Etude du mécanisme d'activation de la transcription en réponse aux rétinoïdes

Le récepteur à l'acide rétinoïque (RAR) est un facteur de transcription qui module l'expression de gènes impliqués dans différents processus biologiques tels que l'homéostasie cellulaire, le développement embryonnaire, la croissance et la reproduction. RAR interagit, en réponse aux rétinoïdes avec une pléthore de protéines corégulatrices qui participent pleinement au mécanisme complexe et très contrôlé de la transcription. Leur implication dans le remodelage de la chromatine, dansla formation du complexe d'initiation de la transcription ainsi que dans l'activation des différents protagonistes, montre l'importance de la contribution des coactivateurs. Au cours de mon doctorat, je me suis intéressé au recrutement de trois principaux complexes coactivateurs : le complexe p160, le complexe médiateur et le complexe SWI/SNF. Leur implication dans le mécanisme de la transcription induite par les rétinoïdes, bien que connue, était faiblement caractérisée. Mes travaux dans les cellules P19 de carcinome embryonnaire de souris et utilisant la technique d'interférence par ARN ont permis de définir le rôle distinct de SRC1 et med1/DRIP205 dans la réponse aux rétinoïdes. D'autre part ces travaux ont abouti à un modèle de recrutement des coactivateurs et de formation du complexe d'initiation de la transcription au niveau du promoteur du gène suppresseur de tumeur RAR 2. Enfin, l'intégration du complexe SWI/SNF dans mon étude a permis de déterminer par des techniques de biologie moléculaire l'interaction physique et fonctionnelle avec le récepteur RAR. Ce travail apporte un éclairage dans la compréhension du mécanisme subtile d'activation de la transcription régulée par les récepteurs aux rétinoïdes.The Retinoic Acid Receptor (RAR) is a ligand activated transcription factor which modulates the expression of retinoic acid-target genes. These genes are implied in fundamental biological processes such as cellular homeostasis, embryogenesis, growth and reproduction. RAR recruits a plethora of coregulators with multiple functions in response to retinoids. These multiprotein complexes are key structural components in the complex and controlled transcription mechanism. Many of them participate in remodeling of chromatin, while otehrs are implied transcription complex formation. This underlines the importance of these coactivators in transcriptional activation. Yet their contribution to RAR-mediated transactivation remains poorly studied. This PhD thesis focused on the recruitment of three coactivator complexes : P160, mediator and SWI/SNF complex. These proteins are implied in distinct steps of the RAR-mediated process. I investigated this question by assessing the respective role of these coactivators by using molecular and cellular biology approaches in the P19 embryonal carcinoma cell line, an appropriate developmental system to study the role of RARs. Results of knock-down of SRC1 and med1 by RNA interference have demonstrated distinct roles of these coactivator complexes in retinoid-induced biological responses. This allowed us to propose a model summarizing complex formation during transcription initiation at the RARBeta2 promoter, a prototypical retinoic acid-regulated gene. Finally the study of the interaction between the ATP-dependent chromatin remodeling SWI-SNF complex and RAR identified us an additional step in the regulation of transcription by retinoids. Characterization of the role of each coactivators provide important information to better understand this complex regulation of RA-induced transcription.LILLE2-BU Santé-Recherche (593502101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

The elongation complex components BRD4 and MLLT3/AF9 are transcriptional coactivators of nuclear retinoid receptors.

International audienceNuclear all-trans retinoic acid receptors (RARs) initiate early transcriptional events which engage pluripotent cells to differentiate into specific lineages. RAR-controlled transactivation depends mostly on agonist-induced structural transitions in RAR C-terminus (AF-2), thus bridging coactivators or corepressors to chromatin, hence controlling preinitiation complex assembly. However, the contribution of other domains of RAR to its overall transcriptional activity remains poorly defined. A proteomic characterization of nuclear proteins interacting with RAR regions distinct from the AF-2 revealed unsuspected functional properties of the RAR N-terminus. Indeed, mass spectrometry fingerprinting identified the Bromodomain-containing protein 4 (BRD4) and ALL1-fused gene from chromosome 9 (AF9/MLLT3), known to associate with and regulates the activity of Positive Transcription Elongation Factor b (P-TEFb), as novel RAR coactivators. In addition to promoter sequences, RAR binds to genomic, transcribed regions of retinoid-regulated genes, in association with RNA polymerase II and as a function of P-TEFb activity. Knockdown of either AF9 or BRD4 expression affected differentially the neural differentiation of stem cell-like P19 cells. Clusters of retinoid-regulated genes were selectively dependent on BRD4 and/or AF9 expression, which correlated with RAR association to transcribed regions. Thus RAR establishes physical and functional links with components of the elongation complex, enabling the rapid retinoid-induced induction of genes required for neuronal differentiation. Our data thereby extends the previously known RAR interactome from classical transcriptional modulators to components of the elongation machinery, and unravel a functional role of RAR in transcriptional elongation

Increased Adipogenesis in Cultured Embryonic Chondrocytes and in Adult Bone Marrow of Dominant Negative Erg Transgenic Mice

<div><p>In monolayer culture, primary articular chondrocytes have an intrinsic tendency to lose their phenotype during expansion. The molecular events underlying this chondrocyte dedifferentiation are still largely unknown. Several transcription factors are important for chondrocyte differentiation. The Ets transcription factor family may be involved in skeletal development. One family member, the <em>Erg</em> gene, is mainly expressed during cartilage formation. To further investigate the potential role of Erg in the maintenance of the chondrocyte phenotype, we isolated and cultured chondrocytes from the rib cartilage of embryos of transgenic mice that express a dominant negative form of Erg (DN-Erg) during cartilage formation. DN-Erg expression in chondrocytes cultured for up to 20 days did not affect the early dedifferentiation usually observed in cultured chondrocytes. However, lipid droplets accumulated in DN-Erg chondrocytes, suggesting adipocyte emergence. Transcriptomic analysis using a DNA microarray, validated by quantitative RT-PCR, revealed strong differential gene expression, with a decrease in chondrogenesis-related markers and an increase in adipogenesis-related gene expression in cultured DN-Erg chondrocytes. These results indicate that Erg is involved in either maintaining the chondrogenic phenotype <em>in vitro</em> or in cell fate orientation. Along with the <em>in vitro</em> studies, we compared adipocyte presence in wild-type and transgenic mice skeletons. Histological investigations revealed an increase in the number of adipocytes in the bone marrow of adult DN-Erg mice even though no adipocytes were detected in embryonic cartilage or bone. These findings suggest that the Ets transcription factor family may contribute to the homeostatic balance in skeleton cell plasticity.</p> </div

Human Leukocyte Antigen-G Is Frequently Expressed in Glioblastoma and May Be Induced in Vitro by Combined 5-Aza-2′-Deoxycytidine and Interferon-γ Treatments

International audienc

Expression of chondrogenic and adipogenic markers during monolayer culture of wt and DN-Erg chondrocytes.

<p>A. Expression of chondrogenic genes <i>Col2a1</i>, <i>Sox9</i>, <i>Col10</i>, and <i>Runx2</i>. Gene expression was evaluated by RT-qPCR. B. Expression of adipogenic genes <i>Adpn</i>, and <i>Pparγ</i>. Gene expression was evaluated by RT-qPCR. The reported target gene: <i>Hprt</i> transcript ratio in chondrocytes was normalised to the target gene:<i>Hprt</i> transcript ratio (set to 1) of freshly plated wt chondrocytes (day 0). Data represent the mean of at least 2 independent chondrocyte cultures from 2 distinct mice for each genotype.</p

Morphological changes in wt and DN-Erg E18.5 chondrocytes in culture.

<p>Chondrocytes from freshly isolated from ribs of wt and DN-Erg transgenic mouse embryos (at E18.5) were cultured for up 20 days and stained with Oil red O. Phase-contrast images at days 0, 3, 9, 15 and 20 (with day 0, the day of plating) are shown (Scale bar, 50 µm).</p

Microarray analysis and gene ontology analysis of signalling pathways.

<p>A. Numbers of genes with differential expression between monolayer culture day 0 and day 20 in wt and DN-Erg embryo (E18.5) chondrocytes. Probe sets were filtered according to a 10-fold change cut-off. B. Hierarchical Clustering (HCl) diagram with clusters genes corresponding to the “extracellular matrix”, “metallopeptidase activity”, “Cartilage condensation and development”, “Ossification” annotations. C. Major signalling pathways predicted using Pathway-Express. Pathways listed are pathways with at least 5 or more genes which expression was modified during culture, as determined by Pathway Express. D. Hierarchical Clustering (HCl) tree with clusters of “Lipid metabolism process” and “Lipid transport” genes.</p

AF9 and BRD4 coactivate RARα in a ligand-independent manner.

<p>(A, B) P19 cells were transfected with the indicated amounts of AF9, sBRD4 or lBRD4 expression vectors for 24 hours with or without 1 µM atRA and <i>Rarβ2</i> gene expression was assayed by RT-QPCR. The basal expression level in non transfected, untreated cells was arbitrarily set to 1 and data were expressed as the mean±SEM (n = 5). *, p<0.05; **, p<0.01; ***, p<0.005. (C) <i>Af9</i> or <i>Brd4</i> knockdowns. (C, upper panel) AF9 or BRD4 expression was assayed by western blot analysis in P19wt, P19^Af9(−) and P19^Brd4(−). (C, lower panel) <i>Rarβ2</i> gene expression in AF9- or BRD4-depleted P19 cells. The time-dependent expression of <i>Rarβ2</i> upon stimulation with 1 µM TTNPB was quantified by RT-QPCR. (D) Exon-specific RT-QPCR assay of the <i>Rarβ2</i> mRNA. Cloned <i>mRarβ2</i> cDNA was used as a standard in PCR reaction, and used to select PCR primer sets displaying a similar efficiency (“Cloned cDNA”). <i>Rarβ2</i> mRNA from either P19^wt, P19^Af9(−) or P19^Brd4(−) was then formally quantified by Q-PCR. **, p<0.01, intra-sample comparison; ^§§, p<0.01, inter-sample comparison. (E) RAR associates to <i>Rarβ2</i> transcribed regions as a function of AF9 and BRD4 levels. P19^wt, P19^Af9(−) or P19^Brd4(−) were treated with 1 µM TTNPB for 1 hour and ChIP assays were performed. The specific enrichment in the different <i>Rarβ2</i> amplicons was assayed by Q-PCR and expressed normalized to background values (myoglobin gene). Data are expressed as the mean±SEM (n = 2). *, p<0.05; **, p<0.01; ***, p<0.005. (F) The AF-1 region of RAR confers DRB sensitivity to RA-induced transcription of the <i>Rarβ2</i> promoter. P19 cells were cotransfected as indicated with expression vectors coding for wtRXRα, wtRARα or ΔAF-1-RARα or ΔAF-2-RARα together with the mRARβ2-Luc reporter gene. Cells were treated 24 hours with 1 µM atRA and/or DRB and luciferase activity was quantified. Basal expression levels were arbitrarily set to 1 and data are expressed as the mean±SEM (n = 6). *, p<0.05; **, p<0.01; ***, p<0.005.</p

The N-terminus of RARα interacts with nuclear proteins.

<p>(A) A nucleus-targeted RARα AF-1 domain acts as a dominant negative receptor. HeLa cells were cotransfected with expression vectors coding for wild type (wt) RXRα, wtRARα, GFP-NLS and GFP-NLS-AF-1 at the indicated ratio together with a m<i>Rarβ2</i> promoter-driven reporter gene (mRARβ2-Luc). Cells were treated overnight with 1 µM atRA and luciferase activity was quantified. Basal expression levels were arbitrarily set to 1 and data are expressed as the mean±SEM (n = 3). *, p<0.05; **, p<0.01; ***, p<0.005. (Right panel) Confocal laser microscopy of transfected HeLa cells. (B) The RARα AF-1 domain is transcriptionally active. HeLa cells were transfected with mRARβ2-Luc and expression vectors coding for wtRXRα, wtRARα, N–terminally ΔAF-1-RARα) or C-terminally truncated ΔAF-2-RARα) RARα. Cell treatment, luciferase assays and calculations are as in (A). (C, D) Isolation and identification of proteins interacting with the AF-1 transactivation motif of RARα. AF-1 fused to GST (GST-AF-1) or GST alone (GST) were immobilized on a matrix and incubated with HeLa nuclear extracts (+HeLa) or buffer alone (Mock). Numbers indicate bands that were subjected to mass spectrometry analysis. (D) The table indicates the name, protein abbreviation, the UniProtKB/TrEMBL entry, percentage of peptide coverage in two representative purifications, and the predicted molecular mass. (E) Target validation by GST pulldown assays. Various domains of RARα were expressed as fusion proteins to GST (left panel) and used as baits for ³⁵S-labeled shortBRD4 (sBRD4), AF9, PAK6 and NAP1L2. CB: Coomassie Blue staining of RAR derivatives adsorbed on glutathione-Sepharose. (F) Interaction of RARα with BRD4 or AF9. FLIM-based FRET fluorescence assays were performed to determine the lifetime of the donor (GFP) in the indicated conditions.</p