Thyroid hormone receptors: the isotype specificity problem Frédéric FLAMANT Neurodevelopment group Institut de Génomique Fonctionnelle de Lyon Ecole Normale Supérieure de Lyon France PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
Learning objective: Finding two good reasons to study TR α 1 and TR β 1/2 Hint1) Receptor-selective ligands would be useful. Hint 2) There are now two genetic diseases: RTH α and RTH β PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
500 My ago: The ancestral TR gene duplication and its consequences. 1) Redundancy: Provides robustness to thyroid hormone signaling 1) Subfunctionalization: changes in expression patterns distributes TR function to two proteins. 2) Neofunctionalization: due to changes in coding sequences one TR can gain new properties. RXR TR β RXR TR α PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
TR 1 is the predominant receptor in brain. P0 P7 P21 Adult TR 1 TR 1 TR 2 Bradley et al., PNAS 1992 PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
Changes in amino ‐ acids sequence suggest subtle differences in the gene regulation properties of TR α 1 and TR β 1/2 TR RXR • Structure heterodimer DR4 5’ AGGCTANNNNAGGTCA 3’ 3’ TCCGATNNNNTCCGAT 5’ PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
Are the properties of TR α 1 and TR β 1 identical? A global comparative analysis of TR α 1 and TR β 1-mediated response to T3 in a neural cell line PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
C17.2 cells can differentiate into neuronal ‐ like cells after serum deprivation 10% serum Nestin Immunostaining 0% Serum Tuj1 immunostaining PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
Stable expression of tagged receptors GSTR α 1 or GSTR β 1 in C17.2 cells = C17.2 α and C17.2 β cells G prot SBP 5’LTR TR α 1/ β 1 IRES EGFP LTR3’ C 17.2 C17.2a C17.2b 95kDa 72kDa 55kDa Western Blot Protein G PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
Digital gene expression • Treat C17.2a or C17.2b cells with T3 • (10 ‐ 7 M, 6h, 12h, 24h, no serum) • Extract RNA samples • Reverse transcribe and prepare cDNA 3’end libraries • Sequence cDNA libraries • (>8x10 6 reads/sample SOLID sequencing) PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
Log2 induction rate In C17.2 β cells TR α repressed TR α and TR β activated TR β activated TR β activated 1 Log2 induction rate TR α repressed 1 TR α activated -1 In C17.2 αα cells -1 TR β repressed TR α activated TR α and TR β repressed TR β repressed PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
Hif2a Klf9 Hr Many genes display receptor ‐ selective response PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
Q-RT-PCR confirmation of TR α 1 selective response Adamtsl4 Aoc3 Slc16a1 Epas1 C17.2 α C17.2 β C17.2 α C17.2 β C17.2 α C17.2 β C17.2 α C17.2 β PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
Which genes are direct TR target? Chromatin affinity purification/sequencing: « ChapSeq » in C17.2 α and C17.2 β cells 2) DNA breakage 1) Cross ‐ linking 3) IgG affinity purification of TRcontaining 2) Sequence DNA fraction (>10 7 reads) complexes AGGTCGATCGATCGATGGGACTAGATCG AGGTCGATCGATCGATGGGACTAGATCG AGGTCGATCGATCGATGGGACTAGATCG AGGTCGATCGATCGATGGGACTAGATCG Y AGGTCGATCGATCGATGGGACTAGATCG AGGTCGATCGATCGATGGGACTAGATCG AGGTCGATCGATCGATGGGACTAGATCG AGGTCGATCGATCGATGGGACTAGATCG AGGTCGATCGATCGATGGGACTAGATCG AGGTCGATCGATCGATGGGACTAGATCG AGGTCGATCGATCGATGGGACTAGATCG AGGTCGATCGATCGATGGGACTAGATCG PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
Genome wide analysis reveals the existence of receptor ‐ specific binding sites PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
Receptor selective response is not due to differential receptor binding PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
T3 signaling getting simpler? TR TR TR TR TR RXR TR TR DR4 ER6 IR0 PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
MEME identifies only the DR4 consensus (MEME.org) PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
DR4 is the only consensus enriched in TR binding sites. X axis: random sequence y axis: TR binding sites. PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
Only evidences for DR4 TR TR TR TR TR RXR TR TR DR4 … but still room for alternatives PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
A recurrent co ‐ occurrence with CTCF occupation PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
T3 target genes in C17.2: any relevance to neurodevelopment? PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
Hypothetical new connection of T3 with glucose metabolism Glucose 2 ATP Epas1 Pfkfb3 Pyruvate Vegf Fatty acids Acetyl CoA O2 Cpt1a Krebs cycle Cox7a1 30 ATP 4NADH PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
A putative regulation of brain sensitivity for hypoxia Epas1 pfkfb3 vegf * * PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
Conclusions: 1) Both different expression patterns and different transactivation properties explain the different function of TR α 1 and TR β 1 2) Receptor selective response is not explained by selective promoter occupancy 3) TR/RXR/DR4/Coactivator complexes may have allosteric properties. 4) Regulation by TR of the hypoxia ‐ pathway could be of physiological relevance in some situations. PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
Neurodevelopment Group IGFL ENS Lyon Past group members: Laure Quignodon Ph.D. (now in Lausanne) Frédéric Picou Ph.D. (Now in Santiago) Eva Romero post ‐ doc (now in Bengalore) Collaborations: Teddy Fauquier post ‐ doc (now in Marseille) Hassan Avci Isabelle Dusart UPMC Paris Present group members: 1) Fabrice Chatonnet post ‐ doc 2) Romain Guyot IR CNRS 3) Sabine Richard CR INRA 4) Suzy Markossian IE INRA 5) Pierre Godement DR CNRS 6) Frédéric Flamant DR INRA Special Thanks to: Denise Aubert PBES Nadine Aguilera PBES PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
A sequential intervention of TR α 1 and TR β 1 during Purkinje cells differentiation. TR 1 L400R TR β 1 337T PO P8 P15 P21 P28 TR α 1 TR β 1 Polarisation Dendritic growth Growth factors secretion Synapses Formation Extra synapses elimination T3 PRESENTATION FROM THE 83rd ANNUAL MEETING OF THE AMERICAN THYROID ASSOCIATION, OCTOBER 16-20, 2013 (Frédéric Flamant)
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