Stimulation of Histone Deacetylase Activity by Metabolites of
Maria Vogelauer‡1, Abigail S. Krall‡1, Matthew A. McBrian‡§2, Jing-Yu Li‡¶, and Siavash K. Kurdistani‡§¶?3
Los Angeles, California 90095
Background: Little is known about potential regulation of non-sirtuin HDACs by cellular metabolites.
Results: HDAC activity is stimulated by conjugated CoA derivatives and NADPH and is inhibited by free CoA.
Conclusion: Cellular metabolites required for anabolism directly stimulate HDAC activity.
Significance: Cellular HDAC activity may be modulated in response to the metabolic state of a cell.
Histone deacetylases (HDACs) function in a wide range of
molecular processes, including gene expression, and are of sig-
nificant interest as therapeutic targets. Although their native
complexes, subcellular localization, and recruitment mecha-
nisms to chromatin have been extensively studied, much less is
known about whether the enzymatic activity of non-sirtuin
HDACs can be regulated by natural metabolites. Here, we show
that several coenzyme A (CoA) derivatives, such as acetyl-CoA,
butyryl-CoA, HMG-CoA, and malonyl-CoA, as well as NADPH
recombinant HDAC1 and HDAC2 in vitro following a mixed
activation kinetic. In contrast, free CoA, like unconjugated
butyrate, inhibits HDAC activity in vitro. Analysis of a large
6-phosphate dehydrogenase (G6PD) to decrease NADPH levels
olites and the exquisite selectivity of NADPH over NADP?,
NADH, and NAD?as an HDAC activator reveal a previously
unrecognized biochemical feature of the HDAC proteins with
well as the development of more specific and potent HDAC
HDACs4catalyze the removal of acetyl groups from lysine
residues of a wide array of substrate proteins in addition to
histones, including transcription factors and other nuclear and
HDACs are classified into class I (HDAC1–3 and -8), class II
and class IV (HDAC11) (1, 2). HDACs are ubiquitously
expressed and play roles in regulation of cell growth, differen-
tiation, and death (1, 3). They show deregulated expression in
pathological states such as cancer and have therefore garnered
significant interest as therapeutic targets (4). There are cur-
rently more than 80 clinical trials underway to determine the
therapeutic efficacies of several HDAC inhibitors that have
shown anti-neoplastic functions in cell culture and animal
studies. Two HDAC inhibitors, Vorinostat and Romidepsin,
have been approved for use in treatment of cutaneous T-cell
lymphoma (5, 6). Other HDAC inhibitors have been used suc-
cessfully as mood stabilizers and anti-epileptics and have
shown promise in treatment of diverse neurological diseases
sclerosis, and spinal muscular atrophy (7, 8). Therefore, it is
ulate the activities of HDACs for the development of more
effective HDAC inhibitors and more informed use of the cur-
rent drugs in the clinic.
HDACs are generally found in multiprotein complexes, the
functions of which can be regulated through several mecha-
nisms. These complexes can be recruited to specific genomic
loci by DNA-binding proteins, thereby bringing HDACs to
taining complexes are localized in both the nucleus and cyto-
plasm but can shuttle between the two subcellular compart-
ments, affecting their accessibility to relevant substrates (12,
13). Modulating the stability of interactions between HDACs
and other members of the complexes in which they reside can
also affect the overall activity of HDAC complexes (14, 15).
Other regulatory mechanisms may affect the HDAC enzy-
matic activity more directly. Acetylation of HDAC1 protein
reduces its enzymatic activity both in vivo and in vitro (16).
Sumoylation of HDAC1 increases its enzymatic activity, and
phosphorylation of HDAC1 stimulates both its activity and
complex formation (17, 18). The catalytic activity of class III
HDACs (Sirtuins) depends on the presence of the oxidized
Because the availability of NAD?is linked to cellular metabo-
* This work was supported in part by a California Institute for Regenerative
Medicine award and Beckman Young Investigator and Howard Hughes
Medical Institute Early Career awards (to S. K. K.).
1Both authors contributed equally to this work.
2Supported in part by a UCLA Genetics Training Grant fellowship.
3To whom correspondence should be addressed. Tel.: 310-794-5194; E-mail:
4The abbreviations used are: HDAC, histone deacetylase; G6PD, glucose-
6-phosphate dehydrogenase; rcf, relative centrifugal force; DHEA,
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 38, pp. 32006–32016, September 14, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
32006 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER38•SEPTEMBER14,2012
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sirtuins (22). Such direct regulation by a metabolic cofactor
may transmit information on the cellular energy state to the
chromosome, influencing nuclear functions such as gene
expression and DNA replication.
However, little is known about direct regulation of non-sir-
of this kind of regulation is the binding and inhibition of
HDAC1 and -2 by the endogenous lipid mediator sphingosine
1-phosphate (23). Because HDACs release free acetate anions
from chromatin, we asked whether intermediates of nitrogen
and carbon metabolism that generate or consume two-carbon
units as acetate directly regulate HDAC activity.
Here, we show that coenzyme A (CoA) derivatives, such as
stimulate the activity of class I HDACs on histones, whereas
free CoA inhibits HDAC activity in vitro. Detailed kinetic anal-
the enzyme for the substrate as well as its catalytic activity.
Inhibition by free CoA is substrate-independent and is charac-
terized by decreased reaction velocity while at the same time
increasing the affinity of the enzyme for histones. We further
as inhibition of NADPH production increases global histone
acetylation. Many of the identified metabolites are products of
catabolic pathways of glucose and amino acids that together
bolic reactions that fuel cell growth and replication. Our data
therefore suggest that cellular HDAC activity may be tightly
linked to cellular biosynthetic capacity. Furthermore, identifi-
cation of natural activators and inhibitors of HDACs that con-
tain a nucleotide-like moiety may enhance our understanding
of structure-function relationship and better inform the design
and development of HDAC inhibitors.
Experiments in Figs. 1–3 were performed with recombinant
HDACs obtained from US Biological. All metabolic com-
pounds were purchased from Sigma as sodium or lithium salts.
Generation of Recombinant WT HDAC1 and HDAC1
Mutants—An EcoRI-HDAC1-His6-SalI fragment was gener-
ated by PCR using a commercially available cDNA for HDAC1
(OriGene). Recombinant HDAC baculovirus was generated
using the Bac-to-Bac expression system (Invitrogen), following
by site-directed mutagenesis on the pFastbac-HDAC1 plasmid
using QuikChange Site-directed mutagenesis kit (Agilent).
Purification of Recombinant HDAC Protein—Sf9 cell pellets
were washed with ice-cold PBS and resuspended in hypotonic
2.5 mM ?-mercaptoethanol, 1? protease inhibitor mixture
(Roche Applied Science)). The cells were incubated on ice for
30 min and then disrupted using a Dounce homogenizer.
Nuclei were spun down at 7000 ? g for 10 min, and the cyto-
plasmic supernatant (containing the majority of the recombi-
trifuged at 22,000 ? g for 30 min to remove debris. The
recombinant protein was purified using the TALON Metal
tions and dialyzed in HDAC storage buffer (20 mM HEPES, 150
mM KCl, 30% glycerol, and 2.5 mM ?-mercaptoethanol).
were grown exponentially in suspension to a density of 6 ? 105
cells/ml. Histones were labeled and extracted as described pre-
protein assay (Pierce), and their molarity was calculated using
the average molecular weight of histones H2A, H2B, H3, and
HDAC Assay—Recombinant HDACs or nuclear extracts
were incubated with3H-labeled histones and metabolic inter-
mediates (quantities as indicated in figures) for 30 min at 37 °C
glycerol, 66.7 mM KCl, and 66.7 ?M EDTA). The reaction was
3.6 M HCl), and the reaction product (3H-labeled acetate) was
extracted with 2 volumes of ethyl acetate. Extracted3H-labeled
acetate was quantified using a liquid scintillation counter
(TriCarb2800TR, PerkinElmer Life Sciences). HDAC assays
with fluorescent substrate were performed using the Fluor de
LysTM-Green HDAC assay kit (Enzo Life Sciences).
confluency. ?5 ? 106cells were washed twice with ice-cold
KOH, pH 7.9, 10 mM KCl, 0.1 mM EDTA, and freshly added
0.4% IGEPAL, 1 mM DTT, 0.5 mM PMSF, and 1? protease
inhibitor mixture (Roche Applied Science)). Nuclei were cen-
trifuged at 16,000 rcf for 3 min at 4 °C and resuspended in 150
?l of Buffer B-NE (20 mM HEPES-KOH, pH 7.9, 400 mM NaCl,
Applied Science)) and incubated at 4 °C for 2 h with vigorous
shaking. Nuclear extract was separated from debris by centrif-
ugation at 16,000 rcf for 10 min at 4 °C and subsequently dia-
lyzed twice for 2 h into Buffer D-NE (20 mM HEPES-KOH, pH
7.9, 100 mM KCl, 0.1 mM EDTA, 20% glycerol) at 4 °C. Protein
concentration was detected by BCA protein assay (Pierce).
Histone Extraction for Western—Cell cultures were grown in
DMEM plus serum to 80% confluency, and 5 ? 106cells were
washed twice with ice-cold PBS and resuspended in 750 ?l of
and 1? protease inhibitors (Roche Applied Science)). After
for 2.5 min at 4 °C and resuspended in 5 pellet volumes of
extraction buffer (432 mN H2SO4, 28 mM ?-mercaptoethanol,
10% glycerol). Histones were extracted for 15 min on ice. After
centrifugation at 16,000 rcf for 10 min, histones were precipi-
centrifugation at 16,000 rcf for 10 min at 4 °C. The pellet was
of lysis buffer (20 mM HEPES-KOH, pH 7.9, 100 mM KCl, 1 mM
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sine 1-phosphate. Science 325, 1254–1257
24. Reinhart, G. D. (2004) Quantitative analysis and interpretation of allos-
teric behavior. Methods Enzymol. 380, 187–203
are components of a yeast histone deacetylase (HDA) complex. J. Biol.
Chem. 271, 15837–15844
26. Wysocka, J., Reilly, P. T., and Herr, W. (2001) Loss of HCF-1-chromatin
association precedes temperature-induced growth arrest of tsBN67 cells.
Mol. Cell. Biol. 21, 3820–3829
27. Cairns, S. P., Robinson, D. M., and Loiselle, D. S. (2008) Double-sigmoid
model for fitting fatigue profiles in mouse fast- and slow-twitch muscle.
Exp. Physiol. 93, 851–862
28. Kaeberlein, M., McDonagh, T., Heltweg, B., Hixon, J., Westman, E. A.,
Substrate-specific activation of sirtuins by resveratrol. J. Biol. Chem. 280,
the form of the contraction curve and the method of temperature coeffi-
cients. J. Physiol. 39, 361–373
30. Bradner, J. E., Mak, R., Tanguturi, S. K., Mazitschek, R., Haggarty, S. J.,
Ross, K., Chang, C. Y., Bosco, J., West, N., Morse, E., Lin, K., Shen, J. P.,
Kwiatkowski, N. P., Gheldof, N., Dekker, J., DeAngelo, D. J., Carr, S. A.,
Schreiber, S. L., Golub, T. R., and Ebert, B. L. (2010) Chemical genetic
strategy identifies histone deacetylase 1 (HDAC1) and HDAC2 as thera-
peutic targets in sickle cell disease. Proc. Natl. Acad. Sci. U.S.A. 107,
(2008) Evolution of the arginase fold and functional diversity. Cell. Mol.
Life Sci. 65, 2039–2055
32. Bressi, J. C., Jennings, A. J., Skene, R., Wu, Y., Melkus, R., De Jong, R.,
O’Connell, S., Grimshaw, C. E., Navre, M., and Gangloff, A. R. (2010)
Exploration of the HDAC2 foot pocket. Synthesis and SAR of substituted
N-(2-aminophenyl)benzamides. Bioorg. Med. Chem. Lett. 20, 3142–3145
33. Vannini, A., Volpari, C., Gallinari, P., Jones, P., Mattu, M., Carfí, A., De
Francesco, R., Steinku ¨hler, C., and Di Marco, S. (2007) Substrate binding
to histone deacetylases as shown by the crystal structure of the HDAC8-
substrate complex. EMBO Rep. 8, 879–884
34. Tian, W. N., Braunstein, L. D., Pang, J., Stuhlmeier, K. M., Xi, Q. C., Tian,
X., and Stanton, R. C. (1998) Importance of glucose-6-phosphate dehy-
drogenase activity for cell growth. J. Biol. Chem. 273, 10609–10617
35. Ziboh, V. A., Dreize, M. A., and Hsia, S. L. (1970) Inhibition of lipid syn-
androsterone. J. Lipid Res. 11, 346–354
36. Levy, H. R. (1979) Glucose-6-phosphate dehydrogenases. Adv. Enzymol.
Relat. Areas Mol. Biol. 48, 97–192
37. Cornish-Bowden, A. (1986) Why is uncompetitive inhibition so rare? A
possible explanation, with implications for the design of drugs and pesti-
cides. FEBS Lett. 203, 3–6
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Maria Vogelauer, Abigail S. Krall, Matthew A. McBrian, Jing-Yu Li and Siavash K.
Stimulation of Histone Deacetylase Activity by Metabolites of Intermediary
doi: 10.1074/jbc.M112.362467 originally published online July 20, 2012
2012, 287:32006-32016. J. Biol. Chem.
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