From the Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, BP 163, 67404 Illkirch Cedex, France
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ABSTRACT |
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The pleiotropic effects of retinoids are mediated
by nuclear receptors that are activated by 9-cis- or
all-trans-retinoic acid to function as
ligand-dependent transcription factors. In a yeast
one-hybrid screen for proteins capable of interacting with native
retinoic acid receptor (RAR), we have isolated the T:G
mismatch-specific thymine-DNA glycosylase (TDG), which initiates the
repair of T:G mismatches caused by spontaneous deamination of
methylated cytosines. Here, we report that TDG can interact with RAR
and the retinoid X receptor (RXR) in a ligand-independent manner, both
in yeast and in vitro. Mapping of the binding sites revealed interaction with a region of the ligand binding domain harboring -helix 1 in both RAR and RXR. In transient transfection experiments, TDG potentiated transactivation by RXR from a direct repeat element spaced by one nucleotide (DR1) and by RXR/RAR
heterodimers from a direct repeat element spaced by five nucleotides
(DR5). In vitro, TDG enhanced RXR and RXR/RAR binding to
their response elements. These data indicate that TDG is not only a
repair enzyme, but could also function in the control of
transcription.
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INTRODUCTION |
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Retinoic acids exert their pleiotropic effects on vertebrate
development and homeostasis by binding to nuclear receptors that function as ligand-dependent transcription factors
regulating the expression of target genes. These receptors belong to a
gene superfamily including the receptors for steroid hormones, thyroid hormone, vitamin D3, and a growing number of the so-called orphan receptors for which no ligands have yet been found (1-3). The receptors for retinoic acids comprise two families, the retinoic acid
receptors (RARs)1 and the
retinoid X receptors (RXRs), each consisting of three isotypes (,
, and
) (4). RARs bind all-trans-retinoic acid (t-RA)
and 9-cis-retinoic acid (9c-RA), whereas RXRs respond
exclusively to 9c-RA. As all nuclear receptors, they share a conserved
modular structure with five (RXRs) or six regions (RARs) denoted as A to E or A to F, respectively. Out of these, the central C region harboring the core of the DNA-binding domain shows the highest homology
among all receptors. The C-terminal E region contains the ligand
binding domain (LBD), which functions as a ligand-dependent transactivation domain (AF-2), and contains surfaces for the homo- and
heterodimerization with other receptors and interaction with various
cofactors. The ligand-dependent activation domain AF-2 in
the E region can synergize with the ligand-independent activation domain AF-1 located in the N-terminal A/B region. RARs and RXRs bind as
dimers to their response elements (RAREs) consisting of two hexameric
motifs (PuG(G/A)(T/A)CA) usually arranged as direct repeats. Although
RXRs can bind on their own as homodimers to direct repeat elements
spaced by one nucleotide (DR1), response elements for RXR/RAR
heterodimers are direct repeats with a spacing of one (DR1), two (DR2),
or five nucleotides (DR5) (4). After binding to their response elements
they modulate transcription in a promoter-specific manner by acting on
the transcription machinery or through remodeling of the chromatin
template. The link to the basic transcription factors and proteins
involved in chromatin remodeling is most likely mediated by direct
protein-protein interactions with cofactors (3, 5, 6). Recently,
several such proteins were cloned and characterized as putative
corepressors like N-CoR (7) and SMRT (8) or coactivators like
Trip1/Sug1 (9, 10), TIF1
(11), RIP140 (12), SRC-1 (13), TIF2/GRIP1
(14-17), AIB1/RAC3/ACTR/p/CIP (18-21), and CBP/p300 (22, 23) which
bind to RAR and/or RXR in a ligand-regulated manner. In addition to these interactions with different cofactors, the AF-1 function of RAR
can be stimulated through binding to the general transcription factor
TFIIH and phosphorylation by Cdk7 (24, 25).
Using a one-hybrid screening in yeast for putative cofactors of RAR, we
report here the isolation of a new splice variant of the T:G
mismatch-specific thymine-DNA glycosylase (TDG), which is required for
the repair of methylated DNA sites. In vertebrates, DNA is most
commonly modified by addition of a methyl group to the carbon 5 position in the pyrimidine ring of cytosines. Changes in
5-methylcytosine methylation patterns have been implicated in various
processes such as control of gene expression (26), chromatin structure
(27), somatic X-chromosomal inactivation in females (28), timing of DNA
replication (29), and genomic imprinting (30). Methylation of cytosines
is essential in vertebrates (31). However, it also causes genome
instability; although not more than 1% of bases are methylated
cytosine in mammalian genomes, they probably cause one third of all
transition mutations responsible for genetic diseases and cancer in
humans (32-34). This is due to the much higher spontaneous deamination
of 5-methylcytosine when compared with cytosine; deamination of
5-methylcytosine leads to T:G mismatches, whereas deamination of
cytosine generates U:G mismatches in the DNA. The repair of U:G and T:G
DNA mismatches is initiated by mismatch-specific DNA glycosylases (35),
which excise in a first and rate-limiting step the mismatched base
leaving an abasic site (AP) that is cleaved by an AP-endonuclease and deoxyphosphodiesterase to create a single nucleotide gap; this gap is
then filled in by DNA polymerase- and finally sealed by DNA ligase
(36). TDG was first characterized as a 55 kDa protein purified from
HeLa cells (37). Further analysis revealed that TDG was not only able
to excise thymine from T:G, but also uracil from U:G DNA mismatches
(38). The cloning of the human TDG cDNA showed that this
glycosylase belongs to a new class of excision repair enzymes with no
significant sequence similarity to the established DNA glycosylase gene
family including the U:G mismatch-specific uracil-DNA glycosylase (UNG)
(39). However, the gene for human TDG contains conserved regions with
greater than 30% sequence identity with an E. coli gene
(MUG) which was found later to encode an U:G mismatch-specific DNA
glycosylase that, in contrast to the human TDG, cannot cut at T:G
mismatches under physiological conditions (40). The recently published
crystal structure of the E. coli MUG, nevertheless, revealed
structural and functional homologies with UNG despite of their low
sequence similarity (41). Interestingly, TDG was initially isolated and
characterized in a yeast two-hybrid screening as a protein that can
interact with the transcription factor c-Jun (42). This interaction,
together with those with RAR and RXR described here points to a
possible function of TDG in transcription beyond its role in
maintenance of methylated CpG sites in DNA, and provides a further link
coupling transcription and DNA repair.
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EXPERIMENTAL PROCEDURES |
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Cloning of mTDG Isoforms--
The Saccharomyces
cerevisiae reporter strain BP-G5 (MAT ura3-
1
his3-
200 leu2-
1 trp1::DR5 RARE-URA3) was derived
from the strain PL1 (MAT
ura3-
1 his3-
200 leu2-
1
trp1::ERE-URA3) (43) by substituting the ERE by one copy
of a DR5 RARE (AGG TCAgcgagAGG TCA). The one-hybrid screening was
performed as described in Ref. 10 using S. cerevisiae strain
BP-G5 expressing full-length hRAR
1 from YEp90, and a VP16-tagged
mouse embryo cDNA library (10). Library plasmids from positive
clones were isolated and sequenced leading to the isolation of a TDG
cDNA encoding the TDGb isoform. A cDNA fragment (286-2567 bp)
was used as a probe to screen a mouse embryo cDNA library (10 days
post coitum) for additional TDG cDNAs. A full-length mTDGa cDNA
was cloned by PCR using a 5'-primer encoding the missing 8 amino acids
(ATG GAC GCG GAG GCC GCG AGA AGC) and the mTDGb cDNA as a
template.
Plasmid Constructs-- For the expression of full-length or various mTDG fragments, the pSG5 vector (44) was used for mammalian cells, the pET15b vector (Novagen) for His-tagged proteins in Escherichia coli, and the pERV vector2 for expression from Vaccinia virus. Reporter gene constructs and expression vectors for RXR and RAR were as described (47-49); Gal4-c-Jun fusion proteins were expressed from a pG4MpolyII vector (45), and other Gal4 fusion proteins were as described (46). Further details on all constructs used in this study are available on request.
Northern Blots and RT-PCR-- Northern blot analyses were performed using a mouse multi-tissue mRNA blot (CLONTECH) and a 32P-labeled fragment of the mTDGb cDNA (286-2567 bp) as described by the manufacturer. RNA preparation form P19 cells and RT-PCRs were as described (47) using the following primers: TDGa-specific 5'-primer: GAG ACC GGC TGC CCG TGT GCC (bp 343-363 in hTDGa cDNA; Ref. 39), TDGb-specific 5'-primer: AGC AGC GTG GGA GGG GCC GAG (bp 1-21 in mTDGb cDNA), common 3'-primer: CCA GGC CCA GGG TAG TGA TGT CC (bp 514-536 in mTDGb cDNA).
Antibodies and Western Blotting-- Anti-TDG monoclonal antibodies were raised against E. coli-expressed mTDGa(32-421) purified on a nickel matrix as described in Ref. 48. Western blots were performed according to standard protocols using 50 µg of protein from whole cell extracts separated on 10% SDS gels. For dephosphorylation experiments, 25 µg of a whole cell extract from MCF7 cells were incubated for 30 min at 37 °C with 20 units of calf thymus intestinal phosphatase (CIP) in a buffer provided by the manufacturer (Boehringer Mannheim). To avoid nonspecific degradation, leupeptin, pepstatin, aposterin, antipain, and chymostatin were added to the reactions at a final concentration of 2.5 µg/ml, and vanadate was used at a concentration of 50 µM.
Two-hybrid System and Transient Transfections--
For the
mapping of the interaction domains in yeast, the LexA system was
applied using the vectors pBTM116 (49) for the cloning of the LexA
fusions and pASV3 (50) for the cloning of the VP16 fusions.
-Galactosidase assays on individual L40 transformants were carried
out as described (46). The expression of all LexA and VP16 fusion
proteins was checked by Western blotting using anti-LexA or
anti-VP16-specific antibodies (data not shown). Transient transfections
in COS cells as well as CAT and
-galactosidase assays were done as
described in Ref. 51. All transfections employed duplicate samples, and
were repeated at least twice. Representative experiments are shown in
the figures.
In Vitro Assays--
In vitro binding studies using
GST-tagged hRAR and mRXR
were carried out as described (11, 52).
Electrophoresis mobility shift assays (EMSAs) were performed using
E. coli-expressed RAR
AB, RXR
AB, Vaccinia virus
expressed mTDGa(32-421), and 32P-labeled double-stranded
oligonucleotides (DR1: 5'-GGG TCA a AGG TCA-3', DR5: 5'-AGG TCA agcttc
AGG TCA-3'). The binding reaction was done in the presence of 1 µg of
bovine serum albumin and protein-DNA complexes and free DNA were then
submitted to non-denaturing gel electrophoresis as described (53).
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RESULTS |
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Isolation and Characterization of a New Splice Variant of
TDG--
Previous studies in yeast have shown that RAR cannot
efficiently induce transcription from a DR5 element without
heterodimerization with RXR (54-56). In a search for additional
partners allowing RAR to transactivate from such an element, the yeast
strain BP-G5 harboring one DR5 element within the URA3 promoter was
designed for a functional one-hybrid screening with full-length RAR
as a bait and a mouse embryo (9.5 to 12.5 days post coitum) cDNA library fused to the acidic activation domain of the viral VP16 protein. Positive clones from this screening were expected not only to
interact with RAR, but also to stabilize its binding to the DR5
element, thus allowing efficient RAR-mediated activation of the
selection marker. Out of 30 positive clones obtained from the
screening, 25 were identified as RXRs. From the five remaining clones,
three encoded for another nuclear receptor, COUP-TFI (57), and two for
TDG, which was initially cloned as a c-Jun-interacting protein (42).
The 2859-bp TDG cDNA clone obtained from the yeast screening
(denoted b isoform, Fig. 1, A
and B) differs between positions 1 and 58 from the
previously published sequences of TDG, here called TDG isoform a (39).
Due to these differences, the mouse TDGb cDNA lacks the codon for
the starting methionine of the TDGa isoform and encodes for a truncated
form of the enzyme missing the first 25 amino acids (Fig.
1B). Inasmuch as, in the TDGb cDNA, no stop codon was
found in front of the first methionine (Fig. 1A), a mouse
embryo (10 days post coitum) cDNA library was screened for longer
mTDG cDNAs. However, this screening did not result in the cloning
of further 5'-extended TDGb cDNA.
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TDG Interacts with RAR and RXR in Yeast and in
Vitro--
Full-length mTDGa was fused to the VP16 acidic activation
domain (denoted AAD) and assayed for interaction with full-length RAR and RXR
fused to LexA in the yeast strain L40 which contains a lacZ reporter gene driven by eight LexA binding sites
(49). Interestingly, while a very weak (~5-fold), but reproducible, increase in
-galactosidase activity was obtained by co-expressing AAD-TDGa with unfused LexA, 335- and 75-fold enhancements were observed
by co-expressing AAD-TDGa with LexA-RXR
and LexA-RAR
, respectively, in the absence of ligand (Fig.
3A). Addition of 9c-RA to
yeast expressing LexA-RXR
and AAD-TDGa or t-RA to yeast expressing
LexA-RAR
and AAD-TDGa diminished by a factor of ~2 and 3, respectively, the -fold enhancement mediated by AAD-TDGa over the AAD
control (Fig. 3A), indicating that TDGa may interact preferentially with the unliganded forms of RXR
and RAR
in yeast. As a representative of the other conserved gene family of DNA glycosylases (see Introduction), the human uracil-specific U:G mismatch-specific DNA glycosylase (hUNG1) (58, 59) was tested for its
ability to interact with RXR
and RAR
in yeast. Similarly to
AAD-TDGa, co-expression of AAD-UNG1 with unfused LexA resulted in a
weak activation of the reporter gene (Fig. 3A). However, no
further stimulation was observed by co-expressing AAD-UNG1 with
LexA-RXR
or LexA-RAR
in either the presence or absence of ligand
(Fig. 3A), indicating that, in contrast to TDGa, UNG1 does
not interact with RXR
and RAR
.
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TDG Enhanced Transactivation by RXR and RXR/RAR--
To
investigate whether the interactions between TDG and either RAR or RXR
could affect the transactivation mediated by RXR homodimers or RXR/RAR
heterodimers, transient transfection experiments with increasing
amounts of TDGa(32-421) were performed in COS cells. Ligand-induced
transactivation by RXR from a single DR1 element in a DR1(G)-tk-CAT
reporter gene enhanced up to 4-fold by addition of TDGa(32-421) (Fig.
5A). In similar transfection assays using a DR5(G)-tk-CAT reporter gene cotransfected with RAR
and RXR
expression vectors, t-RA induced activation from one DR5
element increased by 2-3-fold (Fig. 5B). Control
experiments using a reporter gene construct lacking a RARE indicated
that overexpression of TDG had no effect on transcription from the tk
promoter (data not shown). The truncated TDGa(32-307), which could not
bind to RXR
(see Fig. 4C), did not enhance
transactivation from a DR1(G)-tk-CAT reporter gene (Fig.
5C). Similarly, the N-terminally truncated mutant
TDGa(122-421) was unable to increase transactivation by RXR
,
although it could still interact with RXR in yeast (see Figs.
4C and 5C). This observation points to a
requirement of the N terminus of TDG for enhancement of
receptor-mediated transcription. However, experiments with various TDG
fusion proteins in yeast and mammalian cells did not reveal any
transactivation domain in the N terminus or any other part of TDG (data
not shown).
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TDG Enhanced the Binding of RXR and RXR/RAR to DNA in
Vitro--
EMSAs were performed to investigate whether TDG could
enhance the binding of RXR or RXR/RAR to their response elements.
Liganded RXRAB was studied for its binding to a DR1 element (Fig.
6B) as well as liganded
RXR
AB/RAR
AB heterodimers for their binding to DR1 and DR5
elements in the presence of increasing amounts of Vaccinia
virus-expressed TDGa(32-421) (Fig. 6, A and C).
Although no TDG-receptor complex could be identified in these
experiments, in all cases the addition of TDGa(32-421) led to an
approximately 3-fold increase in DNA-binding by RXR
AB and
RXR
AB/RAR
AB. Under similar EMSA conditions, TDGa(32-421)
could not bind to DR1 or DR5 response elements on its own.
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DISCUSSION |
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In a screening for factors supporting RAR to function as a ligand-dependent transcription factor in yeast, we have identified TDG as a protein that can interact with RAR and RXR in yeast as well as in vitro. In a yeast two-hybrid screening, TDG was previously cloned as a c-Jun interacting protein (42), and later identified as a T:G and U:G mismatch-specific DNA repair enzyme (39).
A new TDG isoform-specific cDNA (called b) has been cloned, which differs in the 5' sequences from the previously published mouse and human cDNAs (denoted isoform a) (39). These cDNAs are likely to correspond to alternative splicing variants of the same transcript. Although Northern blots analysis detected only one RNA species in all mouse tissues tested, transcripts corresponding to the TDG isoforms a and b could be detected in P19 embryonal carcinoma cells. Using monoclonal anti-TDG antibodies, two major TDG species were revealed in whole cell extracts from various mouse, monkey, and human cell lines as well as mouse tissues. These two forms of TDG are probably encoded by the two different transcripts found in P19 cells. Alternatively, these two species could be translated from TDGa mRNA using two different starting methionines as discussed for the human enzyme (39). However, the cloning of additional splice variants from mouse suggests that similar transcripts could also exist in humans. As the first 25 amino acids of the TDGa isoform, which are lacking in TDGb, are dispensable for both the binding to RAR or RXR and the enzymatic activity (40), the functional significance of the two isoforms is unknown. In addition to the two TDGa and TDGb isoforms, Western blot analyses and phosphatase treatment of cell extracts indicated the existence of phosphorylated forms of the TDG isoforms. The significance of this modification is also unknown, although it does not appear to be required for in vitro interactions between TDG and RAR or RXR.
In the present study, we have shown that TDG can interact with RAR and
RXR, both in yeast and in vitro in a ligand-independent manner. Mapping of the binding sites revealed interaction with a region
in the RAR and RXR LBD harboring the -helix 1 and a central region
of TDG between amino acids 122 and 346. An alignment of hTDGa with the
related U:G-specific DNA glycosylase from E. coli (MUG) has
shown that the TDG region from amino acid 122 to 346 is the most
conserved one and that it also contains the enzymatic center of the
glycosylase (40). Interestingly, the same region of TDG appears to be
required for its binding to c-Jun and its homodimerization in
yeast,3 suggesting that it
could participate in various protein-protein interactions.
Transfection of TDG in COS cells increased RXR- and RXR/RAR-mediated
transactivation from reporter genes containing cognate response
elements for the receptors. The observation that the truncated
TDGa(32-307) could not bind to the receptors and failed also to
enhance RXR-induced transcription suggests that the interaction between
TDG and the receptors could be of functional importance. To investigate
the basis for the increased transactivation by RXR and RAR in the
presence of TDG in transient transfection experiments, TDG was fused to
various DNA-binding domains as those of the yeast transcription factor
GAL4, the estrogen receptor, or the E. coli regulator LexA.
Using these fusion proteins in yeast or in transfected COS cells with
cognate reporter genes did not reveal any transcriptional activation
domain in TDG (data not shown). However, TDG was shown to enhance the
DNA binding of either RXR homodimers or RXR/RAR heterodimers to cognate
DR elements, arguing that TDG can enhance RXR- and RXR/RAR-mediated
transactivation by stabilizing their binding to DNA. In this context,
it is interesting to note that the N-terminal part of TDG (amino acids
70-122) is rich in basic amino acids sharing in part similarity with a
HMG box motif (HMG-I/HMG-Y family). This TDG region is required for its
T:G-specific, but not for its U:G-specific DNA glycosylase activity,
suggesting that it contributes to the enzyme selectivity by contacting
DNA (40). Interestingly, HMG-1 was found to stabilize binding of the
progesterone receptor to its target sequences in EMSA (66, 67) and to
enhance transactivation by estrogen receptor at the level of DNA
binding (68). In the case of the progesterone receptor, a model was
proposed in which HMG-1 first bends the DNA, and dissociates from DNA
after formation of a stable receptor-DNA complex (66). The N-terminal
region of TDG may act similarly to support receptor-DNA binding.
Accordingly, a truncated mTDGa(122-421) lacking the basic region could
not enhance the RXR-mediated transactivation in transient transfection
assays, even though it could still interact with the receptors. During
the last years, an increasing number of putative coactivators was
cloned which have been characterized for their binding to nuclear
receptors (5, 6). TDG is the first to be shown to act at the level of
DNA binding. Interestingly, TDG can also interact with the putative
nuclear receptor cofactor TIF1.3 This interaction may
generate additional possibilities through which TDG could affect the
transcriptional activity of nuclear receptors.
The effect of TDG on RXR- and RXR/RAR-dependent transcription was specific to nuclear receptors, since TDG expression had no effect on the transcriptional activity of GAL4 DNA-binding domain fusion proteins containing the activation domains of SP1, AP2, Oct1, or VP16 (data not shown). However, the transcriptional activity of a GAL4-cJun fusion protein was increased by overexpression of TDG.4 This enhancement may involve the previously reported interaction between c-Jun and TDG (42). As a component of the AP1 transcription factor c-Jun can participate in transrepression of RAR and RXR (63). As TDG can interact with RAR and RXR as well as with c-Jun, we investigated whether TDG could be involved in transrepression. TDG overexpression had no significant effect on transrepression of RXR and RAR by AP1 or on repression of AP1 by RAR and/or RXR.5 Thus, transrepression between AP1 and the receptors does not appear to be caused by a competition for limiting amounts of TDG.
DNA repair and transcription are linked by a number of factors
participating in both processes (69) such as the c-Jun and p53
activating A/P endonuclease Ref-1 (70, 71) or the basic transcription/repair factor TFIIH (72). The fact that TDG can interact
with different transcription factors like c-Jun, RAR, RXR, or other
nuclear receptors and a transcription-related protein like TIF1
suggests that TDG could be another DNA repair enzyme which functions
also in transcription. The use of DNA repair factors like TDG in
transcription could therefore be one mechanisms of how to maintain the
integrity of transcribed regions.
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ACKNOWLEDGEMENTS |
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We are very grateful to S.M. Hollenberg for
the yeast strain L40 and the vector pBTM116, H. Nilsen and H.E. Krokan
for the cDNA clone of human UNG1, and J. Jyer for RXRAB and
RAR
AB/RXR
AB heterodimers purified from E. coli.
We thank Y. Lutz for the preparation of monoclonal antibodies against
mTDG, and our colleagues in the yeast and retinoid groups for helpful
discussions.
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FOOTNOTES |
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* This work was supported in part by CNRS, INSERM, the Center Hospitalier Universitaire Régional, the Association pour la Recherche sur le Cancer, the Collège de France, and the Fondation pour la Recherche Médical.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF069519.
INSERM postdoctoral fellow. Present address: Dept. of Obstetrics
and Gynaecology, Catholic University Medical College, 505, Banpo-dong,
Seocho-gu, Seoul 137-040, Republic of Korea.
§ These authors contributed equally to this work and should both be considered as first authors.
¶ Recipient of fellowships from the Human Capital and Mobility Program of the EEC and the Fondation pour la Recherche Médicale. Present address: RIKEN, Tsukuba Life Science Center, 3-1-1 Koyadai, Tsukuba Ibaraki 305, Japan.
Recipient of Marie Curie Long Term Fellowship ERBFMBICT961269
from the European Commission.
** Present address: Novartis Pharma AG, Molecular Genetics Unit, K-681-3.10, 4002 Basel, Switzerland.
To whom correspondence should be addressed.
The abbreviations used are: RAR, retinoic acid receptor; RXR, retinoid X receptor; TDG, thymine-DNA glycosylase; t-RA, all-trans-retinoic acid9c-RA, 9-cis-retinoic acidAAD, acidic activation domainLBD, ligand binding domain AP, abasic siteRT, reverse transcriptionPCR, polymerase chain reactionbp, base pair(s)tk, thymidine kinaseGST, glutathione S-transferaseEMSA, electrophoretic mobility shift assayCIP, calf thymus intestinal phosphatase.
2 E. Remboutsika, unpublished data.
3 M. Harbers, unpublished data.
4 S. Um, unpublished data.
5 S. Um, M. Harbers, A. Benecke, B. Pierrat, R. Losson, and P. Chambon, unpublished data.
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REFERENCES |
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