THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 10, pp.
6608 –6615, March 10, 2006
Down-regulation of Histone Deacetylases Stimulates
Adipocyte Differentiation*□ S
Received for publication, August 15, 2005, and in revised form, November 3, 2005 Published, JBC Papers in Press, January 5, 2006, DOI 10.1074/jbc.M508982200
Eung Jae Yoo1,2, Jun-Jae Chung1,2, Sung Sik Choe2, Kang Ho Kim2, and Jae Bum Kim2,3
From the Department of Biological Sciences and Research Center for Functional Cellulomics, Seoul National University,
Seoul 151-742, South Korea
Specific cell type differentiation is driven by programmed regu-
lation of gene expression, which is the result of coordinated modu-
lation of the transcription machinery and chromatin-remodeling
factors. We present evidence here that the down-regulation of his-
tone deacetylases is an important process during adipocyte differ-
entiation. In 3T3-L1 cells, histone hyperacetylation was selectively
induced at the promoter regions of adipogenic genes during adipo-
cyte differentiation. Interestingly, this was accompanied by a dra-
matic decrease in the expression level of several histone deacety-
lases including HDAC1, -2, and -5 and a reduction in overall histone
deacetylase enzyme activity. Inhibition of histone deacetylase activ-
ity using sodium butyrate resulted in stimulation of adipogenic gene
expression and adipocyte differentiation. Consistently, HDAC1
knock-down promoted adipogenesis whereas HDAC1 overexpres-
sion attenuated adipocyte differentiation in 3T3-L1 cells. Together,
these results suggest that the regulation of not only adipogenic tran-
scription factors, but also chromatin-modifying enzymes is crucial
for the execution of bona fide adipogenesis.
The architecture of the eukaryotic chromatin is dynamically modu-
lated by post-translational modifications of the histones, including
acetylation, methylation, phosphorylation, and ubiquitination (1). The
changes in nucleosome structure influence gene expression by modu-
lating the accessibility of the promoter regions to specific transcription
factors. In particular, acetylation of histones H3 (Lys9 and Lys14) and H4
(Lys8 and Lys12), mediated by histone acetyltransferases (HATs)4 such
as p300, CBP, and P/CAF, is associated with transcriptional activation
(2, 3). Methylation of H3 Lys4, H4 Arg3, and phosphorylation of H3 Ser10
also results in gene activation (4 –7). In contrast, methylation of histone
H3 Lys9 generally correlates with gene repression (8, 9).
Histone deacetylases (HDACs) repress gene expression by deacety-
* This work was supported in part by grants from Stem Cell Research Center of the 21st
Century Frontier Research Program (SC13150), Research Center for Functional Cellu-
lomics of Science Research Center Program, and the National Research Laboratory
Program of Korea Science and Engineering Foundation. The costs of publication of
this article were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
□S
The on-line version of this article (available at http://www.jbc.org) contains supple-
mental Figs. S1–S3.
1
Both authors contributed equally to this work.
2
Supported by BK21 Research Fellowships from the Ministry of Education and Human
Resources Development.
3
To whom correspondence should be addressed: Dept. of Biological Sciences, Research
Center for Functional Cellulomics, Seoul National University, San 56-1, Sillim-Dong,
Gwanak-Gu, Seoul, South Korea 151-742. Tel.: 82-2-880-5852; Fax: 82-2-878-5852;
E-mail: jaebkim@snu.ac.kr.
4
The abbreviations used are: HATs, histone acetyltransferases; HDAC, histone deacety-
lase; C/EBP, CCAAT/enhancer-binding protein; PPAR␥, peroxisome proliferator-acti-
vated receptor ␥; ADD1/SREBP1c, adipocyte determination- and differentiation-de-
pendent factor 1/sterol response element-binding protein 1c; NaB, sodium butyrate;
siRNA, small interference RNA; ChIP, chromatin immunoprecipitation, Pref-1, preadi-
pocyte factor 1; GST, glutathione S-transferase; HEK, human embryonic kidney cells.
6608 JOURNAL OF BIOLOGICAL CHEMISTRY
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
lating histones and other proteins, such as transcription factors (10, 11).
Until now, five yeast and eleven human HDACs have been identified,
which are classified into three classes (12). Class I HDACs consist of
HDAC1, -2, -3, and -8. HDAC 4, -5, -6, -7, -9, and -10 belong to the class
II HDACs. Class III HDACs are members of the sirtuin family of
HDACs (13–17). In addition, a new member of the HDAC family,
HDAC11, has been recently identified (18). Class I HDACs are
expressed ubiquitously, whereas class II HDACs are abundantly
expressed in heart, skeletal muscle, and brain (12). The major function
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of HDACs is to remove acetyl groups from histones, which results in
condensation of the chromatin structure. This, in turn, diminishes the
access of transcription factors to the target DNA and ultimately leads to
transcriptional repression.
Chromatin modifications, notably histone acetylation and deacetyla-
tion, are crucial for the regulation of gene expression and development
in eukaryotes (19, 20). During tissue differentiation, early inductive pro-
cesses that determine cell fate leave traces at particular genes in the form
of histone modifications. HATs and HDACs have been shown to
respond to signals that regulate cell differentiation and directly modu-
late tissue-specific gene expression. For example, studies of myogenesis
have suggested a role for histone-modifying enzymes in coordinating
the activation and repression of genes involved in differentiation proc-
ess (21–23). HDAC1 and members of the class II HDACs including
HDAC4, -5, and -6 inhibit MyoD and Mef2, respectively, to block mus-
cle cell differentiation (24 –27).
Adipogenesis is the process by which undifferentiated precursor cells
differentiate into adipocytes. In vitro studies using 3T3-L1 and 3T3-
F442A cells have elucidated the complex transcriptional cascades that
are essential for the execution of adipocyte differentiation (28, 29).
CCAAT/enhancer-binding proteins (C/EBPs), peroxisome prolifera-
tor-activated receptor ␥ (PPAR␥), and adipocyte determination- and
differentiation-dependent factor 1/sterol response element-binding
protein 1c (ADD1/SREBP1c) have been identified as the key transcrip-
tion factors involved in adipogenesis (30, 31). When preadipocytes are
exposed to differentiation conditions, a complex signaling cascade
induces the expression of previously silent adipogenic genes. Although
it is not clear how these genes are maintained quiescent in preadipo-
cytes, it is possible to speculate that HDACs are involved.
Past research on tissue differentiation has been mainly focused on
understanding the regulatory roles of specific transcription factors. On
the contrary, little is known about the functional roles of coregulators
such as HATs and HDACs during differentiation processes. In this
study we have discovered a novel role of HDACs during adipogenesis.
Our results suggest that the down-regulation of HDACs at the early
stages adipogenesis is a rate-limiting step during adipogenesis. These
findings add to the list of the functional roles of coregulators in regulat-
ing cell differentiation and may provide a new target for discovering
novel therapies for the treatment of fat-related disorders.
VOLUME 281 • NUMBER 10 • MARCH 10, 2006

FIGURE 1. Histone modifications at the pro-
moter regions of adipogenic genes during adi-
pocyte differentiation. ChIP assays were per-
formed using 3T3-L1 cells at different stages of
adipogenesis. Isolated DNA was immunoprecipi-
tated with anti-acetylated histone H3 (K9, A), anti-
acetylated H4 (K8, B), or anti-dimethylated histone
H3 (K9, C) antibodies, and amplified using primers
specific for the promoter regions of the indicated
genes (see “Materials and Methods” for details).
MATERIALS AND METHODS
Cell Culture—3T3-L1 and 3T3-F442A mouse fibroblasts were main-
tained in Dulbecco’s modified Eagle’s medium supplemented with 10%
bovine calf serum (BCS, Invitrogen), with a change of medium every 2–3
days. 3T3-L1 cells were differentiated into adipocytes as previously
described (32). 3T3-F442A cells were differentiated into adipocytes by
replacing the medium with Dulbecco’s modified Eagle’s medium con-
taining 10% fetal bovine serum (FBS, Invitrogen) and 5 ␮g/ml insulin
upon reaching confluence. BOSC and H29D retrovirus packaging cells
were maintained in Dulbecco’s modified Eagle’s medium supplemented
with 10% FBS.
Chromatin Immunoprecipitation (ChIP) Assays—Chromatin was
immunoprecipitated as described previously (33). Conditions for PCR
reactions were as follows: 0.25 ␮M of each primer, 0.1 mM of each dNTP,
1⫻ PCR buffer, 1 unit Nova Taq polymerase (Genenmed), and 0.06
mCi/ml [␣-32P]dCTP in 20 ␮l of reaction volume. PCR primers used
were as follows: ADD1-P5, 5⬘-GGT TGG TAC CAC AGT GAC CG-3⬘;
ADD1-P3, 5⬘-AAT GTG CAA TCC ATG GCT CCG TGG T-3⬘; Adi-
ponectin-P5, 5⬘-GGT GCT GGG AAT TGA ACT CA-3⬘; Adiponectin-
P3, 5⬘-CCT GTT TCC AGG CTT TGG CC-3⬘; aP2-P5, 5⬘-TTC CAG
GTG AGA AAG GCT GC-3⬘; aP2-P3, 5⬘-GTC CAG TGA TCA TTG
CCA GG-3⬘; C/EBP␣-P5, 5⬘-CCC GGG CTC CCT AGT GTT-3⬘;
C/EBP␣-P3, 5⬘-CTG GCT CGC GCC CGC GCA-3⬘; PPAR␥2-P5,
5⬘-CTG TAC AGT TCA CGC CCC TC-3⬘; PPAR␥2-P3, 5⬘-TCA CAC
TGG TGT TTT GTC TAT G-3⬘; Pref-1-P5, 5⬘-CTT TTC GTG GTG
GTT TTC GT-3⬘; Pref-1-P3, 5⬘-GCA AGT CTC AGG AAC CAA
GC-3⬘; GAPDH-f, 5⬘-GTG TTC CTA CCC CCA ATG TG-3⬘;
GAPDH-r, 5⬘-CTT GCT CAG TGT CCT TGC TG-3⬘.
Acid Extraction of Histones and Western Blotting—Histone proteins
were isolated from 3T3-L1 preadipocytes and adipocytes by acid extrac-
tion according to the protocol provided by Upstate Biotechnology. The
proteins were separated by electrophoresis on SDS-polyacrylamide gels
and transferred to polyvinylidene difluoride membranes (Millipore).
Following transfer, the membranes were blocked with milk and probed
with antibodies against HDAC1, -2, -3, -4, -5, -6, -7, -8, PPAR␥, C/EBP␣,
CBP, p300 (Santa Cruz Biotechnology), acetylated histone H3 (Lys9),
dimethylated histone H3 (Lys9), histone H3, acetylated histone H4 (Lys8
and Lys12), and histone H4 (Upstate Biotechnology). Results were visu-
alized with horseradish peroxidase-conjugated secondary antibodies
(Sigma Aldrich) and enhanced chemiluminescence.
HDAC Enzyme Activity Assays—Total cellular HDAC enzymatic
activity was measured using an HDAC assay kit (Upstate Biotechnol-
ogy) according to the manufacturer’s protocol. Briefly, 40 ␮g of total cell
MARCH 10, 2006 • VOLUME 281 • NUMBER 10
HDAC and Adipogenesis
extracts from 3T3-L1 and 3T3-F442A preadipocytes and adipocytes
were incubated with a fluorometric substrate in HDAC assay buffer for
45 min at 30 °C. An activator solution was then added to release the
fluorophore from the deacetylated substrates, and the fluorescence was
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measured in a fluorescence plate reader.
mRNA Analysis—Total RNA was isolated with TRIzol reagent
(Invitrogen) according to the manufacturer’s protocol. For Northern
blotting, 20 ␮g of RNA was denatured in formamide and formaldehyde,
and separated by electrophoresis on formaldehyde-containing agarose
gels. RNA was transferred to Nytran membranes (Schleicher and
Schuell), and the membranes were cross-linked, hybridized, and washed
as described by the manufacturers. Probes were labeled by the random
priming method using the Klenow fragment of DNA polymerase I (Pro-
mega) and [␣-32P]dCTP (Amersham Biosciences). PPAR␥, aP2,
C/EBP␣, and FAS cDNAs were used as probes. For real-time PCR anal-
ysis, cDNA was synthesized by reverse transcription using oligodT and
subjected to PCR amplification with gene-specific primers in the pres-
ence of Cybergreen (Bio-Rad). Relative abundance of mRNA was calcu-
lated after normalization to 18 S ribosomal RNA. Primer sequences for
real-time PCR analyses are available upon request.
Retroviral Infection—BOSC or H29D cells were transfected with
either pBABE, pBABE-HDAC1, pSUPERretro (Oligoengine), or pSUPER-
retro-HDAC1 RNAi (5⬘-GCA GCG TCT CTT TGA GAA C-3⬘) using
Lipofectamine (Invitrogen). 48 h after transfection, the medium con-
taining retroviruses was harvested, filtered, and transferred to 3T3-L1
target cells with polybrene (1 ␮g/ml). Infected cells were selected with 5
␮g/ml puromycin (Sigma) for 7 days.
Transient Transfections and Luciferase Assays—HEK293 cells were
grown in Dulbecco’s modified Eagle’s medium containing 10% bovine
calf serum. A luciferase reporter driven by multiple PPREs (DR1-luc)
was cotransfected with PPAR␥ and HDAC1 expression vectors using
the calcium phosphate method (32). The cells were lysed and assayed for
luciferase activity.
Protein Interaction Analyses—GST-pulldown assays were performed
as previously described (34). Briefly, GST and GST-PPAR␥ fusion pro-
teins were bound to glutathione beads and incubated with 35S- labeled
HDAC1 protein. The beads were washed with TBS and analyzed by
autoradiography after SDS-PAGE. Protein-protein interaction in cells
was also analyzed by co-immunoprecipitation experiments. HDAC1
and PPAR␥ expression vectors were transfected in HEK293 cells using
the calcium phosphate method. After transfection, whole cell extracts
were prepared and incubated with HDAC1 antibody overnight at 4 °C.
The immunocomplexes were isolated with protein A-Sepharose beads
JOURNAL OF BIOLOGICAL CHEMISTRY 6609
HDAC and Adipogenesis
FIGURE 2. Western blot analysis of histone-modifying enzymes in 3T3-L1 (A) and
3T3-F442A (B) cells during adipocyte differentiation. PPAR␥ and C/EBP␣ are markers
of differentiated adipocytes.
(Amersham Biosciences) and washed three times with TBS. The immu-
noprecipitates were separated by SDS-PAGE and immunoblotted using
antibodies against PPAR␥.
RESULTS
Histone Modification at the Promoters of Adipogenic Genes during
Adipocyte Differentiation—The dramatic changes in gene expression
profiles during adipocyte differentiation have been previously described
(35, 36). Notably, the expression of adipogenic genes such as PPAR␥,
C/EBP␣, and ADD1/SREBP1c is increased (28), whereas that of preadi-
pocyte marker genes including preadipocyte factor-1 (Pref-1) and
GATA2 is conversely decreased (37, 38). Although many transcription
factors that participate in adipocyte differentiation have been identified,
the roles of histone-modifying enzymes and chromatin-remodeling fac-
tors during this process have not been thoroughly investigated.
To determine if histone modification is involved in adipogenesis, we
examined the level of histone acetylation and methylation at the pro-
moter regions of adipocyte marker genes. The degree of histone H3 Lys9
acetylation at the promoters of ADD1/SREBP1c, adiponectin, aP2,
C/EBP␣, and PPAR␥ was increased upon differentiation (Fig. 1A). Sim-
ilarly, histone H4 Lys8 acetylation was increased at the promoters of
ADD1/SREBP1c and adiponectin (Fig. 1B). In contrast, histone acetyla-
tion gradually decreased at the promoter of Pref-1, consistent with the
fact that it is down-regulated during adipogenesis (37). On the other
hand, the level of histone H3 Lys9 methylation displayed opposite pat-
terns, decreasing at the promoters of ADD1/SREBP1c and C/EBP␣ and
marginally increasing at the Pref-1 promoter (Fig. 1C). These results
indicate that the up-regulation of adipogenic genes during adipogenesis
is tightly associated with the selective induction of histone hyperacety-
lation at the promoter regions of these genes.
Changes of Histone Modification and Expression of HDACs during
Adipogenesis—Because the steady state of histone acetylation is deter-
mined by the balance between HATs and HDACs, we next examined
the protein levels of several histone-modifying enzymes before and after
differentiation. In 3T3-L1 cells, the expression levels of several HDACs
including HDAC1, -2, -5, and -6 were drastically reduced during differ-
entiation (Fig. 2A) whereas HDAC3, -4, -7, and -8 were either slightly
increased or unchanged (supplemental Fig. S1). The protein levels of
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FIGURE 3. Changes in overall histone acetylation and HDAC activity during adipo-
cyte differentiation. A, changes of histone modification during adipocyte differentia-
tion. B, total cellular HDAC enzyme activity of 3T3-L1 and 3T3-F442A preadipocytes/
adipocytes. Representative results from three independent experiments carried out in
duplicate are shown.
HATs, such as CBP or p300, were shown to be unchanged or slightly
increased, respectively (Fig. 2A). A similar reduction in HDAC1, -2, -3,
-5, -7, and -8 was observed in 3T3-F442A cells, another adipogenic cell
line (Fig. 2B). C/EBP␣ and PPAR␥ were used as control to confirm
proper differentiation.
The finding that the expression of several HDACs is severely
decreased prompted us to examine whether the overall balance would
be shifted in favor of histone acetylation. Indeed, the degree of overall
histone acetylation was increased in adipocytes compared with undif-
ferentiated preadipocytes (Fig. 3A). Methylation of histone H3 lysine 9,
which usually displays an opposite pattern to acetylation, was decreased
in adipocytes. To determine whether histone deacetylase activity is
actually regulated during differentiation, we directly measured HDAC
enzyme activity using the same total cell extracts. As shown in Fig. 3B,
total cellular HDAC activity was reduced to ⬃50⬃60% in both 3T3-L1
and 3T3-F442A cells upon differentiation (Fig. 3B). On the contrary,
total cellular HAT activity was slightly increased in differentiated adi-
pocytes (data not shown). Thus, it is likely that the increase of histone
acetylation during adipogenesis is the result of a combination of
decreased HDAC activity, and to some extent, increased HAT activity.
Such modification of histone tails would probably contribute to the
overall increase of gene expression in adipocytes, which have been
reported to express a larger number of genes than preadipocytes (36).
Effects of HDAC Inhibition on Adipocyte Differentiation—Next, to
determine whether the down-regulation of HDAC activity indeed
affects adipogenesis, we treated 3T3-L1 cells with an inhibitor of
HDACs, sodium butyrate (NaB). Butyrate is a short chain fatty acid that
noncompetitively inhibits the activity of most HDAC isoforms (39, 40).
VOLUME 281 • NUMBER 10 • MARCH 10, 2006

FIGURE 4. Induction of adipocyte differentiation and adipogenic gene expression by the HDAC inhibitor, NaB. A, Oil Red-O staining of differentiated 3T3-L1 cells with or without
NaB treatment. B, Northern blot analysis. Total RNA from 3T3-L1 cells incubated in the absence (left) or presence (right) of NaB (500 ␮M) was isolated at the indicated time points after
induction of differentiation. C, differentiating 3T3-L1 cells were treated with NaB for various time periods and stained with Oil Red-O. Bars indicate the duration of NaB treatment.
Representative data from three separate experiments are shown.
As shown in Fig. 4A chronic treatment of NaB accelerated differentia-
tion and increased lipid accumulation assessed by Oil Red-O staining.
Northern blot analysis revealed that the expression of adipogenic genes
was induced at an earlier time point (Fig. 4B). A similar effect was
observed in C3H10T1/2 cells, a murine mesenchymal fibroblast cell line
that can also be induced to differentiate into adipocytes (supplemental
Fig. S2).
To investigate the time periods at which HDAC inhibition exhibits
the most critical effect on adipogenesis, we treated the cells with NaB for
different periods during adipocyte differentiation. Cells treated with
NaB throughout the entire differentiation period clearly exhibited stim-
ulated adipocyte differentiation compared with control cells (Fig. 4C,
panels II and VIII). Notably, inhibition of the HDACs at the early stage
of adipogenesis (the first 1 or 2 days) seemed to be sufficient to stimulate
adipogenesis (Fig. 4C, panels IV and V), while treatment of NaB at later
periods had little or no effect (Fig. 4C, panels VI and VII). Taken
together, these results indicate that suppression of HDAC activity at the
early stage of adipogenesis may be crucial for adipocyte differentiation.
The Role of HDAC1 during Adipogenesis—Among many HDAC iso-
forms, HDAC1 was most consistently and dramatically down-regulated
during adipogenesis (Fig. 2, A and B). To determine if HDAC1 plays an
important role in adipogenic gene expression, we determined the
recruitment of HDAC1 to the promoter regions of adipogenic genes
using ChIP assays. As expected, recruitment of HDAC1 decreased upon
differentiation in most adipogenic genes including adiponectin, ADD1/
SREBP1c, aP2, C/EBP␣, and PPAR␥, indicating that HDAC1 may be
involved in regulating the expression of these genes (Fig. 5A).
To directly determine whether HDAC1 is specifically involved in
adipocyte differentiation, we generated 3T3-L1 preadipocytes retrovi-
rally overexpressing HDAC1. These cells expressed higher amounts of
HDAC1 (⬃2-fold) compared with mock-infected cells (Fig. 5B). Under
differentiation conditions, the HDAC1-overexpressing cells exhibited
substantially attenuated adipocyte differentiation (Fig. 5C). In addition,
the expression of several adipogenic genes is also decreased in HDAC1-
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HDAC and Adipogenesis
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overexpressing cells, suggesting that HDAC1 suppresses adipogenesis
by down-regulating the expression of adipogenic genes (Fig. 5D).
In a reciprocal experiment, we examined the effect of HDAC1 down-
regulation via siRNA. To test whether the HDAC1-siRNA effectively
reduced HDAC1 expression, HEK293 cells were co-transfected with
FLAG-tagged HDAC1 and HDAC1-siRNA vectors. As expected, over-
expression of HDAC1-siRNA efficiently repressed HDAC1 expression
(Fig. 6A). Next, 3T3-L1 cells overexpressing the retroviral HDAC1-
siRNA were tested for their ability to differentiate into adipocytes. Con-
sistent with the above observations, HDAC1 knock-down 3T3-L1 cells
clearly displayed accelerated differentiation (Fig. 6B) and stimulated
expression of ADD1/SREBP1c, aP2, and PPAR␥ (Fig. 6C). Furthermore,
HDAC1 knockdown led to the increase of histone acetylation at H3
Lys9, H4 Lys8, and H4 Lys12 residues. Conversely, the extent of histone
H3 Lys9 methylation decreased (Fig. 6D). These results provide strong
evidence that HDAC1 controls adipocyte differentiation by regulating
the acetylation of histones and implicate that a threshold level of HDAC
activity is probably required to maintain preadipocytes by silencing adi-
pogenic genes in the undifferentiated state.
Regulation of PPAR␥ Activity by HDAC1—To understand how
HDAC1 affects adipocyte differentiation, we examined whether
HDAC1 could directly regulate the transcriptional activity of PPAR␥, a
master adipogenic transcription factor. In transient transfection assays,
HDAC1 repressed the transcriptional activity PPAR␥ in a dose-depend-
ent manner (Fig. 7A). On the other hand, the activity of ADD1/SREBP1c
was not altered by HDAC1 (data not shown). To see if HDAC1 physi-
cally associates with PPAR␥, we performed GST-pulldown assays using
recombinant GST-PPAR␥ fusion proteins and in vitro translated
HDAC1. HDAC1 bound specifically with purified GST-PPAR␥ but not
GST, suggesting that HDAC1 may directly interact with PPAR␥ (Fig.
7B). To investigate whether this interaction occurs in cells, we isolated
whole cell lysates of HEK293 cells after transfection with plasmids
encoding PPAR␥ and HDAC1. Immunoprecipitation with anti-HDAC1
antibody followed by Western blot assays using anti-PPAR␥ antibody
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HDAC and Adipogenesis
FIGURE 5. Recruitment of HDAC1 to the promot-
ers of adipocyte marker genes and the effect of
HDAC1 overexpression on 3T3-L1 cell differen-
tiation. A, ChIP assays were performed with
␣-HDAC1 antibodies. Band intensities were deter-
mined with LabworksTM (Media Cybernetics). B,
Western blot of pBABE-mock and pBABE-HDAC1
infected 3T3-L1 cells. C, HDAC1 overexpression
inhibits adipocyte differentiation. Oil Red O stain-
ing of HDAC1 overexpressing 3T3-L1 cells differ-
entiated for 6 days. D, real-time qPCR analysis of
adipocyte marker gene expression. *, p ⬍ 0.05.
demonstrates that HDAC1 indeed interacts with PPAR␥ in vivo (Fig.
7C). It is therefore possible to speculate that HDAC1 could modulate
adipocyte differentiation by repressing PPAR␥ activity.
DISCUSSION
Recent studies have demonstrated the involvement of chromatin
remodeling factors in the differentiation of specific cell types including
muscle, neurons, and T cells (41– 43). Although it is well known that
certain transcription factors or cofactors interact with HATs or HDACs
to regulate their target gene expression, there are few reports that the
overall level of histone modifications or expression of histone-modify-
ing enzymes such as HDACs is changed during bona fide differentiation.
Furthermore, little is known about the regulatory role of chromatin
remodeling such as histone modifications in adipocyte differentiation.
In the present study, we sought to identify the regulatory function of
histone modifications in adipocyte differentiation. ChIP assays revealed
that histones are dynamically modified at the promoter regions of adi-
pocyte marker genes indicating its involvement in adipogenesis. More
importantly, we discovered that the protein level of class I and class II
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HDACs significantly decreased upon differentiation (Fig. 2, A and B).
Indeed, the endogenous HDAC enzyme activity measured in adipocytes
was shown to be only half that of preadipocytes (Fig. 3B). Inversely, total
HAT activity of adipocytes was slightly elevated upon differentiation
(data not shown). Therefore, it seems that histone hyperacetylation in
adipocytes, as a result of decreased HDAC activity and increased HAT
activity, leads to enhanced adipogenic gene expression. This increase of
histone acetylation is most likely preferentially targeted to adipogenic
genes by tissue-specific transcription factors and cofactors. This idea is
strongly supported by the fact that PPAR␥, a master adipogenic tran-
scription factor, interacts with several coactivators such as p300, CBP,
TRAP, p160, and PGC1␣ and corepressors like HDAC3 to selectively
regulate the expression of its target genes during adipogenesis (44 – 48).
Because these cofactors are able to interact with specific transcription
factors in a highly specific manner, decondensation of chromatin is
tightly controlled so that it only takes place in a subset of genes in the
appropriate tissues and at the appropriate times.
Stimulation of adipogenesis and adipogenic gene expression by NaB
suggests that the down-regulation of histone deacetylases might be one
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FIGURE 6. Effect of HDAC1 knock-down by
siRNA on 3T3-L1 cell differentiation. A, HDAC1-
specific siRNA inhibits HDAC1 expression. Western
blot analysis of cells cotransfected with
pcDNA-HDAC1-FLAG and pSuper-retro-HDAC1-
siRNA was performed with FLAG antibody. B,
HDAC1 siRNA stimulates adipocyte differentia-
tion. 3T3-L1 cells were infected with retroviruses
containing either pSuper-retro-mock or pSuper-
retro-HDAC1-siRNA. After selection with puromy-
cin, the stably infected cells were differentiated
into adipocytes. C, real-time qPCR analysis of adi-
pocyte marker genes in mock and pSuper-retro-
HDAC1-siRNA infected 3T3-L1 cells. *, p ⬍ 0.05. D,
acid extracts from mock or pSuper-retro-HDAC1-
siRNA-infected cells were subjected to Western
blot analysis with the indicated antibodies.
of the rate-limiting steps during adipocyte differentiation (Fig. 4, A and
B). Interestingly, cells treated with NaB for the first 2 or 3 days, but not
at later stages of differentiation, showed a remarkable stimulation of
adipogenesis (Fig. 3C). These results implicate that a critical determina-
tion step for adipogenesis occurs through histone modification at an
early phase of the differentiation process. This crucial event is probably
associated with tissue- or developmentally specific transcription factors
and/or cofactors responsible for triggering tissue-specific gene expres-
sion with down-regulation of HDAC activity. Similar results have been
obtained with Swiss 3T3 cells (49) and was also observed in C3H 10T1/2
cells (supplemental Fig. S2). These cell lines are less determined to the
adipogenic lineage, suggesting that this phenomenon is not restricted to
a specific cell line.
A remarkable drop in the expression of HDAC1 suggested that this
specific isoform may play a major role in adipogenesis. This idea was
supported by the observation that the amounts of HDAC1 localized to
the promoters of adipogenic genes were clearly decreased after differ-
entiation (Fig. 5A). Gain-of-function and loss-of function studies in
3T3-L1 cells also confirmed the critical role of HDAC1 in adipogenesis.
While we were preparing this article, Wiper-Bergeron et al. (50) dem-
onstrated that HDAC1, which forms a complex with mSin3A and
C/EBP␤, is able to attenuate differentiation by repressing C/EBP␣
expression. In another recent report, Fajas et al. (48) demonstrated that
HDAC3 can suppress PPAR␥ by forming a PPAR␥䡠Rb䡠HD3 complex. It
MARCH 10, 2006 • VOLUME 281 • NUMBER 10
HDAC and Adipogenesis
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is also known that HD3 is a component of a corepressor complex with
NCoR/SMRT that represses PPAR␥. Interestingly, HDAC1 interacted
with PPAR␥ and repressed its transcriptional activity (Fig. 7). Even
though many HDACs are known to bind to transcription factors as part
of a corepressor complex, results from GST-pulldown and co-immuno-
precipitation assays suggest that HDAC1 may bind directly to PPAR␥.
In addition, it should be noted that PPAR␥ and HDAC1 display recip-
rocal expression patterns during adipogenesis (Fig. 2A). Thus, it appears
that activation of PPAR␥ might be regulated by two steps during adipo-
cyte differentiation. First, PPAR␥ activity is stimulated by increases in
protein levels and its endogenous ligands. Second, the down-regulation
of HDAC1 would further promote PPAR␥ activity by relieving it from
repression. Therefore, in addition to the previously described role as a
modulator of C/EBP␤, HDAC1 would also regulate adipocyte differen-
tiation by controlling PPAR␥.
In addition to these findings, which suggest that HDACs interact with
several adipogenic transcription factors to modulate their activities, our
results implicate the importance of the regulation of HDAC expression
during adipocyte differentiation. Although the exact mechanism by
which adipogenic stimuli decrease the level of HDACs is yet to be deter-
mined, it appears that the lowering of HDAC expression results in
decondensation of the chromatin structure around the promoter
regions of adipogenic genes, thereby facilitating the binding of tran-
scription factors to their binding sites (supplemental Fig. S3).
JOURNAL OF BIOLOGICAL CHEMISTRY 6613
HDAC and Adipogenesis
FIGURE 7. Repression of PPAR␥ activity by HDAC1. A, HEK293 cells were transfected
with DR1-luciferase reporter construct and expression vectors for PPAR␥ and HDAC1.
The cells were lysed and assayed for luciferase activity after transfection. B, in vitro trans-
lated 35S-labeled HDAC1 was incubated with GST or GST-PPAR␥ fusion proteins and
analyzed by autoradiography. Gels were stained with Coomassie Brilliant Blue to confirm
equal loading. C, whole cell extracts of HEK293 cells cotransfected with HDAC1 and
PPAR␥ were immunoprecipitated with HDAC1 antibody. The immunocomplexes were
subjected to SDS-PAGE and immunoblotted with PPAR␥ and HDAC1 antibodies.
Our group has also discovered that a very similar phenomenon
occurs during osteoblast differentiation, suggesting that the modulation
of HDAC expression and activity may be a general means of regulating
cell differentiation.5 The fact that NaB accelerated the differentiation of
C3H10T1/2 cells into both adipocytes (supplemental Fig. S1) and osteo-
blasts5 depending on the differentiation condition implies that the
down-regulation of HDACs is a prerequisite process to convert the cells
into a state susceptible to induction of differentiation. However, it does
not seem that the down-regulation of HDAC1 specifies the cell-type
into which the precursor cells will differentiate. Instead, this event
seems be a part of a transition of the undifferentiated cells into a state in
which cell type-specific transcription factors can bind to the chromatin
and activate the expression of cell type-specific marker genes. Further
studies will reveal whether this event is common during the differenti-
ation from mesenchymal stem cells.
The effects of HDAC inhibitors appear to be selective since the
expression of only 2⬃5% of mammalian genes is affected when HDAC
activity is inhibited (51, 52). As an HDAC inhibitor, NaB has many
effects on cultured mammalian cells including inhibition of prolifera-
tion and induction of differentiation and gene expression. For example,
HDAC inhibition with NaB has been shown to inhibit the development
5
H. W. Lee, J. H. Suh, Y. S. Lee, S. Y. Park, and J. B. Kim, manuscript in preparation.
6614 JOURNAL OF BIOLOGICAL CHEMISTRY
of colon cancer by altering gene expression and arresting cell division or
to induce apoptosis in human leukemic lymphoblasts (53). Further-
more, HDAC inhibitors induce the expression of cell cycle kinase inhib-
itor p21, causing growth arrest in transformed cells (54, 55). It is inter-
esting to note that expression of p21 is induced by HDAC inhibitors
since p21 is induced at a very early stage of adipocyte differentiation to
modulate clonal expansion (56). Although the precise mechanisms by
which HDAC inhibitors induce cell cycle arrest via p21 expression dur-
ing adipogenesis remain to be elucidated, it might shed light in under-
standing how histone acetylation and deacetylation are possibly linked
to adipocyte differentiation.
In summary, our results show that the down-regulation of HDAC
expression and the resulting increase of histone acetylation are impor-
tant for adipogenesis. The substantial decrease in HDAC activity results
in preferential histone hyperacetylation at the promoter regions of adi-
pocyte marker genes. It is therefore likely that regulation of HDAC
expression would be an essential process in adipocyte differentiation
and that preadipocytes could initiate the differentiation process only
when cellular level of HDACs falls below a certain threshold level and
also that HDACs are involved in maintaining the preadipocyte pheno-
type. This is a novel viewpoint concerning the regulation of adipogen-
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esis since previous studies have mainly focused on adipogenic transcrip-
tion factors, such as PPAR␥ and C/EBP␣ and their ability to recruit
coregulators to adipogenic gene promoters. Our findings could also
provide a novel means of treating obesity-related diseases. Further stud-
ies will reveal whether this is a general mechanism controlling the dif-
ferentiation of diverse cell types.
Acknowledgments—We thank Dr. W-M. Yang for providing the
pcDNA-HDAC1-FLAG vector. We are grateful to Dr. K. U. Lee and Y. S. Lee for
critically reading the manuscript.
REFERENCES
1. Jenuwein, T., and Allis, C. D. (2001) Science 293, 1074 –1080
2. Ogryzko, V. V., Schiltz, R. L., Russanova, V., Howard, B. H., and Nakatani, Y. (1996)
Cell 87, 953–959
3. Schiltz, R. L., Mizzen, C. A., Vassilev, A., Cook, R. G., Allis, C. D., and Nakatani, Y.
(1999) J. Biol. Chem. 274, 1189 –1192
4. Spencer, V. A., and Davie, J. R. (1999) Gene (Amst.) 240, 1–12
5. Chen, H., Lin, R. J., Xie, W., Wilpitz, D., and Evans, R. M. (1999) Cell 98, 675– 686
6. Wang, H., Huang, Z. Q., Xia, L., Feng, Q., Erdjument-Bromage, H., Strahl, B. D.,
Briggs, S. D., Allis, C. D., Wong, J., Tempst, P., and Zhang, Y. (2001) Science 293,
853– 857
7. Cheung, P., Tanner, K. G., Cheung, W. L., Sassone-Corsi, P., Denu, J. M., and Allis,
C. D. (2000) Mol. Cell 5, 905–915
8. Rea, S., Eisenhaber, F., O’Carroll, D., Strahl, B. D., Sun, Z. W., Schmid, M., Opravil, S.,
Mechtler, K., Ponting, C. P., Allis, C. D., and Jenuwein, T. (2000) Nature 406, 593–599
9. Nakayama, J., Rice, J. C., Strahl, B. D., Allis, C. D., and Grewal, S. I. (2001) Science 292,
110 –113
10. Ayer, D. E. (1999) Trends Cell Biol. 9, 193–198
11. Johnstone, R. W. (2002) Nat. Rev. Drug Discov. 1, 287–299
12. de Ruijter, A. J., van Gennip, A. H., Caron, H. N., Kemp, S., and van Kuilenburg, A. B.
(2003) Biochem. J. 370, 737–749
13. Fischle, W., Emiliani, S., Hendzel, M. J., Nagase, T., Nomura, N., Voelter, W., and
Verdin, E. (1999) J. Biol. Chem. 274, 11713–11720
14. Grozinger, C. M., Hassig, C. A., and Schreiber, S. L. (1999) Proc. Natl. Acad. Sci.
U. S. A. 96, 4868 – 4873
15. Fischle, W., Kiermer, V., Dequiedt, F., and Verdin, E. (2001) Biochem. Cell Biol. 79,
337–348
16. Guardiola, A. R., and Yao, T. P. (2002) J. Biol. Chem. 277, 3350 –3356
17. Kao, H. Y., Lee, C. H., Komarov, A., Han, C. C., and Evans, R. M. (2002) J. Biol. Chem.
277, 187–193
18. Gao, L., Cueto, M. A., Asselbergs, F., and Atadja, P. (2002) J. Biol. Chem. 277,
25748 –25755
19. Berger, S. L. (2002) Curr. Opin. Genet. Dev. 12, 142–148
20. Kadam, S., and Emerson, B. M. (2002) Curr. Opin. Cell Biol. 14, 262–268
21. Puri, P. L., Sartorelli, V., Yang, X. J., Hamamori, Y., Ogryzko, V. V., Howard, B. H.,
VOLUME 281 • NUMBER 10 • MARCH 10, 2006
 HDAC and Adipogenesis
Kedes, L., Wang, J. Y., Graessmann, A., Nakatani, Y., and Levrero, M. (1997) Mol. Cell 40. Sealy, L., and Chalkley, R. (1978) Cell 14, 115–121
1, 35– 45 41. de la Serna, I. L., Carlson, K. A., and Imbalzano, A. N. (2001) Nat. Genet. 27, 187–190
22. McKinsey, T. A., Zhang, C. L., and Olson, E. N. (2001) Curr. Opin. Genet. Dev. 11, 42. Machida, Y., Murai, K., Miyake, K., and Iijima, S. (2001) J. Biochem. (Tokyo) 129,
497–504 43– 49
23. Mal, A., and Harter, M. L. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 1735–1739 43. Avni, O., Lee, D., Macian, F., Szabo, S. J., Glimcher, L. H., and Rao, A. (2002) Nat.
24. Ng, H. H., and Bird, A. (2000) Trends Biochem. Sci 25, 121–126 Immunol. 3, 643– 651
25. Mal, A., Sturniolo, M., Schiltz, R. L., Ghosh, M. K., and Harter, M. L. (2001) EMBO J. 44. Puigserver, P., Wu, Z., Park, C. W., Graves, R., Wright, M., and Spiegelman, B. M.
20, 1739 –1753 (1998) Cell 92, 829 – 839
26. Lemercier, C., Verdel, A., Galloo, B., Curtet, S., Brocard, M. P., and Khochbin, S. 45. Yang, W., Rachez, C., and Freedman, L. P. (2000) Mol. Cell Biol. 20, 8008 – 8017
(2000) J. Biol. Chem. 275, 15594 –15599 46. Takahashi, N., Kawada, T., Yamamoto, T., Goto, T., Taimatsu, A., Aoki, N., Kawasaki,
27. McKinsey, T. A., Zhang, C. L., Lu, J., and Olson, E. N. (2000) Nature 408, 106 –111 H., Taira, K., Yokoyama, K. K., Kamei, Y., and Fushiki, T. (2002) J. Biol. Chem. 277,
28. Rosen, E. D., Walkey, C. J., Puigserver, P., and Spiegelman, B. M. (2000) Genes Dev. 14, 16906 –16912
1293–1307
47. Wallberg, A. E., Yamamura, S., Malik, S., Spiegelman, B. M., and Roeder, R. G. (2003)
29. Morrison, R. F., and Farmer, S. R. (2000) J. Nutr. 130, 3116S-3121S
Mol. Cell 12, 1137–1149
30. Wu, Z., Bucher, N. L., and Farmer, S. R. (1996) Mol. Cell Biol. 16, 4128 – 4136
48. Fajas, L., Egler, V., Reiter, R., Hansen, J., Kristiansen, K., Debril, M. B., Miard, S., and
31. Hamm, J. K., Park, B. H., and Farmer, S. R. (2001) J. Biol. Chem. 276, 18464 –18471
Auwerx, J. (2002) Dev. Cell 3, 903–910
32. Seo, J. B., Moon, H. M., Kim, W. S., Lee, Y. S., Jeong, H. W., Yoo, E. J., Ham, J., Kang,
49. Toscani, A., Soprano, D. R., and Soprano, K. J. (1990) J. Biol. Chem. 265, 5722–5730
H., Park, M. G., Steffensen, K. R., Stulnig, T. M., Gustafsson, J. A., Park, S. D., and Kim,
50. Wiper-Bergeron, N., Wu, D., Pope, L., Schild-Poulter, C., and Hache, R. J. (2003)
J. B. (2004) Mol. Cell Biol. 24, 3430 –3444
33. Seo, J. B., Noh, M. J., Yoo, E. J., Park, S. Y., Park, J., Lee, I. K., Park, S. D., and Kim, J. B. EMBO J. 22, 2135–2145
(2003) Mol. Endocrinol. 17, 1522–1533 51. Van Lint, C., Emiliani, S., and Verdin, E. (1996) Gene Expr. 5, 245–253
34. Lee, Y. S., Lee, H. H., Park, J., Yoo, E. J., Glackin, C. A., Choi, Y. I., Jeon, S. H., Seong, 52. Mariadason, J. M., Corner, G. A., and Augenlicht, L. H. (2000) Cancer Res. 60,
R. H., Park, S. D., and Kim, J. B. (2003) Nucleic Acids Res. 31, 7165–7174 4561– 4572
35. Soukas, A., Socci, N. D., Saatkamp, B. D., Novelli, S., and Friedman, J. M. (2001) J. Biol. 53. Rahmani, M., Yu, C., Dai, Y., Reese, E., Ahmed, W., Dent, P., and Grant, S. (2003)
Chem. 276, 34167–34174 Cancer Res. 63, 8420 – 8427
36. Sadowski, H. B., Wheeler, T. T., and Young, D. A. (1992) J. Biol. Chem. 267, 54. Richon, V. M., Webb, Y., Merger, R., Sheppard, T., Jursic, B., Ngo, L., Civoli, F.,

Downloaded from http://www.jbc.org/ by guest on May 7, 2017

4722– 4731 Breslow, R., Rifkind, R. A., and Marks, P. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93,
37. Smas, C. M., and Sul, H. S. (1993) Cell 73, 725–734 5705–5708
38. Tong, Q., Dalgin, G., Xu, H., Ting, C. N., Leiden, J. M., and Hotamisligil, G. S. (2000) 55. Vrana, J. A., Decker, R. H., Johnson, C. R., Wang, Z., Jarvis, W. D., Richon, V. M.,
Science 290, 134 –138 Ehinger, M., Fisher, P. B., and Grant, S. (1999) Oncogene 18, 7016 –7025
39. Candido, E. P., Reeves, R., and Davie, J. R. (1978) Cell 14, 105–113 56. Morrison, R. F., and Farmer, S. R. (1999) J. Biol. Chem. 274, 17088 –17097

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 ` Yoo et al.

Sup Fig. 1. Western blot analysis of histone modifying enzymes in 3T3-L1 cells during adipocyte
differentiation.

Sup Fig. 2. Stimulation of C3H10T1/2 cell differentiation by NaB. C3H10T1/2 cells were
differentiated into adipocytes in the absence or presence of NaB (500 µM) and stained with Oil Red O.

Sup Fig. 3. Schematic model. The expression of adipocyte marker genes is kept silent by histone
deacetylases. Induction of adipocyte differentiation by hormonal signaling reduces the expression and
activity of histone deacetylases, leading to loosening of the chromatin. This facilitates the binding of
adipogenic transcription factors to their binding sites.

16
Supplementary Figure 1

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Supplementary Figure 2

-NaB +NaB
Supplementary Figure 3

Preadipocytes

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HDAC Me HDAC Me HDAC

Adipogenic
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Adipogenesis
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 Down-regulation of Histone Deacetylases Stimulates Adipocyte Differentiation
Eung Jae Yoo, Jun-Jae Chung, Sung Sik Choe, Kang Ho Kim and Jae Bum Kim
J. Biol. Chem. 2006, 281:6608-6615.
doi: 10.1074/jbc.M508982200 originally published online January 5, 2006

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