, 654 (2009);
et al.Yasukazu Nakahata,
Salvage Pathway by+Circadian Control of the NAD
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on May 1, 2009
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material on Science Online.
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27. We thank S.-H. Yoo and members of the Bass, Imai, and
Takahashi laboratories for discussions. Work was supported
by grants from the National Institute of Diabetes and
Digestive and Kidney Diseases (T32 DK007169) to
K.M.R.; the National Institute on Aging (AG02150), the
Ellison Medical Foundation, and the Longer Life Foundation
to S.I.; NIH (PO1 AG011412), the Chicago Biomedical
Consortium Searle Funds, and the Juvenile Diabetes
Research Foundation to J.B.; and a grant from the National
Institute of Mental Health (P50 MH074924) to J.S.T. J.Y. is
supported by the Japan Research Foundation for Clinical
Pharmacology and Keio University Medical Science Fund,
J.S.T. is an Investigator in the Howard Hughes Medical
Institute and a cofounder of ReSet Therapeutics, and J.S.T.
and J.B. are members of its scientific advisory board. J.B. is
also an adviser and receives support from Amylin
Pharmaceuticals. S.I. holds a patent related to the work
reported in this paper, specifically NAMPT and NMN.
Supporting Online Material
Materials and Methods
Figs. S1 to S6
30 January 2009; accepted 9 March 2009
Published online 19 March 2009;
Include this information when citing this paper.
Circadian Control of the NAD+
Salvage Pathway by CLOCK-SIRT1
Yasukazu Nakahata, Saurabh Sahar, Giuseppe Astarita,
Milota Kaluzova, Paolo Sassone-Corsi*
Many metabolic and physiological processes display circadian oscillations. We have shown
that the core circadian regulator, CLOCK, is a histone acetyltransferase whose activity is
counterbalanced by the nicotinamide adenine dinucleotide (NAD+)–dependent histone deacetylase
SIRT1. Here we show that intracellular NAD+levels cycle with a 24-hour rhythm, an oscillation
driven by the circadian clock. CLOCK:BMAL1 regulates the circadian expression of NAMPT
(nicotinamide phosphoribosyltransferase), an enzyme that provides a rate-limiting step in the
NAD+salvage pathway. SIRT1 is recruited to the Nampt promoter and contributes to the circadian
synthesis of its own coenzyme. Using the specific inhibitor FK866, we demonstrated that NAMPT
is required to modulate circadian gene expression. Our findings in mouse embryo fibroblasts reveal
an interlocked transcriptional-enzymatic feedback loop that governs the molecular interplay
between cellular metabolism and circadian rhythms.
in part, rely on epigenetic control and chromatin
remodeling (4). The circadian regulator CLOCK
has an intrinsic acetyltransferase activity, which
enables circadian chromatin remodeling by acet-
ylating histones (5) and nonhistone proteins,
including its own partner BMAL1 (6). The his-
tone deacetylase (HDAC) that counterbalances
the histone acetyltransferase function of CLOCK
is SIRT1 (7, 8), an enzyme whose activity is de-
pendent on intracellular NAD+levels (9). Although
NAD+is SIRT1’s natural cosubstrate, the reduced
form of NAD+(NADH) and the by-product of
NAD+consumption, nicotinamide (NAM), re-
press the activity of SIRT1 (9) and generate an
enzymatic feedback loop on the HDAC function
of this enzyme. Two main systems determine
NAD+levels in the cell, the de novo biosynthesis
from tryptophan and the NAD+salvage pathway
(10). A critical step of the latter pathway is con-
trolledbytheenzyme nicotinamide phosphoribosyl-
ccumulating evidence reveals intriguing
links between the circadian clock and
cellular metabolism (1–3), which, at least
transferase (NAMPT) (11), also known as visfatin
or pre–B cell colony-enhancing factor (PBEF),
which catalyzes the first step in the biosynthesis
of NAD+from NAM. NAMPT is implicated in
cellular metabolism, senescence, and survival in
response to genotoxic stress (12–14).
Because of the role of SIRT1 in modulating
clock function (7), we reasoned that circadian tun-
ing may be achieved by NAD+oscillating levels.
were serum-entrained, and cellular NAD+levels
were measured by liquid chromatography coupled
to tandem mass spectrometry (LC/MSn) from total
extracts prepared at various time intervals after
serum shock (15). Cellular NAD+showed a re-
producible circadian oscillation of about 2.5-fold
(Fig. 1A). The average NAD+concentration of
25 pmol/mg protein (~60 mM) found in MEFs is
in keeping with recent reports for rat axons using
LC coupled to an ultraviolet detector (9 pmol/mg
protein) (16), mouse erythrocyte using LC/MS/MS
(368 mM) (17), and HEK293 cells using LC with
MS (365 mΜ) (14). Note that cellular NAD+os-
ity and its phase is opposite to that of acetylation
of histone H3 and BMAL1 (7).
NAD+levels did not oscillate in entrained
MEFs originated from the clock/clock mutant
mice (c/c) and from the arrhythmic Cry1/Cry2
double-knockout (KO) mice (cry) (Fig. 1B and
fig. S1), which demonstrated that NAD+oscilla-
tion is driven by the circadian clock. Total cellular
NAM levels measured by LC/MSnalso showed
oscillation, albeit more moderate, with a phase
opposite to that of NAD+. Also NAM oscillation
was abolished in the MEFs from c/c and cry
mice (Fig. 1D). Thus, the circadian clock controls
the levels of these metabolites. This analysis also
provided a clue: NAD+and NAM levels are sig-
nificantly lower in mutant MEFs than in WTcells
(for NAD+only 1.1 pmol/mg protein in c/c MEFs,
about 5% of that in WT MEFs) (Fig. 1, C and
E). This notion points to an involvement of the
NAD+salvage pathway and, because of its dy-
namic regulation, specifically to NAMPT, whose
prominent function in NAD+production has been
demonstrated (18–20). Furthermore, increased flux
through the NAD+salvage pathway is responsible
for sirtuin-dependent responses even under condi-
tions of unaltered steady-state NAD+levels (12).
Next, we analyzed the expression of NAD+
salvage pathway metabolic enzymes. The nico-
tinamide mononucleotide adenylyltransferases,
NMNAT1, NMNAT2, and NMNAT3, are central
in NAD biosynthesis; they catalyze the adenyl-
ation of nicotinamide mononucleotide (NMN) or
nicotinic acid mononucleotide (NaMN) by using
the adenosine monophosphate (AMP) moiety of
adenosine triphosphate to form NAD+or nico-
tinic acid dinucleotide, respectively (Fig. 2A). The
expression of these three enzymes is mildly oscil-
latory (fig. S2). Thus, our attention focused on
NAMPT, which operates asrate-limiting enzyme
in NAD+production within the NAD+salvage
pathway (10, 14, 21). Expression of Nampt is
robustly circadian in livers from WT mice,
whereas oscillation is virtually absent in c/c mice
serum-entrained WT MEFs, paralleling Dbp oscil-
lation. Again, Nampt oscillation is abolished in
c/c MEFs (Fig. 2C).
The Nampt promoter contains three putative,
highly conserved, E-box sequences (CACGTG)
Department of Pharmacology, School of Medicine, Uni-
versity of California, Irvine, Irvine, CA 92697, USA.
*Corresponding author: Paolo Sassone-Corsi, firstname.lastname@example.org
1 MAY 2009VOL 324
on May 1, 2009
(Fig. 3, A and B). Using promoter mutagenesis
and transient expression in cultured cells, we dem-
onstrate that the Nampt promoter is readily ac-
tivated by CLOCK:BMAL1 through the E-boxes
(Fig. 3C). Next, using chromatin immunoprecipi-
tation (ChIP), we showed that CLOCK:BMAL1
(Fig. 3, D and E), consistent with Nampt circa-
dian expression. We have recently demonstrated
(7). Dual cross-linking ChIP assays showed that
SIRT1 binds to the E-boxes in a time-dependent
manner, following the circadian timing of
CLOCK:BMAL1 recruitment (Fig. 3, D and E).
Thus, as previously shown for Dbp and Per2 (7),
CLOCK and SIRT1 contribute to circadian chro-
matin remodeling at the Nampt promoter. As
NAD+intracellular levels directly influence the
HDAC activity of SIRT1 (22–24), an enzymatic-
transcription feedback loop seems to operate, in
which NAD+levels determine the oscillatory
synthesis of NAMPT, the key enzyme in the
To address whether NAMPTis critical in mod-
ulating clock function, we took advantage of
FK866, a low-molecular-weight specific NAMPT
inhibitor. FK866 lowers cellular NAD+and NAM
levels over a prolonged length of time (18) (figs. S3
and S4). Pharmacological inhibition of NAMPT
significantly modifies the circadian expression of
Per2 and Dbp in serum-stimulated MEFs (Fig.
4A) and causes an earlier onset of the circadian
peak for both genes by 3 to 4 hours and increases
the amplitude of oscillation by 30 to 40%. This
effect of FK866 is highly similar to the one
caused by inhibition of SIRT1 (7). Thus, we pre-
dicted that blocking NAMPT would modify
BMAL1 acetylation, a regulatory event critical
to clock function (6). Using the antibody against
acetyl-BMAL1 recently developed in our lab-
oratory (7), we showed that inhibition of NAMPT
induces a considerable increase and a broader
peak of Lys537acetylation (Fig. 4B). This BMAL1
acetylation profile is basically equivalent to the
one observed in MEFs and livers from Sirt1−/−
How intimate is the link between cellular me-
tabolism and the circadian clock? Specifically,
are metabolic pathways regulating the circadian
machinery (3, 25, 26), or are molecular elements
of the clock controlling metabolism (3, 27, 28)?
The findings reported here demonstrate that both
pathways exist in the cell, and each utilizes a
discrete molecular mechanism of control in which
the enzyme NAMPT occupies a pivotal position.
Our results reveal the interlocking of two auto-
regulatory systems, in which a classical transcrip-
tion circadian loop is coupled to an enzymatic
feedback loop (Fig. 4C). These findings point to
the oscillation of NAD+as a key regulatory step in
the modulation of rhythms in metabolic tissues
and peripheral clocks. Note that FK866 may be
used for treatment of diseases that may be im-
plicated in deregulated apoptosis or act as a sensi-
tizer for genotoxic agents (18). Thus, our findings
could have multiple implications including ave-
nues to pharmacological intervention.
References and Notes
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2. C. B. Green, J. S. Takahashi, J. Bass, Cell 134, 728 (2008).
3. K. Eckel-Mahan, P. Sassone-Corsi, Nat. Struct. Mol. Biol.,
4. W. J. Belden, J. C. Dunlap, Cell 134, 212 (2008).
Fig. 1. Circadian oscillation of NAD+is controlled by the clock. (A and B) Cellular NAD+was extracted
from serum-entrained MEFs derived from WT (A), clock/clock mutant mice (c/c), and Cry1/Cry2 double-
KO mice (cry) (B) at indicated time points and analyzed by LC/MSn. Three independent experiments
were performed, and representative results are shown. All data are means T SEM of three independent
samples. (C) Average NAD+levels. (means T SEM of >50 independent samples). (D) Cellular NAM was
extracted from serum-entrained MEFs derived from WT, c/c, and cry mice at indicated time points and
analyzed by LC/MSn. (E) Average NAM levels (means T SEM of >50 independent samples).
Fig. 2. The circadian clock controls Nampt gene expression. (A) Scheme of the NAD+salvage pathway
in mammals. (B) Nampt gene expression in livers from light-entrained WT and c/c mutant mice as
measured by quantitative polymerase chain reaction (Q-PCR). Nampt gene expression at Zeitgeber time
(ZT) 15 in liver from WT mice was set to 1. (C) Nampt and Dbp gene expressions in serum-entrained
MEFs from WT and c/c mutant mice were quantified by Q-PCR. Expression at time 0 in WT MEFs was set
to 1. All data are means T SEM of three independent samples.
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on May 1, 2009
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Fig. 4. NAMPT modulates the circadian clock. (A) Per2 and Dbp gene expression levels in serum-entrained
MEFs treated with 10 nM FK866 or ethanol (EtOH) as control (solvent: ctrl) were analyzed by Q-PCR.
Highest value for each gene in EtOH-treated MEFs was set to 1. All data are means T SEM of three
independent samples. (B) BMAL1 Lys537acetylation profile in serum-entrained MEFs treated with 10 nM FK866 or EtOH. Extracts prepared from
indicated time points and BMAL1 acetylation were monitored (7). Input samples were probed with BMAL1- and GAPDH-specific antibodies. (C) Scheme
of the transcription-enzymatic interplay by which the circadian machinery governs the intracellular levels of NAD+. The NAD+-dependent deacetylase
SIRT1 is thereby controlling the oscillatory synthesis of its own coenzyme.
Fig. 3. Regulation of the Nampt
promoter by CLOCK:BMAL1 and
SIRT1. (A) Schematic diagram of reg-
ulatory elements in human Nampt
promoter. TSS(transcription start site)
truncated forms of Nampt promoter
used in (C), arrows. Putative tran-
scription factor-binding sites are
indicated: HRE, hypoxia response
element; SP1, specificity protein 1;
CRE, cyclic AMP–response element;
AP-1, activator protein 1; GRE, gluco-
corticoid receptor response element.
(B) Conserved E-boxes (capitalized)
are shown. Numbers indicate positions
from human TSS. (C) Schematic dia-
gram of Nampt promoter constructs.
Effect of CLOCK:BMAL1 (+CL/BM;black
bars) on luciferase activity is shown.
Luciferase activity of CLOCK:BMAL1
on the pGL4.10 was set as 1. All
data are means T SEM of three in-
dependent samples. (D) Represent-
ative results of ChIP assays analyzed
by semiquantitative PCR. Dual cross-
linked nuclear extracts isolated from
MEFs after 16 or 24 hours serum
shock and subjected to ChIP assay
with antibodies against SIRT1, CLOCK,
or BMAL1 or no antibody (ctrl). No
antibody and 3′R primers were used
as controls for immunoprecipitation
and PCR, respectively. (E) Quantification of ChIP by Q-PCR. Q-PCR was performed on the samples described in (D). All data are means T SEM of three
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29. Thanks to D. Piomelli for insightful discussions and
help. Thanks also to L. Guarente, K. Yamagata, and
members of the Sassone-Corsi laboratory for help,
reagents, and support. Y.N. was supported by the
Japan Society for the Promotion of Science
Postdoctoral Fellowships for Research Abroad. S.S. was
supported by a postdoctoral fellowship from the
American Heart Association, Western States Affiliate.
Work supported by the Cancer Research Coordinating
Committee of the University of California and NIH
Supporting Online Material
Materials and Methods
Figs. S1 to S4
12 January 2009; accepted 2 March 2009
Published online 12 March 2009;
Include this information when citing this paper.
A Cytidine Deaminase Edits C to U
in Transfer RNAs in Archaea
Lennart Randau,1*† Bradford J. Stanley,1* Andrew Kohlway,1Sarah Mechta,1
Yong Xiong,1† Dieter Söll1,2†
All canonical transfer RNAs (tRNAs) have a uridine at position 8, involved in maintaining tRNA
tertiary structure. However, the hyperthermophilic archaeon Methanopyrus kandleri harbors 30 (out
of 34) tRNA genes with cytidine at position 8. Here, we demonstrate C-to-U editing at this location in
the tRNA’s tertiary core, and present the crystal structure of a tRNA-specific cytidine deaminase,
CDAT8, whichhasthe cytidinedeaminase domain linked toa tRNA-binding THUMP domain.CDAT8 is
specific for C deamination at position 8, requires only the acceptor stem hairpin for activity, and
belongs to a unique family within the “cytidine deaminase–like” superfamily. The presence of this
C-to-U editing enzyme guarantees the proper folding and functionality of all M. kandleri tRNAs.
cytidine (1). In transfer RNA (tRNA), adenosine
deaminases acting on tRNA engage in adenosine-
in the formation of 1-methylinosine found in some
editing in tRNA is seen in plant mitochondrial
(6), eukaryotic, organellar (7), and cytoplasmic
Trypanosoma brucei tRNA (8), but the enzyme(s)
involved in these conversions are unknown.
In Methanopyrus kandleri, only four tRNAs
possessa U8(T8in thetRNAgene),whereasthe
he gene sequence of RNA molecules can
be altered by editing enzymes that cat-
alyze the deamination of adenosine or
other 30 tRNA genes encode C8 (9, 10). The
highly conserved U8 in tRNAs forms a reverse
Hoogsteen tertiary base pair with A14 that sta-
bilizes the sharp kink between the acceptor stem
and base A9 (fig. S1) (11). A U8C mutation is
associated with mitochondrial myopathy in hu-
mans (12) and destabilizes the tRNA structure,
resulting in a molecule unfit in translational initia-
to U in order to support the tertiary interaction
with A14, which is present in all 34 tRNAs.
Sequencing of total small RNA from M.
kandleri revealed that mature tRNAAsp, tRNACys,
and tRNAHisspecies contain U8, whereas their
respective genes have C8 (Fig. 1, A and B).
Furthermore, precursor tRNAAspmolecules con-
taining 5´ leader and 3´ trailer sequences still
have C8, indicating that C-to-U editing occurs at
the RNA level (Fig. 1, A and B). Primer ex-
tension of an oligonucleotide annealed to the
tRNA directly downstream of base 8 with either
mentary to the unedited C8) or 2´-deoxyadenosine
5´-triphosphate (dATP; complementary to the
edited U8) revealed that most of base C8 in
tRNAAspand tRNAHisis edited to U (Fig. 1C).
A computational search for C-to-U editing
enzyme candidates in M. kandleri used the con-
Cys (where X is any amino acid) (14), and yielded
one candidate, the orphan protein MK0935, which
also contained a THUMP domain, shown to me-
diate activity on the tertiary core of tRNA in
and RNAmethylases(15,16). TheMK0935gene
(renamed CDAT8, “cytidine deaminase acting on
tRNA base C8”) was cloned, expressed in
Escherichia coli, and the recombinant enzyme
The C-to-U editing activity of CDAT8 was
verified in vitro by measuring the deamina-
tion of cytidine residues including C8 in
M. kandleri tRNAHistranscribed in the presence
of [a-32P]cytidine 5´-triphosphate (CTP) (Fig. 2,
A and B) (17). To analyze the substrate recog-
minimal tRNA substrates based on M. kandleri
tRNAAsp(Fig. 2C). The smallest substrate (sAsp1
in Fig. 2C) consists only of the acceptor stem mi-
where GGAC represents the variable loop of
tRNAAsp. The slightly larger substrates, sAsp2 and
sAsp3, additionally contained the tRNA T-stem/
loop with bulged-out C8G9 and C8G9GGAC
sequences, respectively. All three minimal con-
structs were substrates for CDAT8, indicating the
dispensability of the tRNA anticodon and D-arm
for activity (Fig. 2D). Although all three constructs
were edited by CDAT8 at 70°C, only sAsp3 was
a substrate at 37°C. These results suggest that
1Department of Molecular Biophysics and Biochemistry,
Yale University, New Haven, CT 06520, USA.2Department
of Chemistry, Yale University, New Haven, CT 06520, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail:
email@example.com (L.R.); firstname.lastname@example.org (D.S.);
Fig. 1. C8-to-U8 editing in vivo. (A)
Sequencing results for the indicated
tRNA gene and reverse transcription
polymerase chain reaction (RT-PCR)
products of circularized precursor tRNA
(pre, with 5´ and 3´ extensions) and
mature tRNA (mat). Only the mature
tRNA displays the edited U8 (T8 in
DNA, underlined). (B) Sequencing
traces of RT-PCR products with base 8
underlined. (C) The majority of tRNAs
are edited to U8 as minisequencing
in the presence of dATP but not dGTP.
VOL 3241 MAY 2009
on May 1, 2009