Induction of chicken ovalbumin upstream promoter-transcription
factor I (
COUP-TFI
) gene expression is mediated
by ETS factor binding sites
Ramiro Salas*, Fabrice G. Petit, Carlos Pipaon, Ming-Jer Tsai and Sophia Y. Tsai
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA
Chicken ovalbumin upstream promoter-transcription factor I
(COUP-TFI, or NR2F1) is an orphan nuclear receptor that
plays a major role in th e development of the nervous system.
We show here that three ETS response elements in the
COUP-TFI promoter mediate its transcription. A reporter
gene containing these ETS binding sites i s activated by Ets-1,
while the same reporter with point mutations on all three
ETS response elements is not. We also s how that Ets-1 binds
to these response elements and that other ETS factors also
transactivate t he COUP-TFI promoter . In a ddition, COUP-
TFI is coexpressed w ith some ETS factors in the mouse
embryo. These results indicate that members o f the ETS
family can activate COUP-TFI gene expression.
Keywords: COUP-TFI; ETS; gene expression; transcription;
orphan r eceptors.
Chicken ovalbumin upstream promoter-transcription
factors (COUP-TFs) are among the best characterized
orphan members of the nuclear receptor superfamily [1].
COUP-TFs have been shown to be negative regulators of
the transcription of many genes [1,2], but can also act as
activators of gene t ranscription [ 1,3]. I n mammals, two
COUP-TF genes have been identi®ed, COUP-TFI and
COUP-TFII. Although t hey have different physiological
functions [4,5], sequence a nalysis a nd molecular studies

Ets-1 t hat lacks this exon is constitutively ac tive [18].
Little is known ab out the upstream signals that regulate
COUP-TFI gene expression. Here we show that Ets-1 and
other ETS factors a re able to trans activate COUP-TFI
expression through a cluster of ETS response elements in t he
promoter. In a ddition, several ETS factors colocalize with
COUP-TFI in different tissues of the developing mouse
embryo.
MATERIALS AND METHODS
Genomic screening
To isolate the mCOUP-TFI promoter, a genomic library
(129SVJ Mouse Genomic Library in the Lambda FIX II
vector, S tratagene) was screened using p art of the 5¢ UT R o f
the mCOUP-TFI gene. T he fragment w as labeled with
32
P-dCTP by random priming (Prime-a-gene kit, Promega).
The g enomic library was used t o infect XL-1 blue bacteria
and standard protocols were used t o perfo rm the screening
[19]. A fter tertiary screening, the phage DNA was isolated
using the k Wizard kit (Promega), cut with NotIand
subcloned into t he pBluescript K SII vector ( Stratagene).
The Genbank accession number for this sequence is
AY055471.
Correspondence to S. Y. Tsai, Department of Molecular and Cellular
Biology, Baylor College of Medicine, One Baylor Plaza, Houston,
Texas 77030, USA. Fax: + 713 798 8227 , Tel.: + 713 798 6251,
E-mail:
Abbreviations: COUP-TFI, chicken ovalbumin upstream promoter-
transcription factor I; DBD, DNA-binding domain; EMSA, electro-
mobility shift assay; DMEM, Dulbecco's modi®ed Eagle's medium.

instructions. Cells were collected 48 h later. Luciferase
activity was measured in a luminometer (Monolight 2010,
Analytical Luminescence Laboratory) according to the
manufacturer's instructions. Protein c ontent was measured
using the Bradford reagent (Bio-Rad).
Electromobility shift assay
For EMSA (electromobility shift assay) studies, proteins
were prepared (with or without [
35
S]Met) from different
DNA constructs using the TNT Coupled reticulocyte lysate
system (Promega), according to the manufacturer's i nstruc-
tions. Probes for EMSA were end-labeled using a
32
P-dCTP
(ICN) and Sequenase enzyme (Amersham USB). EMSAs
were carried out as follows: 2 lL of reticulocyte lysate were
incubated for 10 min at room temperature with 1 lLof
labeled probe (2±3 ´ 10
4
c.p.m.) and 10 pmol of dIdC in
buffer H (20 m
M
Hepes, 1 m
M
MgCl
2
,100m
M
KCl,

listed below (th e ETS core binding sequences are in bold). A
forward: CGGGTACCCTCCGT
TTCCCACTTCTCG; A
for Mut: CGGGTACCCTCCGT
TTCTCACTTCTCG; B
forward: CGGGGTACCTCCCTC
TTCCCCGTCTTCT
CGTTCGTTCG; B for Mut CGGGGTACCTCCCTCT
TCTCCGTC
TTCTCGTTCGTTCG; B reverse GAA
GATCTCGAACGAACGAGAAGACGG
GGAAGAG
GGA; B rev Mut GAAGATCTCGAACGAACG
AG
AAGACGGAGAAGAGGGA; C rev GAAGATCTC
AAGTCAGTCACA
GGAAAAGAGC; C rev Mut
GAAGATCTCAAGTCAGTCACA
AGAAAAGAGC.
In situ
hybridization
The ÔBÕ domains of th e ets-1 and ets-2 genes were used to
prepare p robes a s d escribed previously [21]. The N-terminus
of ERM, ETV1 and PEA3 genes were used to prepare
probes a s described previously [22]. T he full length cDNA of
the mCOU P-TFI genewasusedtopreparetheCOUP-TFI
probe. The templates for probes were subcloned into
pBluescript and RNA probes were p repared f rom linearized
plasmid using T3 RNA polymerase (Promega) and 100 lCi
[a-

computer search for transcription factor binding sites
[25]. Surrounding this element, we identi®ed additional
sequences that resembled ETS binding sites (Fig. 2). To
assess whether Ets-1 regulates COUP-TFI promoter
activity on this region, we cotransfected a reporter contain-
ing the )490/)259 fragment linked to a TATA-Luc
reporter expression v ector, in the presence or absence of
an Ets-1 expression vector. Ets-1 was able to signi®cantly
318 R. Salas et al. (Eur. J. Biochem. 269) Ó FEBS 2002
activate the reporter g ene containing this COUP-TFI
promoter fragment. Then we subdivided that segment into
two s ubfragments ()397/)259 and )490/)408), each
carrying three putative binding sites. The subfragment
)397/)259 is not responsive to Ets-1, w hile the )490/)408
subfragment is stimulated by Ets-1 to a similar extent as
the )490/)259 fragment (Fig. 2A) in a dose-dependent
manner (Fig. 2B). Interestingly, the best putative Ets-1
binding site sequence is located within this fragment.
The three putative Ets-1 response elements within )490
and )408 (ETS-RE) were then named A, B and C, in the
order of 5¢ to 3 ¢. Point mutations (TTCC to TTCT) were
introduced on each or combinations of the three putative
sites. Mutations in one or two sites diminished Ets-1-
dependent reporter activity (Fig. 2C), while mutations in all
three sites abolished the response. These results indicate that
these t hree Ets-1 response elements work in c oncert to
achieve maximum Ets-1-dependent activation.
Ets-1 binds the ETS response elements
in the
mCOUP-TFI

that the antibody may elicit a conformational change that
-6 Kb
-4 Kb
-734
-387
-197
-96
Empty vector
+1
0
10
20
+446
RLU
ETS
A
-734
GTACGCGGGACCGTCCTCCTGCCTACCCCTCCTTTTGCGACCAATCACCTTCGGGAATGGGGTCTCAGTCACACACACC
CCAACACACACACACACACACACACACACACACACACACCACCACCACCACCACCACCACCACCACCACCACCACCAC
CACCACCACCACCACCACACAGCGAGTGAGAGACTCAGTCTCTTCCTCCTCCTCCTCCTCCTCCTCCTCTCCCCCTCCCC
CTCCCCTCCGTTTCCCACTTCTCGTCCCCTCCCCTCCTCCCCTCTCCCTCTTCCCCGTCTTCTCGTTCGTTCGTTTGCTCTT
ETS
TTCCTGT
GACTGACTTGTCCGCACTAACAGCCGCCCCACAACAATATGAGGAGTTACAAATGCTTTATTAATAATCATT
Nkx2. Nxk2.
GAAGCATTGTTTGGAGTTTGAGCATCCTGGGAATAAAAATGATGAAAAAGGAAAAAGAGGATTGATTGGAAAGTTTAT
TTTAAGATCATCTTTGGGATGAATAGGAATCATCGATTCGGATCGAATTTGTGGCAGTAGCTGCAGTTTCATGTGTGTG
C/EBP C/EBP
CTTTGTCGTAATTA
CGCCTCCGAAACTATGATATACTTCAGATTTTTAAATGAGGAGGCTTTTCATAATTATATAAAATGA

Ets-1 speci®c antibody (lane 2), but not by an u nrelated
antibody (lane 13). Increasing amounts (10 and 100-fold
molar excess) of unlabeled site C o ligos were able to compete
for the binding of Ets-1DAE to the Ets-1 consensus binding
site (lanes 4 and 5). In c ontrast, a mutation (TTCC to
TACT) of site C was unable to do so ( lane 6). S imilar
competition experiments were carried out with site s B (lanes
7, 8 and 9) and A (lanes 10, 11 and 12). Very weak
competition could b e detected with s ite A and B oligos, w hile
oligos containing mutations of these sites did not compete at
all ( Fig. 3D). Taken together, these experiments suggest that
Ets-1 is able t o bind t he COUP-TFI promo ter preferentially
at site C. This is not surprising because site C is the most
closely related to the consensus Ets-1 binding site.
ETS factors colocalize with COUP-TFI
on the developing mouse embryo
We performed in situ hybridization studies on mouse
embryos w ith C OUP-TFI and different ETS factors. Mouse
embryos 14.5-days-old-were chosen for these experiments
because t he expression levels of COUP-TFI are high a t this
stage o f d evelopment [ 1]. T he expression patterns of COUP-
TFI, Ets-1, Ets-2, ETV1 a nd PEA3 were studied (Table 1).
There were s everal areas o f coexpression of COUP-TFI and
Ets-1: the mesenchyme of t he bladder ( Fig. 4A±D), the
mesenchyme of the nasal septum (Fig. 4E±H), the cerebral
cortex (Fig. 4I±L), the mesenchyme of vibrissae
(Fig. 4M±P), spleen, and submandibular glands (Table 1).
Ets-2 was found to colocalize with C OUP-TFI on the
mesenchyme of vibrissae (Figs 4M±N,Q±R) and subman-
dibular glands (Table 1). PEA3 w as found to colocalize w ith

indicate that COUP-TFI and ETS factors a re colocalized in
many regions of the developing mouse embryo.
Ets-1
binding
Supershift
Antibody
Competitor
Ets-1∆A-E
C
Cmut
B
Bmut
A
Amut
U
+
-
-

-
-
-
-
-
-
-
10
100
10
10

A
39
52
64
87
126
B
C
Ets-1
binding
Supershift
Antibody
NS

+++
V
Ets-1
Ets-1∆
CE
Ets-1
∆AE
Ets-1 Ets1∆CE
Ets-1∆AE
1
2
3
4
5
6
7

35
S-labeled
methionine using a reticulocyte lysate system.
The translated products were se parated on a
10% SDS/PAGE and autoradiographed. For
the deleted Ets-1, two vectors were used. (C)
Electromobility shift assay (EMSA) of Ets-1,
Ets-1DCE and Ets-1DAE on an Ets-1 con-
sensus binding s ite. Lane 1 (V), v ector control.
Lanes 2 and 3, wild-type Ets-1 w ithout or with
anti-(Ets-1) Ig, respectively. L anes 4 and 5,
Ets-1DCE without and w ith antibodies,
respectively. Lanes 6 and 7, Ets-1DA-E with-
out and with antibodies. NS, n onsp eci®c
binding. (D) EMSA of Ets-1DAE on a con-
sensus Ets-1 binding site and competition
experiments (lane 1, vector control, lanes 2±13
contain Ets-1DAE).Lane2,eectofanti-
(Ets-1) Ig. For competition experiments, 10x
and 100x molar excess (site C, la nes 4 and 5 ;
site B, lanes 7 a nd 8; site A , lanes 10 and 11) or
100x molar excess (mutated site C, lane 6;
mutated site B, lane 9 ; mutated si te A, la ne 12)
of unlabeled oligonucleotide were used. Lane
13, addition of unre lated antibodies have no
eect on E ts-1DAE binding.
Table 1. Expression of COUP-TFI, Ets-1, Ets-2, ETV1 and PEA3 mRNA in 14.5 day old m ouse embryos.
Tissue COUP-TFI Ets-1 Ets-2 ETV1 PEA3
Cortex +++ + +/± + +
Cochlea +++ + + +/± +

ETS family and, as a result, the biological activities of some
of these proteins h ave been studied. ETS factors are
involved in processes such as development [26,27], tumor
progression [8,28], speci®cation of synaptic connectivity [29]
and synapse-speci®c transcription [30]. A lthough there is a
considerable body of research on ETS factors, only a few
target genes have been identi®ed.
In this paper, we have presented data indicating the
coexpression of ETS proteins and COUP-TFI in the same
tissues. Among these ETS factors, Ets-1, Ets-2, ETV1 a nd
PEA3 are coexpressed with COUP-TFI in many different
tissues of the d eveloping mouse embryo suggesting t hat
COUP-TFI may be a target gene of these factors. This
would render a very complex pattern of activation of
COUP-TFI as we showed that m ost ETS factors a re able to
activate the COUP-TFI p romoter. Furthermore, the com-
plexity of this system is also illustrated b y the fact that ETS
factors work t ogether w ith a ccessory proteins [8]. Therefore,
the ®nal eff ect of a particular ETS factor on the promoter
DARK FIELD BRIGHT FIELD BRIGHT FIELDDARK FIELD
COUP-TFI
Ets-1
Ets-2
COUP-TFI
Ets-1
A
B
C
E
D

that this is the effect of endogenous Ets-1 or other ETS
factors in HeLa cells. The activity drop is small probably
because the reporter amount used in transfection experi-
ments is in large excess and there might not be enough
endogenous protein t o reach full activation. In addition, we
demonstrated that all three ETS sites must be occupied and
this would be even more d if®cult when the ETS factors a re
present in a limiting amount. F inally, it is also possible that
sequences closer to the initiation o f transcription are
responsible for a high basal activity.
In transfection experiments, all the ETS factors studied
activated the COUP-TFI promoter, with Ets-1 showing the
strongest effect. It is interesting to note that Ets-1 is also the
factor that showed more regions of coexpression with
COUP-TFI. T herefore, there may be a correlation between
the level of coexpression and the extent of activation in
transfected cells. T he fact that all the ETS factors e xamined
activated the COUP-TFI promoter is not really surprising.
As stated earlier, all ETS factors recognize the same core
motif. As the n eighboring sequences also affect binding, the
consensus binding site is not the same for all these proteins.
Therefore, it is likely that the COUP-TFI promoter might
have evolved to b e more highly r esponsive to some members
of the family, in this case Ets-1, as c ompared to o thers. The
putative correlation between transactivation potential and
mRNA coexpression supports this hypothesis. In addition,
although we studied several ETS factors, there are many
more, and it is possible that some other ETS factors also
colocalize with COUP-TFI. Furthermore, the temporal
expression of COUP-TFI and ETS factors change during

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