largely suppressed in HFD-fed FKOmice (figs.
S10A, S11, and S12). Moreover, JNK deficiency
reduced chemokine expression by macrophages
in vitro under a variety of stimulation conditions
(figs. S10B and S13 to S15). Because tissue
chemokine expression is mediated by both pa-
renchymal cells and macrophages, it is likely that
the reduced chemokine expression detected in
HFD-fed FKOmice is a consequence of both a
primary macrophage defect and a secondary re-
sponse to improved glycemia. Collectively, these
data indicate that reduced chemokine signaling
may contribute to the decreased accumulation of
ATMs in FKOmice. However, no selective defect
in the expression of CCR2 or CCR5 receptors or
therefore represent an intrinsic defect in M1 polar-
ization rather than inefficient M1 ATM recruitment.
derived macrophages (BMDMs) from FWTand
FKOmice (Fig. 4). Treatment with IFN-g causes
M1 differentiation (3) and expression of the cell-
surface marker CD11c (20). Macrophage-specific
as compared with that in control mice (Fig. 4A).
Furthermore, JNK deficiency caused decreased
chemokines (Ccl2 and Ccl5), and cytokine genes
(Il1b, Il6, and Tnfa) in IFN-g–stimulated macro-
detected in lipopolysaccharide (LPS)–stimulated
centration of M1-associated cytokines and che-
FKOmice as compared with FWTmice in vivo
(Fig. 4C). Together, these data demonstrate that
JNK deficiency suppresses M1 polarization. In
contrast, no significant difference in M2 differ-
FKOmice was detected (figs. S16 to S18).
The observation that JNK is required for the
differentiation of pro-inflammatory macrophages
provides an explanation, in part, for previous find-
ings that have implicated JNK in inflammatory
responses (17), including the promotion of TH1
matory macrophages in the liver may account for
the requirement of JNK for the development of
fulminant hepatitis in mouse models (24). JNK-
dependent tissue macrophages may also contribute
to the inflammation associated with insulin resist-
is required for the accumulation of inflammatory
tissue macrophages and insulin resistance caused
by diet-induced obesity. Drug-mediated targeting
of macrophage-expressed JNK therefore repre-
sents apotentialtherapeutic approach to suppress
inflammation that may be applicable to the treat-
ment of inflammatory disorders.
References and Notes
1. K. M. Flegal, B. I. Graubard, D. F. Williamson, M. H. Gail,
JAMA 298, 2028 (2007).
2. M. Qatanani, M. A. Lazar, Genes Dev. 21, 1443 (2007).
3. F. O. Martinez, A. Sica, A. Mantovani, M. Locati,
Front. Biosci. 13, 453 (2008).
4. M. E. Shaul, G. Bennett, K. J. Strissel, A. S. Greenberg,
M. S. Obin, Diabetes 59, 1171 (2010).
5. S. P. Weisberg et al., J. Clin. Invest. 112, 1796 (2003).
6. C. N. Lumeng, J. L. Bodzin, A. R. Saltiel, J. Clin. Invest.
117, 175 (2007).
7. C. N. Lumeng, J. B. DelProposto, D. J. Westcott,
A. R. Saltiel, Diabetes 57, 3239 (2008).
8. D. Patsouris et al., Cell Metab. 8, 301 (2008).
9. K. Kang et al., Cell Metab. 7, 485 (2008).
10. J. I. Odegaard et al., Cell Metab. 7, 496 (2008).
11. J. I. Odegaard et al., Nature 447, 1116 (2007).
12. G. Sabio, R. J. Davis, Trends Biochem. Sci. 35, 490 (2010).
13. G. Solinas et al., Cell Metab. 6, 386 (2007).
14. G. Sabio et al., Science 322, 1539 (2008).
15. S. N. Vallerie, M. Furuhashi, R. Fucho, G. S. Hotamisligil,
PLoS ONE 3, e3151 (2008).
16. J. Hirosumi et al., Nature 420, 333 (2002).
17. R. J. Davis, Cell 103, 239 (2000).
18. A. Mantovani et al., Trends Immunol. 25, 677 (2004).
19. H. Kitade et al., Diabetes 61, 1680 (2012).
20. J. Wang, H. Beekhuizen, R. van Furth, Clin. Exp. Immunol.
95, 263 (1994).
21. D. D. Yang et al., Immunity 9, 575 (1998).
22. C. Dong et al., Science 282, 2092 (1998).
23. C. Dong et al., Nature 405, 91 (2000).
24. M. Das et al., Cell 136, 249 (2009).
Acknowledgments: We thank T. Barrett, V. Benoit, Y. Lee,
and J.-H. Liu for technical assistance; S. Vernia for designing
Taqman probes; and K. Gemme for administrative assistance.
These studies were supported by grants from the National
Institutes of Health (DK090963, DK080756, and CA065861).
The University of Massachussetts Mouse Metabolic Phenotyping
Center is supported by grant DK093000. M.S.H. was
supported by a postdoctoral fellowship (7-10-BETA-02) from
the American Diabetes Association. R.J.D. and J.K.K. are
members of the University of Massachussetts Diabetes and
Endocrinology Research Center (DK032520). R.J.D. and
R.A.F. are Investigators of the Howard Hughes Medical
Institute. Mouse strains described are available to qualified
investigators upon completion of a standard institutional
materials transfer agreement. The data presented in this
manuscript are tabulated in the main paper and in the
Materials and Methods
Figs. S1 to S19
17 July 2012; accepted 6 November 2012
Published online 6 December 2012;
Influence of Threonine Metabolism
on S-Adenosylmethionine and
Ng Shyh-Chang,1,2,3,4,5,6Jason W. Locasale,5,6* Costas A. Lyssiotis,5,6Yuxiang Zheng,5,6Ren Yi Teo,1
Sutheera Ratanasirintrawoot,1,2,3Jin Zhang,1,2,3Tamer Onder,1,2,3Juli J. Unternaehrer,1,2,3
Hao Zhu,1,2,3John M. Asara,5George Q. Daley,1,2,3,4† Lewis C. Cantley5,6†
Threonine is the only amino acid critically required for the pluripotency of mouse embryonic
stem cells (mESCs), but the detailed mechanism remains unclear. We found that threonine and
S-adenosylmethionine (SAM) metabolism are coupled in pluripotent stem cells, resulting in
regulation of histone methylation. Isotope labeling of mESCs revealed that threonine provides a
substantial fraction of both the cellular glycine and the acetyl–coenzyme A (CoA) needed for
SAM synthesis. Depletion of threonine from the culture medium or threonine dehydrogenase (Tdh)
from mESCs decreased accumulation of SAM and decreased trimethylation of histone H3 lysine
4 (H3K4me3), leading to slowed growth and increased differentiation. Thus, abundance of SAM
appears to influence H3K4me3, providing a possible mechanism by which modulation of a
metabolic pathway might influence stem cell fate.
onnections between pluripotency and the
cells (mESCs) are only beginning to be
described (1). Metabolically, mESCs are charac-
terized by a distinct mode of amino acid catabo-
(Thr) oxidation, threonine dehydrogenase (Tdh)
(1). The abundance of Tdh in mESCs is more
(MEFs), and Thr restriction or Tdh inhibition
abolishes mESC growth (1–3). Thus, Thr oxida-
tion appears to be critical for mESCs, but the
relationship between downstream metabolites
and the pluripotent state remains unclear.
To investigate how metabolism is altered
upon reprogramming to the pluripotent state,
1Stem Cell Transplantation Program, Division of Pediatric
Research, Boston Children’s Hospital and Dana-Farber Cancer
Institute, Boston, MA 02115, USA.2Department of Biological
Chemistry and Molecular Pharmacology, Harvard Medical
School, Boston, MA 02115, USA.3Harvard Stem Cell Institute,
Cambridge, MA 02138, USA.4Howard Hughes Medical Insti-
tute, Harvard Medical School, Boston, MA 02115, USA.5De-
partment of Medicine, Division of Signal Transduction, Beth
Israel Deaconess Medical Center, Boston, MA 02215, USA.
6Department of Systems Biology, Harvard Medical School,
Boston, MA 02115, USA.
*Present address: Division of Nutritional Sciences, Cornell
University, Ithaca, NY 14850, USA.
†To whom correspondence should be addressed. E-mail:
firstname.lastname@example.org (G.Q.D.); lewis_cantley@
11 JANUARY 2013 VOL 339
on January 14, 2013
we used liquid chromatography–based tandem
mass spectrometry (LC-MS/MS) (4) to profile
metabolomic changes during reprogramming
into induced pluripotent stem cells(iPSCs). We
transgenes encoding Oct4, Sox2, Klf4, and Myc
(iOSKM) (2, 5) and profiled metabolism during
reprogramming. Hierarchical clustering based
on the abundance of each metabolite revealed
that iPSCs were similar to mESCs and were dis-
tinct from MEFs (Fig. 1, A and B). Inspection of
the top metabolites regulated by OSKM-induced
reprogramming revealed a large number of gly-
colytic intermediates (fig. S1A and table S1),
manyof whichweresignificantly more abundant
within just 4 days of reprogramming (Fig. 1C),
when proliferation wasaccelerated but thecells
were not yet pluripotent (2). Thus, increases in
glycolytic intermediates occurred before the
acquisition of pluripotency (Fig. 1C). The other
top metabolites regulated during reprogramming
are involved in Thr and S-adenosylmethionine
(SAM) metabolism. In contrast to glycolytic in-
termediates, Thr- and SAM-related metabolites
changed late in reprogramming, which suggests
that enhanced Thr and SAM metabolism ac-
companied acquisition of the pluripotent state
(Fig. 1D). Upon reprogramming, some of the
largest decreases were in Thr, Cys, and folate,
and the largest increases were in SAM and
Metabolism is also rewired during mESC
differentiation. In mESCs, the let-7 microRNA
promotes differentiation by suppressing the plu-
ripotency network, whereasLin28a promotes plu-
ripotency by repressing let-7 (6, 7). Hence, we
profiled metabolic changes in mESCs acutely af-
ter dox-induced activation of a transgene encod-
ing let-7 or Lin28a (iLet-7 or iLin28a) while the
manner (fig. S1C), among which were a large
number of Thr and SAM metabolites (Fig. 1E);
that Thr and SAM metabolism are coupled to
We integrated our metabolomics data on
mESCs (table S1) with cDNA microarray data
on mESCs (2) using the KEGG database of
metabolic networks. This analysis showed that
many of the metabolic enzymes that channel Thr
metabolism into SAM metabolism (e.g., Tdh,
Gcat, Gldc, Dhfr, Fol1r, Mat2a/b, and Ahcy) are
Consistent with the enzyme expression pat-
terns, several Thr-SAM pathway inputs such
as Thr, Cys, and folate were less abundant in
mESCs than in MEFs, whereas downstream
outputs such as SAM and cystathionine were
Thus, a Thr-SAM pathway appears to be acti-
vated in mESCs.
To test this pathway, we traced the metabolic
liquid chromatography (HPLC).14C was incor-
used to synthesize these amino acids (Fig. 2B).
In contrast, MEFs incubated with [U-14C]Thr
did not exhibit Thr catabolism (fig. S2A). We
also traced the fate of [U-13C]Thr in mESCs
with LC-MS/MS metabolomics (fig. S2, B to F,
and table S1). mESCs used Thr to synthesize
acetyl-CoA–derived tricarboxylic acid (TCA)
cycle intermediates (Fig. 2, C and D). At steady
state, [U-13C]Thr contributed ~20% of the citrate
via acetyl-CoA, whereas [U-13C]glucose contrib-
uted ~35% via acetyl-CoA (+2 isotopomer). Thus,
Thr contributes significantly to the acetyl-CoA
Fig. 1. TheThr-SAMpathwayisac-
tivated by pluripotency factors. (A)
Heat map showing relative abun-
in the absence of doxycycline treat-
4D); 18 days after dox, whereupon
iPSC clones are fully reprogrammed
using selected reaction monitoring
of metabolite intensities in mESCs
(x axis) versus iPSCs (y axis). Metab-
olite intensities were obtained from
a single SRM transition. (C) SRM
analysis of abundance in glycolytic
intermediates in iOSKM-MEFs dur-
ing reprogramming (n = 3). *P <
0.05, **P < 0.01. (D) SRM analysis
of abundance in Thr-SAM pathway
intermediates in iOSKM-MEFs dur-
ing reprogramming (n = 3). **P <
0.01. (E) SRM analysis of abun-
dance in Thr-SAM pathway inter-
sent SEM from three independent
VOL 33911 JANUARY 2013
on January 14, 2013
pool in mESCs (Fig. 2D). [U-13C]Thr-derived
Gly alsodonated its13C-methyl group toultimate-
ly generate 5-methyltetrahydrofolate (5mTHF)
and SAM (+1 isotopomer), whereas [U-13C]Ser-
derivedGly contributed little to the synthesis of
these metabolites (Fig. 2, C and D). Although
only ~25% of intracellular Gly and 5mTHF, and
~10% of SAM, were labeled by [U-13C]Thr at
steady state, these numbers underestimate the
contribution of Thr to the synthesis of these in-
termediates, because of rapid one-for-one antiport
of intracellular [13C]Gly and [13C]Met for extra-
appearance of large quantities of [13C]Gly and
[13C]Metinthe media: Within 1hour of [13C]Thr
addition to the media, 6% of extracellular Gly and
13% of extracellular Met was13C-labeled, with
no substantial consumption of Gly or Met from
the media (fig. S2G). These data suggest that the
major fraction of the 5mTHF needed for recy-
cling S-adenosylhomocysteine (SAH) to SAM.
To test whether Thr was indeed a major fuel
source for Gly and SAM metabolism, we pro-
filed metabolic changes upon Thr restriction in
medium (DMEM) was removed, but there re-
mained enough Thr in the serum for cellular pro-
viability additively with, and thus independently
of, protein synthesis (fig. S2, H and I). The 80%
drop in intracellular Thr caused intracellular Gly
and the SAM/SAH ratio to decrease steadily, sug-
gesting an imbalance in Gly synthesis and con-
adenosine monophosphate) ratio increased (Fig.
2, E and F), consistent with a compensatory in-
crease in glycolysis. However, some of the TCA
cycle intermediates still decreased (fig. S2J).
Fig. 2. Thr is catabolized to maintain the SAM/SAH ratio in mESCs. (A) Sche-
matic of Thr-SAM pathway activity in pluripotent stem cells, relative to MEFs.
(B) HPLC analysis of14C-labeled amino acids derived from [U-14C]Thr in mESCs
after 24 hours. Scintillation counts per minute (CPM) are plotted against re-
tention time. (C) Fraction of intracellular metabolites derived from [U-13C]Thr
in mESCs over 5 hours, as measured by SRM analysis (n = 3). (D) Steady-state
fraction of intracellular metabolites derived from [U-13C]Thr, [U-13C]Ser,
[U-13C]glucose, or [U-13C]Gln in mESCs after 48 hours, as measured by SRM
analysis(n =3).(E)SRM analysisofmetaboliteabundances inmESCsduring
6 hours of Thr restriction, relative to time zero (n = 3). (F) SRM analysis of
several metabolicratios over a6-hourtime course,relativeto timezero (n = 3).
(G) Feeder-free mESCs were subjected to Thr restriction for 12 hours, then sup-
plemented for 36 hours with the indicated metabolites at the given concentra-
tions.Alkaline phosphatase–positive colonies were quantified and normalized
to mESC colony numbers in normal mESC media (% colonies recovered). X de-
notes relative concentration with respect to DMEM. NAC, N-acetylcysteine; Py,
pyruvate; DMG, dimethylglycine; Bet, betaine; H, hypoxanthine; T, thymidine.
All error bars represent SEM from three independent measurements.
11 JANUARY 2013 VOL 339
on January 14, 2013
of reducing equivalents (Fig. 2F and fig. S2, K
is critically required in mESCs because of its
and because acetyl-CoA produced from Thr could
contribute to this anabolic process (Fig. 2A).
As another test of this model, we attempted
to suppress the effects of Thr restriction on
mESCs by supplementing the culture medium
with downstream metabolites in the Thr-SAM
pathway. Addition of Gly and pyruvate (a source
death after Thr restriction (Fig. 2G). Glucose,
acetate, Ser, Cys, Glu, Gln, N-acetylcysteine, ascor-
bate, or combinations with pyruvate all failed to
prevent mESC death after Thr restriction. How-
ever, a combination of pyruvate with the methyl
donors dimethylglycine or betaine prevented
death from Thr restriction (Fig. 2G); these re-
sults suggest that Thr-derived 5mTHF and its
for mESCs. SAM could not be used because it
cannot cross mammalian plasma membranes
(9). In contrast, 3-deazaadenosine (DZA) (10),
which decreases the SAM/SAH ratio by inhib-
iting SAH hydrolase, did enter mESCs and in-
hibited mESC viability (fig. S2M). Hypoxanthine
after Thr restriction although they could enter
mESCs (11), which suggests that nucleosides
are not important downstream products of Thr-
derived Gly. A downstream product of SAM ca-
ineffective. Thus, Thr appears to be necessary to
generate the optimal balance of Gly and acetyl-
CoA required to synthesize 5mTHF for main-
taining the SAM/SAH ratio in mESCs.
SAM is the universal substrate for all pro-
tein methylation reactions in the cell, and the
SAM/SAH ratio is important for regulating pro-
tein methylation because methyltransferases are
product-inhibited by SAH (Fig. 3A) (13). To test
whether Thr restriction affects protein methyl-
ation, we used a pan–methyl-lysine antibody to
detect differential lysine methylation of proteins
after restriction of Thr, Gly, and Ser. Among the
most abundant methylated proteins (14), histone
H3 showed a drop in methylation upon Thr re-
striction, whereas heat shock protein 8, elon-
gation factor-1a, and actin showed only subtle
for epigenetic plasticity in mESCs, and because
histone H3 lysine 4 trimethylation (H3K4me3)
taining euchromatin (15–18), we tested whether
Thr catabolism regulates H3K4me3 and H3ac
in mESCs. Under milder conditions of Thr re-
(fig. S3A), H3K4me3 dropped after 48 hours,
whereas H3ac remained unchanged (Fig. 3C).
In comparison, MEFs showed no changes in
H3K4me3 nor H3ac upon 0.3X Thr restriction
(Fig. 3C), consistent with our observations that
Thr-SAM metabolism is activated only in the
To test the sensitivity and reversibility of
H3K4me3, we supplemented Thr-restricted
mESCs with various doses of Thr. Although
mESCs lost most of their H3K4me3 after just
6 hours of Thr restriction, refeeding with var-
ious concentrations of Thr for 6 hours reversed
the drop in H3K4me3 (fig. S3B). H3K4me3
could also be restored by adding pyruvate and
Gly to the medium (Fig. 3D). Both pyruvate and
Gly were necessary to restore the SAM/SAH
ratio to a normal state, consistent with the idea
that Thr-derived acetyl-CoA and Gly regulate
H3K4me3 by modulating the SAM/SAH ratio
(Fig. 3E). Conversely, DZA, which decreases
the SAM/SAH ratio, led to a rapid extinction of
H3K4me3 and overrode the effects of pyruvate
which suggests that acetyl-CoA and Gly regulate
the SAM/SAH ratio to modulate H3K4me3. We
thentestedH3methylation on lysines 4,9,27,36,
and 79 in mESCs after restriction of a variety of
were decreased by Thr restriction. H3K4me1,
did not change significantly with any amino acid
(Fig. 3F). Also, a-ketoglutarate did not change
gesting minimal influence on a-ketoglutarate–
dependent histone demethylases. These results
suggest that the SAM/SAH ratio maintained by
Thr catabolism is not required to regulate all pro-
ic lysines such as H3K4me2 and H3K4me3
transferase activity from nuclear lysates further
indicated that the change in H3K4me3 with Thr
concentration was not due to a change in the net
amount of methyltransferase activity (fig.S3D),
supporting the idea that it is the change in the
substrate/product ratio (SAM/SAH) that influ-
enced the amount of H3K4me3.
To test whether the Thr-SAM pathway has
functional consequences on the pluripotency and
differentiation of mESCs, we partially depleted
Fig. 3. Influence of Thr-SAM metabolism on H3K4 meth-
ylation. (A) Schematic of methyltransferase reactions.
(B) Immunoblot analysis of proteins from mESCs with
an antibody against methyl-lysine, after restriction of
the indicated amino acids for 24 hours. (C) Immunoblot
analysis of mESCs and MEFs for H3K4me3 and H3ac when exposed to 3X and 0.3X concentrations for
48 hours. (D) Immunoblot analysis of mESCs for H3K4me3 after Thr restriction (0X) for 6 hours, then
re-fed for 6 hours with indicated metabolites. (E) SAM/SAH ratio after Thr restriction (0X) for 6 hours,
then re-fed for 6 hours with indicated metabolites. (F) Immunoblot analysis of mESCs for H3K4me3,
H3K4me2, H3K4me1, H3K9me3, H3K27me3, H3K36me3, and H3K79me3 after restriction (0X) of
the indicated amino acids for 24 hours.
VOL 339 11 JANUARY 2013
on January 14, 2013
the Tdh enzyme by RNA interference (Fig. 4A
and fig. S4A). In low-Thr conditions, partial Tdh
depletion led to a decrease in mESC growth and
an increase in mESC differentiation (Fig. 4B).
Expression profiling by quantitative reverse tran-
scription polymerase chain reaction (qRT-PCR)
revealed that Tdh depletion led to a decrease
in expression of pluripotency factors, including
Oct4, Sox2, Nanog, Rex1, and Blimp1, and in-
creased expression of thedifferentiation factors
Foxa2 and Sox17 (fig. S4B). Embryoid body
that Tdh depletion led to more rapid extinction
of the pluripotency factor Sox2 and an aberrant
increase in transcription of the differentiation
factors Gata4 and Sox17 (fig. S4C).
The differentiation of mESCs after partial
Tdh depletion was dependent on the amount of
Thr in the culture medium, which suggests that
Tdh was required for its metabolic function
(Fig. 4B). Indeed, Tdh depletion decreased Thr-
SAM flux (Fig. 4C). At steady state, Tdh deple-
ratio (Fig. 4D). Tdh depletion also decreased
H3K4me3 abundance (Fig. 4A). Thus, Thr ca-
tabolism by Tdh appears to be required for
maintenance of the pluripotent epigenetic state.
Tdh in the Thr-SAM pathway are also required
by mESCs, including glycine decarboxylase
(Gldc), which produces methylene-THF from
Gly (19), and methionine adenosyltransferase
(Mat2a), which produces SAM from Met. De-
pletion of Gldc decreased Thr-SAM flux and the
level of H3K4me3, and decreased mESC colony
growth (fig. S4, D to H). In contrast, transient
overexpression of Gldc prevented mESC death
after Thr restriction (fig. S4, I and J), suggesting
that the 5mTHF and SAM downstream of Thr
and Gly are necessary for mESCs. Depletion of
the downstream Mat2a also decreased SAM
synthesis and H3K4me3 levels, and decreased
transient overexpression of the SAH hydrolase
(Ahcy), which decreases SAH and thereby in-
creases the SAM/SAH ratio, prevented mESC
death after Thr restriction (fig. S4, I and J).
complexes—such as Wdr5, Dpy30, and Setd1a—
also improved mESC survival after Thr restric-
tion, supporting the idea that Thr-SAM metab-
olism may affect mESCs in part through effects
Our results show that the Thr-SAM pathway
dependent changes in the SAM/SAH ratio cor-
related with H3K4me3, thus revealing a possible
mechanistic link between cellular metabolism
and the epigenetic state. The unique activity of
Tdhinconverting ThrintobothGlyand acetyl-
CoA appears to optimize the synthesis of SAM
to maintain a high SAM/SAH ratio. H3K4me3
histone H3 lysines to changes in Thr metabo-
lism, possibly because of the high abundance
of mESCs and its rapid turnover (15–18, 20, 21).
H3K4me3 is critical for self-renewal of pluri-
potent stem cells (22). Because dysregulation of
Gly metabolism has been implicated in a variety
of human cancers (4, 19, 23, 24) and because the
in humans (1), our findings may provide insight
into a metabolic pathway that is dysregulated dur-
ing human tumorigenesis.
References and Notes
1. J. Wang et al., Science 325, 435 (2009).
2. T. S. Mikkelsen et al., Nature 454, 49 (2008).
3. P. B. Alexander, J. Wang, S. L. McKnight, Proc. Natl.
Acad. Sci. U.S.A. 108, 15828 (2011).
4. J. W. Locasale et al., Nat. Genet. 43, 869 (2011).
5. M. Stadtfeld, N. Maherali, D. T. Breault, K. Hochedlinger,
Cell Stem Cell 2, 230 (2008).
6. S. R. Viswanathan, G. Q. Daley, R. I. Gregory, Science
320, 97 (2008).
7. C.Melton, R. L.Judson,R. Blelloch, Nature 463,621(2010).
8. H. Eagle, K. A. Piez, M. Levy, J. Biol. Chem. 236, 2039
9. J. M. McMillan, U. K. Walle, T. Walle, J. Pharm.
Pharmacol. 57, 599 (2005).
10. P. S. Backlund Jr., D. Carotti, G. L. Cantoni, Eur. J. Biochem.
160, 245 (1986).
11. V. Valancius, O. Smithies, Mol. Cell. Biol. 11, 1402 (1991).
12. D. Zhang et al., Genes Dev. 26, 461 (2012).
13. Z. Luka, S. H. Mudd, C. Wagner, J. Biol. Chem. 284,
14. H. Iwabata, M. Yoshida, Y. Komatsu, Proteomics 5, 4653
15. A. Gaspar-Maia, A. Alajem, E. Meshorer, M. Ramalho-Santos,
Nat. Rev. Mol. Cell Biol. 12, 36 (2011).
16. V. Azuara et al., Nat. Cell Biol. 8, 532 (2006).
17. X. D. Zhao et al., Cell Stem Cell 1, 286 (2007).
18. G. Pan et al., Cell Stem Cell 1, 299 (2007).
19. W. C. Zhang et al., Cell 148, 259 (2012).
20. R. B. Deal, J. G. Henikoff, S. Henikoff, Science 328, 1161
21. B. M. Zee et al., J. Biol. Chem. 285, 3341 (2010).
22. Y. S. Ang et al., Cell 145, 183 (2011).
23. R. Possemato et al., Nature 476, 346 (2011).
24. M. Jain et al., Science 336, 1040 (2012).
Acknowledgments: We thank members of the Daley and
Cantley labs for helpful discussions. Supported by the NSS
Scholarship from the Agency for Science, Technology and
Research, Singapore (N.S.-C.), the American Cancer Society
and NIH grant 4R00CA168997-02 (J.W.L.), a Damon Runyon
Cancer Research Foundation Amgen fellowship (C.A.L.), NIH
grants RC2HL102815 and U01 HL100001 (G.Q.D.), and
NIH grant R01 GM56203 (L.C.C.). G.Q.D. is an investigator of
the HHMI. G.Q.D., as a co-founder and scientific advisory
board member, holds stock options and receives consulting
fees from iPierian Inc., a biopharmaceutical company that uses
iPS cells in drug discovery against neurologic disease. L.C.C. is
the founder and a member of the board of directors of Agios
Pharmaceuticals, a company that targets metabolic enzymes for
Materials and Methods
Figs. S1 to S4
26 June 2012; accepted 22 October 2012
Published online 1 November 2012;
Fig. 4. Tdh regulates the pluripotency of mESCs. (A) Immunoblot for Tdh, H3K4me3, H3K27me3, and
pan–methyl-lysine in mESCsgrown under 0.1XThr, immediately after depletion of Tdh with two different
short hairpin RNAs (shTdh), compared to a control shRNA targeting luciferase (shLuc). (B) Micrographs of
alkaline phosphatase staining in mESCs after shTdh or shLuc, seeded at clonal density without feeder
MEFs, after 48 hours of culture in 0.1X, 0.3X, and 1X Thr concentrations. (C) Steady-state fraction of
intracellular metabolites derived from [U-13C]Thr in mESCs after 24 hours, as measured by SRM analysis,
after 48 hours of dox induction of shTdh or shLuc (n = 3). (D) SRM analysis of metabolite abundances in
mESCs after 48 hours of dox induction of shTdh or shLuc (n = 3). All error bars represent SEM from three
11 JANUARY 2013VOL 339
on January 14, 2013