REVIEW
Molecular mechanism of chicken ovalbumin upstream
promoter-transcription factor (COUP-TF) actions
Joo-In Park1,3, Sophia Y. Tsai1,2 and Ming-Jer Tsai1,2
1Department of Molecular and Cellular Biology, 2Program of Developmental Biology, Baylor Collegeof Medicine, One Baylor Plaza, Houston, TX, USA(Received for publication on February 27, 2003)

Abstract. Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) are one of themost characterized orphan receptors of the steroid/thyroid hormone receptor superfamily. COUP-TFs
play important roles in the regulation of organogenesis, neurogenesis, and cellular differentiation during embryonic development. COUP-TFs were generally considered to be repressors of transcription,
however, there are growing evidences that COUP-TFs can function as transcription activators. Herewe will review the molecular mechanism of COUP-TFs as repressors and activators. Also, we will
review the known biological function of COUP-TFI during development and differentiation. (Keio J
Med 52 (3): 174–181, September 2003)
Key words: COUP-TF, transcription repressors, transcription activators, neurogenesis, organogenesis

Introduction
The nuclear receptor superfamily comprises a large
group of ligand-activated transcription factors, including receptors for steroids, retinoids, and thyroid hormones.1 Also included are a large number of structurally and functionally related transcription regulatory
proteins termed orphan receptors, for which speciﬁc
ligands have not been deﬁned. Chicken ovalbumin
upstream promoter-transcription factor (COUP-TF) is
one of the most extensively studied orphan receptors.
Two genes called COUP-TFI (also termed EAR3) and
COUP-TFII (also termed ARP-1) have been identiﬁed
in mammals. These are closely related transcription
factors that are expressed in many places and are
involved in the regulation of several important biological processes, such as neurogenesis, organogenesis, cellfate determination, and metabolic homeostasis.1–8 Bothgenes show an exceptional homology and overlapping
expression patterns, suggesting that they may serve redundant functions. However, each factor possesses its
own distinct expression proﬁle during development.8
This review will focus on the advances that have been

made towards understanding the molecular mechanismof COUP-TF actions and biological function of COUPTFI during development and differentiation.
Molecular Mechanism of COUP-TF Actions
Repressor
COUP-TFs homodimerize or heterodimerize withretinoid X receptor (RXR) and a few other nuclear
receptors and bind to a variety of response elementsthat contain imperfect AGGTCA direct or inverted
repeats with various spacings.9,10 Although COUP-TF
was originally characterized as a transcriptional activator of the chicken ovalbumin gene,11 COUP-TFs are
generally considered to be repressors of transcription
for other nuclear hormone receptors such as retinoic
acid receptor (RAR), thyroid hormone receptor (TR),vitamin D receptor (VDR), peroxisome proliferatoractivated receptor (PPAR), and hepatocyte nuclear
factor 4 (HNF4).10,12,13 There are four mechanismsthat account for the repressive effects of COUP-TFs(Fig. 1).

Presented at the 1295th Meeting of the Keio Medical Society in Tokyo, November 25, 2002.
3Present afﬁliation: Department of Biochemistry, Dong-A University College of Medicine
Reprint requests to: Dr. Ming-Jer Tsai, Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston 77030, USA, e-mail: mtsai@bcm.tmc.edu
174

Keio J Med 2003; 52 (3): 174–181

Fig. 1 Molecular mechanism of COUP-TFs as repressors. DR: direct repeats, HRE: hormone response element, MHC: major histocompatibility complex, ApoAI: apolipoprotein AI.

The competition for occupancy of the binding sites:
COUP-TFs have been shown to bind to a variety of direct repeats, including DR1, DR3, DR4, and DR5 of
the AGGTCA motif, which are response elements of
PPAR, VDR, TR, and RAR, respectively.14–16 It has
been demonstrated that COUP-TFs repress the hormonal induction of target genes by PPAR, VDR, TR,
and RAR in transient transfection assays through directcompetition with VDR, TR, and RAR for the available binding sites.14,15 The repression is released byincreasing the expression of RAR, which suggests that
COUP-TFs negatively regulate retinoid responses bycompeting for binding to the retinoid response elements of the target reporter. COUP-TFs have also
been demonstrated to interfere with the transactivation of TR, RXR, and PPAR through a similar mechanism.14–16 In addition, COUP-TFs have been shownto inhibit the transactivation of steroidogenic factor 1(SF1) and HNF4 due to mutually exclusive binding to
the promoter of many genes.17–19 Finally, COUP-TF
has been shown to antagonize ER activation of the lactoferrin and oxytocin promoters by binding to a binding site that overlaps with the estrogen response element.17,20
The competition for RXR: It is well known that
RXR is a universal heterodimeric partner of RAR, TR,
VDR, PPAR, and other orphan receptors.6 Homodimers of RAR, TR, VDR, and PPAR either bindpoorly or not at all to their cognate response elements.
The heterodimeric receptors can bind to the cognate
response elements with high afﬁnity through association
with RXR, thus, enhance the transactivation potentialof this group of receptors. Because the direct repeat

175

recognition sequence is asymmetric, it has been shownthat RXR occupies the 5 0 half-site while the otherpartner binds the downstream 3 0 half-site, which confers the hormone responsiveness.6 RXR can also bindto DR1 elements as a homodimer and as a heterodimer
with RAR and PPAR. The RXR homodimer is anactivator that responds to 9-cis-retinoic acid. RXR/
PPAR heterodimers respond to both 9-cis-retinoic acidand PPAR-speciﬁc ligands. However, RAR/RXR heterodimers, in which RAR binds to the 5 0 -half-site of
DR1 element, are transactivationally inactive. It has
been shown that RAR and TR bind to a corepressor[either silencing mediator for retinoid and thyroid hormone receptors (SMRT) or nuclear receptor corepressor (N-CoR)] in the absence of hormone.21,22
Binding of these corepressors is necessary for receptors
to silence the promoter activity. Upon binding hormone, the corepressor is released, and, thus, silencing
activity of receptors is abolished. However, when RAR/
RXR binds to DR1, the retinoic acid ligand is not able
to release the corepressor from RAR; therefore, RAR/
RXR heterodimer is not able to activate the DR1 reporter.23
Although COUP-TFs exist in solution as homodimers and fail to form stable heterodimers with RXR
in coimmunoprecipitation assays,10,24 they readily form
DNA-binding heterodimers with RXR.10,15,16 Therefore, COUP-TFs are able to sequester the common
heterodimerization partner RXR and reduce the available concentrations of RXR.10,14–16,25 The loss of
RXR indirectly decreases the DNA-binding afﬁnity ofTR, VDR, RAR, and PPAR and thereby interfereswith the potential of this subgroup of receptors totransactivate their target genes.14,15,26 This notion isfurther veriﬁed by the relief of COUP-TF inhibition
when RXR is overexpressed.14 In addition, it has been
demonstrated that COUP-TFs form heterodimers with
TR and RAR and disrupt their functions.13,27,28 Thus,
the ability of COUP-TFs to form heterodimers with
RXR, TR, and RAR may contribute signiﬁcantly to thenegative regulatory role of COUP-TFs in modulating
hormone responsiveness of a large number of receptorsof the TR and RAR subfamily.6
Active repression: Similar to unliganded nuclear hormone receptors, COUP-TFs have been shown to repress basal transcriptional activity of a number of thymidine kinase reporters containing DR3, DR4, or DR5
hormone response elements.10,14 This silencing of basaltranscriptional activity is response element speciﬁc andis unlikely due to squelching of TFIIB, which interacts
with COUP-TFs or other general transcription factors,
since reporter genes lacking COUP-TF binding sites
show little COUP-TF-mediated repression.13 Subsequently, it has been demonstrated that COUP-TFs,

176

similar to TR and RAR, possess an active silencing
domain within the C terminus of the putative ligand
binding domain (LBD). This repressor domain can betransferred to a heterologous GAL4 DNA binding domain (DBD) and can be shown to retain its ability torepress basal transcriptional activity.
In addition, Leng et al. have shown that COUP-TFs
can function as an active repressor in a dose-dependent
manner to inhibit transactivation mediated by acidic(Gal4-RII), glutamine-rich (Gal4-ftzQ), proline-rich(Gal4-CTF1P), and Ser/Thr-rich (Gal4-ZenST) transactivators.13 The active repressor function of COUPTFs is position independent, i.e. the binding sites of
COUP-TFs can either be localized upstream of theactivator binding site or downstream of the reporter
gene without signiﬁcantly affecting the active repression. The fact that COUP-TFs can repress such diverse
groups of transactivators suggests that it is unlikely due
to COUP-TFs directly quenching these transactivators
or interfering with their interaction with their respective targets. Rather, it is likely that COUP-TFs interact
with a common target, a putative corepressor(s) thatmediates their repression. Shibata et al. demonstrated
that corepressors are involved in COUP-TFI-mediatedgene silencing, and that both N-CoR and SMRT canfunction as corepressors for COUP-TFI in mammaliancells.29 Therefore, COUP-TFI can function as a repressor in vivo by utilizing corepressors that are common for members of the TR and RAR subfamily. In
addition, it was discovered that in adenovirus type 12transformed cells, COUP-TF associates with histone
deacetylase through its C-terminal repression domain
and that this association apparently plays an importantrole in the down-regulation of major histocompatibility
complex (MHC) class I transcription.30 Thus, COUPTFs can repress transcription through a mechanism
similar to that described for nuclear receptors RAR/RXR or TR/RXR, which associate with histone deacetylases in the absence of their speciﬁc ligands.31
Transrepression: COUP-TFs can also repress transcription by directly binding to the LBD of nuclear
hormone receptors, a process called transrepression.13,32 Leng et al. have demonstrated that COUPTFI represses transcriptional activity induced by fusion
proteins between the GAL4 DBD and LBD of TR,
RXR, or RAR. Based on these results, they proposeda mechanism for transrepression by COUP-TFs invovling heterodimerization of COUP-TF proteins withother nuclear hormone receptors, such as TR, RAR,
or RXR.13 Therefore, COUP-TFs can be tethered to
DNA in the absence of their cognate response elements via LBD-LBD interactions with other receptors
such as TR, RAR, and RXR to transrepress the liganddependent transactivation of the nuclear receptors.

Park J-I, et al: Molecular mechanism of COUP-TF actions

Fig. 2 Molecular mechanism of COUP-TFs as activators. 1: direct
activation by binding to DNA response element, 2: indirect activationby acting as accessory factors for transcription activation, 3: activation
through protein-protein interaction with a DNA-bound factor. DR:
direct repeats, AF: accessory factor elements, CYP7A: cholesterol 7ahydroxylase, PEPCK: phosphoenolpyruvate carboxykinase.

Also, Achatz et al. reported that transrepression is the
predominant mechanism underlying repressor activityof ARP-1/COUP-TFII, and this mechanism most likely
involves interaction of protein with one or several transcriptional coactivator proteins which are employed byvarious liver-enriched transactivators such as HNF-4,
HNF-3, and C/EBP but not by ubiquitous factors suchas Sp1 or ATF.32
Activator
COUP-TFs can also function as positive regulatorsfor many different genes. Positive regulation by COUPTFs can be carried out by at least three different molecular mechanisms of activation of gene expression by
COUP-TFs (Fig. 2). First, COUP-TF activates transcription by binding to a nuclear receptor DNA response element and directly activating gene expression.
For example, COUP-TFII stimulates the transcriptional
activity of the rat cholesterol 7a-hydroxylase (CYP7A)
promoter by binding to the nucleotide sequence locatedbetween 74 and 54 (relative to the transcription startsite), which contains a direct repeat of two hormone
response element half-sites separated by 4 nucleotides(a DR4).33 Similarly, in the arrestin gene promoter, aDR-7 element mediates the positive transcriptional effect of COUP-TF.34
Second, COUP-TF activates transcription by binding
to a DNA element and indirectly inﬂuencing expression

Keio J Med 2003; 52 (3): 174–181

in the context of several other transcription factors, asin the phosphoenolpyruvate carboxykinase (PEPCK)
gene glucocorticoid response unit (GRU).35,36
Finally, COUP-TF activates transcription throughprotein-protein interaction with a DNA-bound factor,
such as with HNF-4 in the HNF-1a gene promoter,37
with Sp1 in the trout estrogen receptor,38 the phosphoenolpyruvate carboxykinase,35 the vHNF1,39 thehuman immunodeﬁciency virus long terminal repeat,40
and NGFI-A41 genes.
Based on three different molecular mechanisms ofgene activation by COUP-TFs, Sugiyama et al. havecharacterized the domain required for COUP-TFmediated transcriptional activation of PEPCK gene andhave determined that SRC-1 and GRIP1 bind to thisdomain and serve as coactivators.42 Similarly, for the
NGFI-A gene, COUP-TF enhances transcription by
recruiting coactivator SRC-1 through its interaction
with Sp1.41
Recently, Metivier et al. studied the molecular basis
underlying the positive action of COUP-TFI on ERa
activity and suggested a novel pathway in which theformation of a tight ERa-COUP-TFI intermediate
complex through COUP-TFI DBD and ERLBD resulted in an increased recruitment of ERK2/p42MAPK ,phosphorylation of the hERa on Ser 118 and enhanced
its transcriptional activity.43
COUP-TFs can act as repressors or activators ofgene expression. However, the mechanisms underlying
functional duality are unknown. Some of this functional
duality might depend on the repertoire of coregulator
proteins which interact with COUP-TF. Evidence suggests that the function of many nuclear hormone receptors is dependent upon, or modulated by, the actionsof both common and distinct receptor binding cofactorsthat differentially recognize liganded and unliganded
receptors.21,22,44–49 Most of the auxiliary factors so
far identiﬁed act as corepressors (e.g. N-CoR, SMRT,
TRUP, and TRIP1). However, in a few cases, receptorselective, positively acting coactivators (e.g. RIP140,
SRC-1, and CBP/p300) have been identiﬁed. Marcus
et al. have identiﬁed a factor that bound COUP-TFII
in vitro and allowed COUP-TFII to act as a transcriptional activator in mammalian cells. This factor is a
recently reported ligand of the tyrosine kinase signaling
molecule p56 lck . These results, if proved to be correct,
suggest that this factor mediates cross-talk between
mitogenic and nuclear hormone receptor signal transduction pathways.50 In addition, the bifunctional activity of COUP-TF may also depend on the promoter
contexts of target genes. In this respect, a recent
paper describes that corepressor SMRT functions as
a coactivator for TR from a negative hormone response element.51 The mechanism on how this works is
not clear.

177

Fig. 3 Biological functions of COUP-TFI during development anddifferentiation.

Biological Function of COUP-TFI During
Development and Differentiation
The molecular biology and expression studies haverevealed several possible functions of COUP-TFs during development and differentiation. Here we will concentrate our review on COUP-TFI and describe severalof its physiological functions (Fig. 3).
The role of COUP-TFI in neurogenesis
COUP-TFI and COUP-TFII 3 have been extensively
studied, both in terms of biochemical properties andtissue distribution, with a particular emphasis on developmental processes.9 In all the species that were
examined for the presence of COUP-TF during development (from sea urchin to mouse), expression of thisorphan receptor was clearly associated with neurogenesis.34,52–56 Connor et al. demonstrated that COUPTFI overexpression resulted in decreased contact stability between neurites and substrate cells and suggested COUP-TFI is involved in the regulation of cellcell contacts.57 Recent studies using gene targetingsuggest that COUP-TFI is involved in modulationof axonal growth.58,59 Indeed, targeted disruption ofmouse COUP-TFI results in decreased arborization of
spinal nerves,58 in abnormal morphogenesis of theninth cranial nerve and ganglion,58 and in defects in theguidance of axons emanating from thalamic neurons
that normally project to cortical layer IV.59 Finally,
disruption of COUP-TFI in mice results in the improper
brain regionalization. These results strongly suggestthat COUP-TFI is an important component in the regulation of neurogenesis and cellular differentiation
during embryonic development in several organisms.
Several studies have pointed out the possibility that
COUP-TF genes could be part of retinoid signaling
pathways both in vivo and in cell culture systems.60–63
Notably, up-regulation of COUP-TFI and COUP-TFII
genes occurs during the differentiation programs induced by retinoic acid (RA) in mouse teratocarcinoma

178

cells such as P19 embryonal carcinoma (EC) cells.61
Pluripotent P19 EC cells can be induced to differentiate into all three germ layer derivatives (i.e. ectoderm, endoderm, and mesoderm) when appropriate
inducers and culture conditions are used.64–66 WhenP19 cells are grown as aggregates, RA induces aneuroectodemal-like differentiation pathway.64 Therefore, P19 cells have been widely used to screen forgenes involved in neuronal differentiation.67–69 Adam
et al. studied the functional involvement of COUPTFI in RA-induced neuronal differentiation of P19
cells through two approaches: 1) deregulated expression of COUP-TFI, and 2) inactivation of endogenous
COUP-TF by means of a dominant negative COUPTFI mutant. They reported that a too early (or toohigh) expression of wild-type COUP-TFI impedes neural differentiation and inhibition of endogenous COUPTF by expression of a dominant-negative COUP-TFI
mutant results in a strengthening of cell-cell contacts,
decreased axonal growth, and slower migration of neurons. These data provide evidence that COUP-TFs are
regulators of cell adhesion mechanisms required for thedifferentiation of embryonal carcinoma.70
The role of COUP-TFI in neocortex regionalization
Regionalization of the cerebral cortex can be divided
into at least two steps: (1) an early regionalization
phase in which cortical neurons establish their regional
identity through regulating gene expression in a cellautonomous manner and (2) a reﬁnement phase inwhich extrinsic inﬂuences from subcortical areas,
including thalamocortical inputs, shape and maintainthe cortical subdivisions. Recent studies showed that
neocortical regional identity was shifted in Pax6 andEmx2 mutant mice, indicating that Pax6 and Emx2 are
two such intrinsic factors that regulate the regional expression of marker genes and specify cortical identity.71,72 Zhou et al. studied the role of COUP-TFI inearly neocortical regionalization and demonstrated that
COUP-TFI is required for the region-speciﬁc expression of many marker genes, as well as the precise axonal projections between the thalamus and the cortex.
These data indicate that COUP-TFI is a regulatory
factor that works in concert with Pax6 and Emx2 tospecify cortical identity.73
The role of COUP-TFI in organogenesis
Retinoids are known to regulate the expression ofHox genes, which play a major role in pattern formation and bone morphogenesis.74–76 Because COUPTFI is hypothesized to antagonize retinoid function andits expression is known to be regulated by retinoids, it isof interest to assess whether loss of COUP-TFI function

Park J-I, et al: Molecular mechanism of COUP-TF actionsin the null mutants will affect bone formation. Qiu et al.
reported that the left or the right exoccipital bone isprematurely fused to the basioccipital bone in 98% of
the COUP-TF1 null mutants.58 This result suggests that
COUP-TFI plays a major role in the development ofthese bones.
In addition, several other abnormalities including
malformation of hippocampus, faulty axon myelination,and inner ear defects were also observed in the COUPTFI null mutants.58 These results strongly suggest that
COUP-TFI is a crucial component in the regulation ofcellular differentiation during embryonic development
in several organs.
Future Directions
Further characterization of COUP-TFI activity invivo and analysis of its target genes will allow a better
understanding of its role in development and differentiation. Moreover, the role of COUP-TFI beyond theembryonic stage is very intriguing and remains to beelucidated. The discovery by Brian Sauer that the Cre
recombinase of bacteriophage P1 could be used torearrange the eukaryotic genome suggested a mechanism that would remove the restriction against introducing a mutation speciﬁcally into somatic cells.77 The
ﬁrst report of the successful application of this approach, inactivating the DNA polymerase b gene in T
cells, was published in 1994.78 Two lines of mice are
required for the Cre-loxP approach to work. In one
line, the targeted allele is ﬂanked by two loxP sequences, which target the ﬂanked region for excision by
the Cre recombinase. In another line, the expression of
Cre is controlled by a tissue-speciﬁc promoter. Crossing
the two lines of mice leads to an animal lacking thegene only in tissues that express Cre, while leaving thegene functional in all other tissues. The advantages ofsuch a system are obvious: the function of genes necessary for fetal development can be preserved, allowing
the function(s) of the gene to be examined during
adulthood. The perinatal lethality of COUP-TFI nullmutants limit the study of the function of COUP-TFI
after birth. Therefore, the future generation of conditional knockouts using Cre-loxP system to delete
COUP-TFI gene in a particular cell or tissue of interest
will lead to a better understanding the role of COUPTFI.
Acknowledgments: JIP was supported by Dong-A University Research Fund, in 2001. This work was supported by NIH grants to MJT
and SYT.

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