Répression transcriptionnelle du gène TRH

Les hormones thyroïdiennes (HT : T3, T4) exerçant des effets pléiotropes chez les vertébrés, leur synthèse et leur sécrétion doivent être finement contrôlées. Elles agissent elles-mêmes sur leur production, par un système de rétrocontrôle négatif de l’expression des gènes hypothalamique TRH et hypophysaire TSH. Les fondements moléculaires de cette répression transcriptionnelle des gènes TRH et TSH par l’hormone T3, forme biologiquement la plus active des HT, restent méconnus. Certaines caractéristiques de cette régulation commencent toutefois à être identifiées, notamment le rôle spécifique des isoformes TRβ (versus TRα) des récepteurs des HT. La spécificité fonctionnelle de ces isoformes résiderait principalement dans leur extrémité aminoterminale, qui permettrait une interaction différentielle avec certains comodulateurs. L’objectif, aujourd’hui, est de caractériser ces comodulateurs et d’analyser leur contribution à la régulation transcriptionnelle du gène TRH par l’hormone T3.The synthesis and secretion of thyroid hormones (TH: T3, T4) must be strictly regulated. TH act on their own production via a negative feedback system. The synthesis of thyrotropin-releasing hormone (TRH), produced in the hypothalamus, and thyrotropin (TSH) in the pituitary is inhibited at the transcriptional level by TH. TRH and TSH stimulate production of TH. An outstanding, still open, question is the molecular basis of T3-dependent transcription repression of TRH and TSH genes. However, some regulatory components have been identified, with the β-TH receptor (TRβ) playing a specific regulatory role (versus TRα) in the negative feedback effects of T3 on production of TRH and TSH. Moreover, the N-terminus of TRβ is known to be a key element in this regulation. A hypothesis to explain this isoform specificity could be that TRβ and TRα interact differentially with transcriptional comodulators. Thus, it is critical to characterize these comodulators and to analyse their contribution to the transcription regulation of TRH

MECANISMES MOLECULAIRES DE LA REGULATION TRANSCRIPTIONNELLE PHYSIOLOGIQUE DE LA THYREOLIBERINE HYPOTHALAMIQUE (TRH) (ETUDE IN VIVO CHEZ LA SOURIS)

NOTRE ANALYSE A PORTE SUR LE ROLE DES ISOFORMES DES RECEPTEURS AUX HORMONES THYROIDIENNES (TR), TR ET TR, DANS LA REGULATION NEGATIVE INDUITE PAR LA T, SUR LE GENE DE LA THYREOLIBERINE HYPOTHALAMIQUE (TRH). L'UTILISATION DE LA POLYETHYLENIMINE (PEI) A PERMIS PREMIEREMENT, D'INTRODUIRE UN TRANSGENE CONTENANT LE PROMOTEUR TRH COUPLE AU GENE RAPPORTEUR LUCIFERASE (TRH-LUC) ET DEUXIEMMENT, DE SUREXPRIMER DES TRS DIRECTEMENT DANS L'HYPOTHALAMUS DE SOURIS IN VIVO. NOUS MONTRONS QUE LE TRANSGENE TRH-LUC EST REGULE DE MANIERE PHYSIOLOGIQUE : LA TRANSCRIPTION EST AUGMENTEE CHEZ LES ANIMAUX HYPOTHYROIDIENS ; ET REPRIMEE CHEZ CEUX TRAITES A LA T 3. EN CE QUI CONCERNE LES EFFETS DES TRS, SEULES LES DIFFERENTES ISOFORMES TR (1/2) SUREXPRIMEES DANS L'HYPOTHALAMUS INDUISENT UNE REPRESSION TRANSCRIPTIONNELLE SIGNIFICATIVE DU TRANSGENE TRH-LUC EN PRESENCE DE LA T 3. LA SUREXPRESSION DE TR1, LA BLOQUE. AINSI, TR MAIS NON TR JOUE UN ROLE SPECIFIQUE DANS LA REGULATION PHYSIOLOGIQUE DU GENE TRH. DE PLUS, LES EFFETS TRANSCRIPTIONNELS DES TRS SONT CORRELES AVEC DES EFFETS SUR LA T 4 CIRCULANTE. ENSUITE, NOUS AVONS ANALYSE LA BASE STRUCTURALE DE LA SPECIFICITE DE FONCTION DES TRS DANS CETTE REGULATION. L'UTILISATION DE CHIMERES TR CORRESPONDANT A L'ECHANGE DE DOMAINES A/B, C/D ET E ENTRE LES ISOFORMES TR1 ET 1 DE RAT (, , , ) MONTRE QUE LA REGION N-TERMINALE CONFERE UNE ACTION SPECIFIQUE DE L'ISOFORME TR DANS LA REPRESSION TRANSCRIPTIONNELLE DU GENE TRH PAR LA T 3. EN EFFET, LES CHIMERES AVEC UNE REGION N-TERMINALE (, ) BLOQUENT CETTE REPRESSION ALORS QUE LES CHIMERES AVEC UNE REGION N-TERMINALE (, ) L'INDUISENT. CEPENDANT, L'ANALYSE PAR RETARD SUR GEL MONTRE QUE CETTE SPECIFICITE N'EST PAS TROUVEE POUR LA RECONNAISSANCE D'UN TRE NEGATIF APPARTENANT AU GENE TRH, SUGGERANT QUE LE CONTEXTE CELLULAIRE ET LES INTERACTIONS PROTEINE-PROTEINE IN VIVO SONT IMPLIQUEES DANS CETTE SPECIFICITE. POUR CETTE RAISON, NOUS AVONS CIBLE LE ROLE QUE PEUVENT JOUER LES CO-REPRESSEURS NUCLEAIRES. NCOR ET SMRT DANS LA REGULATION NEGATIVE INDUITE PAR LE LIGAND. NOUS AVONS UTILISE LES TECHNIQUES D'HYBRIDATION IN SITU (HIS) ET DE NORTHERN POUR SUIVRE L'EXPRESSION DES CO-REPRESSEURS DANS L'HYPOTHALAMUS. L'HIS MONTRE DES ARNM NCOR/SMRT DANS LES MEMES REGIONS QUE LES TRS, SUGGERANT UN ROLE GENERAL DE CES CO-REPRESSEURS DANS LES MECANISMES DE TRANSMISSION DU SIGNA INDUITS PAR LES TRS. EN REVANCHE, BIEN QUE LES ARNM NCOR SOIENT EXPRIMES DANS LES NOYAUX PARAVENTRICULAIRES DE L'HYPOTHALAMUS, ILS SONT ABSENTS AU NIVEAU DES NEURONES A TRH. DE PLUS, NOS ETUDES FONCTIONNELLES IN VIVO, MONTRENT QUE NCOR A UN EFFET INHIBITEUR SUR LA REPRESSION DU TRANSGENE TRH-LUC PAR LA T 3. AINSI, NOUS CONCLUONS QUE L'EXPRESSION DU CO-REPRESSEUR NUCLEAIRE NCOR EST INCOMPATIBLE AVEC LA REGULATION PHYSIOLOGIQUE NEGATIVE DU GENE TRH PAR LA T 3.PARIS-BIUSJ-Thèses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Homology Modeling of Class A G-Protein-Coupled Receptors in the Age of the Structure Boom in resolved structures

International audienc

Evolution of chemokine receptors is driven by mutations in the sodium binding site.

Chemokines and their receptors (members of the GPCR super-family) are involved in a wide variety of physiological processes and diseases; thus, understanding the specificity of the chemokine receptor family could help develop new receptor specific drugs. Here, we explore the evolutionary mechanisms that led to the emergence of the chemokine receptors. Based on GPCR hierarchical classification, we analyzed nested GPCR sets with an eigen decomposition approach of the sequence covariation matrix and determined three key residues whose mutation was crucial for the emergence of the chemokine receptors and their subsequent divergence into homeostatic and inflammatory receptors. These residues are part of the allosteric sodium binding site. Their structural and functional roles were investigated by molecular dynamics simulations of CXCR4 and CCR5 as prototypes of homeostatic and inflammatory chemokine receptors, respectively. This study indicates that the three mutations crucial for the evolution of the chemokine receptors dramatically altered the sodium binding mode. In CXCR4, the sodium ion is tightly bound by four protein atoms and one water molecule. In CCR5, the sodium ion is mobile within the binding pocket and moves between different sites involving from one to three protein atoms and two to five water molecules. Analysis of chemokine receptor evolution reveals that a highly constrained sodium binding site characterized most ancient receptors, and that the constraints were subsequently loosened during the divergence of this receptor family. We discuss the implications of these findings for the evolution of the chemokine receptor functions and mechanisms of action

Feedback on hypothalamic TRH transcription is dependent on thyroid hormone receptor N terminus.

The beta thyroid hormone receptor (TRbeta), but not TRalpha1, plays a specific role in mediating T(3)-dependent repression of hypothalamic TRH transcription. To investigate the structural basis of isoform specificity, we compared the transcriptional regulation and DNA binding obtained with chimeric and N-terminally deleted TRs. Using in vivo transfection assays to follow hypothalamic TRH transcription in the mouse brain, we found that TRbeta1 and chimeras with the TRbeta1 N terminus did not affect either transcriptional activation or repression from the rat TRH promoter, whereas N-terminally deleted TRbeta1 impaired T(3)-dependent repression. TRalpha1 or chimeras with the TRalpha1 N terminus reduced T(3)-independent transcriptional activation and blocked T(3)-dependent repression of transcription. Full deletion of the TRalpha1 N terminus restored ligand-independent activation of transcription. No TR isoform specificity was seen after transcription from a positive thyroid hormone response element. Gel mobility assays showed that all TRs tested bound specifically to the main negative thyroid hormone response element in the TRH promoter (site 4). Addition of neither steroid receptor coactivator 1 nor nuclear extracts from the hypothalamic paraventricular nuclei revealed any TR isoform specificity in binding to site 4. Thus N-terminal sequences specify TR T(3)-dependent repression of TRH transcription but not DNA recognition, emphasizing as yet unknown neuron-specific contributions to protein-promoter interactions in vivo

Human subcutaneous adipose tissue Glut 4 mRNA expression in obesity and type 2 diabetes

International audienceCellular resistance to insulin caused by reduced glucose transport and metabolism is a primary defect leading to the development of metabolic disease. While the etiology of insulin resistance is multifactorial, reduced insulin action is associated with impaired activity of the glucose transporter GLUT4 in insulin-sensitive tissues. Yet, the role of adipose tissue GLUT4 deregulation in the pathogenesis of insulin resistance, obesity, and diabetes is still unclear. In this study, we assessed the relative GLUT4 level in human subcutaneous adipose tissue from obese, diabetic, and diabetic obese versus control subjects, using a real-time PCR method. GLUT4 mRNA levels were considerably decreased among type 2 diabetic patients compared with those of the controls (P < 0.01), whereas no such difference was found between obese and normal-weight controls. Multiple linear regressions analysis in both diabetic non-obese and diabetic obese groups showed a negative correlation between GLUT4 mRNA expression and both markers of obesity or insulin resistance (P < 0.01). However, in obese group, GLUT4 was inversely associated only with HOMA-IR (P < 0.01). Our findings showed that adipose GLUT4 gene expression changes were more related to insulin resistance and type 2 diabetes rather than to obesity

TRα2 or TRα1 gain or loss of function held endogenous <i>Trh</i> levels constant.

<p><b>A</b>: <b>TRα2 or TRα1 overexpression does not interfere with endogenous </b><b><i>Trh</i></b><b> mRNA levels.</b> Euthyroid 2-days old mice were transfected with 100 ng of empty pSG5 (ct), pSG5-TRα1 (TRα1) or pSG5-TRα2 (TRα2). Hypothalami were dissected at either 3 (3 days old) or 5 (5 days old) days of age (corresponding to 1 day and 3 days post transfection, respectively). Two µg of totRNA were reverse-transcribed and qPCR were performed. Endogenous <i>Trh</i> mRNA levels are not significantly modified by TRα2 or TRα1 overexpression in either 3 or 5-day old mice as compared to controls (Ct) <i>18S</i> mRNA were used as endogenous control. The whole experiment was repeated twice. <b>B</b>: <b>TRα2 or TRα1 knockdown does not interfere with endogenous </b><b><i>Trh</i></b><b> mRNA levels.</b> Euthyroid 2-days old mice were transfected with 400 ng of empty pCMV-H1 (Ct), pCMV-H1-TRα1 (shTRα1) or a mixture of 200 ng sh1TRα2 and 200 ng sh2TRα2 vectors (shTRα2). Hypothalami were dissected 1.5 days post transfection (3.5 days old mice). Endogenous <i>Trh</i> mRNA levels are not significantly modified by TRα2 or TRα1 knockdown as compared to control. <i>Gapdh</i> mRNA was used as an endogenous control. The whole experiment was repeated twice.</p

TRα2 shows dominant negative activity on positively and negatively regulated T₃ target genes <i>in vivo</i>.

<p><b>A</b>: <b>TRα2 exerts dominant negative activity on positively </b><b><i>ME-tk-luc</i></b><b> transcription.. </b><i>ME-tk-luc</i> transcription was measured in hypothyroid (PTU) 2 days old mice treated with T₃ (2.5 µg/g b.w.) (PTU+T3) or saline (PTU), 18 h after hypothalamic injection of 1 µg reporter construct and 100 ng expression vector (empty pSG5 (Ct) or pSG5-TRα1 (TRα1) or pSG5-TRα2 (TRα2)). Transcription from <i>ME-tk-luc</i> is significantly increased in the presence of T₃ when TRα1 is overexpressed (as compared to Ct) (p<0.001). In contrast TRα2 overexpression significantly increases basal, T₃-independent <i>ME</i> transcription as compared to Ct and TRα1 (p<0.001), but addition of T₃ does not modify transcription further. <b>B</b>: <b>TRα2 exerts dominant negative activity on negatively </b><b><i>Trh-luc</i></b><b> transcription.. </b><i>Trh-luc</i> transcription was measured in hypothyroid (PTU) 2 days old mice as described above (100 ng expression vector and 1 µg reporter gene, <i>Trh-luc</i> per pup). Transcription from a <i>Trh-luc</i> construct is significantly decreased both in absence (PTU) and presence of T₃ (PTU+T3) when TRα1 is overexpressed (as compared with Ct). In contrast, overexpression of TRα2 has no effect on T₃-independent <i>Trh</i> transcription, but blocks its T₃-dependent repression. SEMs are given, n≥10 per point. In each case, the whole experiment was repeated twice giving similar results. *, p<0.05; **, p<0.01; ***, p<0.001.</p