Identification of Transcripts Initiated from an Internal Promoter in the c-erbA{alpha} Locus That Encode Inhibitors of Retinoic Acid Receptor-{alpha} 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 d’Oncologie Moléculaire (V.L.) Institut Pasteur 59019 Lille, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The thyroid hormone receptor-coding locus, c-erbA{alpha}, 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{Delta}{alpha}1 and TR{Delta}{alpha}2 [mRNA for isoform {alpha}1 and {alpha}2 of the T3 receptor (TR), respectively], whose transcription is initiated from an internal promoter located within intron 7 of the c-erbA{alpha} 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{alpha} locus. TR{Delta}{alpha}1 and TR{Delta}{alpha}2 mRNAs encode N-terminally truncated isoforms of T3R{alpha}1 and T3R{alpha}2, respectively. The protein product of TR{Delta}{alpha}1 antagonizes the transcriptional activation elicited by T3 and retinoic acid. This protein inhibits the ligand-induced activating functions of T3R{alpha}1 and 9-cis-retinoic acid receptor-{alpha} but does not affect the retinoic acid-dependent activating function of retinoic acid receptor-{alpha}. We predict that these truncated proteins may work as down-regulators of transcriptional activity of nuclear hormone receptors in vivo.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha} locus encodes T3R{alpha}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{alpha}1 are the members of the RXR family (3).

The mechanism of the transcriptional activity of T3R{alpha}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{alpha}1 is regulated by products of the c-erbA{alpha} locus itself. TR{alpha}2 RNA results from differential splicing of the TR{alpha} primary transcript (12). The T3R{alpha}2 protein lacks T3-binding and AF-2 domains but retains a DNA-binding domain and can exert dominant negative activity over T3R{alpha}1 (13). The inhibition mediated by T3R{alpha}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{alpha}2 is still unknown, although its mRNA is expressed at higher levels than TR{alpha}1 in all tissues. N-truncated T3R{alpha}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{alpha}1 transcripts (15). These short proteins are in the C-terminal part of T3R{alpha}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{alpha}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{alpha} 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{alpha} 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{alpha}1 and RAR{alpha}.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of New Transcripts from the c-erbA{alpha} Locus
ES cells containing an inactivated c-erbA{alpha} 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. 1AGo and Ref. 17), and the neoR gene driven by the ß-actin promoter. Heterozygous cell lines (c-erbA{alpha}±) were grown at high G418 concentration (18) to select for homozygous clones in which both alleles of the c-erbA{alpha} gene were disrupted (c-erbA{alpha}-/-). Two independent c-erbA{alpha}-/- 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{alpha}-/- ES cell lines. The I7E8 RNA probe covering intron 7 and exon 8 was used (Fig. 2AGo). After a 24-h exposure, only one protected fragment of 238 nucleotides, corresponding to TR{alpha} RNA, could be detected in wild type cells under these conditions. This fragment was not detected in the RNA of mutant c-erbA{alpha}-/- ES cells (Fig. 2BGo, lane Cl10 and Cl20). The absence of TR{alpha} transcripts was further confirmed by RT-PCR using oligonucleotide 5S positioned in exon 5 and oligonucleotides 1A or 2A positioned in exons 9 (TR{alpha}1-specific) and exon 10 (TR{alpha}2-specific) as primers (see position in Fig. 1BGo). No amplification occurred in c-erbA{alpha}-/- ES cells (Fig. 2DGo)



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Figure 1. Modifications of the Structure of the Genomic Locus of the c-erbA{alpha} Gene and Position of Oligonucleotides

The position of exons and introns is extrapolated from the human c-erbA{alpha} gene structure (19 ). Exons are numbered 1 to 10. A, Modifications introduced in the c-erbA{alpha} locus by the homologous recombination. Short horizontal arrows indicate the position of each oligonucleotide. (a) Structure of the wild type locus. Vertical arrows indicate transcription stops. ß, ß-Actin promoter. TK, HSV thymidine kinase gene; ATG, translation initiation codon of T3R{alpha}1 and T3R{alpha}2 proteins. (b) Structure of the homologous recombination vector. (c) Structure of the modified locus. B, Structure of exons 5 to 10 of the c-erbA{alpha} gene. P, PstI sites used in different cloning procedures (see text). {alpha}c represents the part of exon 9 that is common to TR{alpha}1 and TR{alpha}2 isoforms and {alpha}1 ({alpha}2) represents the part of exon 9 (10 ) that is specific to {alpha}1 ({alpha}2) isoform. Primer names are indicated under arrows. Sequences are given in Materials and Methods.

 


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Figure 2. Identification of New Transcripts Generated from the c-erbA{alpha} 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{alpha} mRNA. B, RNase protection with 20 µg total RNA from wild type (CGR8) or c-erbA{alpha}-/- (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{alpha}-/- (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.

 
When RT-PCR was performed using oligonucleotide 8S (exon 8) as primer in 5'-position (see position in Fig. 1BGo), amplification occurred in both c-erbA{alpha}-/- clones and wild type ES cells (Fig. 2DGo), 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{alpha}1-like (called TR{Delta}{alpha}1) and TR{alpha}2-like (called TR{Delta}{alpha}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{alpha}-/- ES cells.

A single DNA fragment was amplified from two independent c-erbA{alpha}-/- 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{alpha}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{alpha} 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. 2AGo). 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{alpha} RNA (Fig. 2CGo). 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{alpha}-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{Delta}{alpha}1 and TR{Delta}{alpha}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{alpha} 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{alpha} 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. 3AGo). These data suggest that transcripts initiated in intron 7 may be present in all vertebrates.



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Figure 3. TR{Delta}{alpha} Transcripts Are Initiated in Intron 7

A, Amplification of a TR{Delta}{alpha}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{alpha} 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.

 
Identification of a Promoter within the c-erbA{alpha} 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. 3BGo, 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{alpha} 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. 3CGo). Comparison between human and mouse sequences showed two conserved blocks (Fig. 3CGo). 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{alpha}1 and TR{alpha}2 Short Transcripts
Using 3'-RACE methodology, we cloned the 3'-end of TR{Delta}{alpha}1 and TR{Delta}{alpha}2 RNAs using mutant c-erbA{alpha}-/- 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. 1BGo) to selectively amplify cDNAs initiated in intron 7. A second round was performed with either oligonucleotides 1S or 2S to specifically amplify {alpha}1 or {alpha}2 isoforms, respectively. Products of amplification displayed 100% homology with TR{alpha}1 or TR{alpha}2 in the coding regions. 3'-Noncoding regions were homologous to those described for rat or human TR{alpha}1 (gb M18028 and emb X55005) and TR{alpha}2 RNAs (emb X07409 and emb X55066). Antisense primers IXA and XA (Fig. 1BGo) were designed from the 3'-untranslated portions of TR{Delta}{alpha}1 and TR{Delta}{alpha}2 RNAs, respectively, and used together with VIIS to amplify full-length TR{Delta}{alpha}1 and TR{Delta}{alpha}2 RNAs by RT-PCR. Each amplification generated one single band, establishing the existence of TR{Delta}{alpha}1 and TR{Delta}{alpha}2 RNAs in both c-erbA{alpha}-/- and wild type ES cells (data not shown). Three clones obtained from each amplification product had identical sequences to the previously published mouse TR{alpha}1 and TR{alpha}2 RNA, respectively (20) with the exception of the 5'-short intronic sequence. We can therefore conclude that the c-erbA{alpha} gene encodes two related RNAs, TR{Delta}{alpha}1 and TR{Delta}{alpha}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{alpha}1 and TR{alpha}2 transcripts in Fig. 4Go. Assuming that TR{Delta}{alpha}1 and TR{Delta}{alpha}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. 4AGo). 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{Delta}{alpha}1) and 1.5 kb (TR{Delta}{alpha}2) were detected in addition to the 5.5-kb (TR{alpha}1) and 2.5-kb (TR{alpha}2) transcripts (Fig. 4BGo, lane ES) in ES cells. The ratio of the different isoforms was TR{Delta}{alpha}1 = 0.5, TR{alpha}1 = 1, TR{Delta}{alpha}2 = 2, TR{alpha}2 = 10. The shorter transcripts were not detected in liver (Fig. 4BGo, lane L).



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Figure 4. The TR{alpha} and TR{Delta}{alpha} Transcripts

A, Schematical representation of the transcripts generated by the c-erbA {alpha} locus. Gray staining: region common to {alpha}1 and {alpha}2 isoforms. Light hatched blocks indicate region specific to {alpha}1-isoforms. Heavy hatched blocks show region specific to {alpha}2-isoforms. Black blocks indicate 5'-end specific to TR{Delta}{alpha} mRNAs. Upstream region from the c-erbA{alpha} gene and the 5'-end of the TR{alpha} mRNAs are not shown (horizontal dashed blocks), and 3'- untranslated regions of RNAs are shown as lines. B, Identification of all TR{alpha} 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).

 
Expression of TR{Delta}{alpha} RNAs Is Regulated in Mouse Tissues and During ES Cells’ Differentiation
The patterns of expression of TR{Delta}{alpha}1, TR{alpha}1, TR{Delta}{alpha}2, and TR{alpha}2 mRNAs were analyzed in different mouse tissues, using semiquantitative RT-PCR (Fig. 5AGo). As expected, the TR{alpha} transcripts were detected in all tissues. TR{alpha}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{alpha}2 was found to be expressed predominantly in brain, gut, heart, lung, kidney, and muscle. In contrast, TR{Delta}{alpha} transcripts displayed a more restricted pattern of expression. The highest amount of TR{Delta}{alpha}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{Delta}{alpha}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{alpha} and TR{Delta}{alpha} 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{alpha} and TR{Delta}{alpha} 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{alpha} RNAs increased (Fig. 5BGo). 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{Delta}{alpha} transcripts may be important in maintaining the ES cells in an undifferentiated state.



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Figure 5. Pattern of Expression of TR{Delta}{alpha} 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{alpha}1, TR{alpha}2, TR{Delta}{alpha}1, TR{Delta}{alpha}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{Delta}{alpha}2 cDNAs, 35 cycles to amplify TR{Delta}{alpha}1 and TR{alpha}2 cDNAs, and 30 cycles to amplify TR{alpha}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. 2AGo) for RNase protection analysis. The positions of specific bands are indicated. The gel was exposed for 4 h to detect the c-erbA{alpha} transcripts and 30 h to detect the TR{Delta}{alpha} transcripts.

 
Translation of TR{Delta}{alpha}1 and TR{Delta}{alpha}2 RNAs in HeLa Cells
Both TR{Delta}{alpha}1 and TR{Delta}{alpha}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. 3AGo). Interestingly, both codons are highly conserved in fish, Xenopus, chicken, mouse, rat, and human (22). The reading frames are identical to those of TR{alpha}1 and TR{alpha}2. The predicted proteins would be N-terminally truncated forms of T3R{alpha}1 or T3R{alpha}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{alpha} proteins (Fig. 6Go 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).



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Figure 6. Structure of the Proteins Produced by the c-erbA{alpha} Locus

Numbers refer to amino acids of the T3R{alpha}1 and T3R{alpha}2 proteins. 256/259 indicates the putative positions of the NH2-terminal methionine of p16{alpha}1 and p26{alpha}2. 369 and 319 are the positions of the stop codons introduced to generate N-terminally truncated mutants of p16{alpha}1. DBD, DNA-binding domain; HR, hinge region; T3, ligand binding domain; {tau}3/AF2, {tau}3 and AF2 domains. Gray staining indicates region common to {alpha}1- and {alpha}2-isoforms. Light hatched blocks show region specific to {alpha}1-isoforms. Heavy hatched blocks indicate region specific to {alpha}2-isoforms.

 
All our attempts to detect the endogenous products in ES cells have been unsuccessful. Like their full-length counterparts T3R{alpha}1 and T3R{alpha}2, the endogenous proteins translated from the TR{Delta}{alpha}1 and TR{Delta}{alpha}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{alpha}1 cannot be detected in ES cells either. To assess the ability of the two TR{Delta}{alpha} transcripts to encode the predicted proteins, the TR{Delta}{alpha}1 and TR{Delta}{alpha}2 cDNAs were cloned into pSG5 expression vector (pSGTR{Delta}{alpha}1 and pSGTR{Delta}{alpha}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{Delta}{alpha}1 RNA encodes one single polypeptide of approximately 16 kDa, called p16{alpha}1, and TR{Delta}{alpha}2 RNA yields one single polypeptide of approximately 26 kDa, called p26{alpha}2 (Fig. 7AGo). The half-life of these proteins in HeLa cells was estimated in pulse-chase experiments to be 1 h for p16{alpha}1 and 2–3 h for p26{alpha}2 (Fig. 7BGo). The cellular localization of transiently expressed p16{alpha}1 and p26{alpha}2 was investigated by in situ immunofluorescence in HeLa cells. p16{alpha}1 exhibited both cytoplasmic and nuclear localization (Fig. 7CGo, panel 1) whereas p26{alpha}2 was predominantly in the cytoplasm (Fig. 7CGo, panel 2). These localizations differ from that of nuclear receptors like T3R{alpha}1, which exhibit a strict nuclear localization (data not shown).



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Figure 7. Expression of TR{Delta}{alpha} cDNAs in HeLa Cells

A, Detection of the p16{alpha}1 and p26{alpha}2 proteins in whole cell lysates of HeLa cells transfected with 2 µg of pSGTR{Delta}{alpha}1 or pSGTR{Delta}{alpha}2 expression vectors, respectively. The No. 21 antibody is used to detect the truncated proteins. B, Pulse chase experiment measuring the half-life of the p16{alpha}1 and p26{alpha}2 proteins. C, Detection of p16{alpha}1 (1 ) and p26{alpha}2 (2 ) in HeLa cells transfected with 1 µg of either pSGTR{Delta}{alpha}1 or pSGTR{Delta}{alpha}2 expressing vectors, using No. 21 antibody. Bar, 5 mm.

 
Functional Interference of p16{alpha}1 and p26{alpha}2 with T3Rs and RAR{alpha} in Vivo
Artificial constructs that express C-terminal portions of the T3R{alpha}1 receptor have been shown to behave as repressors of the transactivation mediated either by T3R{alpha}1 or by RAR{alpha} (26). Therefore, we investigated the possibility that the p16{alpha}1 and p26{alpha}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{alpha}1, as compared with the well described inhibition exerted by T3R{alpha}2. One or 4 µg of pSGTR{Delta}{alpha}1, pSGTR{Delta}{alpha}2, or pSGTR{alpha}2 expression vectors were cotransfected with an expression vector for T3R{alpha}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. 8AGo show that both p16{alpha}1 and p26{alpha}2 inhibited the transcription mediated by T3R{alpha}1 in a dose-dependent manner. This repressing activity was comparable to the one of TR{alpha}2. These inhibitory activities required the translation of the cDNAs because another mutated construct, pSGTR{Delta}T, which does not enable the production of p16{alpha}1 but generates TR{Delta}{alpha}1 transcripts, was unable to repress the transcriptional activation by T3.



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Figure 8. Effect of p16{alpha}1 and p26{alpha}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{alpha}1 and p26{alpha}2 on the transcriptional activity of liganded T3R{alpha}. The transcriptional activity of T3R{alpha}1 was measured by transfecting HeLa cells with 0.8 µg of IR0 globinCAT (IR0; Ref. 41) together with 1 µg of pSGTR{alpha}1 (provided by Dr B. Vennström, Sweden). One or 4 µg of translationally silent vector ({Delta}T) or of vector encoding T3R{alpha}2, p16{alpha}1 (p16), or p26{alpha}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{alpha}1 and p26{alpha}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{Delta}{alpha}1 (open bars), or pSGTR{Delta}{alpha}2 (dotted bars) was cotransfected. C, Mapping of the inhibitory activity within p16{alpha}1. The experimental conditions are as in panel A. Four micrograms of pSG5 vector carrying either mutant TR{Delta}T (-), mutant 256–369, mutant 256–319, or mutant 320–408 were cotransfected. D, Effect of p16{alpha}1 toward T3R{alpha}1, T3Rß1, and RAR{alpha}. The transcriptional activity of T3R{alpha}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{alpha} was measured by transfecting HeLa cells with 0.2 µg pSGRAR{alpha} (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{Delta}T (0) or pSGTR{Delta}{alpha}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.

 
We checked whether this inhibition could be the result of nonspecific effects on the transcriptional machinery (Fig. 8BGo). Whereas p16{alpha}1 did not significantly modify the activity of either SV40, CMV, or ß-actin promoters, p26{alpha}2 strongly repressed all promoters. Therefore, p16{alpha}1 behaved as a specific antagonist of the T3-dependent T3R{alpha}1 activity, but p26{alpha}2 had a more general inhibitory effect toward transcription.

To map the inhibitory activity of p16{alpha}1, we generated truncated variants of this protein (see Fig. 6Go for the position of truncations) and investigated their ability to repress the transcriptional activity of T3R{alpha}1. The efficient production of all variant proteins in transfected cells was checked by Western blot analysis (data not shown). Figure 8CGo shows that a mutant protein that retained the N-terminal portion common to p16{alpha}1 and p26{alpha}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 (256–319), which lacked most of the heptad repeats involved in homodimerization of the receptors, inhibited T3R{alpha}1. This protein retained a domain previously identified as {tau}3 (31). In contrast, the complementary C-terminal domain (320–410) displayed no inhibition.

We investigated the repressing activity of p16{alpha}1 toward different nuclear receptors associated with different hormone response elements, in the presence of their ligand (Fig. 8DGo). A strong repression of T3R{alpha}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{alpha} was efficiently repressed.

Mechanism of Action of p16{alpha}1
We investigated the mechanism by which p16{alpha}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{alpha}1 with either of these three steps. To assay the effect of p16{alpha}1 on DNA binding, we tested its activity on the ligand-independent transcriptional repression of RAR{alpha} and T3R{alpha}1. Figure 9AGo shows that the active repression mediated by these unliganded receptors on a IR0 response element was not affected by p16{alpha}1. In agreement with these data, in vitro binding of T3R{alpha}1 to DR4 or palindromic response elements was unaffected by p16{alpha}1, as assayed by gel shift experiments (data not shown). These data rule out a squelching activity of p16{alpha}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{alpha}1 on Ligand-Independent Repression, Heterodimerization, and Transcriptional Activation

A, Effect of p16{alpha}1 on the repression activity of unliganded receptor. Transfection experiments are as in Fig. 8Go, A or C, except that no ligand was added. Zero or 4 µg of p16{alpha}1 expressing vector pSGTR{Delta}{alpha}1 was cotransfected. Four micrograms of pSGTR{Delta}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{alpha}1 on the heterodimerization between RAR{alpha} or T3R{alpha}1 and RXR{alpha}: 0.4 µg of (UAS)4TKCAT reporter plasmid was cotransfected with 0.25 µg of pCMXGal4-RAR{alpha} (GalRAR) or pSVGal4-TR{alpha}1 (GalT3R) in the presence or absence of 0.5 µg of pCMXVP16-RXR{alpha} (VPRXR). C, Effect of p16{alpha}1 on the activating functions of T3R{alpha}1, RAR{alpha}, and RXR{alpha}: 0.4 µg of (UAS)4TKCAT reporter plasmid was cotransfected with 0.25 µg of either pCMXGal4-RAR{alpha} (GalRAR), pCMXGal4-RXR{alpha} (GalRXR), or pSVGal4-T3R{alpha} (GalT3R). Two micrograms of pSGTR{Delta}{alpha}1 and 2 µg of pSGTR{Delta}T (2 ), or 4 µg of pSGTR{Delta}{alpha}1 (4 ), were cotransfected. Four micrograms of pSGTR{Delta}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{alpha}1 on RAR{alpha}/RAR{alpha} homodimers and RAR{alpha}/RXR{alpha} heterodimers. The activity of RAR{alpha}/RAR{alpha} homodimers was measured using 1 µg pSGRAR{alpha} and 0.8 µg IR0 Globin CAT; the activity of RAR{alpha}/RXR{alpha} heterodimers was measured by cotransfection of 1 µg pSGRAR{alpha}, 0.3 µg pSG RXR{alpha}, and 0.8 µg IR0 Globin CAT. Four micrograms of either pSGTR{Delta}T (0) or pSGTR{Delta}{alpha}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{alpha}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{alpha} or T3R{alpha}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 9BGo 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{alpha}1. Therefore, p16{alpha}1 was unable to prevent the interaction between RAR{alpha} and RXR{alpha} or T3R{alpha}1 and RXR{alpha}. This is consistent with data from electromobility shift assays that showed that this protein could not prevent or modify the binding of T3R{alpha}1-RXR{alpha} heterodimers to IR0 or DR4 response elements (data not shown).

Finally, we tested the effect of p16{alpha}1 on the activating functions of T3R{alpha}1, RAR{alpha}, and RXR{alpha}. Therefore, we used chimeric proteins consisting of the hinge and ligand-binding regions of the T3R{alpha}1, RAR{alpha}, and RXR{alpha} 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{alpha}1 (Fig. 9CGo). In contrast, the activation of Gal4RAR by retinoic acid was unaffected. These data suggest that p16{alpha}1 does not affect the activating function of RAR{alpha} but interferes with the transcriptional activation domains, or activating functions, of T3R{alpha}1 and RXR{alpha}.

The absence of inhibition of the activating function of RAR{alpha} is in apparent contradiction with data presented in Fig. 8DGo showing that the activity of the full-length receptor on different response elements could be at least partially inhibited by p16{alpha}1. We repeated the experiment described in Fig. 8DGo, using 1 µg pSGRAR{alpha} (Fig. 9DGo). We assumed that, in these new conditions, RAR{alpha} homodimers were the predominant active complex. Given this assumption, we expected that raising the amount of RXR{alpha} would result in the preferential formation of heterodimers, leading to an enhanced activation of the IR0 (27). Indeed, cotransfection of 1 µg pSGRAR{alpha} and 0.3 µg pSGRXR{alpha} produced a 2.2-fold enhanced RA-mediated activation of IR0, as compared with the activation observed with RAR{alpha} alone. Interestingly, p16{alpha}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{alpha}1 can antagonize the ligand-dependent activation of RAR{alpha}, but only when it acts as RAR-RXR heterodimer.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
All the cDNAs reported to date for the c-erbA{alpha} locus correspond to mRNAs initiated by the c-erbA{alpha} promoter (5): TR{alpha}1, coding for a T3 receptor, and TR{alpha}2, coding for a protein that does not bind T3. We have cloned and characterized two new transcripts of the c-erbA{alpha} locus: TR{Delta}{alpha}1 and TR{Delta}{alpha}2, which begin in intron 7 and are otherwise identical to the 3'-part of TR{alpha}1 and TR{alpha}2, respectively. These transcripts were first identified in c-erbA{alpha}-/- 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{alpha} gene inactivation in c-erbA{alpha}-/- 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{Delta}{alpha} RNAs through this promoter is further suggested by the differences observed in the patterns of expression of the TR{Delta}{alpha} and TR{alpha} transcripts. The opposite variation of their amount during the differentiation of ES cells suggests that the regulation of the c-erbA{alpha} promoter is different from that of intron 7 promoter. The mRNAs generated by the intron 7 promoter are spliced to generate the {alpha}1 or {alpha}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{alpha} proteins of 27 to 46 kDa had previously been described in chicken (15). These proteins can exert a dominant negative activity toward T3R{alpha}1 and RAR{alpha} transcriptional activation (26). We have shown that the product of the TR{Delta}{alpha}1 mRNA that we have identified, p16{alpha}1, also has a dominant negative activity toward T3R{alpha}1, T3Rß1, and RAR{alpha}. This protein does not affect receptor-independent transcription. In contrast, analyzing the function of p26{alpha}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{alpha}2 (29). However, we cannot exclude the possibility that p26{alpha}2 may also have a specific inhibitory action toward nuclear receptors, as was shown for T3R{alpha}2 (14). The exact mechanism responsible for the inhibitory activity of p16{alpha}1 is still unclear. Our data rule out the possibility that p16{alpha}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{alpha} is not affected by p16{alpha}1 is consistent with the absence of inhibition of RAR{alpha}/RAR{alpha} homodimers. Heterodimerization of RAR{alpha} with RXR{alpha} 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{alpha}1. Since the proper activating function of RAR{alpha} is not inhibited, this suggests that, in heterodimers, the activating function of RXR{alpha} is the main target of p16{alpha}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{alpha}1 that contains amino acids 256 to 319 is sufficient to perform inhibition. This region contains four heptad repeats and the {tau}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 {tau}3-domain, which is hidden within the T3R{alpha}1 protein (32), is able to bind and sequester a coactivator when exposed at the N-terminus of p16{alpha}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{alpha}1. Alternatively, the {tau}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{alpha}1 and the target factor may take place either in the nucleus or in the cytoplasm because p16{alpha}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{alpha}1.

The biological functions of the proteins p16{alpha}1 and p26{alpha}2 are unknown. Our data suggest that p16{alpha}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{alpha}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{alpha} locus that includes exon 8 have been generated, thus preventing the synthesis of both TR{alpha} and TR{Delta}{alpha} 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{alpha}-/- cells, producing TR{Delta}{alpha} but no TR{alpha} 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{alpha}-/- cells. We propose that p16{alpha}1 and p26{alpha}2 could be such inhibitors. It could be argued that the ratio between the pSGTR{Delta}{alpha} and pSGTR{alpha}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{alpha}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{alpha}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{alpha}1 and p26{alpha}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{Delta}{alpha} 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{gamma} (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{alpha}1 is a novel example of this mechanism of regulation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}LBD, Gal4DBD-hRXR{alpha}LBD, and VP16-RAR{alpha} 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 40–6055 and RXR-specific ligand Ro25–7386 are gifts from Dr. Michael Klaus from Hoffman LaRoche (Nutley, NJ).

Homologous Recombination and Production of Homozygous ES Cells
The replacement vector, {alpha}HRVTK, was constructed by sequential insertion of genomic fragments of the mouse c-erbA{alpha} 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. 1AGo), 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 {alpha}HRV. A XhoI-SalI HSV-TK fragment isolated from MC1TK (gift of Dr Capecchi) vector was cloned into SalI in {alpha}HRV to generate the targeting vector {alpha}HRVTK (Fig. 1AGo).

ES cells were electroporated with 40 µg {alpha}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{alpha} 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. 1Go, 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{Delta}{alpha}1 and TR{Delta}{alpha}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{Delta}{alpha}1 and TR{Delta}{alpha}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{Delta}{alpha}1 or TR{Delta}{alpha}2 from pBSK by BamHI and EcoRI and inserting them in the corresponding sites of pSG5 (Stratagene). A TGA codon was introduced in TR{Delta}{alpha}1 using the in vitro mutagenesis kit (CLONTECH) and oligonucleotide CTGCTGATGAAG TAGACTGACCTCCGC to allow the production of the 256–369 protein. To build the translation mutant pSGTR{Delta}T, a fragment containing the entire coding sequence of TR{Delta}{alpha}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{alpha}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. 1BGo) 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{alpha}-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 [{alpha}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 229–325 of the chicken T3R{alpha}1 protein was kindly provided by J. Ghysdael (Orsay, France). It recognizes a C-terminal epitope common to the T3R{alpha}1 and T3R{alpha}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{Delta}{alpha} Products
HeLa cells were transfected with 1 µg of p16{alpha}1- and p26{alpha}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 0–2 h or 0–5 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 d’Italie, 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. Back

Received for publication December 2, 1996. Revision received April 21, 1997. Accepted for publication April 29, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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