol], 50 mM Tris-Hcl the circadian clock by directing the degradation of cryptochrome proteins.
pH 8, 1 mM dithiothreitol [DTT]). Reactions were carried out in the presence Science 316, 900–904.
or absence of 100 mM NAD+ for 3 hr at 30.
Cardone, L., Hirayama, J., Giordano, F., Tamaru, T., Palvimo, J.J., andSassone-Corsi, P. (2005). Circadian clock control by SUMOylation of SUPPLEMENTAL DATA BMAL1. Science 309, 1390–1394.
Cheng, H.L., Mostoslavsky, R., Saito, S., Manis, J.P., Gu, Y., Patel, P., Bron- Supplemental Data include six figures, one table, and two movies and can be son, R., Appella, E., Alt, F.W., and Chua, K.F. (2003). Developmental defects found with this article online at and p53 hyperacetylation in Sir2 homolog (SIRT1)-deficient mice. Proc. Natl.
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Acute Otitis Media: For the treatment of acute otitis media in pediatric patients due to suscep- tible strains of Streptococcus pneumoniae or Haemophilus influenzae when in the judgment ofthe physician sulfamethoxazole and trimethoprim offers some advantage over the use of other Susceptibility Testing Methods: antimicrobial agents. To date, there are limited data on the safety of repeated use of BACTRIM