Transcriptional networks andchromatin remodeling controllingadipogenesisRasmus Siersbæk, Ronni Nielsen and Susanne Mandrup

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark

Review

Glossary

DHS site (DNase I hypersensitive site): genomic chromatin region that is

hypersensitive to DNase I digestion compared to the surrounding DNA.

Enhanceosome: enhancer-associated protein complex that drives transcrip-

tional activation.

HOT region (high-occupancy target region): genomic region that is bound by

>8 transcription factors in whole animal studies.

Hotspot: genomic region that is co-occupied by multiple transcription factors

in a given cell (similar to MTL).

MTL (Multiple transcription factor loci): genomic regions that are co-occupied

by multiple transcription factors in a given cell (similar to hotspots).

DR1 (direct repeat 1): two similar DNA sequences of 6 bp in the same

orientation separated by 1 bp.

Adipocyte differentiation is tightly controlled by a tran-scriptional cascade, which directs the extensive repro-gramming of gene expression required to convertfibroblast-like precursor cells into mature lipid-ladenadipocytes. Recent global analyses of transcription fac-tor binding and chromatin remodeling have revealed‘snapshots’ of this cascade and the chromatin landscapeat specific time-points of differentiation. These studiesdemonstrate that multiple adipogenic transcription fac-tors co-occupy hotspots characterized by an open chro-matin structure and specific epigenetic modifications.Such transcription factor hotspots are likely to representkey signaling nodes which integrate multiple adipogenicsignals at specific chromatin sites, thereby facilitatingcoordinated action on gene expression.

Transcriptional waves controlling adipogenesisThe molecular mechanisms controlling the conversion ofpreadipocytes to mature adipocytes (adipogenesis) havereceived much attention in the past decades due to themassive increase in the prevalence of obesity and obesity-related diseases. Adipogenesis is controlled by a tightlyregulated transcriptional cascade where the transcriptionfactors activate or repress the expression of each other in asequential manner. Key players in this transcriptionalcascade include CCAAT/enhancer-binding protein (C/EBP) family members (i.e. C/EBPa, C/EBPb, and C/EBPd)and the nuclear receptor peroxisome proliferator-activatedreceptor g (PPARg), which have been shown by both invitro and in vivo studies to be important regulators ofadipocyte differentiation [1]. The adipogenic cascade canbe divided into at least two waves of transcription factorsthat drive the adipogenic program. The first wave is initi-ated by adipogenic stimuli that activate several earlyadipogenic factors including C/EBPb/d, Kru ppel-like fac-tors (KLFs), cAMP response element binding protein(CREB), early growth response 2 (Krox20), and sterolregulatory element-binding protein 1c (SREBP-1c)(Figure 1; Box 1). These transcription factors in turninduce expression of the second wave of transcriptionfactors, of which PPARg and C/EBPa are the most impor-tant, and these key adipogenic factors then induce expres-sion of the gene program that leads to the mature adipocytephenotype [1–3] (Figure 1; Box 1). Recently, genome-wide

Corresponding author: Mandrup, S. (s.mandrup@bmb.sdu.dk).

56 1043-2760/$ – see front matter � 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.t

studies have mapped the binding profiles of multiple adi-pogenic transcription factors of both the first and secondtranscription factor wave. These analyses revealed a noveldirect crosstalk between transcription factors at sharedgenomic target sites. Here, we review recent advances inthe understanding of chromatin remodeling events associ-ated with adipocyte differentiation and discuss how thesefindings have provided novel insights into how adipogenictranscription factor networks orchestrate adipocyte differ-entiation.

Early adipocyte differentiationA limited number of signaling pathways including bonemorphogenic protein (BMP) and growth differentiationfactor (GDF) signaling have been shown to regulate thecommitment of mesenchymal stem cells to the adipocytelineage and the differentiation of adipocyte precursor cellsin vivo [4]. Thus, most of our knowledge about the molecu-lar mechanisms that control adipocyte differentiationcomes from studies of in vitro preadipocyte cell-line modelsor primary cell cultures. These model cell systems can beinduced to differentiate into mature adipocytes using astandard hormonal cocktail containing insulin, a cAMP-elevating agent, glucocorticoids (e.g. dexamethasone), andgrowth factors (e.g. from serum in the differentiation me-dia) [5]. A multitude of studies using these in vitro modelsystems have revealed the core transcriptional cascade (i.e.the sequential induction of key adipogenic transcriptionfactors) that controls adipocyte differentiation [3,5,6].However, the precise molecular mechanisms throughwhich these various transcription factors control differentdevelopmental stages of differentiation are only beginning

PPRE (PPAR response element): a DR1 element where PPAR can bind directly

and activate transcription.

em.2011.10.001 Trends in Endocrinology and Metabolism, February 2012, Vol. 23, No. 2

Adipogenesis

PPARγ

C/EBPβ

C/EBPδ

GR

STAT5A/B

KLF5

KLF4

CREB

Krox20

C/EBPα

KLF15

GATA2/3

CHOP10

PPARγ ligand

Adipocytephenotype

KLF2

Insulin

Glucocorticoids

cAMP

FBS

FOXO1

LXRSREBP-1c

RORα

RARTR

Wnt10b β-cateninCOUP-TFII

IRF4

TRENDS in Endocrinology & Metabolism

Figure 1. Transcriptional cascade controlling adipocyte differentiation. Early adipogenic transcription factors (green) are activated directly by the adipogenic hormone

cocktail consisting of glucocorticoids, insulin, a cAMP-elevating agent, and fetal bovine serum (FBS). This first wave of adipogenic factors directly induces the late

adipogenic factors (blue), which coordinately induce genes linked to the adipocyte phenotype. Anti-adipogenic factors (orange) act on various adipogenic factors to inhibit

adipocyte differentiation. Solid lines indicate regulation of gene expression, dashed lines indicate regulation of activity, and solid lines with arrowheads at both ends

indicate that transcription factors are found in the same complex. Abbreviations: C/EBP homologous protein, CHOP10; retinoic acid receptor, RAR; RAR-related orphan

receptor a, RORa; forkhead box O1, FOXO1; thyroid hormone receptor, TR; liver X receptor, LXR; chicken ovalbumin upstream promoter transcription factor II, COUP-TFII.

Review Trends in Endocrinology and Metabolism February 2012, Vol. 23, No. 2

to be elucidated. Recently, global approaches haveemerged as powerful tools to study transcription factoraction at a genome-wide level. These studies have greatlyincreased our understanding of the molecular mecha-nisms through which adipogenic transcription factorsregulate adipocyte differentiation at different develop-mental stages. Importantly, extensive crosstalk between

Box 1. Pro-adipogenic transcription factors

C/EBPs (CCAAT/enhancer-binding proteins): these belong to the

basic-leucine zipper (bZIP) family of transcription factors and exist in

six different isoforms, four of which [i.e. a, b, d and z (CHOP-10)] have

been demonstrated to play a role in adipocyte differentiation [57].

C/EBP family members bind as homo- or heterodimers to DNA [57].

CREB (cAMP response element binding protein): belongs to the

bZIP transcription factor family and binds to DNA as a homodimer or

as a heterodimer with other members of the family. Is activated by

phosphorylation upon adipogenic hormone stimulation and is

required for differentiation [58,59].

GR (glucocorticoid receptor): member of the steroid receptor family

of nuclear receptors. Binds as a homodimer to DNA. An important

regulator of early adipogenesis [18,21].

KLFs (Kruppel-like factors): zinc-finger proteins containing three

zinc-fingers separated by conserved spacers of seven amino acids

(TGEKP(Y/F)X) [60–62]. This family of transcription factors contains

both repressors and activators of transcription [62]. Seven of the 17

known KLF proteins have been shown to play a role in different

phases of adipocyte differentiation (i.e. KLF2 [11] and -3 [63] inhibit

differentiation, whereas KLF4 [12], -5 [64], -6 [65], -9 [66], and -15 [67]

stimulate differentiation) (Figure 1).

Krox20 (early growth response 2, Egr2): a member of the Egr family

of zinc finger transcription factors. Is required in the early phase of

adipocyte differentiation [10].

transcription factors is emerging as an important mecha-nism through which various signaling pathways are inte-grated at the genomic level.

Adipogenic signaling pathways converge at

transcription factor hotspots during early differentiation

Addition of the standard hormone cocktail of adipogenic

LXRs (liver X receptors): members of the nuclear receptor super-

family. These exist in two isoforms, LXRa and -b, that bind to DNA as

heterodimers with RXR. Regulate lipid metabolism in mature

adipocytes [68–70].

PPARg (peroxisome proliferator-activated receptor g): member of

the nuclear receptor superfamily. PPARg exists in two different

isoforms (PPARg1 and PPARg2) that bind to DNA together with the

retinoic X receptor (RXR) and are activated by fatty acids and their

derivatives. Play a pivotal role in adipocyte differentiation both in vivo

and in vitro [25,26].

SREBP-1c (sterol regulatory element-binding protein-1c): belongs

to the basic helix-loop-helix leucine zipper (bHLH-Zip) family of

transcription factors. Promotes adipocyte differentiation [71].

STAT5A/B (signal transducer and activator of transcription 5A/B):

belong to the STAT family of transcription factors and bind to DNA as

homo- or heterodimers. STAT5A is particularly important for efficient

adipocyte differentiation whereas STAT5B plays a more auxiliary role

[20,72,73].

TR (thyroid hormone receptor): member of the nuclear receptor

superfamily. Binds to DNA either as a homodimer or as a heterodimer

with RXR. Plays a role in adipocyte differentiation both in vitro and in

vivo [74,75].

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Box 2. Chromatin remodeling and associated techniques

Chromatin is inherently repressive of transcription due to the limited

accessibility of the DNA. Transcription factor binding to DNA is

therefore often associated with remodeling of the chromatin template

to increase accessibility of DNA. This increase in chromatin accessi-

bility associated with protein binding can be detected as an increase

in the local susceptibility to nucleolytic attack. In addition to

disrupting the local nucleosome structure, protein binding is usually

also associated with ‘activating’ histone marks on nucleosomes in the

vicinity. The advent of deep sequencing platforms in recent years has

resulted in an almost explosive increase in the number of genome-

wide maps of different histone modifications in various cell types.

This has also resulted in the identification of multiple distinct

chromatin states with different functions [76–79].

DNase I hypersensitive (DHS) regions are ‘sites of action’ in the

genome (e.g. active promoters and enhancers) that have an increased

DNA accessibility compared to surrounding sequences due to local

remodeling of the chromatin structure. DNase I analysis involves

limited DNase digestion of isolated nuclei followed by analysis of

digested DNA by southern blotting, qPCR, or more recently by deep

sequencing (DHS-seq), which allows unbiased genome-wide map-

ping of all DHS sites in a given cell type [80–83].

Formaldehyde-assisted isolation of regulatory elements (FAIRE) is an

alternative and simpler method that can be used to study the chromatin

structure within cells [84,85]. This method involves isolation of

nucleosome-depleted chromatin regions using sequential phenol–

chloroform extractions. FAIRE in combination with deep sequencing

(FAIRE-seq) and DHS-seq identify distinct but overlapping profiles of

open chromatin [86]; however, the structural and functional differences

between FAIRE sites and DHS sites are currently unknown.

Chromatin immunoprecipitation (ChIP) can be used to identify

interactions between a protein of interest (e.g. a transcription factor)

and chromatin or to study the location of specific histone modifica-

tions (e.g. H3K27ac). This method involves protein–DNA crosslinking

(typically accomplished by fixing the cells in a formaldehyde solution)

followed by DNA fragmentation and immunoprecipitation of the

protein of interest using specific antibodies. DNA from the immuno-

precipitated protein–DNA complexes can be purified and analyzed by

various methods. Real-time qPCR can be used to analyze ChIP’ed

DNA in a gene-by-gene manner, whereas global platforms such as

microarray (ChIP-chip) and more recently deep sequencing (ChIP-seq)

allow unbiased analyses of the ChIP’ed DNA resulting in genome-

wide binding profiles of the protein of interest.

Review Trends in Endocrinology and Metabolism February 2012, Vol. 23, No. 2

inducers to preadipocyte cell cultures (e.g. 3T3-L1 and 3T3-F442A cells) leads to the activation of multiple early pro-adipogenic and repression of anti-adipogenic transcriptionfactors (Figure 1). C/EBPb and d have been widely recog-nized for a long time as key early pro-adipogenic transcrip-tion factors that are involved in initiating the adipogenicprogram [7–9]. More recently, Krox20 [10] and members ofthe KLF family [11,12] have been demonstrated to beimportant early regulators of adipogenesis as well throughtheir ability to induce expression of C/EBPb and PPARg

(Figure 1). Glucocorticoids are important adipogenic indu-cers at least in vitro [13] and alterations in glucocorticoidsignaling in vivo are associated with changes in fat massand adipocyte metabolism [14–17]. However, the preciserole of the glucocorticoid receptor (GR) during the adipo-cyte differentiation process has remained largely unex-plored. Lazar and coworkers recently showed that GR isindeed necessary for adipocyte differentiation in vitro [18].Using ChIP-seq (Box 2) to map transcription factor bindingsites at a genome-wide scale, they demonstrated that GRbinds transiently and cooperatively with C/EBPb to sever-al thousand genomic regions six hours after induction ofdifferentiation. These regions are transiently marked byH3K9ac early in differentiation, possess enhancer-likefeatures such as binding of the coactivators p300 (histoneacetyltransferase) and Med1 (Mediator subunit), and func-tion as enhancers in reporter assays, indicating that theyare in fact transiently active enhancer regions. This sug-gests that GR and C/EBPb drive adipocyte differentiationin response to external adipogenic stimuli through cooper-ative binding to transient enhancer regions.

STAT5A is activated by growth hormone-induced tyro-sine phosphorylation within few hours after stimulationwith adipogenic inducers, and it has been reported to berequired for efficient adipocyte differentiation [19,20]. Itwas also reported that STAT5A forms a complex with GRin the nucleus in the early phase of differentiation [19],suggesting that STAT5A may be recruited to GR-associat-ed putative enhancers. Using ChIP-seq analysis, we haverecently shown that STAT5A and GR do in fact co-occupy

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multiple chromatin regions early in adipogenesis [21].Remarkably, we identified nearly 1000 transcription factorhotspots (see Glossary), where STAT5A, GR, C/EBPb/d,and the nuclear receptor RXR bind cooperatively fourhours after induction of differentiation [21]. Binding ofmultiple transcription factors at such regions was shownto be functionally correlated with expression of early in-duced genes, suggesting that these regions are likely torepresent important regulatory nodes (i.e. enhancers)where multiple adipogenic signals converge early in adi-pogenesis (Figure 2). Most of the identified hotspots have atransiently open chromatin configuration suggesting thatthey are specifically active during the early phase of dif-ferentiation, where they regulate cell-cycle progression aswell as late-acting transcription factors (e.g. PPARg)(Figure 3, left). Taken together, these findings indicatethe existence of a previously unrecognized high degree ofcrosstalk between multiple early adipogenic transcriptionfactors directly on the chromatin template, which mayserve to integrate various adipogenic signals and therebyfine-tune the transcriptional program accordingly.

C/EBPb is an important early transcriptional coordinator

C/EBPb is known to be required for adipocyte differentia-tion both in vivo and in vitro [7,8]. Lane and coworkershave previously shown that C/EBPb is highly induced andphosphorylated by MAPK within a few hours after adipo-genic hormone stimulation; however, subsequent phos-phorylation of C/EBPb by GSK3b after a 14 h time-lagdramatically increases the affinity of C/EBPb for nakedDNA [9]. Interestingly, using ChIP (Box 2) to study inter-actions between C/EBPb and selected chromatin regionswithin cells, Imbalzano and colleagues showed that bothC/EBPb and -d are already able to associate with DNA in achromatin context within the first few hours after theinduction of differentiation [22], in other words before theirreported phosphorylation by GSK3b. Genome-wide profil-ing of C/EBPb and -d binding during the first hours ofadipogenesis have extended these findings and shown thatboth of these C/EBPs associate with several thousand

RNAPIIIIIIIIIIRNAPIIRNAPIIRNAPIIRNAPIIRNAPIRNAPIRNAPIRNAPIRNAPIRNAPIRNAPIRNAPIRNAPIRNAPIRNAPIRNAPIRNAPIRNAPIRNAPIRNAPRNAPRNAPRNAPRNAPRNAPRNAPRNAPRNAPRNAPRNAPRNAPRNAPRNAPRNAPRNAPRNAPRNAPNAPNAPNAPNAPNAPNAPNAPNAPNAPNAPNAPNAP

PIC

GRGRC/EBPβ

C/EBPδSTAT5A RXR:?

Hotspot Promoter

Adipogenic signals

TRENDS in Endocrinology & Metabolism

Figure 2. Model of integration of signaling pathways at transcription factor hotspots. Multiple early adipogenic signals activate a variety of adipogenic transcription factors,

which associate with transcription factor hotspots. This allows integration of adipogenic signals on the chromatin template. Transcription factors associated with such

hotspots coordinate the assembly of a functional enhanceosome and activate transcription of target genes. Abbreviations: pre-initiation complex, PIC; RNA polymerase II,

RNAPII.

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chromatin regions at these early time-points as describedabove [18,21]. This suggests that particular features ofchromatin (e.g. histone modifications) may assist bindingof C/EBPs to DNA in a chromatin context within cellsduring the first hours of adipogenesis. Remarkably, profil-ing of C/EBPb binding in preadipocytes demonstrates thatC/EBPb actually associates with multiple chromatinregions even before the induction of differentiation,suggesting that C/EBPb may also play a role in the

1. Wave

2. Wave

PreadipocyteC/EBPβ/δ

GR

STAT5A

Enhancer

Earlyenhanceosomeeen

Enhancer

Earlyenhanceosomeeen

Enhancer

Lateenhanceosome

Figure 3. Model for establishment of early and late enhanceosomes. (Left) The first wa

enhanceosomes at transcription factor hotspots. This is associated with a transiently op

induce chromatin remodeling and form early enhanceosomes at transcription factors

adipogenic factors, which coordinate the assembly of late enhanceosomes at the sam

remodeling and the assembly of late enhanceosomes at late hotspots. TFs, transcriptio

preadipocyte state [18,21]. Interestingly, C/EBPb seemsto mark a subset of early transcription factor hotspotsbefore the initiation of differentiation, and before thebinding of other transcription factors and full-scale chro-matin remodeling of these hotspots [21]. By doing so it mayact as a pioneering factor for the formation of these hot-spots as well as for the binding of early adipogenic tran-scription factors to other sites. Consistent with this,knockdown of C/EBPb in preadipocytes interferes with

4-24 hours

Day 2-6

PPARγ:RXR

C/EBPα

Cell cycleRibosomeTranslationCellular growthGlucose catabolismLate-acting TFs

Lipid metabolismGlucose metabolismLipid bindingMitochondriaTransport

Protein X/Y

Enhancer

Lateenhanceosome

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ve of adipogenic transcription factors coordinates the transient formation of early

en chromatin structure. (Middle) Early transcription factors recruit coregulators to

hotspots. These putative enhancer regions are subsequently ‘inherited’ by late

e hotspots. (Right) Late adipogenic transcription factors direct de novo chromatin

n factors.

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the recruitment of GR, STAT5A and RXR to early targetsites [21]. Taken together, these studies indicate thatC/EBPb associates with specific putative enhancer regionsboth in the preadipocyte state and in the initial stage ofdifferentiation and that C/EBPb, in addition to its well-established role as activator of PPARg and C/EBPa ex-pression, plays an important role in the establishment ofthe early enhanceosomes.

Early hotspots are ‘inherited’ by late adipogenic

transcription factors

Imbalzano and coworkers have previously demonstratedthat the PPARg2 promoter is remodeled within a few hoursafter induction of adipocyte differentiation using the DN-ase I hypersensitive (DHS) assay (Box 2) [23]. This remo-deling event was shown to be dependent on C/EBPb and c-Fos as well as PKA signaling. Interestingly, PPARg2 is notexpressed until later in adipogenesis (days 1–2), suggest-ing that remodeling of the PPARg2 promoter takes placebefore transcriptional activation of this gene [23,24].

We have recently used DHS-seq (Box 2) to map chroma-tin remodeling events associated with 3T3-L1 adipocytedifferentiation on a global scale [21]. Remarkably, thechromatin landscape is extensively remodeled duringthe first hours after initiation of the adipogenic program,and this coincides with the recruitment of multiple earlytranscription factors (e.g. C/EBPs, GR, STAT5A, and RXR).Chromatin remodeling events generally correlate wellwith transcriptional changes of nearby genes; however,we also identified numerous loci where chromatin remo-deling precedes the transcriptional activation of nearbygenes, as previously reported for PPARg2 [23,24]. Thesefindings suggest that early chromatin remodeling primesgenomic regions for late adipogenic signals (i.e. late-actingtranscription factors, coregulators or post-translationalmodifications). Our finding that C/EBPb binds to manyPPARg binding sites before PPARg suggests that pioneer-ing complexes containing C/EBPb remodel the chromatinstructure at putative enhancers to assist subsequent bind-ing of PPARg in late adipogenesis [21]. This would indicatethat early enhanceosomes directly affect late transcrip-tional events by regulating chromatin accessibility(Figure 3, middle).

The late adipogenic programExtensive crosstalk between the key late adipogenic

transcription factors PPARg and C/EBPa

The first wave of early transcription factors describedabove directly activates expression of the second wave ofadipogenic factors, of which the most important players arePPARg and C/EBPa (Figure 1). Interestingly, multipleearly transcription factor hotspots are found in the vicinityof the PPARg gene, suggesting that several adipogenicsignaling pathways orchestrate the transcriptional induc-tion of this important nuclear receptor during adipogenesis[21]. PPARg has been known for a long time to be essentialfor adipogenesis both in vitro and in vivo [25,26]; however,the molecular mechanisms underlying PPARg action dur-ing adipocyte differentiation have only been studied for alimited number of target genes [27–32]. Interestingly,genome-wide profiling of PPARg throughout adipocyte

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differentiation using ChIP-seq [33] and in mature adipo-cytes by ChIP-chip [34,35], ChIP-PET [36] and ChIP-seq[37,38] demonstrated that PPARg binding is specificallyenriched in the vicinity of most induced adipocyte genes(including genes linked to lipid and glucose metabolism).These findings indicate that PPARg, in addition to being akey regulator of adipogenesis per se, is also a direct activa-tor of the entire genomic reprogramming important foracquisition of the mature adipocyte phenotype. Interest-ingly, de novo motif analyses revealed, in addition to theDR-1-type PPAR response element (PPRE) (see Glossary),a striking enrichment of the C/EBP binding motif atPPARg binding sites [33,34]. C/EBPa ChIP-chip [34] andChIP-seq [38] confirmed extensive colocalization (35–60%)of PPARg and C/EBPa, suggesting that these two keyadipogenic factors cooperate directly on the chromatintemplate to induce the adipocyte gene program. Intrigu-ingly, one of the early adipogenic C/EBP family members,C/EBPb, is also expressed in mature adipocytes, althoughat a lower level than during early adipocyte development,and this C/EBP subtype showed a similar binding patternto C/EBPa in the mature adipocyte [34]. Depletion ofC/EBPa/b or PPARg in mature adipocytes demonstratedthat both PPARg and C/EBPs are required for maintaininghigh expression of several adipocyte genes in the vicinity ofshared PPARg–C/EBPa binding sites [34]. Consistent withthese findings, shared binding sites were enriched in thevicinity of highly induced, compared to modestly induced,adipocyte genes, suggesting that PPARg–C/EBPa coopera-tivity results in a massive induction of nearby target genes,also on a global scale [39]. Taken together, these studiesindicate extensive direct crosstalk between the two keyadipogenic transcription factors, PPARg and C/EBPa, onchromatin. Interestingly, however, depletion of eitherPPARg or C/EBPa does not affect chromatin binding ofthe other factor to the shared sites that were tested [34],suggesting a limited mutual dependence between the twofactors in binding to chromatin in the mature adipocyte.Cumulatively, these results suggest that PPARg andC/EBPa are part of a late adipogenic transcription factornetwork that participates in the assembly of cofactor com-plexes at these shared sites to cooperatively induce tran-scriptional activation of adipocyte genes (Figure 3, right).

Conservation of PPARg and C/EBPa binding events in

adipocytes

Comparative genome-wide profiling of PPARg andC/EBPa binding in mouse and human adipocytes revealedlimited conservation of binding events between the twospecies (approximately 10–20% of mouse binding sites areconserved in human adipocytes) [37,38,40]. This low in-terspecies conservation of binding events is consistentwith findings for other factors in other cell types [41].Interestingly, however, there is extensive colocalizationof PPARg and C/EBPa both in mouse and human adipo-cytes, and sites that are co-occupied by these transcriptionfactors in mouse are more likely to be retained in humanadipocytes compared to sites where only one of the factorsbind [38]. These findings showed that the extensive co-occupancy of PPARg and C/EBPa at putative enhancerregions is conserved between mouse and human, and

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suggest that co-occupancy between these factors is func-tionally important for adipocyte differentiation and func-tion.

Comparative analyses focusing on gene regulation fur-thermore suggest that the genes associated with PPARg

binding are significantly more conserved between mouseand human than the actual binding events, indicating thatPPARg targets the same gene programs controlling lipidand glucose metabolism in both species in part throughnon-conserved binding sites [37,40]. These findings areconsistent with a high degree of turnover of PPARg bindingsites (i.e. a new binding event replaces an old one duringthe course of evolution) in the vicinity of adipocyte genes[38].

Other late-acting regulators of adipogenesis

In addition to PPARg and C/EBPa, several other transcrip-tion factors, including KLF15 and sterol regulatory ele-ment-binding protein (SREBP)-1c, have been shown toregulate the late phase of adipogenesis (Figure 1; Box1). Even though no genome-wide binding profiles of anyof these late transcription factors have yet been reported, itis highly likely that they bind to genomic chromatinregions that are hypersensitive to DNase I and that aremodified by activating histone marks – as is the case forPPARg and C/EBPa. In addition to transcription-activat-ing factors, repressive transcription factors are also likelyto bind to remodeled sites. Interestingly, Rosen and col-leagues recently employed DHS site analysis in combina-tion with qPCR to identify novel late transcriptionalregulators of adipocyte differentiation [42]. The authorsdetermined the DNase I susceptibility of 268 conservedregions within 50 kb of 27 known adipocyte genes andidentified 32 differentiation-dependent DHS sites (i.e.regions increasing in accessibility during adipogenesis).Based on motif analysis of the sequences in these regions,they identified interferon regulatory factors (IRFs) as novelnegative regulators of differentiation that may function asa ‘brake’ on adipogenesis through binding to DHS sites inthe later stages of differentiation. Consistently, in re-sponse to a high-fat diet, adipocyte-specific IRF4 knockoutmice have significantly increased fat mass compared towild-type mice as a result of adipocyte hypertrophy, andthis is probably related to the function of IRF4 as arepressor and activator of lipogenesis and lipolysis, respec-tively [43]. Importantly, these findings also indicate thattranscriptional repressors bind to DHS sites, as do acti-vating transcription factors. This further highlights thepotential of DHS site analysis to identify ‘sites of action’ inthe genome without any a priori knowledge of the tran-scriptional events taking place at these sites.

Crosstalk between late adipogenic transcription factorsand activating histone marksMultiple epigenetic modifiers have been shown to regulateadipocyte differentiation at various stages of differentia-tion [44–47]. Even though it is currently unknown to whatextent these effects on adipocyte differentiation are medi-ated entirely through the catalytic domain of the coregu-lators, these findings point to an important role ofepigenetic modifications in regulating adipogenesis. For

extensive reviews on this topic we refer to recent reviews[48,49].

We will focus here on recent studies investigating globalassociations between epigenetic marks and the function ofadipogenic transcription factors. Global analyses of theactivating histone marks H3K27ac and H3K9ac duringadipocyte differentiation and in mature adipocytes, respec-tively, revealed that both marks are highly enriched atPPARg binding sites [34,37,50]. Interestingly, ectopic ex-pression of PPARg in preadipocytes increases H3K9aclevels at a few selected PPARg binding sites [50]. Similarly,acute ectopic expression of PPARg2 in NIH-3T3 cells in-duced H3 and H4 acetylation at an enhancer in the vicinityof the well-characterized PPARg target gene aP2 [51].These findings indicate that PPARg can induce depositionat target sites of at least some activating histone marks.However, most PPARg binding sites in mature adipocytesare actually located in regions that are already enriched inH3K27ac in the preadipocyte state, well before PPARg

induction [37]. This suggests that this mark may be im-portant for directing PPARg binding. Importantly, howev-er, PPARg binding sites that show �fivefold increasedlevels of H3K27ac during differentiation are more highlycorrelated with induced genes than PPARg sites that arealready modified before induction of differentiation [37].This suggests that, even though most PPARg binding sitesin mature adipocytes are marked by H3K27ac already inthe preadipocyte state, the PPARg binding events thatdirect acetylation of H3K27 (and presumably of otherhistone tail residues such as H3K9) during differentiationmay be functionally more important for the regulation ofadipocyte genes.

Sakai and coworkers recently demonstrated thatPPARg directly induces expression of Setd8, a SET-do-main-containing histone methyl transferase (HMT), whichis required for adipogenesis and is expressed at elevatedlevels in two mouse models of obesity [35]. Setd8 mono-methylates H4K20 at multiple adipocyte genes includingthe PPARg gene itself, and changes in the levels of thishistone mark were found to correlate with changes in geneexpression during differentiation. Based on these findings,the authors propose that adipocyte differentiation is con-trolled by a positive feedback loop in which PPARg inducesexpression of Setd8, which in turn activates PPARg ex-pression by methylating H4K20.

Taken together, these genome-wide studies indicateimportant functions of epigenetic marks in the transcrip-tional reprogramming of gene expression during adipogen-esis. Detailed molecular studies are necessary to delineatethe precise molecular mechanisms through which differenthistone marks regulate the binding and activity of adipo-genic transcription factors.

Transcription factor hotspots in other model systemsThe recent explosion in genome-wide maps of transcriptionfactor binding has revealed several examples where differ-ent transcription factors associate with the same genomicregions. Pu and colleagues recently reported that multiplecardiac transcription factors co-occupy several thousandgenomic regions in cardiomyocytes [52]. Interestingly, asubset of these putative enhancer regions were found to be

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active in transient reporter assays both in vivo and in vitro,suggesting that they represent functionally importantregulatory sites involved in coordinating cardiac geneexpression. Multiple signaling pathways have also beenfound to converge at specific genomic regions termed mul-tiple transcription factor-binding loci (MTL) in embryonicstem cells [53]. Several transcription factors seem to coor-dinate the assembly of enhanceosomes at these loci andregulate stem cell-specific gene expression. Taken togeth-er, these studies, and the recent studies on adipocytedifferentiation described above, indicate that transcriptionfactors do not work in isolated transcriptional programsbut instead function as part of transcription factor net-works that integrate multiple signaling pathways at spe-cific genomic regions. This extensive co-occupancy oftranscription factors in the genome may represent animportant avenue to achieve cell type-specific transcrip-tional responses to activation of a particular transcriptionfactor [50,54].

The modENCODE consortium, which has set out tocharacterize the genome of the model organisms Drosophi-la melanogaster and Caenorhabditis elegans, has recentlyidentified genomic regions of approximately 400 bp termedhigh occupancy target (HOT) regions (see Glossary) that, ata whole-organism level, are occupied by at least 15 and 8transcription factors, in C. elegans and D. melanogaster,respectively [55,56]. Notably, genes containing HOTregions in their promoter are ubiquitously expressed andhave an essential developmental function, whereas expres-sion of genes associated with ‘factor-specific’ binding sites(occupied by a subset of 1–4 of the investigated transcrip-tion factors) is generally restricted to a few tissues [55]. Itshould be noted that these results were obtained withwhole animal samples and that the HOT regions thereforeprobably represent regulatory regions that are targeted bydifferent factors in different cell types. By contrast, hot-spots and MTL are genomic regions co-occupied by multi-ple transcription factors in the same cell at a specific timepoint. However, it is likely that many of the HOT regionswill also turn out to be hotspots at a cellular level.

Concluding remarksThe adipocyte differentiation systems constitute excellentmodel systems for studying the establishment of enhan-ceosomes during development. The 3T3-L1 preadipocytecell line is particular well-suited due to its highly synchro-nous and efficient differentiation into mature adipocytes inresponse to adipogenic stimuli. Given the extent of tran-scription factor co-occupancy described in this cell line,cooperation between multiple transcription factors in co-ordinating reprogramming of gene expression associatedwith adipocyte differentiation is likely to be functionallyhighly important. Thus, future studies should aim at iden-tifying the molecular mechanisms underlying the estab-lishment of enhanceosomes at adipogenic transcriptionfactor hotspots to obtain a better understanding of thetranscriptional control of adipogenesis and of the molecu-lar mechanisms regulating enhancer activity in general.Specifically, it will be interesting to elucidate the sequenceof transcription factor and coregulator binding to hotspotsto identify the factor(s) that initiate formation of active

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enhanceosomes at these sites. It will also be of greatimportance to determine the role of chromatin structureand histone modifications in the establishment of activeenhanceosomes. Finally, little is known about which othertranscription factors co-occupy chromatin together withPPARg and C/EBPa in the late adipogenic wave of tran-scription factors. We predict that these transcription fac-tors, similar to the first wave of adipogenic transcriptionfactors, cooperate in hotspots, and we propose that theaction of each individual factor is highly dependent oncooperating factors. Given the clinical importance ofPPARg in the treatment of insulin resistance, it will beinteresting to identify these cooperating factors and deter-mine whether modulation of their activities can be used tofine-tune PPARg activity in adipocytes as well as in othercell types.

AcknowledgmentsWe thank members of the Mandrup laboratory for comments to themanuscript. This work was supported by the Danish Natural ScienceResearch Council and the Novo Nordisk Foundation.

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