Cell Metabolism
Article
EBF2 Determines and Maintains
Brown Adipocyte Identity
Sona Rajakumari,1,2,6 Jun Wu,4,6 Jeff Ishibashi,1,2 Hee-Woong Lim,1,3 An-Hoa Giang,4 Kyoung-Jae Won,1,3
Randall R. Reed,5 and Patrick Seale1,2,*
1Institute
for Diabetes, Obesity, and Metabolism
of Cell and Developmental Biology
3Department of Genetics
Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA
4Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA
5Center for Sensory Biology, Department of Molecular Biology and Genetics, Department of Neuroscience, Johns Hopkins
University School of Medicine, Baltimore, MD 21205, USA
6These authors contributed equally to this work
*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.cmet.2013.01.015
2Department
SUMMARY
The master transcription factor Pparg regulates the
general differentiation program of both brown and
white adipocytes. However, it has been unclear
whether Pparg also controls fat lineage-specific
characteristics. Here, we show that early B cell
factor-2 (Ebf2) regulates Pparg binding activity to
determine brown versus white adipocyte identity.
The Ebf DNA-binding motif was highly enriched
within brown adipose-specific Pparg binding sites
that we identified by genome-wide ChIP-Seq. Of
the Ebf isoforms, Ebf2 was selectively expressed in
brown relative to white adipocytes and was bound
at brown adipose-specific Pparg target genes.
When expressed in myoblasts or white preadipose
cells, Ebf2 recruited Pparg to its brown-selective
binding sites and reprogrammed cells to a brown
fat fate. Brown adipose cells and tissue from Ebf2deficient mice displayed a loss of brown-specific
characteristics and thermogenic capacity. Together,
these results identify Ebf2 as a key transcriptional
regulator of brown fat cell fate and function.
INTRODUCTION
Adipose tissues play an integral role in regulating systemic
metabolism and energy homeostasis. White adipocytes store
excess energy in the form of triglycerides for future need. By
contrast, brown adipocytes metabolize lipid and glucose to
produce heat in a process known as nonshivering thermogenesis (Cannon and Nedergaard, 2004). Brown adipocytes are
packed with specialized mitochondria that contain Uncoupling
Protein-1 (Ucp1) in their inner membrane. When activated,
Ucp1 dissipates the electrochemical gradient used for ATP
synthesis, resulting in increased heat production.
In rodents, brown adipose tissue (BAT) is a major site for coldinduced thermogenesis, which is essential for protection against
562 Cell Metabolism 17, 562–574, April 2, 2013 ª2013 Elsevier Inc.
hypothermia. BAT is also activated by overfeeding in mice as a
physiological countermeasure to limit weight gain (Rothwell
and Stock, 1979). Indeed, mouse strains that have increased
BAT activity and/or increased numbers of brown-like (beige)
adipocytes within their WAT are lean and healthy (Boström
et al., 2012; Cederberg et al., 2001; Chiang et al., 2009;
Guerra et al., 1998; Kopecký et al., 1996; Scimè et al., 2005;
Seale et al., 2011). By contrast, mice lacking brown and/or
beige fat activity are susceptible to obesity (Feldmann et al.,
2009; Lowell et al., 1993).
A natural role for BAT in adult humans was doubted until
recently. Through the use of PET imaging, significant deposits
of Ucp1-expressing adipocytes that take up large amounts of
glucose and lipid were identified in most, if not all, adult humans
(Cypess et al., 2009; Nedergaard et al., 2007; Ouellet et al., 2012;
Saito et al., 2009; van Marken Lichtenbelt et al., 2009; Virtanen
et al., 2009). Moreover, human BAT activity is inversely correlated
with body mass, suggesting that natural variations in BAT function may contribute to the regulation of body weight in humans.
Thus, there is great interest in identifying factors and pathways
that control brown fat cell development and/or function.
Many transcriptional factors have been shown to regulate
the brown adipocyte phenotype. Notable among these are
peroxisome proliferator-activated receptor gamma coactivator
1-alpha (Pgc1a) and PR-domain containing protein-16
(Prdm16). Pgc1a is a transcriptional coactivator that is especially
involved in the acute thermogenic response of brown fat cells to
b-adrenergic stimuli (Kleiner et al., 2012; Lin et al., 2004; Puigserver et al., 1998; Uldry et al., 2006). However, Pgc1a is not
required for determination of brown adipocytes, nor is it necessary for the differentiation-linked induction of Ucp1 and other
brown fat-specific genes (Lin et al., 2004; Uldry et al., 2006).
Prdm16 is a large zinc finger-containing protein that binds
and coregulates Pparg, Ppara, c/EBPb, and Pgc1a to induce
brown fat-specific genes (Hondares et al., 2011; Kajimura
et al., 2009; Seale et al., 2008; Seale et al., 2007). However,
Prdm16 expression is induced quite late during the adipogenesis
process (Seale et al., 2007) and thus probably does not regulate
lineage commitment per se.
Brown and white adipocytes both express a core set of
transcriptional factors that are believed to regulate common
Cell Metabolism
Ebf2 Controls Brown Fat Cell Fate
aspects of the differentiation process. A central component of
this pathway is peroxisome proliferator-activated receptor-g
(Pparg), which is required for the development of all types of
fat cells (Barak et al., 1999; Nedergaard et al., 2005; Rosen
et al., 1999; Tontonoz et al., 1994). However, whether Pparg
also plays a fundamental role in controlling the brown or white
fate of adipocytes had not been assessed.
Here, we used chromatin immunoprecipitiation analysis combined with massively parallel sequencing (ChIP-seq) to identify
Pparg binding sites in BAT and WAT. We found a surprisingly
large number of binding sites that were fat depot selective. Motif
analyses identified the consensus binding site for early B cell
factor (Ebf) as being highly enriched in BAT-specific Pparg
binding regions. Ebfs have been shown to regulate the initiation
of adipogenesis in white fat cells (Akerblad et al., 2002; Jimenez
et al., 2007), but their role in brown versus white fat cell determination had not been evaluated. We found that Ebf2 was
expressed at much higher levels in brown relative to white
adipose cells and tissue. Cell culture and genetic studies in
mice revealed that Ebf2 recruits Pparg to brown fat-selective
gene targets, including Prdm16, to determine brown identity.
RESULTS
The DNA Motif for Early B Cell Factor Is Enriched
at BAT-Specific Pparg Binding Sites
Pparg regulates the adipogenic process in all fat cells and
binds to thousands of loci during white adipocyte differentiation
(Lefterova et al., 2008; Mikkelsen et al., 2010; Nielsen et al.,
2008). We asked whether Pparg has unique transcriptional
targets in BAT and WAT. To do this, we performed genomewide ChIP-seq analysis of Pparg binding in interscapular
BAT and epididymal WAT (eWAT) from 8-week-old 129Sv
mice. Although 80% of binding sites were common to BAT and
eWAT, there were 3,500 and 4,700 sites that were highly
specific to BAT and eWAT, respectively (Figure 1A). ChIP-seq
tracks show representative examples of target genes bound by
Pparg in (1) both depots (e.g., Lipe, Fabp4), (2) eWAT only
(Rarres2), and BAT only (Ucp1, Ppara, Cpt1b and Prdm16)
(Figure 1B and Figure 5A). We also used ChIP-qPCR in independent tissue samples to confirm the differential binding of
Pparg at select sites (Figure 1C). These results reveal that
10% of Pparg binding sites in BAT and eWAT are depot
selective.
We then performed motif analysis to determine which, if any,
transcription factor binding sites were associated with BAT- or
WAT-specific Pparg binding regions. As anticipated, the DR-1
motif, which is bound directly by Pparg, was the highest-scoring
motif in both tissue types. The next most enriched motif in
BAT-selective Pparg sites was the binding sequence for Ebf
(Figure 1D), which prompted us to examine the role of Ebfs in
brown adipocyte differentiation.
Ebf2 Is Selectively Expressed in Brown Relative to White
and Beige Adipocytes
There are four members of the Ebf protein family (Ebf1–Ebf4),
which could potentially bind the ‘‘Ebf’’ motif. By qPCR, we found
that Ebf2 and Ebf3 mRNAs were expressed at higher levels in
BAT relative to both epididymal and inguinal WAT (Figure 2A).
Ebf4 was either expressed at very low levels or absent in the
fat depots we examined. In established cell lines, only Ebf2
transcripts were dramatically enriched (6-fold) in brown (four
independent lines) relative to white adipocytes (3T3-L1, 3T3F442A, 10T1/2) (Figure 2B). Interestingly, the brown-selective
expression of Ebf2 was apparent at the preadipocyte stage,
though its expression also rose during the differentiation process
(see Figures S1A and S1B online).
Thermogenic adipocytes, known as beige or brite (brown-inwhite) cells develop within WAT in response to various stimuli,
including b-adrenergic agonists. These beige adipocytes have
many of the characteristics of brown fat cells but are derived
from a separate cellular lineage (Seale et al., 2008). Wu et al.
recently cloned out several beige and white preadipocyte cell
lines from the inguinal WAT of mice (Wu et al., 2012). Interestingly, we found that Ebf2 was expressed at similar levels in beige
and white adipocytes lines, much lower than its levels in brown
adipocytes, cloned by the same procedures (Figure S1C). These
results suggest that Ebf2 is preferentially expressed in ‘‘classic’’
brown adipocytes relative to other types of adipocytes.
We also examined Ebf protein levels in adipose cells and
tissue by western blotting using commercially available antibodies. First, we assayed the Ebf-isoform specificity by probing
lysates from C2C12 cells that had been transiently transfected
with Flag-tagged versions of Ebf1, Ebf2, or Ebf3 (Figure S1D).
The Ebf2 antibody was highly specific, whereas Ebf1 and Ebf3
antibodies crossreacted to a small degree with the other Ebf
isoforms. In agreement with mRNA analysis, Ebf2 and Ebf3
protein levels were dramatically higher in BAT relative to
eWAT, but Ebf2 protein levels were also very highly enriched in
brown relative to white (3T3-L1) adipocytes (Figure 2C).
The next logical question was whether Ebf2 binds at/near
brown-specific genomic targets of Pparg in BAT. Using ChIPqPCR, we found that Ebf2 was enriched by 6- to 15-fold at
several BAT-specific Pparg binding sites relative to control
regions (Figure 2D). There was much less binding of Ebf2 with
Pparg binding sites that are common between WAT and
BAT (Fabp4, Cd36), and no detectable enrichment of Ebf2 at
WAT-specific Pparg binding sites (Agt, Apoc2, Rarres2). These
results show that Ebf2 is expressed selectively in BAT and binds
to BAT-specific Pparg gene targets.
Ebf2 Expression Stimulates a Brown Adipose-Specific
Differentiation Program
Brown fat cells originate from Myf5- and Pax7-expressing
precursors that also give rise to skeletal muscle (Lepper and
Fan, 2010; Seale et al., 2008). To examine whether Ebf2 is
sufficient to drive brown adipocyte differentiation, we first used
retrovirus to express Ebf2 or control vector in C2C12 cells,
a committed muscle precursor cell line. Ebf2- and controlexpressing cultures were grown to confluence and then induced
to undergo adipocyte differentiation. Strikingly, the Ebf2expressing cultures underwent very efficient conversion into
lipid-containing adipocytes, whereas control cultures were
devoid of adipocytes (Figure 3A). Ebf2 expression stimulated
adipogenesis in C2C12 cells to almost the same extent as retroviral Pparg2 expression (data not shown). Consistent with its
effects on cell morphology, Ebf2 increased the expression
levels of general adipocyte-selective genes, such as Pparg and
Cell Metabolism 17, 562–574, April 2, 2013 ª2013 Elsevier Inc. 563
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Ebf2 Controls Brown Fat Cell Fate
A
B
C
D
Figure 1. The Ebf Motif Is Highly Enriched at BAT-Specific Pparg Binding Sites
(A) ChIP for Pparg followed by massively parallel sequencing and genome alignment was used to profile binding sites in mouse eWAT and BAT.
(B) Representative Pparg ChIP-sequencing tracks in BAT (brown) and eWAT (blue) at common, BAT-specific, and WAT-specific sites.
(C) Independent ChIP-qPCR validation of Pparg binding in BAT and eWAT at select sites (mean ± SD; n = 3; *p < 0.05, **p < 0.01).
(D) Motif analyses of BAT- and eWAT-specific Pparg binding regions.
AdipoQ (Figure 3B). However, unlike Pparg2, Ebf2 also activated
the brown fat-specific gene program, including high levels
of Ucp1 and Cidea (Figure 3C and Figure S2). Finally, Ebf2
expression also enabled adipocytes to acutely increase their
564 Cell Metabolism 17, 562–574, April 2, 2013 ª2013 Elsevier Inc.
expression levels of Pgc1a and Ucp1 in response to the panb-adrenergic agonist, isoproterenol (Figure 3C and Figure S2).
The powerful adipogenic action of Ebf2 in C2C12 cells made
it difficult to isolate the effects of Ebf2 on the induction of
Cell Metabolism
Ebf2 Controls Brown Fat Cell Fate
Figure 2. Ebf2 Is Selectively Expressed in
Brown Relative to White Adipose Cells and
Tissue
A
(A) mRNA levels of Ebf1, Ebf2, and Ebf3 in eWAT,
ingWAT (inguinal), and BAT (mean ± SD; n = 3;
**p < 0.01).
(B) mRNA levels of Ebf1, Ebf2, and Ebf3 in a panel
of white and brown adipose cell lines (mean ± SD;
n = 3; **p < 0.01).
(C) Western blot analysis of Ebf1, Ebf2, and Ebf3
protein expression in representative samples from
eWAT, BAT (left panel), and adipocytes (day 7)
from white (3T3-L1) and brown (BAT 1) preadipocytes. Tbp was used for a loading control.
(D) ChIP-qPCR analysis of Ebf2 binding in BAT at
sites bound by Pparg in BAT and WAT (common),
BAT only, and WAT only (mean ± SD; n = 3–5).
B
C
D
brown-specific genes. We thus wanted to investigate the function of Ebf2 in preadipose cells that are naturally competent to
undergo adipocyte differentiation. The stromal vascular fraction
(SVF) of WAT contains bona fide preadipocytes that differentiate
into white adipocytes. We expressed Ebf2 or vector control
in primary SVF cultures isolated from inguinal WAT of 8- to
12-week-old male mice. Cells transduced with Ebf2 virus
expressed 4-fold higher levels of Ebf2 mRNA relative to its
endogenous levels in brown fat cells. Six days after inducing
differentiation, both Ebf2 and control cultures contained mostly
mature, lipid-filled adipocytes (Figure 3D).
The molecular phenotype of adipocytes from Ebf2- and
control-expressing SVF cells was analyzed in greater detail by
qPCR-based gene expression analysis. We found that Ebf2 expression resulted in a mild increase in the levels of some general
adipocyte genes, including AdipoQ and Fabp4 (Figure 3E).
Strikingly however, Ebf2 very strongly increased the expression
levels of many brown adipocyte-specific genes, including
a near 500-fold increase in Ucp1, and 4- to 10-fold increases
in Prdm16, Pgc1a, Ppara, and Cidea relative to control cultures
(Figure 3F). Additionally, Ebf2 increased the mRNA levels of
several
mitochondrial
components,
including Cytochrome-c (Cyc), Cox5b,
Cox8b, and Cox7a1 (Figure 3G). Isoproterenol treatment further increased the
expression of thermogenic genes, like
Ucp1 and Pgc1a, to much higher levels
in Ebf2- compared to control-expressing
cells (Figure 3H). Finally, immunofluorescence analysis showed that expression
of Ebf2 greatly increased the levels of
Ucp1 protein under unstimulated conditions (Figure 3I).
We also found that Ebf2 robustly and
consistently induced the thermogenic
gene program when expressed in other
types of fat precursors, including beige
and white preadipose cell lines isolated
from inguinal WAT (Figure S3), and in
the classic 3T3-L1 white adipocyte line
(Figure S4). In beige cells, we found that
Ebf2 stimulated adipogenic differentiation and suppressed the
expression of several beige-specific genes (Ear2, Tmem26,
CD137, and SP100).
Ebf2 Is Required for the Maintenance of Brown
Fat Cell Fate
The requirement for Ebf2 in brown adipocyte differentiation was
investigated using siRNA-mediated gene knockdown. First,
we used lentiviral vectors to express a shRNA targeting Ebf2
or a control Scrambled (Scr) shRNA in mouse brown
preadipocytes. The sh-Ebf2 construct efficiently depleted Ebf2
mRNA levels in brown preadipocytes (Figure S5A) without
reducing the levels of other Ebf isoforms (data not shown).
Whereas control sh-Scr cells underwent efficient morphological
differentiation and accumulated lipid droplets, loss of Ebf2
completely blocked differentiation (Figure S5B). Consistent
with this, Ebf2-depleted cells failed to induce general adipogenic
genes including Pparg, Fabp4, and AdipoQ as well as brownspecific markers, like Prdm16 and Cidea (Figure S5C).
The complete differentiation block caused by shRNAmediated loss of Ebf2 in preadipocytes confounded a meaningful
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Ebf2 Controls Brown Fat Cell Fate
A
D
G
B
E
C
F
H
I
Figure 3. Ebf2 Expression Drives a Brown Fat-Specific Differentiation Program
(A) Ctl- and Ebf2-expressing C2C12 myoblasts were induced to differentiate into adipocytes followed by staining of triglycerides with oil red O.
(B and C) The above cultures were analyzed for their expression levels of Pparg and AdipoQ (B) and Ucp1 levels ± isoproterenol treatment (C) (n = 3).
(D–I) Ctl- and Ebf2-expressing white preadipocytes were induced to differentiate into adipocytes followed by staining with oil red O (D). (E–H) mRNA levels
of Ebf2 and general adipocyte markers (E), brown-selective genes (F), mitochondrial genes (G), and Ucp1 and Pgc1a ± isoproterenol treatment (H) (n = 3–5 pools/
construct). (I) Bodipy staining of triglycerides (green) and Ucp1 immunostaining (red). All expression data are mean ± SD; *p < 0.05; **p < 0.01.
analysis of brown-specific pathways (that are only expressed
in mature adipocytes). Therefore, we performed knockdown
experiments in mature brown adipocytes. On day 6 of differentiation, brown adipocytes were electroporated with a control Scr
siRNA or an Ebf2 siRNA. Forty-eight to seventy-two hours later,
Scr- and Ebf2 siRNA-treated cultures were morphologically
indistinguishable, each composed of well-differentiated adipocytes (Figure 4A). siEbf2 reduced Ebf2 mRNA levels by 50%
(Figure 4B) and caused a much more dramatic loss of Ebf2
protein levels (Figure 4E). Importantly, the depletion of Ebf2 in
adipocytes did not affect Pparg levels (mRNA or protein) or the
mRNA levels of general adipocyte genes AdipoQ and Fabp4
(Figures 4C and 4E). Reduction of Ebf2 did, however, cause
a very specific decrease in the expression levels of a wide array
566 Cell Metabolism 17, 562–574, April 2, 2013 ª2013 Elsevier Inc.
of brown-specific and mitochondrial genes, including an 50%
and an 90% reduction in Prdm16 and Ucp1 mRNA levels,
respectively (Figure 4D). Loss of Ebf2 also caused a dramatic
reduction in Ucp1 protein levels (Figure 4E).
Ebf2 and Pparg Cooperate to Activate Transcription of
Prdm16
The binding of Ebf2 at Pparg target sites suggested a functional
interaction between these two factors. Prdm16 is a transcriptional
coactivator that plays a pivotal role in brown fat cell differentiation
(Kajimura et al., 2008, 2009; Seale et al., 2007, 2008). We noted
that ectopic expression of Ebf2 greatly increased Prdm16 levels
in fat cells (Figure 3), whereas loss of Ebf2 reduced Prdm16 levels
(Figure 4); this raised the question of whether Ebf2 directly
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Ebf2 Controls Brown Fat Cell Fate
A
B
Figure 4. Ebf2 Is Required to Maintain the
Expression of Brown-Specific Genes in
Brown Adipocytes
C
(A) Phase contrast micrographs of brown adipocyte cultures 2 days after treatment with an
Ebf2-specific siRNA or a siSCR control.
(B–D) mRNA levels of Ebf2 (B), general fat
markers (C), and brown-specific and mitochondrial genes (D).
(E) Western blot analysis of Ebf2, Pparg, Ucp1, and
tubulin (loading control) protein levels. Expression
data are mean ± SD; n = 3; **p < 0.01.
D
regulates Prdm16 transcription. Upon re-examination of the
Pparg ChIP-seq data, we uncovered a strong BAT-specific
Pparg binding site located 25 kb upstream of the Prdm16
promoter (Figure 5A). Using ChIP qPCR, we found that both
Pparg and Ebf2 bind to this region in BAT, but not in eWAT
(Figure 5B).
The Pparg/Ebf2 binding region in Prdm16 was cloned
upstream of a minimal thymidine kinase promoter and firefly
luciferase reporter gene. We then expressed Ebf2 and/or Pparg
with its partner, Rxra, in combination with the Prdm16-luciferase
reporter gene in NIH 3T3 cells. The reporter gene had very
minimal activity on its own and only a slightly increased activity
in response to either Ebf2 alone or Pparg and Rxra (Figure 5C).
However, coexpression of Ebf2 with Pparg and Rxra led to
a very dramatic rise in transcription (70-fold over ctl) (Figure 5C). This synergistic activation of transcription was not due
to an increase in protein levels of either factor (Figure 5D).
Ebf2 is expressed in brown preadipocytes prior to the
differentiation-linked increase in Pparg and Prdm16 expression (Figure S1). We thus wondered whether Ebf2 was recruited
to the Prdm16 locus before Pparg. To answer this, we performed
ChIP-qPCR for Ebf2 and Pparg during a time course of
brown fat cell differentiation. We found that Ebf2 was already
bound to Prdm16, but not to Fabp4, in preadipocytes (day 0);
the binding at Prdm16 increased further during differentiation
(Figure 5E). By contrast, Pparg was not detectable at Prdm16
until day 4. These results suggest that Ebf2 binds to brown
fat gene targets before (and independently of) Pparg.
Ebf2 Recruits Pparg to BAT-Selective Gene Targets
The above experiments raised the question of whether Ebf2
facilitates the binding Pparg to its BAT-specific target sites.
To test this, we used Pparg-deficient fibroblasts expressing
retroviral Pparg2 in order to hold Pparg levels constant.
E
These cells were then transduced with
control- or Ebf2-expressing retrovirus
and induced to undergo adipocyte
differentiation for 7 days. Gene expression analysis confirmed that Ebf2
robustly stimulated the brown-specific
gene program in these cells (data not
shown). Remarkably, ChIP-qPCR analysis showed that exogenous Pparg2
interacted strongly with BAT-specific
targets, such as Prdm16 and Ucp1 in
Ebf2-expressing but not in control-expressing cultures (Figure 6A). Conversely, we found that siRNA-mediated reduction
of Ebf2 in brown adipocytes caused a significant decrease in
Pparg binding at several BAT-specific sites, including Ucp1,
Ppara, and Prdm16, with no change in binding at BAT/WAT
common sites or WAT-specific sites (Figure 6B).
We also examined the effect of Ebf2 expression on the chromatin binding activity of Pparg in primary SVF cells from WAT.
Here, we found that Ebf2 expression caused a slight increase in
the binding of Pparg to Fabp4, a target in both BAT and WAT (Figure 6C). However, Ebf2 expression increased Pparg binding by
2- to 4-fold at brown-specific targets, including Prdm16, while
significantly reducing Pparg binding at WAT-specific target sites
(Agt and Rarres2) (Figure 6C). Altogether, these results suggest
that Ebf2 enables Pparg to bind to its BAT-specific target sites.
Ebf2 Is Required for BAT Development in Mice
To determine whether Ebf2 is important for brown fat development in vivo, we analyzed BAT in wild-type and Ebf2-deficient
mouse embryos. Ebf2-deficient mice are born in normal
Mendelian ratios but fail to thrive and typically die soon after birth
(Corradi et al., 2003; Wang et al., 2004). Due to the systemic
illness that occurs postnatally in Ebf2-knockout (KO) animals,
we analyzed BAT in late-stage mouse embryos. By hematoxylin
and eosin (H&E) staining, the presumptive BAT from the Ebf2deficient embryos at E18 days postcoitum (dpc) was reduced
in size and had a very unusual morphology (Figure 7A). Specifically, the KO tissue was very weakly stained with eosin, suggesting a dramatic reduction in mitochondrial density.
Gene expression and western blot analysis confirmed that
Ebf2 mRNA (data not shown) and protein (Figure S6A) were
ablated in the knockout tissue. Interestingly, Ebf2 KO tissue
expressed normal levels of the panadipocyte markers Pparg,
AdipoQ, and Fabp4 (Figure 7B, left side). However, there was
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Ebf2 Controls Brown Fat Cell Fate
A
C
B
D
E
an almost complete loss of brown fat-specific gene expression in
Ebf2-KO tissue, including an 85% reduction in Prdm16
levels and a more than 95% reduction in Ucp1 and Cidea levels
(Figure 7B, center). Mitochondrial gene expression was also
severely decreased in Ebf2-deficient BAT (Figure 7B, right).
Interestingly, we noted that Ebf2 deficiency also caused a milder
decrease in mitochondrial gene levels in embryonic heart, but
not in skeletal muscle or brain (Figure S6B). Finally, we found
that Ebf2-deficient BAT expressed higher levels of many whitespecific gene markers, including Resistin (Retn), Nnmt, Igf1,
and Gpr64 (Figure 7C). These results reveal a profound genetic
requirement for Ebf2 in determining BAT identity.
We then assessed whether the BAT phenotype in Ebf2deficient mice was due to fat cell-autonomous defects. To
address this, we isolated brown preadipocytes from Ebf2+/+,
Ebf2+/ , and Ebf2 / embryos at E17 dpc and subjected them
to in vitro differentiation. Cultures of all three genotypes underwent efficient morphological differentiation into lipid-containing
adipocytes as shown by oil red O staining (Figure 7D). Gene
568 Cell Metabolism 17, 562–574, April 2, 2013 ª2013 Elsevier Inc.
Figure 5. Ebf2 and Pparg Cooperatively
Activate Transcription
(A) Pparg ChIP-seq tracks from BAT (brown) and
WAT (blue) at the Prdm16 locus.
(B) ChIP-qPCR analysis of Ebf2 and Pparg binding
to the 25 kb Prdm16 region (red box in A) in
eWAT and BAT.
(C) Transcriptional activity of the 25 kb region of
Prdm16 or control reporter construct (pGL4.24)
in response to expression of Ebf2, Pparg/Rxra, or
the combination of Ebf2 and Pparg/Rxra in NIH
3T3 cells (n = 4; mean ± SD).
(D) Western blot analysis of Ebf2 and Pparg levels
in NIH 3T3 lysates that were used for transcription
assays in (C).
(E) ChIP-qPCR analysis of Ebf2 and Pparg binding
to the 25 kb Prdm16 region and Fabp4 during
a time course of brown adipocyte differentiation.
expression analysis showed that Ebf2
mRNA was undetectable in Ebf2 / cells
and reduced by 60% in Ebf2+/ cultures
(Figure 7E). Reduction of Ebf2 did not
alter the levels of either Ebf1 or Ebf3 (Figure 7E). Ebf2+/+, Ebf2+/ , and Ebf2 /
cultures expressed similar levels of
general adipocyte markers (Figure 7F).
However, the expression of brownspecific and mitochondrial genes was
severely reduced by the decrease or
loss of Ebf2, including an 85%–90%
reduction in the levels of Prdm16, Ucp1,
and Cidea in KO relative to wild-type cells
(Figure 7G and Figure S6C). Ebf2-deficient cultures also expressed greatly
elevated levels (4- to 6-fold higher than
wild-type) of white adipocyte-specific
genes (Figure 7H). Interestingly, ectopic
overexpression of Ebf1, Ebf2, or Ebf3
brown preadipocytes
into Ebf2 /
rescued the differentiation-linked expression of brown genes
to levels approximately five times higher than seen in uninfected,
wild-type cells (Figure S7). These results demonstrate that Ebf2
regulates brown adipose identity in a cell-autonomous manner.
Brown adipocytes produce heat through a catecholaminestimulated activation of Ucp1 and a resulting increase in
uncoupled respiration (Cannon and Nedergaard, 2004). To
examine whether Ebf2 is critical for brown adipocyte function,
we measured the respiratory activity of cultured Ebf2+/ and
Ebf2 / brown adipocytes using a Clark electrode. At day 7 of
differentiation, Ebf2+/ cells had slightly higher levels of basal
and maximal (FCCP-stimulated) respiration (Figure 7I and Figure S6D). Importantly, however, norepinephrine (NE) stimulation
elicited a very dramatic rise (approximately doubling) in the O2
consumption of Ebf2+/ cells and had no effect in Ebf2 / cells
(Figure 7I). This NE-stimulated respiration in Ebf2+/ cells was
largely insensitive to oligomycin and was thus uncoupled (Figure S6D). These studies indicate that Ebf2 is essential for the
proper respiratory function of brown adipocytes.
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Ebf2 Controls Brown Fat Cell Fate
A
B
C
Figure 6. Ebf2 Promotes the Binding of Pparg to Its Brown Fat-Selective Gene Targets
(A) ChIP-qPCR analysis of Pparg binding in adipocytes from Pparg-deficient cells that were first transduced with Pparg retrovirus, followed by transduction with
either a control (ctl) or Ebf2-expressing retrovirus.
(B) ChIP-qPCR analysis of Pparg binding in Scrambled (Ctl) or Si-Ebf2-treated brown adipocytes.
(C) ChIP-qPCR analysis of Pparg binding in mature adipocytes from control (Ctl) and Ebf2-expressing white preadipocytes. All data are normalized to background
binding (at nonspecific Insulin and 18 s rRNA loci) and presented as mean ± SD, n > 3.
DISCUSSION
Pparg is the master regulator of adipocyte differentiation in
brown and white fat cells, even though these cells belong to
separate lineages. This raised intriguing questions: Did Pparg
play a major role in lineage-specific programming? And if so,
how?
We found that Pparg binds to thousands of loci differentially in
BAT and eWAT. Consistent with our results, the Mandrup lab
recently reported that in vitro-differentiated adipocytes also
display depot-selective Pparg binding profiles (Siersbæk et al.,
2012). In both studies, Pparg binding is highly correlated with
depot-selective gene expression, suggesting that Pparg plays
a broad and unanticipated role in executing brown and white
fat cell-selective gene programs.
BAT-specific target sites of Pparg are also specifically bound
by Ebf2 in brown adipocytes. Our studies suggest that Ebf2 acts
as a pioneer factor in brown fat cells to ‘‘mark’’ genes for the
later recruitment of Pparg and transcriptional activation. This is
reminiscent of the role of Ebf1 in B cells, which has been found
to promote the demethylation of some of its target genes to
poise genes for their later activation by Pax5 and other transcription factors (Maier et al., 2004; Treiber et al., 2010). In fat cells,
we also noted that Ebf2 expression reduced Pparg binding
at white fat-specific loci, which could explain the repressive
effects of Ebf2 on white-specific gene transcription. Whether
this is due to squelching because Pparg protein is limiting or
occurs via the action of a downstream Ebf2-target gene(s) is
not yet known.
Interestingly, neither Ebf2 nor Pparg alone could efficiently
activate transcription from a putative enhancer in Prdm16, which
contains both Ebf and Pparg binding motifs. Ebf2 binding may
induce structural changes to DNA and/or chromatin that facilitate or stabilize Pparg binding. It is also possible that Ebf2 has
additional effects on the Pparg transcriptional complex, such
as promoting coactivator recruitment and/or displacing corepressors, to stimulate transcriptional activity. Interestingly,
Ebf1 interacts with the Swi/Snf chromatin remodeling complex,
which is necessary for Ebf-mediated transcriptional activation
from certain B cell-specific enhancers (Gao et al., 2009). Further
studies will be needed to unravel the mechanistic details of how
Ebf2 and Pparg synergize to drive transcription in fat cells.
Cell Metabolism 17, 562–574, April 2, 2013 ª2013 Elsevier Inc. 569
Cell Metabolism
Ebf2 Controls Brown Fat Cell Fate
A
B
C
D
G
E
H
F
I
Figure 7. Ebf2 Is Required for BAT Development in Mice
(A) Hematoxylin and eosin (H&E) staining of representative sections from the interscapular region of wild-type and Ebf2-deficient embryos at E18 dpc.
(B and C) BAT from Ebf2+/ and Ebf2 / embryos (E18) was examined by real-time PCR analysis for mRNA levels of general adipocyte genes, brown-specific
genes, and mitochondrial genes (B); and WAT-selective genes (C) (n = 5–8 embryos/genotype).
(D) Brown fat precursors from Ebf2+/+, Ebf2+/ , and Ebf2 / embryos were differentiated into adipocytes in culture and stained with oil red O.
(legend continued on next page)
570 Cell Metabolism 17, 562–574, April 2, 2013 ª2013 Elsevier Inc.
Cell Metabolism
Ebf2 Controls Brown Fat Cell Fate
Prdm16 drives brown fat-specific gene expression in all
known types of thermogenic adipocytes (Seale et al., 2011;
Seale et al., 2007). However, molecular events that control
the depot-selective and differentiation-linked expression of
Prdm16 had not been identified. We reveal here that Ebf2 and
Pparg act upstream of Prdm16 in the transcriptional program
of classic brown fat cells. It seems likely that activation of
Prdm16 is critical for the action of Ebf2 in brown fat determination. However, Ebf2 also interacts directly with other major
brown fat-specific genes, including Ucp1, suggesting that Ebf2
controls both Prdm16-dependent and -independent pathways.
We propose that Prdm16, once induced by Ebf2, provides
a feedforward mechanism to amplify and reinforce the expression of brown fat-specific genes.
The presence of fatty tissue in Ebf2 / animals, rather than
a complete absence of BAT, suggests that other, unknown
factors specify preadipose cell fate. One possibility is that
Ebf1 and/or Ebf3 preserves preadipocyte commitment in the
absence of Ebf2 in vivo. Surprisingly, in cultured brown preadipocytes, shRNA-mediated reduction of Ebf2 completely
blocked adipogenesis. This result is consistent with previous
studies showing that Ebf2 is required for early events during
the differentiation of 3T3-L1 cells (Jimenez et al., 2007) and
raises the possibility that other Ebfs can compensate for the
chronic but not acute loss of Ebf2 in preadipocytes.
Importantly, the fat tissue in Ebf2 null animals was completely
devoid of brown character. This is despite the fact that Ebf1
and especially Ebf3 are expressed in brown adipocytes and
have the ability to stimulate the brown fat gene program when
ectopically expressed in cultured precursors. The specific
requirement for Ebf2 is likely dictated by the timing and
levels of its expression during brown adipocyte development/
differentiation. Alternatively, Ebf2 may have isoform-specific
actions in brown fat cells which can be conferred by other isofroms through high (superphysiological) levels of expression.
The loss of thermogenic gene programming in Ebf2-deficient
brown adipocytes was associated with a profound defect in
respiratory function, much like that seen in Ucp1-deficient brown
adipocytes (Matthias et al., 2000). The loss of brown fat-like
respiratory activity predicts that Ebf2-deficient BAT would be
incapable of adaptive thermogenesis in response to high-fat
diet or cold. Unfortunately, however, we are not yet able to
assess the physiological consequence of Ebf2 action in adult
BAT, including effects on energy balance, due to the severe
systemic effects caused by whole-body Ebf2 deficiency in our
mouse model.
Genetic and developmental studies have shown that there
are at least two lineages of thermogenic adipocytes: (1) classic
brown adipocytes (in BAT) and (2) beige adipocytes (a.k.a. brite
adipocytes) that develop within WAT (Petrovic et al., 2010; Seale
et al., 2008; Xue et al., 2007). In inguinal WAT, beige adipocytes
develop from committed preadipocytes and only express high
levels of thermogenic genes upon stimulation with b-adrenergic
agonists (Wu et al., 2012). We found that Ebf2 expression was
enriched in brown but not beige adipocytes relative to white
adipocytes. Moreover, Ebf2 expression in beige or white
adipocyte lines increased thermogenic gene levels in the
absence of b-adrenergic stimulation. Ebf2 may thus provide
a brown fat-specific mechanism for inducing high levels of
thermogenic gene/protein levels during differentiation under
basal conditions.
There is great hope that the thermogenic action of brown/
beige adipocytes can be used therapeutically to reduce obesity
and/or metabolic disease. To effectively increase brown fat
mass and/or function, it will be important to identify treatments
that stimulate a broad and specific program of differentiation.
Our work suggests that the binding of Pparg to its brown fatspecific gene targets is fundamental for proper brown adipocyte
development. Thus, searching for the upstream signals and
factors that induce Ebf2 expression and activity in fat precursors
will reveal important biological insights and may identify new
therapeutic options.
EXPERIMENTAL PROCEDURES
Animals
All animal experiments were performed according to procedures approved by
the University of Pennsylvania’s Institutional Animal Care and Use Committee.
Ebf2-deficient mice were described previously (Wang et al., 2004).
ChIP-Seq
High-throughput sequencing was done by the Functional Genomics Core of
the Penn Diabetes Research Center using the Illumina Genome Analyzer IIx
or HiSeq 2000, and the reads were aligned to the mm8 genome assembly
genome using Bowtie. In each ChIP-seq sample, all the duplicate reads
were removed except for one before downstream analysis. ChIP-seq data is
available at Gene Expression Omnibus (accession code GSE43763).
Peak Calling and Motif Analysis
To identify depot-specific Pparg peaks, we performed peak calling for one
depot sample as foreground and the other sample as background, and vice
versa. All the peak calling was performed using Homer with options, peak
size 200 bp and minimum distance 200 bp (size 200, minDist 200), and then
1 rpm cutoff was applied. Common Pparg peaks were defined as follows. First,
Pparg peaks were called for each depot using a matching input sample, and
then all the peaks were pooled and overlapping peaks were merged. Among
these peaks, any peak that overlaps with the depot-specific Pparg peaks
was eliminated, and the remaining peaks were defined as common Pparg
peaks. It should be noted that our definition of common peak here is naive
in the sense that some peaks might show different peak height between
different depots. However, this should not be a problem in this work because
our primary target is to identify depot-specific (especially BAT-specific) Pparg
binding sites.
Cell Culture
Primary brown and white preadipocytes were fractionated from mice fat
depots using collagenase and dispase digestion as described previously
(Seale et al., 2007). Immortalized brown fat preadipocytes and PPARg /
fibroblasts have been described elsewhere (Rosen et al., 2002). Murine clonal
beige and white cells from inguinal WAT were cultured and differentiated as
described (Wu et al., 2012). For adipocyte differentiation assays, immortalized
brown fat or primary preadipocytes were grown to confluence, and cells
were treated with induction medium containing 10% FBS, 0.5 mM
(E–H) The above cultures were analyzed by real-time qPCR for their expression levels of Ebfs (E), general adipocyte markers (F), brown-specific genes (G), and
white-selective genes (H) (n = 3 pools [>5 mice per pool]).
(I) Oxygen consumption in cultured brown adipocytes from Ebf2+/ and Ebf2 / before and after stimulation with norepinephrine (NE) (mean ± SD; n = 3).
Expression data are mean ± SD, *p < 0.05; **p < 0.01.
Cell Metabolism 17, 562–574, April 2, 2013 ª2013 Elsevier Inc. 571
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Ebf2 Controls Brown Fat Cell Fate
isobutylmethylxanthine, 125 nM indomethacin, 1 mM dexamethosone, 20 nM
insulin, and 1 nM T3. After 48 hr, cells were switched to medium containing
10% FBS, 1 nM T3, and 20 nM insulin until harvest. To stimulate thermogenesis, cells were incubated with 10 mM isoproterenol for 3 hr.
Oxygen Consumption Assays
Primary brown preadipocytes were induced to undergo adipogenesis. At day 7
of differentiation, adipocytes were trypisinized and counted, and oxygen
consumption was measured using a Strathkelvin Clark-type electrode. NE
(1 mM) (Sigma), 1 mM oligomycin (Sigma), or 2 mg of FCCP (Sigma) was added
acutely to the chamber to activate Ucp1, block state III respiration, and induce
uncoupling, respectively.
Viral Vectors and Production
Full-length coding sequences for mouse Ebf1 (GeneID 13591), Ebf2 (GeneID
13592), and Ebf3 (GeneID 13593) were amplified from BAT mRNA and
cloned into pMSCV-puro retroviral vector (Stratagene) or pcDNA3.1
(Invitrogen). The siRNA and double-stranded DNA sequence encoding the
shRNA targeting Ebf2-knockdown were purchased from (Dharmacon). For
retrovirus production, 293T cells (Kinsella and Nolan, 1996) were transfected with 15 mg retroviral vectors along with packaging plasmids at
70% confluence by calcium phosphate coprecipitation. The viral supernatant was harvested every 24 hr for 3 days and concentrated using SW28
ultracentrifuge rotor at 26,000 rpm. For retroviral transduction, cells were
incubated overnight with viral supernatant supplemented with 8 mg/ml
polybrene.
Real-Time PCR
Total RNA from cultured cells or tissues was isolated using the TRIzol method
(Invitrogen) combined with QIAGEN RNeasy minicolumns. For real-time PCR
analysis, RNA was reverse transcribed using the ABI high-capacity cDNA
synthesis kit and used in quantitative PCR reactions containing SYBR green
fluorescent dye (ABI) using an ABI9300 PCR machine. Tata-binding protein
(Tbp) served as an internal control to normalize samples. Primers are listed
in Table S1.
Western Blot Analysis
Cells or tissues were lysed in radioimmunoprecipitation assay (RIPA) lysis
buffer containing 0.5% NP-40, 0.1% sodium deoxycholate, 150 mM NaCl,
50 mM Tris-Cl (pH 7.5), protease inhibitor cocktail (Complete; Roche), and
1 mM phenylmethylsulfonyl fluoride (PMSF). The protein content was
measured using a detergent-compatible (DC) protein assay kit (Bio-Rad).
Lysates or nuclear fractions were resolved onto bis-Tris NuPAGE gels
(Invitrogen), transferred to PVDF membrane (Millipore), and probed with antiEbf1(Santa Cruz Biotechnology), anti-Ebf2 (R&D systems), anti-Ebf3 (Santa
Cruz Biotechnology), anti-EBF (D-8) (Santa Cruz Biotechnology), anti-Pparg
(E-8) (Santa Cruz Biotechnology), anti-Ucp1 (R&D Systems), anti-FLAG
(Mouse polyclonal), anti-TBP (Abcam), and anti-tubulin (Santa Cruz Biotechnology) antibodies.
Oil Red O Staining
After 6–8 days of differentiation, cells were washed once in PBS and fixed
with freshly prepared 4% formaldehyde for 15 min and stained using oil red
O solution for 30 min.
Chromatin Immunoprecipitation
Fat depots from 129 3 1/SvJ male mice (8 weeks old) were harvested and
washed with PBS. Fat tissues were minced in DMEM and crosslinked with
1% formaldehyde for 15 min at room temperature. Crosslinking was quenched
using 125 mM glycine for 5 min. Tissues were then washed with PBS and
chromatin immunoprecipitation (ChIP) dilution buffer containing 0.1% SDS,
50 mM Tris, 140 mM NaCl, 1% Triton X-100, 0.1% Na-Deoxycholate, 1 mM
PMSF complete protease inhibitor cocktail (Roche), and 1 mM EDTA.
Chromatin was sheared by sonication, and immunoprecipitation was carried
out as described (Ishibashi et al., 2012). The protein-DNA complexes were
immunoprecipitated using 2.5 mg of antibody (sc-2027; Santa Cruz) and
isolated with protein A Sepharose beads, washed and eluted with 1% SDS–
0.1 M NaHCO3. Reverse crosslinking was performed for overnight incuba-
572 Cell Metabolism 17, 562–574, April 2, 2013 ª2013 Elsevier Inc.
tion at 65 C, followed by Proteinase K digestion and purification of DNA
fragments using PCR purification columns (QIAGEN). Eluted DNA was
analyzed by real-time PCR on a 7900 machine (ABI) using SYBR green
chemistry (ABI, Affymetrix). Target enrichment was calculated as percent
input and normalized to 18S for nonspecific background signal. Primer
sequences used for this study are found in Table S2.
Transcription Assays
Luciferase reporter plasmids were constructed by PCR cloning C57Bl/6
genomic fragments corresponding to Pparg-bound regions into the pGL4.24
vector (Promega). pGL4-based reporters were cotransfected into NIH 3T3
cells (ATCC) in multiwell plates along with Renilla luciferase (internal control),
Ebf2, and/or Pparg2 expression plasmids using Lipofectamine 2000 (Invitrogen). Cells were harvested into passive lysis buffer (Promega) at 48 hr
posttransfection, and dual luciferase activity was assayed with a Synergy HT
plate reader (BioTek). Reporter luciferase activity was normalized to the
internal Renilla control activity.
Statistical Analysis
All data are reported as mean ± SD unless otherwise noted in the figure
legends. Student’s t test was used to calculate significance (*p < 0.05;
**p < 0.01) using Excel or Prism software packages.
SUPPLEMENTAL INFORMATION
Supplemental Information includes seven figures and one table and can be
found with this article at http://dx.doi.org/10.1016/j.cmet.2013.01.015.
ACKNOWLEDGMENTS
We thank Drs. Mitch Lazar and Dave Steger for expert guidance with ChIP
studies, Dr. Mary Selak for help with oxygen consumption assays, Li Huang
for expert technical assistance, and members of the Seale lab for helpful
discussions. We are grateful to the Functional Genomics Core of the Penn
Diabetes and Endocrinology Research Center (DK19525) for high-throughput
sequencing and data analyses. J.W. is supported by an NIH grant
(1K01DK094824). This work was supported by NIDDK/NIH award R00
DK081605, NIGMS/NIH award DP2OD007288, and a Searle Scholars Award
to P.S.
Received: October 16, 2012
Revised: January 3, 2013
Accepted: January 23, 2013
Published: March 14, 2013
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Article EBF2 Determines and Maintains Brown Adipocyte Identity Cell Metabolism