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P Chambon

Publications and source records attributed to P Chambon.

At least 19 recordsLinked to original sources
PubMed1992

Homeotic transformation of the occipital bones of the skull by ectopic expression of a homeobox gene.

Murine Hox genes have been postulated to play a role in patterning of the embryonic body plan. Gene disruption studies have suggested that for a given Hox complex, patterning of cell identity along the antero-posterior axis is directed by the more 'posterior' (having a more posterior rostral boundary of expression) Hox proteins expressed in a given cell. This supports the 'posterior prevalence' model, which also predicts that ectopic expression of a given Hox gene would result in altered structure only in regions anterior to its normal domain of expression. To test this model further, we have expressed the Hox-4.2 gene more rostrally than its normal mesoderm anterior boundary of expression, which is at the level of the first cervical somites. This ectopic expression results in a homeotic transformation of the occipital bones towards a more posterior phenotype into structures that resemble cervical vertebrae, whereas it has no effect in regions that normally express Hox-4.2. These results are similar to the homeotic posteriorization phenomenon generated in Drosophila by ectopic expression of genes of the homeotic complex HOM-C (refs 7-10; reviewed in ref. 3).

Animals↗
PubMed1992

All-trans and 9-cis retinoic acid induction of CRABPII transcription is mediated by RAR-RXR heterodimers bound to DR1 and DR2 repeated motifs.

Two cooperating retinoic acid response elements (RAREs) in the cellular retinoic acid-binding protein II (CRABPII) gene mediate differential transcriptional transactivation by retinoic acid receptors (RARs) and retinoid X receptors (RXRs) in P19 embryonal carcinoma cells. RARE1 and RARE2 are direct repeats (DR) of two motifs separated by 2 bp (DR2) and 1 bp (DR1), respectively, and bind RAR-RXR heterodimers more efficiently than homodimers. Using all-trans and 9-cis RA, which differentially activate RARs and RXRs, and RAR and RXR dominant-negative mutants, RAR-RXR heterodimers bound to RARE1 and RARE2 are shown to be responsible for CRABPII promoter transactivation, arguing against a unique DR spacing specifying recognition by RARs. Within heterodimers, RAR and RXR independently and differentially transactivate, depending on the specific RARE. Consistent with these results, 9-cis RA increases CRABPII mRNA levels more efficiently than all-trans RA. In contrast, all-trans and 9-cis RA have identical effects on induction of RAR beta 2 transcripts.

Base Sequence↗
PubMed1992

Promoter context- and response element-dependent specificity of the transcriptional activation and modulating functions of retinoic acid receptors.

Using several naturally occurring and synthetic retinoic acid (RA)-responsive reporter genes, we show that the patterns of transcriptional activation by various retinoic acid receptor (RAR) and retinoid X receptor (RXR) forms vary according to the nature of the RA response element and the context of the stimulated promoter. We demonstrate the presence of autonomous, ligand-inducible, and promoter context-dependent transactivation functions (AF-2s) located in the C-terminal region of all RARs and RXRs. In addition, promoter context-specific modulating transactivation functions are associated with the N-terminal A and B regions of RARs and RXRs. We also show that these transactivation and modulating functions exhibit response-element specificity. The modulating functions display a marked specificity in their cooperation with the AF-2 transactivation functions, cooperation that depends on the receptor origin of the modulating and transactivation functions and the promoter context of the RA-responsive gene, thus accounting for the specific transactivation properties of RAR and RXR types.

Animals↗
PubMed1992

Ligand dependence of estrogen receptor induced changes in chromatin structure.

To determine whether the human estrogen receptor requires ligand to bind to its cognate estrogen receptor element (ERE) in vivo, we have examined the structure of chromatin at a chromosomally integrated ERE-URA3 reporter gene in yeast, and the influence of ligand bound and ligand free estrogen receptors on that structure. Using indirect end-labelling to map DNaseI and micrococcal nuclease sensitive sites, we found that receptor induced alterations in chromatin structure were completely dependent upon the presence of estradiol. These same alterations in chromatin structure were induced by a truncated estrogen receptor with both TAF-1 and TAF-2 transactivation functions deleted, suggesting that DNA binding per se disrupts chromatin structure. These results support models in which the estrogen receptor requires ligand to bind to the ERE in vivo.

Chromatin↗
PubMed1992

Cloning of the 62-kilodalton component of basic transcription factor BTF2.

Cloning of the mammalian basic transcription factors serves as a major step in understanding the mechanism of transcription initiation. The 62-kilodalton component (p62) of one of these transcription factors, BTF2 was cloned and overexpressed. A monoclonal antibody to this polypeptide inhibited transcription in vitro. Immunoaffinity experiments demonstrated that the 62-kilodalton component is closely associated with the other polypeptides present in the BTF2 factor. Sequence similarity suggests that BTF2 may be the human counterpart of RNA polymerase II initiation factor b from yeast.

Amino Acid Sequence↗
PubMed1992

Characterization of a retinoic acid responsive element isolated by whole genome PCR.

We have used whole PCR in an attempt to isolate novel retinoic acid (RA) responsive genes. We cloned several small genomic fragments from total human DNA containing putative retinoic acid responsive elements (RAREs) selected by direct binding to the retinoic acid receptor alpha (RAR alpha). We report here that an oligonucleotide containing a sequence from one of the cloned human DNA fragments, and referred to as alpha 1, functions as an authentic RARE. It is shown that both RAR alpha and RAR beta produced in Cos cells as well as in vitro translated RAR alpha bind directly and sequence-specifically to the alpha 1RARE. By mutational analysis it is demonstrated that the alpha 1RARE consists of an imperfect direct repeat of the estrogen- and thyroid hormone-related AGGTCA half-site motif separated by a 5 bp spacer. The orientation and spacing of the half-site repeats are shown to play a critical role in RAR recognition. When cloned upstream of a TK-Luc reporter, the alpha 1RARE is shown to confer responsiveness to RA in an orientation-independent fashion in F9 and CV-1 cells. The magnitude of the RA response mediated by the alpha 1RARE differed in these cell lines.

Base Sequence↗
PubMed1992

Promoter specificity of the two transcriptional activation functions of the human oestrogen receptor in yeast.

We have previously demonstrated that the human oestrogen receptor (hER) contains two transcriptional activation functions located in the N-terminal region (TAF-1) and in the hormone binding domain (TAF-2), which can act both independently and synergistically in a promoter- and cell-specific manner in animal cells. We have also demonstrated that hER can activate transcription from chimaeric oestrogen-responsive GAL1 promoters in yeast, and shown that transcriptional activation was due to TAF-1, whereas TAF-2 showed little, if any, transcriptional activity on these promoters. By using a more complex promoter derived from the URA3 gene, we now show that TAF-2 is also active in yeast, and that the activities of TAF-1 and TAF-2 are promoter-context-specific in yeast. We also confirm that the agonistic activity of 4-hydroxytamoxifen (OHT) can be ascribed to the activity of TAF-1.

Base Sequence↗
PubMed1992

RAR-beta 4, a retinoic acid receptor isoform is generated from RAR-beta 2 by alternative splicing and usage of a CUG initiator codon.

An isoform of retinoic acid receptor beta, RAR-beta 4, has been identified. RAR-beta 4 is expressed under the control of the same retinoic acid-responsive promoter as RAR-beta 2. RAR-beta 4, which is generated by alternative splicing from the same primary transcripts as RAR-beta 2, is initiated by a non-AUG codon, CUG. The amino acid sequence of RAR-beta 4 in regions B-F is identical to that of the other RAR-beta isoforms beta 1, beta 2, and beta 3. However, the RAR-beta 4 A region is much shorter (4 amino acids long) than those of these isoforms. RAR-beta 4 exhibits a tissue-specific pattern of expression and distinct transcriptional activation properties when compared with the other RAR-beta isoforms.

Animals↗
PubMed1992

The initiation accuracy of the SV40 early transcription is determined by the functional domains of two TATA elements.

To locate the boundaries of the TATA element in the SV40 early promoter, point mutations have been constructed such as to cover the whole T + A-rich region of the replication origin. The effects of these mutations on the rate of transcription in vivo show that this region actually contains two TATA elements I and II, each independently directing the accurate initiation of transcription from a specified set of start sites, EES1 and EES2, respectively. The sequence of TATA element I fits best with the compiled 'consensus' sequence found in eukaryotic gene promoters and is the most efficient in directing transcription initiation. Mutations which improve this fit can still increase the rate of transcription, confirming the theory of a correlation between the nucleotide sequence of a TATA element and its functional efficiency. Moreover, some mutations which simultaneously modify the angle of DNA curvature in the T + A-rich promoter region and the rate of transcription reveal a correlation between DNA bending and transcription initiation.

Base Sequence↗
PubMed1992

A far upstream estrogen response element of the ovalbumin gene contains several half-palindromic 5'-TGACC-3' motifs acting synergistically.

We have identified an estrogen-responsive enhancer element (DH3 ERE) in the estrogen-induced DNAase I-hypersensitive region III of the chicken ovalbumin gene, which is located approximately 3.3 kb upstream from the mRNA start site and does not contain palindromic ERE. Four TGACC half-palindromic motifs, separated from each other by more than 100 bp, are responsible for conferring estrogen inducibility either to the proximal ovalbumin gene promoter or to heterologous promoters. Thus, widely spaced half-palindromic ERE motifs can act synergistically. Each half-palindromic motif was shown to bind the estrogen receptor (ER) with a low efficiency in vitro. However, two widely spaced half-palindromic motifs bound the ER cooperatively, much more efficiently than expected from binding to isolated half-ERE motifs. The ovalbumin promoter half-palindromic ERE motif located close to the TATA box was required for the activity of the distal DH3 ERE, but could be replaced by the binding sites of other transactivators.

Animals↗
PubMed1992

In vitro activity of the transcription activation functions of the progesterone receptor. Evidence for intermediary factors.

The human progesterone receptor (hPR) is a ligand-dependent transcription factor which contains two distinct transcription activation functions (TAFs). The full-length hPR and its individual TAFs were overexpressed in the baculovirus system and tested in a HeLa cell-derived in vitro transcription system. hPR stimulated transcription in a ligand-independent manner. When the two TAFs fused to the DNA-binding domain of GAL4 were tested, only the constitutive TAF-1 was functional in vitro, strongly suggesting that the transcriptional activity of baculovirus-expressed hPR comes solely from TAF-1. The GAL-TAF-1 activator was found to self-squelch without affecting basal transcription. A partially purified fraction relieved this self-squelching and, moreover, stimulated transcriptional activation by GAL-TAF-1, while having no influence on basal transcription. These results strongly suggest that the transcriptional activity of GAL-TAF-1 requires a factor(s) distinct from the general transcription factors.

Animals↗
PubMed1992

A single amino acid that determines the sensitivity of progesterone receptors to RU486.

The progesterone analog RU486, an abortifacient, inhibits the action of progestins in humans but not in chickens or hamsters. Substitution of cysteine at position 575 by glycine in the hormone binding domain (HBD) of the chicken progesterone receptor (cPR) generated a cPR that binds RU486 and whose activity is antagonized by that compound. In fact, all receptors that bind RU486 have a glycine at the corresponding position. The hamster PR, like cPR, has a cysteine. Only glycine--not methionine or leucine--at position 575 allowed binding of RU486 to cPR. Substitution of this glycine by cysteine in the human PR (hPR) abrogated binding of RU486 but not that of an agonist. The corresponding mutation in the human glucocorticoid receptor resulted in a loss of binding of both dexamethasone and RU486. Examination of a series of 11 beta-substituted steroids showed that antagonism is not an intrinsic property of an antihormone, because one hPR antagonist acted as an agonist for a mutated hPR. The positioning of an aromatic 11 beta-substitution in the PR HBD appears to be critical for generating agonistic or antagonistic activity.

Amino Acid Sequence↗
PubMed1992

Class II (B) general transcription factor (TFIIB) that binds to the template-committed preinitiation complex is different from general transcription factor BTF3.

A class II (B) general transcription factor of 34 kDa has been purified from HeLa cells to apparent homogeneity. This factor appears to be transcription factor IIB (TFIIB), since it binds in vitro to template-committed preinitiation complexes formed between a template containing the TATA box/cap-site elements of the adenovirus type 2 major late promoter (Ad2MLP) and recombinant human or yeast TFIID (previously called BTF1) expressed in Escherichia coli. DNase I footprint studies show an extended pattern of protection of Ad2MLP TATA box/cap-site sequences when TFIIB is bound to template-committed complexes, even though TFIIB does not bind on its own to the template in the absence of TFIID. We also show that TFIIB is different from BTF3 by a number of criteria.

Amino Acid Sequence↗
PubMed1992

Structure, localization and transcriptional properties of two classes of retinoic acid receptor alpha fusion proteins in acute promyelocytic leukemia (APL): structural similarities with a new family of oncoproteins.

Acute promyelocytic leukemia (APL) is due to a chromosomal t(15;17) translocation which involves a novel human gene, Myl, (also named PML) and the retinoic acid (RA) receptor alpha (RAR-alpha) gene. We report here the characterization of Myl and of the reciprocal MylRAR (PMLRAR) and RARMyl (RARPML) fusion transcripts which are found in two classes of APL patients. Myl displays similarities with a new family of proteins of which some members are fused to protooncogenes in the transforming proteins RFP-ret and T18. The speckled nuclear localization of Myl, as well as its sequence homology with the 52 kDa component of the RO/SSA ribonucleoprotein particle, suggest that Myl may be present in a ribonucleoprotein complex. In contrast to both Myl and RAR-alpha whose localization is essentially nuclear in the presence or absence of RA, MylRAR which is largely cytoplasmic in the absence of RA appears to be translocated to the nucleus in the presence of RA. Myl and MylRAR can associate in vitro and this association is mediated by a coiled coil in the Myl sequence. In vivo this association results in a colocalization of Myl and MylRAR which is identical to that of MylRAR alone. Studies of activation of transcription from the promoters of several RA target genes indicate that MylRARs have altered transcription activation properties when compared with RAR-alpha. Most notably, MylRAR represses markedly the activity of some RA target promoters in the absence of RA. Western blot analyses of patient samples show that MylRAR is expressed to a much higher level than wild type RAR-alpha originating from the normal allele. Taken together, these results suggest that MylRAR may interfere in a dominant manner with both Myl and RAR functions.

Alleles↗
PubMed1992

The acidic transcriptional activator GAL-VP16 acts on preformed template-committed complexes.

The action of the chimeric acidic transcriptional activator GAL-VP16 has been investigated by performing a series of kinetic experiments using the detergent Sarkosyl as well as monoclonal antibodies which specifically inhibit GAL-VP16 DNA binding and transcriptional activation. GAL-VP16 binds to recognition site rapidly, remains bound after transcriptional initiation and is required to maintain stimulated levels of reinitiation. GAL-VP16 action, which appears to result in an increase in the number of preinitiation complexes formed, occurs after the formation of template-committed complexes composed of promoter-bound TFIIA (STF) and a partially purified TFIID fraction conferring GAL-VP16 responsiveness on a reconstituted basal transcription system. This TFIID fraction cannot be replaced by TFIIB or cloned TFIID. Our results suggest that GAL-VP16 activates step(s) in preinitiation complex assembly occurring after TFIID has bound.

Antibodies, Monoclonal↗
PubMed1992

Transcriptional enhancer factor (TEF)-1 and its cell-specific co-activator activate human papillomavirus-16 E6 and E7 oncogene transcription in keratinocytes and cervical carcinoma cells.

The human papillomavirus (HPV)-16 oncogenes, E6 and E7, are transcribed preferentially in keratinocytes and cervical carcinoma cells due to a 5' enhancer. An abundant peptide binding to a 37 nt enhancer element was purified from human keratinocytes by sequence-specific DNA chromatography. This protein was identified as transcriptional enhancer factor (TEF)-1 by complex mobility, binding to wild-type and mutant SV40 and HPV-16 enhansons and antigenic reactivity with two anti-TEF-1 antibodies. TEF-1 is cell-specific, but its transactivation also depends on a limiting, cell-specific TEF-1 'co-activator'. We show that both TEF-1 and the TEF-1 co-activator are active in human keratinocytes and essential for HPV-16 transcription. TEF-1 binding in vivo was necessary for HPV-16 P97 promoter activity. Excess TEF-1 and chimeric GAL4-TEF-1 specifically inhibited the P97 promoter by 'squelching', indicating that HPV-16 transcription also requires a limiting TEF-1 co-activator. TEF-1 and the TEF-1 co-activator functions mirrored HPV-16 transcription by their presence in keratinocytes and cervical carcinoma cells and their absence from lymphoid B-cells, but also functioned in liver cells where the HPV-16 promoter is inactive. TEF-1 and its associated co-activator are thus part of a complex mechanism which determines the restricted cell range of the HPV-16 E6 and E7 oncogene promoter.

Antibodies↗