21 results on '"Lamribet K"'
Search Results
2. Comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation
- Author
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Anoud, M., primary, Delagoutte, E., additional, Helleu, Q., additional, Brion, A., additional, Duvernois-Berthet, E., additional, As, M., additional, Marques, X., additional, Lamribet, K., additional, Senamaud, C., additional, Jourdren, L., additional, Adrait, A., additional, Heinrich, S., additional, Toutirais, G., additional, Hamlaoui, S., additional, Gropplero, G., additional, Giovannini, I., additional, Ponger, L., additional, Gèze, M., additional, Blugeon, C., additional, Coute, Y., additional, Guidetti, R., additional, Rebecchi, L, additional, Giovannangeli, C., additional, De Cian, A., additional, and Concordet, J-P., additional
- Published
- 2024
- Full Text
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3. Comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation
- Author
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Anoud, M., primary, Delagoutte, E., additional, Helleu, Q., additional, Brion, A., additional, Duvernois-Berthet, E., additional, As, M., additional, Marques, X., additional, Lamribet, K., additional, Senamaud, C., additional, Jourdren, L., additional, Adrait, A., additional, Heinrich, S., additional, Toutirais, G., additional, Hamlaoui, S., additional, Gropplero, G., additional, Giovannini, I., additional, Ponger, L., additional, Gèze, M., additional, Blugeon, C., additional, Coute, Y., additional, Guidetti, R., additional, Rebecchi, L, additional, Giovannangeli, C., additional, De Cian, A., additional, and Concordet, J-P., additional
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- 2023
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4. CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair
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Charpentier, M., Khedher, A., Menoret, S., Brion, A., Lamribet, K., Dardillac, E., Boix, C., Perrouault, L., Tesson, L., Geny, S., De Cian, A., Itier, J, Anegon, I., Lopez, B., Giovannangeli, C., Concordet, J., Structure et Instabilité des Génomes (STRING), Muséum national d'Histoire naturelle (MNHN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Translational Sciences [Paris] (SANOFI), SANOFI Recherche, Genetic and Cellular Engineering in Immunology and Regenerative Medicine (Team 2 - U1064 Inserm - CRTI), Centre de Recherche en Transplantation et Immunologie (U1064 Inserm - CRTI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Stabilité Génétique et Oncogenèse (UMR 8200), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), ANRT, ANR-11-INBS-0014,TEFOR,Transgenèse pour les Etudes Fonctionnelles sur les Organismes modèles(2011), ANR-16-CE18-0012,STaHR,Stimulation de la Recombinaison Homologue pour la Thérapie Génique(2016), ANR-14-LAB5-0008,SOURIRAT,Nouveaux outils pour la création de rongeurs génétiquement modifiés(2014), ANR-11-LABX-0016,IGO,Immunothérapies Grand Ouest(2011), ANR-10-IBHU-0005,CESTI (TSI-IHU),Centre Européen des Sciences de la Transplantation et de l'Immunothérapie (TSI-IHU)(2010), Le Bihan, Sylvie, Infrastructures - Transgenèse pour les Etudes Fonctionnelles sur les Organismes modèles - - TEFOR2011 - ANR-11-INBS-0014 - INBS - VALID, Stimulation de la Recombinaison Homologue pour la Thérapie Génique - - STaHR2016 - ANR-16-CE18-0012 - AAPG2016 - VALID, Laboratoires communs organismes de recherche publics – PME/ETI - Nouveaux outils pour la création de rongeurs génétiquement modifiés - - SOURIRAT2014 - ANR-14-LAB5-0008 - LABCOM - VALID, Laboratoires d'excellence - Immunothérapies Grand Ouest - - IGO2011 - ANR-11-LABX-0016 - LABX - VALID, and Instituts Hospitalo-Universitaires B - Centre Européen des Sciences de la Transplantation et de l'Immunothérapie (TSI-IHU) - - CESTI (TSI-IHU)2010 - ANR-10-IBHU-0005 - IBHU - VALID
- Subjects
[SDV.MHEP] Life Sciences [q-bio]/Human health and pathology ,Science ,lcsh:Q ,lcsh:Science ,[SDV.MHEP]Life Sciences [q-bio]/Human health and pathology - Abstract
International audience; In genome editing with CRISPR-Cas9, transgene integration often remains challenging. Here, we present an approach for increasing the efficiency of transgene integration by homology-dependent repair (HDR). CtIP, a key protein in early steps of homologous recombination, is fused to Cas9 and stimulates transgene integration by HDR at the human AAVS1 safe harbor locus. A minimal N-terminal fragment of CtIP, designated HE for HDR enhancer, is sufficient to stimulate HDR and this depends on CDK phosphorylation sites and the multimerization domain essential for CtIP activity in homologous recombination. HDR stimulation by Cas9-HE, however, depends on the guide RNA used, a limitation that may be overcome by testing multiple guides to the locus of interest. The Cas9-HE fusion is simple to use and allows obtaining twofold or more efficient transgene integration than that with Cas9 in several experimental systems, including human cell lines, iPS cells, and rat zygotes.
- Published
- 2018
5. Identification des cibles génomiques du récepteur minéralocorticoïde (cistrome) dans des cellules rénales humaines : implication dans la régulation transcriptionnelle des gènes régulés par l’aldostérone
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Le Billan, F., primary, Khan, J.A., additional, Lamribet, K., additional, Bouligand, J., additional, Viengchareun, S., additional, Fagart, J., additional, and Lombès, M., additional
- Published
- 2015
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6. Rôle anti-thermogénique du récepteur minéralocorticoïde dans le tissu adipeux brun in vitro et in vivo : trans-répression de la protéine UCP1
- Author
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Kuhn, E., primary, Lamribet, K., additional, Viengchareun, S., additional, Le Menuet, D., additional, Fève, B., additional, and Lombès, M., additional
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- 2015
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7. Functional analysis of endoglin mutations from hereditary hemorrhagic telangiectasia type 1 patients reveals different mechanisms for endoglin loss of function
- Author
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Mallet, C., primary, Lamribet, K., additional, Giraud, S., additional, Dupuis-Girod, S., additional, Feige, J.-J., additional, Bailly, S., additional, and Tillet, E., additional
- Published
- 2014
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8. L’hypotonicité extracellulaire stimule l’expression rénale du récepteur minéralocorticoïde et augmente la réponse à l’aldostérone
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Lema, I., primary, Amazit, L., additional, Lamribet, K., additional, Blanchard, A., additional, Lombès, M., additional, Cherradi, N., additional, and Viengchareun, S., additional
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- 2014
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9. Mécanismes inédits du contrôle post-transcriptionnel de l’expression rénale du récepteur minéralocorticoïde par l’hypotonicité : conséquences physiopathologiques sur la balance hydro-électrolytique
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Lema, I., primary, Amazit, L., additional, Lamribet, K., additional, Blanchard, A., additional, Lombès, M., additional, Cherradi, N., additional, and Viengchareun, S., additional
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- 2014
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10. Mécanismes inédits du contrôle post-transcriptionnel de l’expression rénale du récepteur minéralocorticoïde par l’hypotonicité : conséquences physiopathologiques sur la balance hydro-é...
- Author
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Lema, I., Amazit, L., Lamribet, K., Blanchard, A., Lombès, M., Cherradi, N., and Viengchareun, S.
- Abstract
Introduction La balance hydrosodée et la pression artérielle sont régulées par l’aldostérone et le récepteur minéralocorticoïde (MR), un facteur de transcription dont l’expression rénale varie au cours du développement et est altérée dans certaines néphropathies. Nous venons de montrer que les variations de tonicité contrôlent l’expression rénale du MR par des mécanismes post-transcriptionnels [1] . L’hypertonicité (500 mOsm/L) induit l’expression de Tis11b, une protéine de liaison à l’ARN, qui accélère la dégradation du transcrit MR et réduit la signalisation relayée par le MR. Nous avons aussi démontré que l’augmentation urinaire du flux sodé par restriction hydrique ou diurétiques chez la souris réprime l’expression rénale de MR [2] . Matériels et méthodes Les études sont réalisées dans les cellules tubulaires rénales KC3AC1. Résultats Nous montrons que l’hypotonicité extracellulaire (150 mOsm/L) stimule (×4) l’expression du MR (messager et protéine) grâce au recrutement d’une autre protéine de liaison à l’ARN, HuR, sur des motifs riches en adénosines et uraciles (ARE) présents dans la région 3′-UTR du transcrit MR. La contre-expression de la protéine HuR par des siRNA, prévient l’augmentation d’expression du MR induite par l’hypotonicité, suggérant que HuR stabilise le transcrit MR. L’analyse par microscopie haut débit de la localisation subcellulaire de HuR a mis en évidence une redistribution rapide (2 min), transitoire et réversible de HuR dans le compartiment cytoplasmique en réponse à l’hypotonicité. Les interactions moléculaires de HuR avec la région 3′-UTR du MR et les conséquences fonctionnelles de la surexpression rénale de MR sont en cours de caractérisation. Nous explorerons aussi la relevance physiopathologique de ces modulations de l’expression rénale du MR in vivo en soumettant des souris à des conditions mimant une hypotonicité extracellulaire (gavage hydrique ou traitement par un antagoniste sélectif du récepteur V2R responsable d’un diabète insipide néphrogénique). Discussion et conclusion L’identification de ces mécanismes de régulation post-transcriptionnelle d’un récepteur stéroïdien, modulant sélectivement la signalisation minéralocorticoïde devrait améliorer notre compréhension de la physiopathologie des maladies rénales, de l’hypertension ou des résistances minéralocorticoïdes. Ce travail pourrait déboucher sur la mise en œuvre de nouvelles stratégies thérapeutiques. [ABSTRACT FROM AUTHOR]
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- 2014
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11. Comparative transcriptomics reveal a novel tardigrade-specific DNA-binding protein induced in response to ionizing radiation.
- Author
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Anoud M, Delagoutte E, Helleu Q, Brion A, Duvernois-Berthet E, As M, Marques X, Lamribet K, Senamaud-Beaufort C, Jourdren L, Adrait A, Heinrich S, Toutirais G, Hamlaoui S, Gropplero G, Giovannini I, Ponger L, Geze M, Blugeon C, Couté Y, Guidetti R, Rebecchi L, Giovannangeli C, De Cian A, and Concordet JP
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- Animals, Humans, Gene Expression Profiling, DNA Damage, Radiation Tolerance genetics, Tardigrada genetics, Tardigrada metabolism, Radiation, Ionizing, DNA Repair, DNA-Binding Proteins metabolism, DNA-Binding Proteins genetics, Transcriptome
- Abstract
Tardigrades are microscopic animals renowned for their ability to withstand extreme conditions, including high doses of ionizing radiation (IR). To better understand their radio-resistance, we first characterized induction and repair of DNA double- and single-strand breaks after exposure to IR in the model species Hypsibius exemplaris . Importantly, we found that the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in tardigrades' radio-resistance. To identify novel tardigrade-specific genes involved, we next conducted a comparative transcriptomics analysis across three different species. In all three species, many DNA repair genes were among the most strongly overexpressed genes alongside a novel tardigrade-specific gene, which we named Tardigrade DNA damage Response 1 ( TDR1 ). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and preserve chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade-specific gene conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping cope with high levels of DNA damage inflicted by IR., Competing Interests: MA, ED, QH, AB, ED, MA, XM, KL, CS, LJ, AA, SH, GT, SH, GG, IG, LP, MG, CB, YC, RG, LR, CG, AD, JC No competing interests declared, (© 2024, Anoud, Delagoutte, Helleu et al.)
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- 2024
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12. UCP1 transrepression in Brown Fat in vivo and mineralocorticoid receptor anti-thermogenic effects.
- Author
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Kuhn E, Lamribet K, Viengchareun S, Le Menuet D, Fève B, and Lombès M
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- Adipocytes chemistry, Adipocytes physiology, Adipogenesis physiology, Aldosterone pharmacology, Animals, Cell Differentiation physiology, Cell Line, Cold Temperature, Energy Metabolism physiology, Gene Expression drug effects, Gene Expression physiology, Humans, Male, Mice, Mice, Transgenic, Promoter Regions, Genetic physiology, RNA, Messenger analysis, Receptors, Mineralocorticoid genetics, Thermogenesis genetics, Transcription, Genetic drug effects, Tretinoin pharmacology, Adipose Tissue, Brown metabolism, Receptors, Mineralocorticoid physiology, Thermogenesis physiology, Transcription, Genetic physiology, Uncoupling Protein 1 genetics
- Abstract
Objectives: The mineralocorticoid receptor (MR), a hormone-activated transcription factor, besides its role in controlling hydroelectrolytic homeostasis, exerts pro-adipogenic and anti-thermogenic effects, inhibiting mitochondrial-uncoupling protein UCP1 expression in brown adipocytes. The aim of this study was to gain insight into the molecular mechanisms by which MR participates in such metabolic regulation., Methods: We evaluated in vivo MR effects on cold-induced UCP1 expression in MR-overexpressing mice. Expression profiles of several transcriptional coregulators were analyzed during differentiation of the brown adipocyte T37i cell line. Given that UCP1 expression is inversely controlled by catecholamines/retinoic acid and corticosteroids, we investigated the mechanisms of MR's inhibitory effect on UCP1 transcription in T37i cells. Chromatin immunoprecipitation (ChIP) experiments enabled us to explore MR interaction with UCP1 promoter regions., Results: Cold-induced UCP1 expression was blunted in the brown fat of MR-overexpressing mice. Along with induction of increasing mRNA levels for specific adipocyte markers during T37i differentiation, MR coactivator transcript levels significantly increased in intermediate states of differentiation, whereas expression of MR corepressors transiently increased approximately 2-fold. Such a simultaneous transient peak in coregulator expression is consistent with physiologically relevant cooperation occurring during brown adipogenesis. ChIP demonstrated that, after retinoic acid stimulation and aldosterone exposure, MR and PPARγ concomitantly bind to specific UCP1 promoter motifs., Conclusion: Our studies demonstrate that MR exerts a pivotal metabolic role by controlling energy expenditure, and provide novel information on how MR participates in the regulation of brown adipocyte function., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)
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- 2019
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13. Chromosomal Translocation Formation Is Sufficient to Produce Fusion Circular RNAs Specific to Patient Tumor Cells.
- Author
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Babin L, Piganeau M, Renouf B, Lamribet K, Thirant C, Deriano L, Mercher T, Giovannangeli C, and Brunet EC
- Abstract
Circular RNAs constitute a unique class of RNAs whose precise functions remain to be elucidated. In particular, cancer-associated chromosomal translocations can give rise to fusion circular RNAs that play a role in leukemia progression. However, how and when fusion circular RNAs are formed and whether they are being selected in cancer cells remains unknown. Here, we used CRISPR/Cas9 to generate physiological translocation models of NPM1-ALK fusion gene. We showed that, in addition to generating fusion proteins and activating specific oncogenic pathways, chromosomal translocation induced by CRISPR/Cas9 led to the formation of de novo fusion circular RNAs. Specifically, we could recover different classes of circular RNAs composed of different circularization junctions, mainly back-spliced species. In addition, we identified fusion circular RNAs identical to those found in related patient tumor cells providing evidence that fusion circular RNAs arise early after chromosomal formation and are not just a consequence of the oncogenesis process., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
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14. RNA-binding protein HuR enhances mineralocorticoid signaling in renal KC3AC1 cells under hypotonicity.
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Lema I, Amazit L, Lamribet K, Fagart J, Blanchard A, Lombès M, Cherradi N, and Viengchareun S
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- 3' Untranslated Regions genetics, Active Transport, Cell Nucleus genetics, Aldosterone metabolism, Gene Expression Regulation genetics, HEK293 Cells, Humans, Immediate-Early Proteins metabolism, Kidney physiology, Osmosis physiology, Promoter Regions, Genetic genetics, Protein Serine-Threonine Kinases metabolism, RNA Processing, Post-Transcriptional genetics, RNA, Messenger metabolism, Transcription, Genetic genetics, ELAV-Like Protein 1 metabolism, Kidney metabolism, Mineralocorticoids metabolism, Osmotic Pressure physiology, RNA-Binding Proteins metabolism, Receptors, Mineralocorticoid metabolism, Signal Transduction physiology
- Abstract
Mineralocorticoid receptor (MR) mediates the sodium-retaining action of aldosterone in the distal nephron. Herein, we decipher mechanisms by which hypotonicity increases MR expression in renal principal cells. We identify HuR (human antigen R), an mRNA-stabilizing protein, as an important posttranscriptional regulator of MR expression. Hypotonicity triggers a rapid and reversible nuclear export of HuR in renal KC3AC1 cells, as quantified by high-throughput microscopy. We also identify a key hairpin motif in the 3'-untranslated region of MR transcript, pivotal for the interaction with HuR and its stabilizing function. Next, we show that hypotonicity increases MR recruitment onto Sgk1 promoter, a well-known MR target gene, thereby enhancing aldosterone responsiveness. Our data shed new light on the crucial role of HuR as a stabilizing factor for the MR transcript and provide evidence for a short autoregulatory loop in which expression of a nuclear receptor transcriptionally regulating water and sodium balance is controlled by osmotic tone.
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- 2017
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15. HuR-Dependent Editing of a New Mineralocorticoid Receptor Splice Variant Reveals an Osmoregulatory Loop for Sodium Homeostasis.
- Author
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Lema I, Amazit L, Lamribet K, Fagart J, Blanchard A, Lombès M, Cherradi N, and Viengchareun S
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- Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Crystallography, X-Ray, ELAV-Like Protein 1 metabolism, Exons, Feedback, Physiological, Furosemide pharmacology, Homeostasis genetics, Humans, Introns, Kidney drug effects, Mice, Models, Molecular, Osmolar Concentration, Osmotic Pressure, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Receptors, Mineralocorticoid metabolism, Sodium, Dietary administration & dosage, Structural Homology, Protein, Water Deprivation, Water Intoxication genetics, Water Intoxication metabolism, Water Intoxication physiopathology, Alternative Splicing, ELAV-Like Protein 1 genetics, Kidney metabolism, Osmoregulation genetics, Receptors, Mineralocorticoid genetics, Sodium, Dietary metabolism
- Abstract
Aldosterone and the Mineralocorticoid Receptor (MR) control hydroelectrolytic homeostasis and alterations of mineralocorticoid signaling pathway are involved in the pathogenesis of numerous human diseases, justifying the need to decipher molecular events controlling MR expression level. Here, we show in renal cells that the RNA-Binding Protein, Human antigen R (HuR), plays a central role in the editing of MR transcript as revealed by a RNA interference strategy. We identify a novel Δ6 MR splice variant, which lacks the entire exon 6, following a HuR-dependent exon skipping event. Using isoform-specific TaqMan probes, we show that Δ6 MR variant is expressed in all MR-expressing tissues and cells and demonstrate that extracelullar tonicity regulates its renal expression. More importantly, this splice variant exerts dominant-negative effects on transcriptional activity of the full-length MR protein. Collectively, our data highlight a crucial role of HuR as a master posttranscriptional regulator of MR expression in response to osmotic stress. We demonstrate that hypotonicity, not only enhances MR mRNA stability, but also decreases expression of the Δ6 MR variant, thus potentiating renal MR signaling. These findings provide compelling evidence for an autoregulatory feedback loop for the control of sodium homeostasis through posttranscriptional events, likely relevant in renal pathophysiological situations.
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- 2017
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16. CRISPR-Cas9-guided oncogenic chromosomal translocations with conditional fusion protein expression in human mesenchymal cells.
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Vanoli F, Tomishima M, Feng W, Lamribet K, Babin L, Brunet E, and Jasin M
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- CRISPR-Cas Systems, DNA Breaks, Double-Stranded, Humans, Mesenchymal Stem Cells cytology, Mesenchymal Stem Cells metabolism, Oncogene Proteins, Fusion genetics, Promoter Regions, Genetic, Gene Editing methods, RNA-Binding Protein EWS genetics, Translocation, Genetic, WT1 Proteins genetics
- Abstract
Gene editing techniques have been extensively used to attempt to model recurrent genomic rearrangements found in tumor cells. These methods involve the induction of double-strand breaks at endogenous loci followed by the identification of breakpoint junctions within a population, which typically arise by nonhomologous end joining. The low frequency of these events, however, has hindered the cloning of cells with the desired rearrangement before oncogenic transformation. Here we present a strategy combining CRISPR-Cas9 technology and homology-directed repair to allow for the selection of human mesenchymal stem cells harboring the oncogenic translocation EWSR1-WT1 found in the aggressive desmoplastic small round cell tumor. The expression of the fusion transcript is under the control of the endogenous EWSR1 promoter and, importantly, can be conditionally expressed using Cre recombinase. This method is easily adapted to generate any cancer-relevant rearrangement.
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- 2017
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17. The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1.
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Rataj F, Planel S, Desroches-Castan A, Le Douce J, Lamribet K, Denis J, Feige JJ, and Cherradi N
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- 3' Untranslated Regions, Animals, COS Cells, Cell Culture Techniques, Chlorocebus aethiops, Cyclic AMP, Cyclic AMP-Dependent Protein Kinases metabolism, Endoribonucleases metabolism, Humans, Intracellular Signaling Peptides and Proteins metabolism, Mutagenesis, Site-Directed, Phosphorylation, Protein Interaction Domains and Motifs, RNA Recognition Motif Proteins metabolism, RNA Stability physiology, RNA, Messenger metabolism, RNA-Binding Proteins metabolism, Trans-Activators metabolism, Tristetraprolin metabolism, TATA-Binding Protein Associated Factors genetics, TATA-Binding Protein Associated Factors metabolism
- Abstract
TPA-inducible sequence 11b/butyrate response factor 1 (TIS11b/BRF1) belongs to the tristetraprolin (TTP) family of zinc-finger proteins, which bind to mRNAs containing AU-rich elements in their 3'-untranslated region and target them for degradation. Regulation of TTP family function through phosphorylation by p38 MAP kinase and Akt/protein kinase B signaling pathways has been extensively studied. In contrast, the role of cAMP-dependent protein kinase (PKA) in the control of TTP family activity in mRNA decay remains largely unknown. Here we show that PKA activation induces TIS11b gene expression and protein phosphorylation. Site-directed mutagenesis combined with kinase assays and specific phosphosite immunodetection identified Ser-54 (S54) and Ser-334 (S334) as PKA target amino acids in vitro and in vivo. Phosphomimetic mutation of the C-terminal S334 markedly increased TIS11b half-life and, unexpectedly, enhanced TIS11b activity on mRNA decay. Examination of protein-protein interactions between TIS11b and components of the mRNA decay machinery revealed that mimicking phosphorylation at S334 enhances TIS11b interaction with the decapping coactivator Dcp1a, while preventing phosphorylation at S334 potentiates its interaction with the Ccr4-Not deadenylase complex subunit Cnot1. Collectively our findings establish for the first time that cAMP-elicited phosphorylation of TIS11b plays a key regulatory role in its mRNA decay-promoting function., (© 2016 Rataj, Planel, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)
- Published
- 2016
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18. Finerenone Impedes Aldosterone-dependent Nuclear Import of the Mineralocorticoid Receptor and Prevents Genomic Recruitment of Steroid Receptor Coactivator-1.
- Author
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Amazit L, Le Billan F, Kolkhof P, Lamribet K, Viengchareun S, Fay MR, Khan JA, Hillisch A, Lombès M, Rafestin-Oblin ME, and Fagart J
- Subjects
- Active Transport, Cell Nucleus drug effects, Blotting, Western, Cell Line, Cell Nucleus drug effects, Cell Nucleus metabolism, Chromatin Immunoprecipitation, Dose-Response Relationship, Drug, Down-Regulation drug effects, Epithelial Sodium Channels genetics, HEK293 Cells, Humans, Kinetics, Microscopy, Fluorescence, Mutation, Promoter Regions, Genetic genetics, Protein Binding drug effects, Receptors, Mineralocorticoid genetics, Signal Transduction drug effects, Spironolactone pharmacology, Transcriptional Activation drug effects, Aldosterone pharmacology, Naphthyridines pharmacology, Nuclear Receptor Coactivator 1 metabolism, Receptors, Mineralocorticoid metabolism
- Abstract
Aldosterone regulates sodium homeostasis by activating the mineralocorticoid receptor (MR), a member of the nuclear receptor superfamily. Hyperaldosteronism leads todeleterious effects on the kidney, blood vessels, and heart. Although steroidal antagonists such as spironolactone and eplerenone are clinically useful for the treatment of cardiovascular diseases, they are associated with several side effects. Finerenone, a novel nonsteroidal MR antagonist, is presently being evaluated in two clinical phase IIb trials. Here, we characterized the molecular mechanisms of action of finerenone and spironolactone at several key steps of the MR signaling pathway. Molecular modeling and mutagenesis approaches allowed identification of Ser-810 and Ala-773 as key residues for the high MR selectivity of finerenone. Moreover, we showed that, in contrast to spironolactone, which activates the S810L mutant MR responsible for a severe form of early onset hypertension, finerenone displays strict antagonistic properties. Aldosterone-dependent phosphorylation and degradation of MR are inhibited by both finerenone and spironolactone. However, automated quantification of MR subcellular distribution demonstrated that finerenone delays aldosterone-induced nuclear accumulation of MR more efficiently than spironolactone. Finally, chromatin immunoprecipitation assays revealed that, as opposed to spironolactone, finerenone inhibits MR, steroid receptor coactivator-1, and RNA polymerase II binding at the regulatory sequence of the SCNN1A gene and also remarkably reduces basal MR and steroid receptor coactivator-1 recruitment, unraveling a specific and unrecognized inactivating mechanism on MR signaling. Overall, our data demonstrate that the highly potent and selective MR antagonist finerenone specifically impairs several critical steps of the MR signaling pathway and therefore represents a promising new generation MR antagonist., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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19. Cistrome of the aldosterone-activated mineralocorticoid receptor in human renal cells.
- Author
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Le Billan F, Khan JA, Lamribet K, Viengchareun S, Bouligand J, Fagart J, and Lombès M
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- Aldosterone metabolism, Cell Line, Gene Expression Regulation physiology, Humans, Kidney cytology, Receptors, Mineralocorticoid genetics, Signal Transduction physiology, Aldosterone pharmacology, Gene Expression Regulation drug effects, Kidney metabolism, Receptors, Mineralocorticoid biosynthesis, Response Elements physiology, Signal Transduction drug effects
- Abstract
Aldosterone exerts its effects mainly by activating the mineralocorticoid receptor (MR), a transcription factor that regulates gene expression through complex and dynamic interactions with coregulators and transcriptional machinery, leading to fine-tuned control of vectorial ionic transport in the distal nephron. To identify genome-wide aldosterone-regulated MR targets in human renal cells, we set up a chromatin immunoprecipitation (ChIP) assay by using a specific anti-MR antibody in a differentiated human renal cell line expressing green fluorescent protein (GFP)-MR. This approach, coupled with high-throughput sequencing, allowed identification of 974 genomic MR targets. Computational analysis identified an MR response element (MRE) including single or multiple half-sites and palindromic motifs in which the AGtACAgxatGTtCt sequence was the most prevalent motif. Most genomic MR-binding sites (MBSs) are located >10 kb from the transcriptional start sites of target genes (84%). Specific aldosterone-induced recruitment of MR on the first most relevant genomic sequences was further validated by ChIP-quantitative (q)PCR and correlated with concomitant and positive aldosterone-activated transcriptional regulation of the corresponding gene, as assayed by RT-qPCR. It was notable that most MBSs lacked MREs but harbored DNA recognition motifs for other transcription factors (FOX, EGR1, AP1, PAX5) suggesting functional interaction. This work provides new insights into aldosterone MR-mediated renal signaling and opens relevant perspectives for mineralocorticoid-related pathophysiology., (© FASEB.)
- Published
- 2015
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20. Functional analysis of endoglin mutations from hereditary hemorrhagic telangiectasia type 1 patients reveals different mechanisms for endoglin loss of function.
- Author
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Mallet C, Lamribet K, Giraud S, Dupuis-Girod S, Feige JJ, Bailly S, and Tillet E
- Subjects
- Animals, Antigens, CD chemistry, Cell Line, Cell Membrane metabolism, Endoglin, Gene Expression, Growth Differentiation Factor 2, Growth Differentiation Factors metabolism, Humans, Mice, Phenotype, Protein Binding, Protein Multimerization, Protein Transport, Receptors, Cell Surface chemistry, Telangiectasia, Hereditary Hemorrhagic diagnosis, Antigens, CD genetics, Antigens, CD metabolism, Mutation, Receptors, Cell Surface genetics, Receptors, Cell Surface metabolism, Telangiectasia, Hereditary Hemorrhagic genetics, Telangiectasia, Hereditary Hemorrhagic metabolism
- Abstract
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant inheritable vascular dysplasia caused by mutations in genes encoding either endoglin or activin receptor-like kinase-1 (ALK1). Functional significance of endoglin missense mutations remains largely unknown leading to a difficult discrimination between polymorphisms and pathogenic mutations. In order to study the functional significance of endoglin mutations and to help HHT1 diagnosis, we developed a cellular assay based on the ability of endoglin to enhance ALK1 response to bone morphogenetic protein 9 (BMP9). We generated and characterized 31 distinct ENG mutants reproducing human HHT1 missense mutations identified in patients of the Molecular Genetics Department in Lyon. We found that 16 mutants behaved like wild-type (WT) endoglin, and thus corresponded to benign rare variants. The 15 other variants showed defects in BMP9 response and were identified as pathogenic mutations. Interestingly, two mutants (S278P and F282V) had lost their ability to bind BMP9, identifying two crucial amino acids for BMP9 binding to endoglin. For all the others, the functional defect was correlated with a defective trafficking to the cell surface associated with retention in the endoplasmic reticulum. Further, we demonstrated that some intracellular mutants dimerized with WT endoglin and impaired its cell-surface expression thus acting as dominant-negatives. Taken together, we show that endoglin loss-of-function can result from different mechanisms in HHT1 patients. We also provide a diagnostic tool helping geneticists in screening for novel or conflicting ENG mutations., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
- Published
- 2015
- Full Text
- View/download PDF
21. Hypertonicity compromises renal mineralocorticoid receptor signaling through Tis11b-mediated post-transcriptional control.
- Author
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Viengchareun S, Lema I, Lamribet K, Keo V, Blanchard A, Cherradi N, and Lombès M
- Subjects
- 3' Untranslated Regions, Animals, HEK293 Cells, Humans, Mice, RNA Processing, Post-Transcriptional, Sodium metabolism, Water Deprivation, Butyrate Response Factor 1 metabolism, Kidney metabolism, Nuclear Proteins metabolism, RNA-Binding Proteins metabolism, Receptors, Mineralocorticoid metabolism, Water-Electrolyte Imbalance metabolism
- Abstract
The mineralocorticoid receptor (MR) mediates the Na(+)-retaining action of aldosterone. MR is highly expressed in the distal nephron, which is submitted to intense variations in extracellular fluid tonicity generated by the corticopapillary gradient. We previously showed that post-transcriptional events control renal MR abundance. Here, we report that hypertonicity increases expression of the mRNA-destabilizing protein Tis11b, a member of the tristetraprolin/ZFP36 family, and thereby, decreases MR expression in renal KC3AC1 cells. The 3'-untranslated regions (3'-UTRs) of human and mouse MR mRNA, containing several highly conserved adenylate/uridylate-rich elements (AREs), were cloned downstream of a reporter gene. Luciferase activities of full-length or truncated MR Luc-3'-UTR mutants decreased drastically when cotransfected with Tis11b plasmid, correlating with an approximately 50% shorter half-life of ARE-containing transcripts. Using site-directed mutagenesis and RNA immunoprecipitation, we identified a crucial ARE motif within the MR 3'-UTR, to which Tis11b must bind for destabilizing activity. Coimmunoprecipitation experiments suggested that endogenous Tis11b physically interacts with MR mRNA in KC3AC1 cells, and Tis11b knockdown prevented hypertonicity-elicited repression of MR. Moreover, hypertonicity blunted aldosterone-stimulated expression of glucocorticoid-induced leucine-zipper protein and the α-subunit of the epithelial Na(+) channel, supporting impaired MR signaling. Challenging the renal osmotic gradient by submitting mice to water deprivation, diuretic administration, or high-Na(+) diet increased renal Tis11b and decreased MR expression, particularly in the cortex, thus establishing a mechanistic pathway for osmotic regulation of MR expression in vivo. Altogether, we uncovered a mechanism by which renal MR expression is regulated through mRNA turnover, a post-transcriptional control that seems physiologically relevant., (Copyright © 2014 by the American Society of Nephrology.)
- Published
- 2014
- Full Text
- View/download PDF
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