Identification of Transcripts Initiated from an Internal Promoter in the c-erbA
Locus That Encode Inhibitors of Retinoic Acid Receptor-
and Triiodothyronine Receptor Activities
Olivier Chassande1,
Alexandre Fraichard1,
Karine Gauthier,
Frédéric Flamant,
Claude Legrand,
Pierre Savatier,
Vincent Laudet and
Jacques Samarut
Laboratoire de Biologie Moléculaire et Cellulaire (A.F.,
O.C., K.G., F.F., C.L., P.S., J.S.) Centre Nationale de la
Recherche Scientifique UMR 49, Institut Nationale de la Recherche
Agronomique LA 913 Ecole Normale Supérieure de Lyon 69364
Lyon Cedex 07, France
1 Unité dOncologie
Moléculaire (V.L.) Institut Pasteur 59019 Lille,
France
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ABSTRACT
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The thyroid hormone receptor-coding locus,
c-erbA
, generates several mRNAs originating from a single primary
transcript that undergoes alternative splicing. We have identified for
the first time two new transcripts, called TR
1 and TR
2
[mRNA for isoform
1 and
2 of the T3
receptor (TR), respectively], whose transcription is initiated from an
internal promoter located within intron 7 of the c-erbA
gene. These
two new transcripts exhibit tissue-specific patterns of expression in
the mouse. These two patterns are in sharp contrast with the expression
patterns of the full-length transcripts generated from the c-erbA
locus. TR
1 and TR
2 mRNAs encode N-terminally truncated
isoforms of T3R
1 and T3R
2, respectively. The protein product of
TR
1 antagonizes the transcriptional activation elicited by
T3 and retinoic acid. This protein inhibits the
ligand-induced activating functions of T3R
1 and
9-cis-retinoic acid receptor-
but does not affect the
retinoic acid-dependent activating function of retinoic acid
receptor-
. We predict that these truncated proteins may work as
down-regulators of transcriptional activity of nuclear hormone
receptors in vivo.
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INTRODUCTION
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Thyroid hormone T3 plays important roles during
vertebrate development, especially during amphibian metamorphosis (1)
and neurogenesis in mammals (2). T3 is known to control the
transcription of specific genes through binding to nuclear receptors
(T3Rs; Ref. 3). Two loci encode the nuclear receptors of
T3. The c-erbA
locus encodes T3R
1 whereas the
c-erbAß locus encodes T3Rß1 and T3Rß2 isoforms (4). These
receptors belong to a family of nuclear receptors (5) including
retinoic acid receptors (RAR), 9-cis-retinoic acid receptors
(RXR), vitamin D3 receptors (VDR), peroxisome
proliferator-activated receptors. All these receptors contain a
DNA-binding domain and a ligand-binding domain and mediate
ligand-dependent transcriptional control of target genes. Activation or
repression of transcription requires binding of the receptors to
specific recognition sequences in target genes. Homodimerization and
heterodimerization among the various members of the nuclear receptor
family determine the target genes and repression/activation activity.
Heterodimerization allows cross-talk between different signaling
pathways (6). The preferred partners of T3R
1 are the members of the
RXR family (3).
The mechanism of the transcriptional activity of T3R
1 seems to
involve complex interactions with many other nuclear proteins such as
transcription factor IIB (TFIIB) (7), corepressors and coactivators
(reviewed in Refs. 8, 9), or cointegrators (10, 11). The
transcriptional activity of T3R
1 is regulated by products of the
c-erbA
locus itself. TR
2 RNA results from differential splicing
of the TR
primary transcript (12). The T3R
2 protein lacks
T3-binding and AF-2 domains but retains a DNA-binding
domain and can exert dominant negative activity over T3R
1 (13). The
inhibition mediated by T3R
2 is at least partly due to competition
for the DNA-binding sites and requires dephosphorylation of serine
residues whithin the C-terminal part of the protein (14). The
physiological function of T3R
2 is still unknown, although its mRNA
is expressed at higher levels than TR
1 in all tissues. N-truncated
T3R
1 proteins have been detected in chicken embryonic blood and are
thought to result from an initiation of translation at an internal site
on the TR
1 transcripts (15). These short proteins are in the
C-terminal part of T3R
1, and some of them also lack the DNA-binding
domain. They have dominant negative activities that require the ninth
heptad located in the C-terminal part of T3R
1. This ninth heptad is
known to be necessary for heterodimerization (16). Such proteins have
never been identified in mammals. With the use of ES cells in which the
c-erbA
gene had been inactivated by homologous recombination, we
identified for the first time endogenous transcripts generated from an
internal promoter of the mouse c-erbA
locus. These new transcripts
were also detected in wild type ES cells. They exhibit specific
patterns of expression in the mouse, and they direct the synthesis of
proteins that down-regulate transactivation elicited by T3R
1 and
RAR
.
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RESULTS
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Identification of New Transcripts from the c-erbA
Locus
ES cells containing an inactivated c-erbA
gene were generated
by homologous recombination. For this purpose, exon 2 was disrupted
after the second base of the ATG initiation codon by inserting a
plasmid region containing the Lac Z gene, two transcription
stops, polyadenylation signals (Fig. 1A
and Ref. 17),
and the neoR gene driven by the ß-actin promoter.
Heterozygous cell lines (c-erbA
±) were grown at high
G418 concentration (18) to select for homozygous clones in which both
alleles of the c-erbA
gene were disrupted
(c-erbA
-/-). Two independent
c-erbA
-/- clones were isolated and used
for subsequent analysis. To check that both alleles of the gene were
inactivated, a RNAse protection experiment was performed using 20 µg
total RNA either from wild type or from the two
c-erbA
-/- ES cell lines. The I7E8 RNA
probe covering intron 7 and exon 8 was used (Fig. 2A
).
After a 24-h exposure, only one protected fragment of 238 nucleotides,
corresponding to TR
RNA, could be detected in wild type cells under
these conditions. This fragment was not detected in the RNA of mutant
c-erbA
-/- ES cells (Fig. 2B
, lane Cl10 and
Cl20). The absence of TR
transcripts was further confirmed by RT-PCR
using oligonucleotide 5S positioned in exon 5 and oligonucleotides 1A
or 2A positioned in exons 9 (TR
1-specific) and exon 10
(TR
2-specific) as primers (see position in Fig. 1B
). No
amplification occurred in c-erbA
-/- ES
cells (Fig. 2D
)

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Figure 2. Identification of New Transcripts Generated from
the c-erbA Gene in ES Cells
A, Structure of the I7E8 probe used for the RNase protection
experiments. 238 is the size of the fragment protected by the TR
mRNA. B, RNase protection with 20 µg total RNA from wild type (CGR8)
or c-erbA -/- (Cl10, Cl20) ES cells. C,
RNase protection with 10 µg polyA+ RNA from CGR8 cells.
D, RT-PCR with RNA from wild type (CGR8) and
c-erbA -/- (Cl10, Cl20) ES cells. Thirty
five cycles (95 C, 15 sec; 56 C, 15 sec; 72 C, 30 sec) were carried out
using oligonucleotides 5S and 1A, 8S and 1A, 5S and 2A, and 8S and
2A.
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When RT-PCR was performed using oligonucleotide 8S (exon 8) as primer
in 5'-position (see position in Fig. 1B
), amplification occurred in
both c-erbA
-/- clones and wild type ES
cells (Fig. 2D
), using either 1A or 2A oligonucleotide as primer in the
3'-position. These observations led us to suggest that another RNA was
transcribed from an origin located upstream of exon 8 and was processed
to generate both TR
1-like (called TR
1) and TR
2-like (called
TR
2) isoforms. To identify the 5'-end and origin of these
transcripts, 5'-rapid amplification of cDNA ends (RACE) experiments
were performed using RNAs either from wild type or from
c-erbA
-/- ES cells.
A single DNA fragment was amplified from two independent
c-erbA
-/- ES cell clones and from the wild
type cells (data not shown). Sequence analysis of this fragment
revealed that the 3'-end showed 100% identity with the sequence of the
mouse c-erbA
1 cDNA. The alignment of the two sequences was
interrupted at a position corresponding to the junction between exons 7
and 8 (19). Further PCR cloning and sequencing of mouse intron 7 showed
that the first 33 nucleotides of the amplified cDNA originated from the
3'-end of intron 7. These results suggested that a transcript
containing sequences from intron 7 of the c-erbA
gene was produced
in ES cells. To test whether this transcript was initiated in intron 7,
ribonuclease (RNAse) protection assays using the I7E8 probe were
performed (Fig. 2A
). With 10 µg of polyA+ RNA as starting
material, a triplet of new protected fragments was detected, in
addition to the 238-nucleotide fragment protected by the TR
RNA
(Fig. 2C
). The size of the protected RNA fragments ranged between 292
and 310 nucleotides, corresponding to an extension of 54 to 72
nucleotides at the 5'-end compared with the TR
-specific protected
fragment. These fragments were only detected in RNAs extracted from ES
cells and from lungs (data not shown), in agreement with RT-PCR results
showing that the highest levels of TR
1 and TR
2 are detected
in these tissues (see below). These results show that a new mRNA is
transcribed from position -74/-54 upstream from the intron 7/exon 8
boundary in the c-erbA
locus.
The presence of transcripts initiated within intron 7 was further
assessed by RT-PCR amplification of RNA from different species, using
an oligonucleotide complementary to the 3'-end of c-erbA
intron 7
from mouse, human, or chicken. A cDNA was amplified from human
promonocytes U937, 7-day-old chicken embryos, and mouse ES cells (Fig. 3A
). These data suggest that transcripts initiated in
intron 7 may be present in all vertebrates.

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Figure 3. TR Transcripts Are Initiated in Intron 7
A, Amplification of a TR 1 RNA in human (Hu), chicken (Ch), and
mouse (Mo). The size of the PCR products is indicated in base pairs on
the left. Oligonucleotides used for RT-PCR with RNA from
ES cells are VIIS and 1A. Cycle parameters are 95 C, 15 sec; 56 C, 15
sec; and 72 C, 15 sec. Thirty three cycles were performed.
Oligonucleotides used for RT-PCR with U937 RNA are hVIIS and 1A. Cycle
parameters are 95 C, 15 sec; 56 C, 15 sec; and 72 C, 15 sec. Thirty
three cycles were performed. Fifty nanograms of reverse transcribed
U937 RNA were used in both experiments. Oligonucleotides used for
RT-PCR with 7-day-old chicken embryo RNA are cVIIS and c9A. Cycle
parameters are 95 C, 15 sec; 65 C, 15 sec; and 72 C, 15 sec. Thirty
five cycles were performed. Fifty nanograms of reverse transcribed
polyA+ RNA were used in the reaction. B, Intron 7 promoter
activity: The PstI-PstI c-erbA genomic
fragment was cloned upstream from a CAT expression vector in sense (S)
or antisense (AS) orientation. ES, HeLa, or COS7 cells were transfected
with 5 µg of each plasmid. Cells were harvested 48 h later and
analyzed by CAT-ELISA. The results were normalized: 100% is the
relative amount of CAT detected with 0.2 µg of CMV-CAT plasmid. C,
Sequence of intron 7: The last 26 nucleotides of exon 7 and the 60
first nucleotides of exon 8 are indicated in small capital
characters. The sequence of the intron 7 is written in
capital letters. Consensus binding sites are
boxed. The sequence of oligonucleotide VIIS is
underlined and arrowed. The
PstI site within intron 7 is underlined.
The segment carrying the transcription initiation sites is
overlined with a dashed line. Numbers +43 and +52
indicate the position of the putative ATG codons within exon 8. The
human sequence is indicated in lowercase letters, and
homologies with the mouse sequence are indicated by
stars.
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Identification of a Promoter within the c-erbA
Locus
The putative promoter activity of intron 7 was then investigated
in a transient expression experiment. For this purpose, a DNA fragment
containing the last 276 nucleotides of mouse intron 7 and the first 236
nucleotides of exon 8 was inserted upstream from the chloramphenicol
acetyltransferase (CAT) coding sequence. Its promoter activity was
tested by transfection into ES, HeLa, and COS7 cells, in comparison
with a cytomegalovirus (CMV) promoter. As shown in Fig. 3B
, the intron
7 in the sense orientation (S) efficiently promoted the transcription
of the CAT gene in the three cell lines tested. In contrast, no
significant expression was found when intron 7 was inserted in the
antisense orientation. These data establish the existence of a
transcriptional promoter within intron 7 of the mouse c-erbA
gene.
Sequence analysis of mouse intron 7 revealed consensus binding
sites for the transcription factors AP-1, CAAT binding transcription
factor (CTF), Sp1, the glucocorticoid receptor, and the protooncogene
c-Ets (Fig. 3C
). Comparison between human and mouse sequences showed
two conserved blocks (Fig. 3C
). Seventy three percent identity was
found between human and mouse in the last 62 bp of intron 7, probably
corresponding to conserved splicing signals. Strikingly, a
42-nucleotide stretch located at position -130/-89 (referring to the
intron 7/exon 8 boundary) in the mouse sequence showed 75% identity
with a human -180/-138 fragment. These homologies suggest that the
promoter activity of intron 7 may be conserved in humans.
Molecular Cloning of TR
1 and TR
2 Short Transcripts
Using 3'-RACE methodology, we cloned the 3'-end of TR
1 and
TR
2 RNAs using mutant c-erbA
-/- ES
cells RNA as starting material. A first round of PCR amplification was
carried out using oligonucleotide VIIS as the 5'-primer (see position
in Fig. 1B
) to selectively amplify cDNAs initiated in intron 7. A
second round was performed with either oligonucleotides 1S or 2S to
specifically amplify
1 or
2 isoforms, respectively. Products of
amplification displayed 100% homology with TR
1 or TR
2 in the
coding regions. 3'-Noncoding regions were homologous to those described
for rat or human TR
1 (gb M18028 and emb X55005) and TR
2 RNAs (emb
X07409 and emb X55066). Antisense primers IXA and XA (Fig. 1B
) were
designed from the 3'-untranslated portions of TR
1 and TR
2
RNAs, respectively, and used together with VIIS to amplify full-length
TR
1 and TR
2 RNAs by RT-PCR. Each amplification generated
one single band, establishing the existence of TR
1 and TR
2
RNAs in both c-erbA
-/- and wild type ES
cells (data not shown). Three clones obtained from each amplification
product had identical sequences to the previously published mouse
TR
1 and TR
2 RNA, respectively (20) with the exception of the
5'-short intronic sequence. We can therefore conclude that the
c-erbA
gene encodes two related RNAs, TR
1 and TR
2, from
an internal promoter located in intron 7. The structure of these
transcripts is depicted and compared with the known structures of the
TR
1 and TR
2 transcripts in Fig. 4
. Assuming that
TR
1 and TR
2 lack the first 1170 nucleotides of their
full-length counterparts, their length can be predicted to be 4.5 kb
and 1.5 kb, respectively (Fig. 4A
). This prediction was checked by
Northern blot analysis of polyA+ RNA extracted from ES
cells, using a mixture of antisense oligonucleotides located within
exons 8 and 9 as probes. Two transcripts of 4.5 (TR
1) and 1.5 kb
(TR
2) were detected in addition to the 5.5-kb (TR
1) and 2.5-kb
(TR
2) transcripts (Fig. 4B
, lane ES) in ES cells. The ratio of the
different isoforms was TR
1 = 0.5, TR
1 = 1,
TR
2 = 2, TR
2 = 10. The shorter transcripts were not
detected in liver (Fig. 4B
, lane L).

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Figure 4. The TR and TR Transcripts
A, Schematical representation of the transcripts generated by the
c-erbA locus. Gray staining: region common to 1 and 2
isoforms. Light hatched blocks indicate region specific
to 1-isoforms. Heavy hatched blocks show region
specific to 2-isoforms. Black blocks indicate 5'-end
specific to TR mRNAs. Upstream region from the c-erbA gene and
the 5'-end of the TR mRNAs are not shown (horizontal dashed
blocks), and 3'- untranslated regions of RNAs are shown as
lines. B, Identification of all TR related
transcripts in ES cells (ES) and mouse liver (L) by Northern blot
analysis.The oligonucleotides used for hybridization were 9A, 15A, and
3A (located in exons 9, 9, and 8, respectively). The membrane was
autoradiographed for 4 days with a XAR5 film (Kodak, Rochester, NY).
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Expression of TR
RNAs Is Regulated in Mouse Tissues and
During ES Cells Differentiation
The patterns of expression of TR
1, TR
1, TR
2, and
TR
2 mRNAs were analyzed in different mouse tissues, using
semiquantitative RT-PCR (Fig. 5A
). As expected, the
TR
transcripts were detected in all tissues. TR
1 displayed a
predominant expression in gut, heart, lung, muscle, and brain and a
lower expression in spleen, liver, testis, thymus, and ES cells. TR
2
was found to be expressed predominantly in brain, gut, heart, lung,
kidney, and muscle. In contrast, TR
transcripts displayed a more
restricted pattern of expression. The highest amount of TR
1 was
found in lung. Lower amounts were detected in gut and in ES cells, and
no transcript was detected in muscle, spleen, thymus, liver, and
testis. The highest amount of TR
2 was found in ES cells and in
lung. Lower amounts were observed in gut and brain, and no transcript
was detected in kidney, muscle, spleen, liver, and testis. These data
show that the TR
and TR
RNAs have distinct patterns of
expression, suggesting that their transcription may be differentially
controlled. To test this hypothesis, we examined the variations in the
expression of TR
and TR
transcripts during the differentiation
of ES cells. Using a RNAse protection assay, we showed that 4 days
after the induction of the differentiation by removing leukemia
inhibiting factor, the transcripts initiated in intron 7 were no more
detectable, whereas the level of TR
RNAs increased (Fig. 5B
). These
results were confirmed by RT-PCR analysis (data not shown). They bring
further evidence for independent controls of the activities of both
promoters. Moreover, these data suggest that the products of the
TR
transcripts may be important in maintaining the ES cells in an
undifferentiated state.

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Figure 5. Pattern of Expression of TR Transcripts in
the Mouse
A, RT-PCR analysis of the expression pattern of long and short
transcripts in adult mouse tissues and in ES cells. Pairs of
oligonucleotides used to amplify TR 1, TR 2, TR 1, TR 2,
and HPRT cDNAs are 5S/1A, 5S/2A, VIIS/1A, VIIS/2A, and HPRTs/HPRTa.
Fifty nanograms of reverse-transcribed RNA were used in each
amplification reaction. Forty cycles were performed to amplify
TR 2 cDNAs, 35 cycles to amplify TR 1 and TR 2 cDNAs, and
30 cycles to amplify TR 1 cDNAs (95 C, 15 sec, 56 C, 15 sec, 72 C, 20
sec). Twenty eight cycles (95 C, 15 sec; 52 C, 15 sec; 72 C, 15 sec)
were performed to amplify HPRT cDNA. B, Brain; G, gut; H, heart; K,
kidney; Li, liver; Lu, lung; M, muscle; S, spleen; Te, testis; Th,
thymus; ES, ES cells. B, RNase protection analysis of RNA from
undifferentiated or differentiated ES cells. Cells were grown in the
presence (+LIF) or absence (-LIF) for 4 days. Ten micrograms of polyA+
RNA obtained from each culture were incubated with a I7E8 probe
(cf. Fig. 2A ) for RNase protection analysis. The
positions of specific bands are indicated. The gel was exposed for
4 h to detect the c-erbA transcripts and 30 h to detect
the TR transcripts.
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Translation of TR
1 and TR
2 RNAs in HeLa Cells
Both TR
1 and TR
2 cDNAs display open reading frames
starting from two potential translation initiation codons located 43
and 52 nucleotides, respectively, downstream from the first nucleotide
of exon 8 (see position in Fig. 3A
). Interestingly, both codons are
highly conserved in fish, Xenopus, chicken, mouse, rat, and
human (22). The reading frames are identical to those of TR
1 and
TR
2. The predicted proteins would be N-terminally truncated forms of
T3R
1 or T3R
2 proteins, with molecular weights of 16,000 and
26,000, respectively. They would lack the DNA-binding domain, the hinge
region, the nuclear-addressing signals, and the N-terminal part of the
ligand- binding domain of the full-length T3R
proteins (Fig. 6
and Refs. 23, 24). Because none of these truncated
proteins would retain a complete ligand-binding domain, they would have
lost the capacity to bind T3 (25).
All our attempts to detect the endogenous products in ES cells have
been unsuccessful. Like their full-length counterparts T3R
1 and
T3R
2, the endogenous proteins translated from the TR
1 and
TR
2 RNAs are probably expressed at low level in most tissues. In
addition, the available antibodies do not have a sufficient affinity
because endogenous T3R
1 cannot be detected in ES cells either. To
assess the ability of the two TR
transcripts to encode the
predicted proteins, the TR
1 and TR
2 cDNAs were cloned into
pSG5 expression vector (pSGTR
1 and pSGTR
2, respectively),
which does not contain any translation initiation signals, and
transfected into HeLa cells. Analysis of the cellular proteins by
Western blotting revealed that TR
1 RNA encodes one single
polypeptide of approximately 16 kDa, called p16
1, and TR
2 RNA
yields one single polypeptide of approximately 26 kDa, called p26
2
(Fig. 7A
). The half-life of these proteins in HeLa cells
was estimated in pulse-chase experiments to be 1 h for p16
1 and
23 h for p26
2 (Fig. 7B
). The cellular localization of transiently
expressed p16
1 and p26
2 was investigated by in situ
immunofluorescence in HeLa cells. p16
1 exhibited both cytoplasmic
and nuclear localization (Fig. 7C
, panel 1) whereas p26
2 was
predominantly in the cytoplasm (Fig. 7C
, panel 2). These localizations
differ from that of nuclear receptors like T3R
1, which exhibit a
strict nuclear localization (data not shown).
Functional Interference of p16
1 and p26
2 with T3Rs and RAR
in Vivo
Artificial constructs that express C-terminal portions of the
T3R
1 receptor have been shown to behave as repressors of the
transactivation mediated either by T3R
1 or by RAR
(26).
Therefore, we investigated the possibility that the p16
1 and p26
2
proteins might be natural antagonists of nuclear receptors. We first
examined the effect of these proteins on the T3-dependent
transcriptional activity of T3R
1, as compared with the well
described inhibition exerted by T3R
2. One or 4 µg of pSGTR
1,
pSGTR
2, or pSGTR
2 expression vectors were cotransfected with
an expression vector for T3R
1. Transcriptional activity was revealed
from a cotransfected reporter plasmid in which the CAT gene is driven
by a core globin promoter downstream from a palindromic
T3-responsive element (inverted repeat; IR0). Data
presented in Fig. 8A
show that both p16
1 and p26
2
inhibited the transcription mediated by T3R
1 in a dose-dependent
manner. This repressing activity was comparable to the one of TR
2.
These inhibitory activities required the translation of the cDNAs
because another mutated construct, pSGTR
T, which does not enable the
production of p16
1 but generates TR
1 transcripts, was unable
to repress the transcriptional activation by T3.

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Figure 8. Effect of p16 1 and p26 2 on the
Transcriptional Activity of Different Promoters and Nuclear Receptors
on Their Responsive Elements in HeLa Cells
All results are expressed as the percentage of CAT amount. A, Effect of
p16 1 and p26 2 on the transcriptional activity of liganded T3R .
The transcriptional activity of T3R 1 was measured by transfecting
HeLa cells with 0.8 µg of IR0 globinCAT (IR0; Ref. 41) together with
1 µg of pSGTR 1 (provided by Dr B. Vennström, Sweden). One or
4 µg of translationally silent vector ( T) or of vector encoding
T3R 2, p16 1 (p16), or p26 2 (p26) was cotransfected. Each
transfection mixture was completed with pSG5 plasmid up to 4 µg of
SV40-containing plasmid. Activities were measured after addition of
10-7 M T3 for 24 h in 1%
serum. B, Specificity of the activities of p16 1 and p26 2: 0.1
µg of CMV-CAT, 2 µg of ß-actin CAT, or 2 µg of SV40-CAT were
used in the transfections; 4 µg of pSG5 (solid bars),
pSGTR 1 (open bars), or pSGTR 2 (dotted
bars) was cotransfected. C, Mapping of the inhibitory activity
within p16 1. The experimental conditions are as in panel A. Four
micrograms of pSG5 vector carrying either mutant TR T (-), mutant
256369, mutant 256319, or mutant 320408 were cotransfected. D,
Effect of p16 1 toward T3R 1, T3Rß1, and RAR . The
transcriptional activity of T3R 1 on the DR4 response element was
measured by cotransfecting 1 µg pSGTRa1 and 0.4 µg malic enzyme TRE
TK CAT (DR4; Ref. 41). The activity of T3Rß1 was measured by
transfecting 1 µg pSGTRß1 together with either 0.8 µg IR0 globin
CAT or 0.4 µg malic enzyme TRE TK CAT plasmids. The transcriptional
activity of RAR was measured by transfecting HeLa cells with 0.2 µg
pSGRAR (provided by Professor P. Chambon, Strasbourg, France) and
0.8 µg IR0 globinCAT, or 0.4 µg RAREß TKCAT (DR5, provided by
Professor P. Chambon). In every assay, 4 µg of either pSGTR T (0)
or pSGTR 1 (4 ) were cotransfected. The transfected cells were incubated in the presence or absence of 10-7 M
T3 or 10-6 M
all-trans-RA for 24 h. In the absence of hormone,
the relative amount of CAT detected was very low (<1% of the relative
amount in presence of hormone). The results of the experiment presented
in this figure have been reproducible in at least four independent
experiments.
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We checked whether this inhibition could be the result of
nonspecific effects on the transcriptional machinery (Fig. 8B
). Whereas
p16
1 did not significantly modify the activity of either SV40, CMV,
or ß-actin promoters, p26
2 strongly repressed all promoters.
Therefore, p16
1 behaved as a specific antagonist of the
T3-dependent T3R
1 activity, but p26
2 had a more
general inhibitory effect toward transcription.
To map the inhibitory activity of p16
1, we generated truncated
variants of this protein (see Fig. 6
for the position of truncations)
and investigated their ability to repress the transcriptional activity
of T3R
1. The efficient production of all variant proteins in
transfected cells was checked by Western blot analysis (data not
shown). Figure 8C
shows that a mutant protein that retained the
N-terminal portion common to p16
1 and p26
2 (amino acids 256 to
369) was still a potent repressor. This protein does not contain the
ninth heptad repeat necessary for heterodimerization of receptors with
RXR (30). Moreover, a shorter truncation mutant (256319), which
lacked most of the heptad repeats involved in homodimerization of the
receptors, inhibited T3R
1. This protein retained a domain previously
identified as
3 (31). In contrast, the complementary C-terminal
domain (320410) displayed no inhibition.
We investigated the repressing activity of p16
1 toward different
nuclear receptors associated with different hormone response elements,
in the presence of their ligand (Fig. 8D
). A strong repression of
T3R
1 was observed in the context of the malic enzyme T3
response element (DR4-type). The activity of T3Rß1 was repressed both
in the context of the IR0 and the DR4 response element. The activity of
endogenous RARs on the RARß RA response element (DR5-type) was only
partially (60%) but reproducibly inhibited. In contrast, the
RA-dependent activation of the IR0 obtained by the cotransfection of
0.2 µg of pSGRAR
was efficiently repressed.
Mechanism of Action of p16
1
We investigated the mechanism by which p16
1 could repress the
activities of the receptors. The transcriptional activity of the
receptors requires binding to DNA, heterodimerization, and activation
of transcription. We tested in vivo the possible
interference of p16
1 with either of these three steps. To assay the
effect of p16
1 on DNA binding, we tested its activity on the
ligand-independent transcriptional repression of RAR
and T3R
1.
Figure 9A
shows that the active repression mediated by
these unliganded receptors on a IR0 response element was not affected
by p16
1. In agreement with these data, in vitro binding
of T3R
1 to DR4 or palindromic response elements was unaffected by
p16
1, as assayed by gel shift experiments (data not shown). These
data rule out a squelching activity of p16
1 toward the receptors,
which would either prevent binding of the receptors to DNA or their
nuclear transport.

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Figure 9. Effect of p16 1 on Ligand-Independent Repression,
Heterodimerization, and Transcriptional Activation
A, Effect of p16 1 on the repression activity of unliganded
receptor. Transfection experiments are as in Fig. 8 , A or C, except
that no ligand was added. Zero or 4 µg of p16 1 expressing vector
pSGTR 1 was cotransfected. Four micrograms of pSGTR T were used
as control. The results are expressed as the percentage of the amount
of CAT enzyme produced under the control of the IR0 globin promoter. B,
Effect of p16 1 on the heterodimerization between RAR or T3R 1
and RXR : 0.4 µg of (UAS)4TKCAT reporter plasmid was
cotransfected with 0.25 µg of pCMXGal4-RAR (GalRAR) or
pSVGal4-TR 1 (GalT3R) in the presence or absence of 0.5 µg of
pCMXVP16-RXR (VPRXR). C, Effect of p16 1 on the activating
functions of T3R 1, RAR , and RXR : 0.4 µg of
(UAS)4TKCAT reporter plasmid was cotransfected with 0.25
µg of either pCMXGal4-RAR (GalRAR), pCMXGal4-RXR (GalRXR), or
pSVGal4-T3R (GalT3R). Two micrograms of pSGTR 1 and 2 µg of
pSGTR T (2 ), or 4 µg of pSGTR 1 (4 ), were cotransfected. Four
micrograms of pSGTR T (0) were co-transfected in the control
experiments. The cells were treated by 10-8 M
of all-trans-retinoic acid (RA) or of
9-cis-RA. D, Effect of p16 1 on RAR /RAR
homodimers and RAR /RXR heterodimers. The activity of
RAR /RAR homodimers was measured using 1 µg pSGRAR and 0.8
µg IR0 Globin CAT; the activity of RAR /RXR heterodimers was
measured by cotransfection of 1 µg pSGRAR , 0.3 µg pSG RXR ,
and 0.8 µg IR0 Globin CAT. Four micrograms of either pSGTR T (0) or
pSGTR 1 (4 ) were cotransfected. The cells were treated by
10-6 M all-trans- retinoic acid
(RA). The results of the experiment presented in this figure have been
reproducible in at least four independent experiments.
|
|
We then tested the possibility that p16
1 could interfere with the
heterodimerization of the receptors with RXR. Therefore, we used a
double hybrid system in which a Gal4 DNA-binding domain fused to either
RAR
or T3R
1 ligand-binding domain was coexpressed in HeLa cells
together with a VP16-activating domain fused to RXR ligand-binding
domain. Their transcriptional activity was revealed in the presence of
a reporter vector consisting of a CAT gene under the control of four
Gal4 binding sites upstream of a minimal thymidine kinase (TK) promoter
(UAS4CAT). Figure 9B
shows that, whereas unliganded
Gal4-RAR or Gal4T3R alone did not exhibit a significant transcriptional
activity, coexpression of either of them with VP16-RXR produced a
strong activation, which could not be inhibited by p16
1. Therefore,
p16
1 was unable to prevent the interaction between RAR
and RXR
or T3R
1 and RXR
. This is consistent with data from
electromobility shift assays that showed that this protein could not
prevent or modify the binding of T3R
1-RXR
heterodimers to IR0 or
DR4 response elements (data not shown).
Finally, we tested the effect of p16
1 on the activating functions of
T3R
1, RAR
, and RXR
. Therefore, we used chimeric proteins
consisting of the hinge and ligand-binding regions of the T3R
1,
RAR
, and RXR
receptors fused to the DNA-binding domain of
Gal4 (27). These chimera were coexpressed with a UAS4CAT
vector. The activation of Gal4T3R or Gal4RXR by their respective
ligands was efficiently inhibited by p16
1 (Fig. 9C
). In contrast,
the activation of Gal4RAR by retinoic acid was unaffected. These data
suggest that p16
1 does not affect the activating function of RAR
but interferes with the transcriptional activation domains, or
activating functions, of T3R
1 and RXR
.
The absence of inhibition of the activating function of RAR
is in
apparent contradiction with data presented in Fig. 8D
showing that the
activity of the full-length receptor on different response elements
could be at least partially inhibited by p16
1. We repeated the
experiment described in Fig. 8D
, using 1 µg pSGRAR
(Fig. 9D
). We
assumed that, in these new conditions, RAR
homodimers were the
predominant active complex. Given this assumption, we expected that
raising the amount of RXR
would result in the preferential formation
of heterodimers, leading to an enhanced activation of the IR0 (27).
Indeed, cotransfection of 1 µg pSGRAR
and 0.3 µg pSGRXR
produced a 2.2-fold enhanced RA-mediated activation of IR0, as compared
with the activation observed with RAR
alone. Interestingly, p16
1
was unable to inhibit the activity of homodimers but reduced the
activity of the heterodimers by about 60%, as was observed using the
DR5 response element. We conclude that p16
1 can antagonize the
ligand-dependent activation of RAR
, but only when it acts as RAR-RXR
heterodimer.
 |
DISCUSSION
|
---|
All the cDNAs reported to date for the c-erbA
locus correspond
to mRNAs initiated by the c-erbA
promoter (5): TR
1, coding for a
T3 receptor, and TR
2, coding for a protein that does not
bind T3. We have cloned and characterized two new
transcripts of the c-erbA
locus: TR
1 and TR
2, which
begin in intron 7 and are otherwise identical to the 3'-part of TR
1
and TR
2, respectively. These transcripts were first identified in
c-erbA
-/- ES cells, but they were
subsequently cloned from wild type ES cells. They were also found in
several wild type mouse tissues. They were detected in chicken and in
human, suggesting that they are probably present in all vertebrates.
These data show unambiguously that these isoforms are natural products
of this locus and that they are not the result of c-erbA
gene
inactivation in c-erbA
-/- ES cells.
The promoter responsible for the transcription of these isoforms is
located in intron 7, and we confirmed its activity in several cell
types. The existence of a specific transcriptional control of the
expression of the TR
RNAs through this promoter is further
suggested by the differences observed in the patterns of expression of
the TR
and TR
transcripts. The opposite variation of their
amount during the differentiation of ES cells suggests that the
regulation of the c-erbA
promoter is different from that of intron 7
promoter. The mRNAs generated by the intron 7 promoter are spliced to
generate the
1 or
2 isoforms, suggesting that the mechanisms that
control the splicing do not require the presence of sequences upstream
of exon 8.
N-terminally truncated T3R
proteins of 27 to 46 kDa had previously
been described in chicken (15). These proteins can exert a dominant
negative activity toward T3R
1 and RAR
transcriptional activation
(26). We have shown that the product of the TR
1 mRNA that we have
identified, p16
1, also has a dominant negative activity toward
T3R
1, T3Rß1, and RAR
. This protein does not affect
receptor-independent transcription. In contrast, analyzing the function
of p26
2 is hindered by the fact that it can repress the activity of
all promoters tested to date, suggesting a broad range of action. This
might involve a nonspecific inhibition of general transcription factors
as was previously hypothesized for T3R
2 (29). However, we cannot
exclude the possibility that p26
2 may also have a specific
inhibitory action toward nuclear receptors, as was shown for T3R
2
(14). The exact mechanism responsible for the inhibitory activity of
p16
1 is still unclear. Our data rule out the possibility that
p16
1 may prevent the receptors from forming heterodimers with RXRs
or from binding to DNA. They rather suggest that it exerts its action
by preventing the activity of a coactivator normally recruited by T3R
or RXR. The fact that the activating function of RAR
is not affected
by p16
1 is consistent with the absence of inhibition of
RAR
/RAR
homodimers. Heterodimerization of RAR
with RXR
generates a complex in which both partners undergo interdependent
conformational changes in response to different ligands. The
transcriptional activities of this complex can be at least partially
antagonized by p16
1. Since the proper activating function of RAR
is not inhibited, this suggests that, in heterodimers, the activating
function of RXR
is the main target of p16
1. To date, no cofactor
of nuclear receptors that interacts with T3Rs and RXRs, but does not
bind to RARs, has been identified. Using C-terminally truncated
mutants, we have shown that the region of p16
1 that contains amino
acids 256 to 319 is sufficient to perform inhibition. This region
contains four heptad repeats and the
3 domain that provides
transcriptional activity when fused to the Gal4 DNA-binding domain
(31). However, no protein is known to interact with this region. A
possibility would be that the
3-domain, which is hidden within the
T3R
1 protein (32), is able to bind and sequester a coactivator when
exposed at the N-terminus of p16
1. If this coactivator is one of the
factors normally recruited by AF-2 in the presence of ligand, then it
will not be available in the presence of p16
1. Alternatively, the
3-domain within the receptor may be exposed in some circumstances
during the processing of the protein and recruit a factor essential for
its activity. The interaction between p16
1 and the target factor may
take place either in the nucleus or in the cytoplasm because p16
1
can be found within both compartments. This interaction might result,
for example, in sequestering of the coactivator in the cytoplasm.
Identification of this target factor will be necessary to further
understand the mechanisms of inhibition by p16
1.
The biological functions of the proteins p16
1 and p26
2 are
unknown. Our data suggest that p16
1 may block some of the functions
of the nuclear receptors, such as transcriptional activation of target
genes, without affecting other functions such as the activities of
aporeceptors. In contrast, p26
2 may have a broader spectrum of
repressive activities. We propose that these truncated proteins could
work as negative regulators of nuclear receptors activity in
vivo. ES cells harboring a homozygous deletion of the c-erbA
locus that includes exon 8 have been generated, thus preventing the
synthesis of both TR
and TR
transcripts (32a). Interestingly
the authors have reported that these cells are more responsive to RA
treatment than wild type ES cells, whereas our
c-erbA
-/- cells, producing TR
but no
TR
mRNAs, behave as wild type ES cells (our unpublished data).
Therefore this enhanced response to RA could be explained by the
disappearance of an inhibitor of the transcriptional activation pathway
of the RARs that is still present in our
c-erbA
-/- cells. We propose that p16
1
and p26
2 could be such inhibitors. It could be argued that the ratio
between the pSGTR
and pSGTR
1 vectors that must be reached to
obtain inhibition in transient expression experiments in HeLa cells is
very high as compared with the respective levels of endogenous
transcripts in ES cells. According to the coactivator hypothesis, the
amount of p16
1 necessary to achieve inhibition within a given cell
must be proportional to the amount of target coactivator present in
this cell. This amount is probably different according to the cell type
considered. Therefore, a smaller amount of p16
1 may be required to
obtain full inhibition in ES cells than in HeLa cells, if we assume
that the amount of coactivator is lower in ES cells than in HeLa
cells.
We propose that the function of p16
1 and p26
2 in ES cells could
be to prevent the activation of T3 or RA receptors by low
levels of exogenous ligands. They could therefore participate in the
maintenance of ES cells in the undifferentiated state. This hypothesis
is supported by the observation that TR
transcripts are expressed
in undifferentiated ES cells but disappear upon differentiation as the
level of long transcripts increases.
The activity of truncated proteins as potent inhibitors of their
full-length counterpart is now well established. For example, the same
locus generates a transcriptional activator LAP (liver-enriched
transcriptional activator protein) but also a truncated isoform LIP
(liver-enriched transcriptional inhibitory protein) which behaves as an
inhibitor of LAP (33). In the CREM (cAMP response element modulator)
locus an inhibitory truncated isoform is produced from an internal
promoter (34). In the nuclear hormone receptor family several recently
cloned truncated products exhibit inhibitory properties. VDR0 (vitamin
D receptor) (35) and RAR
(36) are inhibited by the truncated
isoforms VDR1 and RARßm382, respectively. In both cases, activators
and repressors are produced by the same locus. Inhibition of T3R and
RAR activities by the truncated protein p16
1 is a novel example of
this mechanism of regulation.
 |
MATERIALS AND METHODS
|
---|
Cells, Vectors, Transfections, and Reagents
CGR8 (ES) cells were cultured as previously described (37). HeLa
cells were transfected using a calcium phosphate procedure, as
previously described (38). Quantitative determination of CAT enzyme was
carried out using the CAT-ELISA kit (Boehringer Mannheim, Meylan,
France) and a kinetic microplate reader (Molecular Devices, Menlo Park,
CA). Gal4DBD-hRAR
LBD, Gal4DBD-hRXR
LBD, and VP16-RAR
expression
vectors are gifts from Dr R. Evans (27). pCAT4, in which four copies of
the Gal4-binding sites are cloned upstream of a thymidine kinase
minimal promoter, is a gift from Dr. Busslinger. RAR-specific ligand Ro
406055 and RXR-specific ligand Ro257386 are gifts from Dr. Michael
Klaus from Hoffman LaRoche (Nutley, NJ).
Homologous Recombination and Production of Homozygous ES
Cells
The replacement vector,
HRVTK, was constructed by sequential
insertion of genomic fragments of the mouse c-erbA
gene into a
modified pGNA vector (17). The pGNAß plasmid was first generated by
replacing the Rous sarcoma virus long terminal repeat driving the
neoR gene by a rat ß-actin promoter (kindly provided by
R. Beddington, Mill Hill, England). The genomic fragments were cloned
from a 129/SV mouse genomic library (gift from Dr J. P. Magaud, Lyon,
France) using an exon 1-specific probe. The 5'-fragment was synthesized
by PCR amplification using oligonucleotides RS and RA as primers (Fig. 1A
), then cloned into pGNAß to generate pGNAß5'. The 3'-fragment
was first selected as a subgenomic fragment hybridizing to an exon
2-specific oligonucleotide and treated with Bal31 nuclease to remove
remaining exon 2 sequences. Digestion products were inserted into
pGNAß5' to generate
HRV. A XhoI-SalI HSV-TK
fragment isolated from MC1TK (gift of Dr Capecchi) vector was cloned
into SalI in
HRV to generate the targeting vector
HRVTK (Fig. 1A
).
ES cells were electroporated with 40 µg
HRVTK vector linearized by
SalI digestion (250 V and 500 µFarads). Cells were
selected in 250 mg/ml G418. Neo-resistant clones were picked up,
amplified, and screened by PCR and Southern blot. Two independent
heterozygous clones were cultured in 2 mg/ml G418 to generate
homozygous cells (18). G418-resistant clones were screened for the
disruption of both alleles of the c-erbA
gene by Southern
blotting.
RT-PCR Analysis
RNAs were extracted by a guanidinium thiocyanate/acidic phenol
method. Oligonucleotides were from Genset (Paris, France). Their
positions are displayed in Fig. 1
, and their sequences are given below.
Reverse transcription was performed as follows, unless specified: 1
µg total RNAs and 0.5 µg random primers (Promega, Madison, WI) were
mixed in 10 µl, heated to 68 C for 5 min, and cooled to 37 C. Ten
microliters of polymerization mix (100 mM Tris-Cl, pH 8.3,
150 mM KCl, 20 mM dithiothreitol, 6
mM MgCl2, 0.5 mM each
deoxynucleoside triphosphate) were added, and the mixture was incubated
at 37 C for 1 h. The reaction was stopped by heating at 68 C for 5
min. PCR was performed using Goldstar thermostable polymerase
(Eurogentech, Seraing, Belgium) on a Perkin Elmer (Norwalk, CT) 9600
thermocycler. The reaction was performed in Goldstar reaction buffer
containing 2 mM MgCl2 and using 100 ng of each
oligonucleotide and 0.3 U of enzyme in a final volume of 50 µl. The
thermocycler was preheated to 95 C before the introduction of the
reaction mix. The cycle parameters are indicated in the figure
legends.
Molecular Cloning of TR
1 and TR
2 cDNAs, Intron 7, and
Subclonings
5'-Ends of messengers were cloned with the 5'-RACE kit
from CLONTECH (Palo Alto, CA) according to the instructions of the
manufacturer. The primer 9A was used for reverse transcription. The
oligonucleotide 15A was used together with the anchor primer for PCR
amplification. After an initial denaturation step of 5 min at 95 C, the
first round of amplification was at 95 C for 30 sec, 62 C for 15 sec,
and 72 C for 20 sec, 35 times. The second round was performed with 0.2
µl of the first amplification mix, using the same parameters and 30
cycles. PCR products were analyzed by Southern blot using
oligonucleotide 8S as a probe. The positive DNA fragment was purified
and cloned into the SmaI site of pBSK (Stratagene. La Jolla,
CA) before sequence analysis. 3'-Ends were amplified using 5'-anchored
PCR. A modified oligodT primer (ACTATCGATTCTGGAACCTTCAGAGG
(T)18GACACGT) was used for the cDNA synthesis. The first
round of PCR amplification was run with VIIS as 5'-primer and P3'
(CTATCGATTCTGGAACCTTCAGAGG) as 3'-primer; 35 cycles were performed at
95 C for 30 sec, at 58 C for 10 sec, and at 72 C for 20 sec. The second
round was performed with 0.2 µl of the first product, with 1S or 2S
as 5'-primer. Thirty two cycles were run at 95 C for 30 sec, at 58 C
for 10 sec, and at 72 C for 15 sec. The PCR products were cloned into
pBSK and sequenced. For TR
1 and TR
2 cDNA cloning, the
amplification parameters were 95 C for 30 sec, 56 C for 10 sec, and 72
C for 30 sec and 35 cycles were run. The PCR amplification products
were cloned into pBSK in SmaI. Expression vectors were
constructed by excising TR
1 or TR
2 from pBSK by
BamHI and EcoRI and inserting them in the
corresponding sites of pSG5 (Stratagene). A TGA codon was introduced in
TR
1 using the in vitro mutagenesis kit (CLONTECH) and
oligonucleotide CTGCTGATGAAG TAGACTGACCTCCGC to allow the production of
the 256369 protein. To build the translation mutant pSGTR
T, a
fragment containing the entire coding sequence of TR
1 was
amplified by PCR and cloned in the BamHI site of a pSGFlag
vector so that the reading frame prevents the translation of the
p16
1 protein. Mouse intron 7 was amplified by PCR reaction with
oligonucleotides 7S and 15A as primers and 100 ng of genomic DNA from
ES cells. Thirty cycles were performed at 95 C for 30 sec, at 58 C for
10 sec, and at 72 C for 30 sec. The product was cloned into pBSK
(Stratagene). CAT expression vectors were constructed by inserting a
512-bp PstI-PstI fragment (Fig. 1B
) into pBAS CAT
(Promega). DNA sequencing was performed on an Applied Automatic
Sequencer (Perkin Elmer).
Purification of polyA+ RNAs, RNase Protection Assay, and Northern
Blot
PolyA+ RNAs were purified using the "Message
Maker" kit from R&D systems (Minneapolis, MN). RNase protection
experiments were performed using the RNase protection assay system from
Promega. The vector used to synthesize the TR
-specific probe was
derived by cloning a PstI fragment containing intron 7 and
exon 8 into the PstI site of pBSK. The antisense RNA probe
was synthesized using T7 RNA polymerase (Promega) and
[
32P]UTP (specific activity 800 Ci/mmol).
Hybridizations were carried out overnight at 50 C. Hybridization
mixtures were digested with 1 U of RNAse ONE at room temperature for
1 h. Northern blots were carried out using the glyoxal
denaturation technique and alkaline transfer onto Hybond
N+. Hybridization buffer (4xSSC, 0.25% low-fat milk, 1%
SDS, 10 mM sodium phosphate, pH 6.8, and 0.1 mg/ml
denatured herring sperm DNA) was supplemented with 1% diethyl
pyrocarbonate and incubated overnight at 56 C. Membranes were
preincubated 1 h in this solution, and hybridization was performed
overnight at 54 C using 5 ng/ml of an equimolar mixture of 9A, 3A, and
15A end-labeled oligonucleotides. RNA quantification was made with a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
In Situ Immunofluorescence and Western Blotting
The No. 21 antibody raised against peptide 229325 of the
chicken T3R
1 protein was kindly provided by J. Ghysdael (Orsay,
France). It recognizes a C-terminal epitope common to the T3R
1 and
T3R
2 proteins. Immunofluorescence was performed as previously
described (37). The No. 21 antibody was diluted 1:1000 in Dako diluent
(Dako, Copenhagen, Denmark) before use. Western blotting were carried
out as previously described (40). Membranes were incubated 2 h
with the No. 21 antibody diluted 1:1000.
Half-Life Measurement of TR
Products
HeLa cells were transfected with 1 µg of p16
1- and
p26
2-expressing vectors. Thirty six hours after transfection, cells
were labeled with 10 µCi of 35S cell labeling mix
(PRO-MIX, Amersham, Arlington Heights, IL) for 45 min, washed three
times in PBS, then further cultured in 35S-free medium for
02 h or 05 h. Cells were scraped off the dish in ice-cold PBS,
pelleted, and frozen in liquid nitrogen until used.
Sequences of Oligonucleotides
7S: GCCAGTCACCTATTGTCTCCATGC; 1S:
CCACTCTTCCTGGAGGTCTTTGA
RS: GAGTGTCGACCTTGTCCTGAAACTGGC;
RA: GAGGGTACCTTCACTTCAATTCCATCCAG
XA: GGAGCTTGGCCACGAGTGGCAT;
IXA: GGTCCCAGAGACTCTAGAACTTG
2S: GCAGCTTGAGCAGCAGCTTGGTG;
8S: CTGCCTTGCGAAGACCAGATC
VIIS: CTCTGTGATCCTGCTGTTCCACAG;
hVIIS: CTCTGTGGCCCTGCCGCTCCACAG
cVIIS1: GTAATGGGGAGGTGTGTGGGGTATGG;
cVIIS2: AGCCGCTGACAGTGCGCCCCACAG
c9A:
CTATCTTATCCACGCAGATCAGCCCCGTCCGG; 9A:
GCGGTGGTTGACGTAGTGCTC
15A: CAGCCTGCAGCAGAGCCACTTCCGT;
5S: GCTGTGCCGCTTCAAGAAGTGCA
1A: CGACTTTCATGTGGAGGAAG;
2A: CCTGAACAACATGCATTCCGA
3A: TGCGGTGGTTGACGTAGTGCTC
HPRT: GCTGGTGAAAAGGACCTCT;
HPRTa: CACAGGACTAGAACACCTGC
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank Dr. Austin Smith (Edinburgh, U.K.) for
the gift of the cell line CGR8.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Olivier Chassande, Laboratorie de Biologie Moleculaire and Cellulaire, Ecole Normale Superieure de Lyon, UMR 49, Allee dItalie, 69364 Lyon Cedex 07, France.
This work was supported by research grants from the Association pour la
Recherche Contre le Cancer (ARC), the Ligue Nationale Française
Contre le Cancer, the Association Française de Lutte Contre la
Myopathie (AFM), and the Federation Nationale de Lutte Contre le Cancer
(FNLCC).
1 The first two authors have contributed equally to this work and they
should be considered as first authors. 
Received for publication December 2, 1996.
Revision received April 21, 1997.
Accepted for publication April 29, 1997.
 |
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