Biomedical subjects

P Chambon

Publications and source records attributed to P Chambon.

At least 73 records · Page 4Linked to original sources

PubMed2000

A Brg1 null mutation in the mouse reveals functional differences among mammalian SWI/SNF complexes.

Mammalian SWI/SNF complexes utilize either brahma (Brm) or brahma-related gene 1 (Brg1) catalytic subunits to remodel nucleosomes in an ATP-dependent manner. Brm was previously shown to be dispensable, suggesting that Brm and Brg1 are functionally redundant. To test this hypothesis, we have generated a Brg1 null mutation by gene targeting, and, surprisingly, homozygotes die during the periimplantation stage. Furthermore, blastocyst outgrowth studies indicate that neither the inner cell mass nor trophectoderm survives. However, experiments with other cell types demonstrate that Brg1 is not a general cell survival factor. In addition, Brg1 heterozygotes are predisposed to exencephaly and tumors. These results provide evidence that biochemically similar chromatin-remodeling complexes have dramatically different functions during mammalian development.

Animals↗

PubMed2000

Crystal structure of a heterodimeric complex of RAR and RXR ligand-binding domains.

The crystal structure of a heterodimer between the ligand-binding domains (LBDs) of the human RARalpha bound to a selective antagonist and the constitutively active mouse RXRalphaF318A mutant shows that, pushed by a bulky extension of the ligand, RARalpha helix H12 adopts an antagonist position. The unexpected presence of a fatty acid in the ligand-binding pocket of RXRalpha(F318A is likely to account for its apparent "constitutivity." Specific conformational changes suggest the structural basis of pure and partial antagonism. The RAR-RXR heterodimer interface is similar to that observed in most nuclear receptor (NR) homodimers. A correlative analysis of 3D structures and sequences provides a novel view on dimerization among members of the nuclear receptor superfamily.

Amino Acid Sequence↗

PubMed2000

Spatio-temporal distribution of cellular retinoid binding protein gene transcripts in the developing and the adult cochlea. Morphological and functional consequences in CRABP- and CRBPI-null mutant mice.

The expression patterns of the mouse cellular retinoid binding protein genes were investigated by in situ hybridization analysis in the inner ear from 10.5 days post coïtum (dpc) up to the adult stage. The cellular retinoic acid binding protein II (CRABPII) and cellular retinol binding protein I (CRBPI) were present in a widespread and abundant pattern in cochlear structures during embryogenesis. Expression of the cellular retinoic acid binding protein I (CRABPI) is restricted during development in Kölliker's organ whilst cellular retinol binding protein II (CRBPII) is only visible after birth with a ubiquitous distribution in most regions of the cochlea including nervous components. No CRABP or CRBP transcripts were observed in the auditory receptors. Morphological observations of CRBPI- and CRABPI/CRABPII-null mutant fetus at 18.5 dpc do not show any structural modification at the level of the organ of Corti. Furthermore, electrophysiological tests performed by measuring distorsion-product otoacoustic emissions and auditory brainstem evoked responses did not present significant alteration of the auditory function for the different types of mutants. The expression of retinoid binding proteins in cochlear structures during embryogenesis could suggest important roles for these proteins during ontogenesis and morphogenesis of the inner ear. Despite these observations, morphological and functional data from mutant mice did not present obvious modifications of the cochlear structures and auditory thresholds. It is therefore unlikely that CRABPs and CRBPI are directly involved in development of the cochlea and hair cell differentiation.

Age Factors↗

PubMed2000

Synergy between estrogen receptor alpha activation functions AF1 and AF2 mediated by transcription intermediary factor TIF2.

The activation function AF2 in the ligand-binding domain of estrogen receptors ER alpha and ER beta signals through the recruitment of nuclear receptor coactivators. Recent evidence indicates that coactivators, such as the transcription intermediary factor TIF2, also bind to and transactivate the N-terminal AF1 function of the two ERs. We have generated TIF2 mutant proteins that are deficient in either AF1 or AF2 interaction and use these mutants to investigate the relative contribution of both AFs to TIF2 recruitment and transactivation. We observe that TIF2 is capable of interacting simultaneously with both the isolated N- and C-terminus of ER alpha in transfected mammalian cells and in vitro, indicating that TIF2 can bridge both receptor domains. The concomitant interaction of TIF2 with both AFs results in synergistic activation of transcription. Thus, synergy between ER alpha AF1 and AF2 is a result of the cooperative recruitment of TIF2 and/or other members of the p160 coactivator family.

Animals↗

PubMed2000

Targeted somatic mutagenesis in mouse epidermis.

Gene targeting in the mouse is a powerful tool to study mammalian gene function. The possibility to efficiently introduce somatic mutations in a given gene, at a chosen time and/or in a given cell type will further improve such studies, and will facilitate the generation of animal models for human diseases. To create targeted somatic mutations in the epidermis, we established transgenic mice expressing the bacteriophage P1 Cre recombinase or the tamoxifen-dependent Cre-ER(T2) recombinase under the control of the human keratin 14 (K14) promoter. We show that LoxP flanked (floxed) DNA segments were efficiently excised in epidermal keratinocytes of K14-Cre transgenic mice. Furthermore, Tamoxifen administration to adult K14-Cre-ER(T2) mice efficiently induced recombination in the basal keratinocytes, whereas no background recombination was detected in the absence of ligand treatment. These two transgenic lines should be very useful to analyse the functional role of a number of genes expressed in keratinocytes.

Animals↗

PubMed2000

Mice bearing deletions of retinoic acid receptors demonstrate reduced lung elastin and alveolar numbers.

In mammals, including rats and mice, the development of pulmonary alveolar septa is primarily limited to late gestation and the early periods of postnatal life. Before this time, the rat lung contains a relatively large supply of endogenous retinyl ester that, together with its metabolite retinoic acid, has been shown to increase elastin gene expression and the number of alveoli. We have hypothesized that mice bearing a deletion of one or more genes encoding for retinoic acid receptors (which are DNA binding proteins that alter transcription of retinoic acid-responsive genes) may demonstrate abnormalities in retinoid-mediated alveolar formation. Our studies demonstrate that the absence of the retinoic acid receptor-gamma (RARgamma) is associated with a decrease in the steady-state level of tropoelastin messenger RNA in a subpopulation of lung fibroblasts at Postnatal Day 12. RARgamma gene deletion also resulted in a decrease in whole lung elastic tissue and alveolar number, and an increase in mean cord length of alveoli (L(m)) at Postnatal Day 28. The additional deletion of one retinoid X receptor (RXR)alpha allele resulted in a decrease in alveolar surface area and alveolar number, and an increase in L (m). These data indicate that RARgamma is required for the formation of normal alveoli and alveolar elastic fibers in the mouse, and that RAR/RXR heterodimers are involved in alveolar morphogenesis.

Animals↗

PubMed2000

RXRalpha overexpression in cardiomyocytes causes dilated cardiomyopathy but fails to rescue myocardial hypoplasia in RXRalpha-null fetuses.

Retinoid X receptor alpha-null (RXRalpha-null) mutants exhibit hypoplasia of their ventricular myocardium and die at the fetal stage. In the present study, we wished to determine whether transgenic re-expression of RXRalpha in mutant cardiac myocytes could rescue these defects. Two transgenic mouse lines specifically overexpressing an RXRalpha protein in cardiomyocytes were generated, using the cardiac alpha-myosin heavy chain (alpha-MHC) promoter. Breeding the high copy number transgenic line onto an RXRalpha-null genetic background did not prevent the myocardial hypoplasia and fetal lethality associated with the RXRalpha(-/-) genotype, even though the transgene was expressed in the ventricles as early as 10. 5 days post-coitum. These data suggest that the RXRalpha function involved in myocardial growth may correspond to a non-cell-autonomous requirement forsignal orchestrating the growth and differentiation of myocytes. Interestingly, the adult transgenic mice developed a dilated cardiomyopathy, associated with myofibrillar abnormalities and specific deficiencies in respiratory chain complexes I and II, thus providing an additional model for this genetically complex disease.

Animals↗

PubMed2000

Thyroid hormone resistance and increased metabolic rate in the RXR-gamma-deficient mouse.

Vitamin A and retinoids affect pituitary-thyroid function through suppression of serum thyroid-stimulating hormone (TSH) levels and TSH-beta subunit gene expression. We have previously shown that retinoid X receptor-selective (RXR-selective) ligands can suppress serum TSH levels in vivo and TSH-beta promoter activity in vitro. The RXR-gamma isotype has limited tissue distribution that includes the thyrotrope cells of the anterior pituitary gland. In this study, we have performed a detailed analysis of the pituitary-thyroid function of mice lacking the gene for the RXR-gamma isotype. These mice had significantly higher serum T4 levels and TSH levels than did wild-type (WT) controls. Treatment of RXR-gamma-deficient and WT mice with T3 suppressed serum TSH and T4 levels in both groups, but RXR-gamma-deficient mice were relatively resistant to exogenous T3. RXR-gamma-deficient mice had significantly higher metabolic rates than did WT controls, suggesting that these animals have a pattern of central resistance to thyroid hormone. RXR-gamma, which is also expressed in skeletal muscle and the hypothalamus, may have a direct effect on muscle metabolism, regulation of food intake, or thyrotropin-releasing hormone levels in the hypothalamus. In conclusion, the RXR-gamma isotype appears to contribute to the regulation of serum TSH and T4 levels and to affect peripheral metabolism through regulation of the hypothalamic-pituitary-thyroid axis or through direct effects on skeletal muscle.

Animals↗

PubMed2000

A role for Akt in mediating the estrogenic functions of epidermal growth factor and insulin-like growth factor I.

This study examines whether the serine/threonine protein kinase, Akt, is involved in the cross-talk between epidermal growth factor (EGF) and insulin-related growth factor I (IGF-I) receptors and ER-alpha. Treatment of MCF-7 cells with either EGF or IGF-I resulted in a rapid phosphorylation of Akt and a 14- to 16-fold increase in Akt activity, respectively. Akt activation was blocked by inhibitors of phosphatidylinositol 3-kinase, but not by an inhibitor of the ribosomal protein kinase p70S6K. Stable transfection of cells with a dominant negative Akt mutant blocked the effects of EGF and IGF-I on ER-alpha expression and activity, whereas stable transfection of cells with a constitutively active Akt mutant mimicked the effects of EGF and IGF-I. In the latter cells, there was a decrease in the amount of ER-alpha protein and messenger RNA (70-80%) and an increase in the amount of progesterone receptor protein, messenger RNA (4- to 9- and by 3.5- to 7-fold, respectively) and pS2 (3- to 5-fold). Coexpression of wild-type ER-alpha and the dominant negative Akt mutant in COS-1 cells also blocked the growth factor-stimulated activation of ER-alpha, but coexpression of the wild-type receptor with the constitutively active Akt mutant increased ER-alpha activity. Receptor activation was blocked by an antiestrogen. Studies using mutants of ER-alpha demonstrated that Akt increased estrogen receptor activity through the amino-terminal activation function-1 (AF-1). Serines S104 S106, S118, and S167 appear to play a role in the activation of ER-alpha by Akt.

Animals↗

PubMed2000

Retinoids stimulate leptin synthesis and secretion in human syncytiotrophoblast.

The syncytiotrophoblast (ST), which forms the outer layer of the chorionic villi, is the endocrine unit of the human placenta. Bathing in the maternal blood of the intervillous space, the ST secretes its hormonal products directly into the maternal circulation. Leptin is expressed in the ST and is secreted into the maternal circulation. However, its regulation and physiological role during pregnancy remain poorly known. In the present work we used the in vitro model of human cytotrophoblast differentiation into ST to study the effect of physiological and synthetic retinoids on leptin synthesis and secretion. Using specific antibodies we first illustrated by immunocytochemistry the expression of retinoic acid (RA) receptor alpha and retinoid X receptor alpha (RXRalpha) in ST. We then observed that leptin messenger ribonucleic acid and protein expression increased with in vitro ST formation. The 9-cis isomer of RA and the synthetic retinoid specific for RXRs (BMS 649) stimulated leptin messenger ribonucleic acid expression and secretion. In contrast, all-trans-RA and a RA alpha-specific ligand had no effect. These results suggest that retinoids regulate leptin expression and highlight a role for RXRalpha in this process.

Adult↗

PubMed2000

The AF-1 and AF-2 activating domains of retinoic acid receptor-alpha (RARalpha) and their phosphorylation are differentially involved in parietal endodermal differentiation of F9 cells and retinoid-induced expression of target genes.

Retinoic acid (RA) induces the differentiation of F9 cells cultured as monolayers into primitive endodermal-like cells, whereas a combination of RA and cAMP leads to parietal endodermal differentiation. In RA receptor alpha-null F9 cells (RARalpha-/- cells), RA still efficiently triggers RARgamma-mediated primitive endodermal differentiation, but parietal endodermal differentiation is markedly delayed. To investigate the role of RARalpha1 activation functions AF-1 and AF-2 and of their phosphorylation sites during RA- and cAMP-induced parietal differentiation, cell lines reexpressing WT or mutated RARalpha1 were established in RARalpha-/- cells. We have found that the protein kinase A (PKA) phosphorylation site and the AF-2AD core (helix 12) of RARalpha1 are required for efficient parietal endodermal differentiation, whereas the AF-1 proline-directed kinase phosphorylation site is dispensible. Interestingly, deletion of the AF-1 activating domain (the A/B region), but not of the AF-2AD core, generates a dominant negative mutant that abrogates primitive endodermal differentiation when expressed in RARalpha-/- cells. We also show that the RARalpha AF-1 and AF-2 activation functions, but not their phosphorylation sites, are involved in the induction of RA-responsive genes in a differential promoter context-dependent manner.

Animals↗

PubMed2000

Retinoic acid synthesis and hindbrain patterning in the mouse embryo.

Targeted disruption of the murine retinaldehyde dehydrogenase 2 (Raldh2) gene precludes embryonic retinoic acid (RA) synthesis, leading to midgestational lethality (Niederreither, K., Subbarayan, V., Dolle, P. and Chambon, P. (1999). Nature Genet. 21, 444-448). We describe here the effects of this RA deficiency on the development of the hindbrain and associated neural crest. Morphological segmentation is impaired throughout the hindbrain of Raldh2-/- embryos, but its caudal portion becomes preferentially reduced in size during development. Specification of the midbrain region and of the rostralmost rhombomeres is apparently normal in the absence of RA synthesis. In contrast, marked alterations are seen throughout the caudal hindbrain of mutant embryos. Instead of being expressed in two alternate rhombomeres (r3 and r5), Krox20 is expressed in a single broad domain, correlating with an abnormal expansion of the r2-r3 marker Meis2. Instead of forming a defined r4, Hoxb1- and Wnt8A-expressing cells are scattered throughout the caudal hindbrain, whereas r5/r8 markers such as kreisler or group 3/4 Hox genes are undetectable or markedly downregulated. Lack of alternate Eph receptor gene expression could explain the failure to establish rhombomere boundaries. Increased apoptosis and altered migratory pathways of the posterior rhombencephalic neural crest cells are associated with impaired branchial arch morphogenesis in mutant embryos. We conclude that RA produced by the embryo is required to generate posterior cell fates in the developing mouse hindbrain, its absence leading to an abnormal r3 (and, to a lesser extent, r4) identity of the caudal hindbrain cells.

Aldehyde Oxidoreductases↗

PubMed2000

Mice lacking the transcriptional corepressor TIF1beta are defective in early postimplantation development.

TIF1beta, a member of the transcriptional intermediary factor 1 family, has been reported to function as a corepressor for the large class of KRAB domain-containing zinc finger proteins of the Krüppel type. To address the biological function of TIF1beta, we have generated TIF1beta-deficient mice by gene disruption. TIF1beta protein was detected in wild-type but not TIF1beta(-/-) blastocysts. Homozygous mutant embryos, which developed normally until the blastocyst stage and underwent uterine implantation, were arrested in their development at the early egg-cylinder stage at about embryonic day (E) 5.5 and were completely resorbed by E8.5. Taken together, these results provide genetic evidence that TIF1beta is a developmental regulatory protein that exerts function(s) essential for early postimplantation development.

Alleles↗

PubMed2000

Effect of single and compound knockouts of estrogen receptors alpha (ERalpha) and beta (ERbeta) on mouse reproductive phenotypes.

The functions of estrogen receptors (ERs) in mouse ovary and genital tracts were investigated by generating null mutants for ERalpha (ERalphaKO), ERbeta (ERbetaKO) and both ERs (ERalphabetaKO). All ERalphaKO females are sterile, whereas ERbetaKO females are either infertile or exhibit variable degrees of subfertility. Mast cells present in adult ERalphaKO and ERalphabetaKO ovaries could participate in the generation of hemorrhagic cysts. Folliculogenesis proceeds normally up to the large antral stage in both ERalphaKO and ERbetaKO adults, whereas large antral follicles of ERalpha+/-ERbetaKO and ERalphabetaKO adults are markedly deficient in granulosa cells. Similarly, prematurely developed follicles found in prepubertal ERalphaKO ovaries appear normal, but their ERalphabetaKO counterparts display only few granulosa cell layers. Upon superovulation treatment, all prepubertal ERalphaKO females form numerous preovulatory follicles of which the vast majority do not ovulate. The same treatment fails to elicit the formation of preovulatory follicles in half of the ERbetaKO mice and in all ERalpha+/-/ERbetaKO mice. These and other results reveal a functional redundancy between ERalpha and ERbeta for ovarian folliculogenesis, and strongly suggest that (1) ERbeta plays an important role in mediating the stimulatory effects of estrogens on granulosa cell proliferation, (2) ERalpha is not required for follicle growth under wild type conditions, while it is indispensable for ovulation, and (3) ERalpha is also necessary for interstitial glandular cell development. Our data also indicate that ERbeta exerts some function in ERalphaKO uterus and vagina. ERalphabetaKO granulosa cells localized within degenerating follicles transform into cells displaying junctions that are unique to testicular Sertoli cells. From the distribution pattern of anti-Müllerian hormone (AMH) in ERalphabetaKO ovaries, it is unlikely that an elevated AMH level is the cause of Sertoli cell differentiation. Our results also show that cell proliferation in the prostate and urinary bladder of old ERbetaKO and ERalphabetaKO males is apparently normal.

Animals↗

PubMed2000

Retinoid signaling is essential for patterning the endoderm of the third and fourth pharyngeal arches.

The requirement of retinoic acid (RA) in the initial formation of the pharyngeal arches was investigated by treating headfold-stage mouse embryos with a pan-RAR antagonist in vitro and in vivo. This results in a complete absence of mesenchyme, arteries, nerves and epibranchial placodes of the 3rd and 4th pharyngeal arches, complete agenesis of the 3rd and 4th pouches and consistent lack of the 6th arch artery. Mesodermally derived endothelial cells are absent from the 3rd and 4th pharyngeal arch region and the distribution domain of EphA2 transcripts in mesodermal cells is shifted caudally. In situ hybridization with CRABPI, kreisler and EphA4 probes and the pattern of expression of a Wnt1-lacZ transgene show that neural crest cells (NCC) normally destined to the 3rd and 4th arches migrate ectopically. Most interestingly, the appearance of the 3rd and 4th arches is prevented by the antagonist only during a very narrow window of time, which does not correspond to the period of post-otic NCC migration. Both the timing of appearance and the nature of the defects in RAR antagonist-treated embryos indicate that migrating NCC and mesodermal cells destined to the caudal pharyngeal arches do not represent primary targets of RA action. Alterations in the endodermal expression pattern of Hoxa1, Hoxb1, Pax1, Pax9, Fgf3 and Fgf8 in response to the antagonist-induced block in RA signal transduction demonstrate for the first time that RA signaling is indispensable for the specification of the pharyngeal endoderm and suggest that this signaling is necessary to provide a permissive environment locally for the migration of NCC and mesodermal cells. Our study also indicates that the formation of the 2nd pharyngeal arch and that of the 3rd and 4th pharyngeal arches probably involve distinct RA-dependent developmental processes.

Animals↗

PubMed2000

The conserved amphipatic alpha-helical core motif of RARgamma and RARalpha activating domains is indispensable for RA-induced differentiation of F9 cells.

In monolayers cultures, retinoic acid (RA) induces the differentiation of F9 embryonal carcinomal (EC) cells into primitive endoderm-like cells, while a combination of RA and dibutyryl cAMP leads to parietal endoderm-like differentiation. Knock out of all RARgamma isoforms (RARgamma(-/-) line) drastically impairs primitive and subsequent parietal endodermal differentiation and affects the induction of many endogenous RA-responsive genes. Using lines that reexpress RARgamma2 or overexpress RARalpha1 lacking their AF-2AD core (RARgammadeltaAF2 and RARalphadeltaAF2, respectively), we show that this conserved amphipatic alpha-helical motif (helix 12) of the ligand binding domain, and therefore the activation function AF-2 of both receptors, is required for the induction of differentiation and target gene expression upon RA treatment of F9 EC cells. We also show that these deletion mutants behave as dominant negatives.

Amino Acid Sequence↗

PubMed2000

Stage-dependent responses of the developing lung to retinoic acid signaling.

Morphological analysis of vitamin A-deficient rat fetuses and of retinoic acid receptor (RAR and RXR) mutant mice have demonstrated that retinoic acid (RA) is essential for lung development. To gainfurther insight into RA signaling pathways during primary lung budformation and lung branching, we have investigated the effects of RA and of a pan-RAR antagonist in cultures of whole embryos and lung explants. Treatment of E8.0 embryos with the pan-RAR antagonist inhibits the formation of the primitive respiratory system. On the other hand, treatment of E11.75 and E12.5 lung explants with RA inhibits branching morphogenesis, whereas treatment with the pan-RAR antagonist at the same developmental stages stimulates formation of distal buds. The inhibitory effect of RA on branching is strongly decreased in RARbeta null lungs, while enhancement of budding by the pan-RAR antagonist is not affected by an RARgamma null mutation. Additionally, cellular retinol binding protein one (CRBPI) null lungs are more sensitive than wild type lungs to the pan-RAR antagonist-induced stimulation of branching. These data indicate that retinoid signaling is indispensable for the formation of primary lung buds and the oesophagotracheal septum from the primitive foregut. They also suggest that at the pseudoglandular stage, RA signaling through RARbeta, but not RARgamma, inhibits distal bud formation thereby promoting the formation of conducting airways. Moreover, the level of CRBPI in the pseudoglandular lung appears to participate in the control of branching morphogenesis.

Animals↗

PubMed1999

Interaction with members of the heterochromatin protein 1 (HP1) family and histone deacetylation are differentially involved in transcriptional silencing by members of the TIF1 family.

Mammalian TIF1alpha and TIF1beta (KAP-1/KRIP-1) are related transcriptional intermediary factors that possess intrinsic silencing activity. TIF1alpha is believed to be a euchromatic target for liganded nuclear receptors, while TIF1beta may serve as a co-repressor for the large family of KRAB domain-containing zinc finger proteins. Here, we report an association of TIF1beta with both heterochromatin and euchromatin in interphase nuclei. Co-immunoprecipitation of nuclear extracts shows that endogenous TIF1beta, but not TIF1alpha, is associated with members of the heterochromatin protein 1 (HP1) family. However, in vitro, both TIF1alpha and TIF1beta interact with and phosphorylate the HP1 proteins. This interaction involves a conserved amino acid motif, which is critical for the silencing activity of TIF1beta but not TIF1alpha. We further show that trichostatin A, an inhibitor of histone deacetylases, can interfere with both TIF1 and HP1 silencing. The silencing activity of TIF1alpha appears to result chiefly from histone deacetylation, whereas that of TIF1beta may be mediated via both HP1 binding and histone deacetylation.

Amino Acid Sequence↗