Offre de thèse
EIFFEL : Détermination du rôle du FUS et du complexe multicomposant ARN-protéine MiCEE dans l'organisation fonctionnelle du génome au cours de la fibrose pulmonaire idiopathique.
Date limite de candidature
16-06-2024
Date de début de contrat
01-10-2024
Directeur de thèse
BARRETO Guillermo
Encadrement
Prof. Dr. Gergana Dobreva Anatomy and Developmental Biology, CBTM; 68167 Mannheim, Germany. European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University; 68167 Mannheim, Germany.
Type de contrat
école doctorale
équipe
Equipe 2 : Ingénierie Moléculaire, Cellulaire, Thérapeutique et Glycosyl Transférasescontexte
Idiopathic pulmonary fibrosis (IPF) is the most common interstitial pulmonary disease showing a prevalence of 20 new cases per 100,000 persons and year 2. Due to increased lung injuries following exposure to air pollution as a consequence of industrialization, this figure is increasing 3. A central event in IPF is an abnormal proliferation and migration of fibroblasts into the alveolar space following lung injury. While fibroblasts under normal circumstances are important to wound healing and connective tissue maintenance, their activity in the fibrotic lung is dysregulated. This results in the formation of fibroblastic foci, which consist of highly proliferative fibroblasts, immune cells and excessive extra cellular matrix (ECM) protein deposits, such as fibronectin (FN1) and collagen (COL1A1). The consequences of these processes are disproportionate levels of scar tissue, alterations of the alveolar framework, changes in the lung epithelium structure, stiffening of functional lung tissue, loss of the gas exchange function of the lung and a dramatic decrease of oxygen saturation in the blood 4. As a result, IPF patients die within 4 years following diagnosis, mostly due to respiratory failure. Current treatments for IPF aim to ameliorate patient symptoms and to delay disease progression. Therapies targeting the causes of IPF and exceeding disease management remain yet to be identified. Myofibroblasts are the key effector cell type that promotes connective tissue remodeling following injury. Myofibroblasts are contractile activated fibroblasts expressing α-smooth muscle actin (ACTA2) to facilitate wound closure 5. Following wound healing, myofibroblasts can dedifferentiate into resting cells such as lipofibroblasts 6. Persistent activation of myofibroblasts, however, causes excessive deposition of ECM proteins in the interstitial space leading to fibroblastic foci 7. Precise control of gene expression is essential for both expansions of activated fibroblasts following injury as well as dedifferentiation into resting fibroblasts after wound healing. During dedifferentiation of activated fibroblast into resting fibroblast, transcriptional induction of lineage specific genes and repression of pro-fibrotic genes allows for the resolution of fibroblastic foci. The nuclear genome in eucaryotic cells consists of DNA molecules packaged into thread-like structures known as chromosomes, which are built of chromatin. Thus, chromatin is the physiological template for biological processes in the nucleus of eucaryotic cells, including gene transcription. Transcriptional regulation directly corresponds to the mechanisms of how chromatin can be structurally arranged making it accessible for the transcription machinery 8. These mechanisms regulating chromatin structure and transcription comprise histone modifications, histone deposition, nucleosome remodeling, DNA methylation, ncRNAs, secondary structures of nucleic acids, among others 9-16. In addition, an increasing number of recent publications based on integrative analysis of multi-omics studies implementing NGS technologies, chromosome conformation capture-based methods, and super-resolution microscopy have provided comprehensive and multilevel insights into 3D genome organization emphasizing its role during transcriptional regulation 17. Chromatin is hierarchically organized at different levels including chromosomal territories, compartments, and self-interacting topologically associating domains (TADs) jointly giving rise to a highly dynamic 3D genome organization 18. The structure of the genome is linked to its function as shown by extensive correlations between chromatin condensation and related gene transcription. For example, chromatin shows condensed regions, referred to as heterochromatin (by convention, transcriptionally “inactive”), and less condensed regions, referred to as euchromatin (transcriptionally “active”). Interestingly, staining methods during interphase have demonstrated that chromosomes are not randomly localized within the interphase nucleus. Rather, regions preferentially occupied by specific chromosomes define chromosome territories and constitute a major feature of nuclear organization 19. The functionally coordinated interaction among (1) specific proteins, (2) different RNA subtypes, and (3) defined genome loci confers the discretely organized yet at the same time restricted tethering of the genetic material to regions in the nucleus with specialized function, so called nuclear sub-compartments 20,21. Thus, the structure and function of each nuclear sub-compartment are defined by the composition of and dynamic interactions among these 3 components. Biologically regulated phase separation has emerged as a key organizing principle underlying the formation of nuclear sub-compartments, which do not have membranous boundaries. Nuclear sub-compartments may thus be defined as micron-scale biomolecular aggregates delimited by intracellular phase separation between liquid, gel and solid states 22. They locally concentrate specific biomolecules and thereby potentiate biological processes catalyzed by them. Mechanistically, phase separation is driven by multivalent interactions among tyrosine residues from prion-like domains (named due to the similarity to the yeast prion protein), and arginine residues from RNA-binding proteins, promoted by low salt concentrations or RNA 23. Thus, biomolecular condensates are physically stabilized and maintained by scaffold proteins and nucleic acids 24. Scaffold proteins from the FUS family proteins may functionally self-associate to form liquid or gel-like aggregates that result in structures with reduced dynamic 25. FUS is an ubiquitously expressed, predominantly nuclear, RNA-binding protein. Various functions have been ascribed to FUS, primarily involving RNA metabolism and processing. We have previously shown that the function of the ncRNA-protein complex MiCEE is disrupted in human lung fibroblasts (hLF) from patients with IPF due to hyperactive EP300 11. In addition, as shown in the preliminary results for this research proposal below, reduced FUS levels in IPF correlate with pathogenic EP300 activation and HDAC inactivation. We will expand on these findings using high-resolution mass spectrometry and NGS technologies in primary hLFs derived from control donors and IPF patients (Ctrl hLF and IPF hLF). The relevance of the results obtained in hLF will be confirmed in human precision-cut lung slices (hPCLS) of control donors and IPF patients. Our results will provide the molecular basis for designing therapies of IPF exceeding symptomatic or supportive treatment.spécialité
Sciences de la Vie et de la Santé - BioSElaboratoire
IMoPA - Ingénierie Moléculaire et Physiopathologie Articulaire
Mots clés
Structure de la chromatine, Réglementation de la transcription, Les ARN non codants, Fusionné dans le sarcome, Fibrose pulmonaire idiopathique
Détail de l'offre
La fibrose pulmonaire idiopathique (FPI) est la maladie pulmonaire interstitielle la plus courante, avec une prévalence de 20 nouveaux cas pour 100 000 personnes par an. Les patients atteints de FPI décèdent dans les 4 années suivant le diagnostic, principalement en raison d'une insuffisance respiratoire. Les traitements actuels de la FPI améliorent les symptômes des patients et retardent la progression de la maladie. Nous avons publié deux articles marquants sur le complexe multicomposants ARN-protéine MiCEE (Singh I et al ; 2018 Nat Genet), qui est dérégulé dans la fibrose pulmonaire en raison de l'EP300 hyperactif (Rubio K et al ; 2019, Nat Comm). Des résultats préliminaires supplémentaires ont montré que la réduction des niveaux de FUS dans la fibrose est corrélé à l'activation pathogène d'EP300 et à l'inactivation de l'HDAC. Nous proposons, dans le cadre du projet de thèse proposé, d'approfondir ces résultats par des études multi-omiques intégratives en utilisant les technologies de séquençage de nouvelle génération (NGS) dans les fibroblastes pulmonaires primaires dérivés de donneurs témoins et de patients IPF (Ctrl hLF et IPF hLF). Ce travail mènera à une meilleure compréhension des mécanismes moléculaires sous-jacents à la FPI, fournissant ainsi la base de thérapies curatives ciblant les causes de la FPI.
Keywords
Chromatin structure, Transcription regulation, non-coding RNAs, Fused in sarcoma, Idiopathic pulmonary fibrosis
Subject details
Idiopathic pulmonary fibrosis (IPF) is the most common interstitial lung disease showing a prevalence of 20 new cases per 100,000 persons per year. Due to increased lung injury following exposure to air pollution as a consequence of industrialization, this figure is increasing. IPF patients die within 4 years following diagnosis mostly due to respiratory failure. Current treatments of IPF ameliorate patient symptoms and delay disease progression. We published two landmark papers on the multicomponent RNA–protein complex MiCEE (Singh I et al; 2018 Nat Genet), which is dysregulated in lung fibrosis due to hyperactive EP300 (Rubio K et al; 2019, Nat Comms). Additional preliminary results showed that reduced FUS levels in fibrosis correlate with pathogenic EP300 activation and HDAC inactivation. For this research proposal, we will delve further into these findings by performing integrative multi-omics studies using next generation sequencing (NGS) technologies in primary lung fibroblasts derived from control donors and IPF patients (Ctrl hLF and IPF hLF). The relevance of the results obtained in hLF will be confirmed in human precision-cut lung slices (hPCLS) of Ctrl donors and IPF patients. This work will lead to a better understanding of the molecular mechanisms underlying IPF providing the basis for curative therapies targeting the causes of IPF.
Profil du candidat
Connaissance et expérience pratique des techniques de laboratoire de biologie moléculaire et de biochimie analysant l'ADN, l'ARN et les protéines.
Connaissance de base de l'analyse de données NGS (RNA-seq et ChIP-seq).
Anglais parlé et écrit couramment. Testez le TOEFL avec plus de 70% des points à obtenir.
Candidate profile
Knowledge and practical experience with molecular biology and biochemistry laboratory techniques analyzing DNA, RNA and proteins.
Basic knowledge of NGS-data analysis (RNA-seq and ChIP-seq).
Fluently spoken and written English. Test TOEFL with more than 70% of the points to obtain.
Référence biblio
References
1 Lowe, L. A., Yamada, S., Kuehn, M. R. & May;128(10):1831-43. Genetic dissection of nodal function in patterning the mouse embryo. Development 128, 1831-1843 (2001).
2 Noble, P. W., Barkauskas, C. E. & Jiang, D. Pulmonary fibrosis: patterns and perpetrators. The Journal of clinical investigation 122, 2756-2762, doi:10.1172/JCI60323 (2012).
3 Navaratnam, V. et al. The rising incidence of idiopathic pulmonary fibrosis in the U.K. Thorax 66, 462-467, doi:10.1136/thx.2010.148031 (2011).
4 Coward, W. R., Saini, G. & Jenkins, G. The pathogenesis of idiopathic pulmonary fibrosis. Therapeutic advances in respiratory disease 4, 367-388, doi:10.1177/1753465810379801 (2010).
5 Xia, H. et al. Identification of a cell-of-origin for fibroblasts comprising the fibrotic reticulum in idiopathic pulmonary fibrosis. The American journal of pathology 184, 1369-1383, doi:10.1016/j.ajpath.2014.01.012 (2014).
6 Glasser, S. W. et al. Mechanisms of Lung Fibrosis Resolution. The American journal of pathology 186, 1066-1077, doi:10.1016/j.ajpath.2016.01.018 (2016).
7 Nanthakumar, C. B. et al. Dissecting fibrosis: therapeutic insights from the small-molecule toolbox. Nat Rev Drug Discov 14, 693-720, doi:10.1038/nrd4592 (2015).
8 Misteli, T. The Self-Organizing Genome: Principles of Genome Architecture and Function. Cell 183, 28-45, doi:10.1016/j.cell.2020.09.014 (2020).
9 Singh, I. et al. High mobility group protein-mediated transcription requires DNA damage marker gamma-H2AX. Cell research 25, 837-850, doi:10.1038/cr.2015.67 (2015).
10 Singh, I. et al. MiCEE is a ncRNA-protein complex that mediates epigenetic silencing and nucleolar organization. Nature genetics 50, 990-1001, doi:10.1038/s41588-018-0139-3 (2018).
11 Rubio, K. et al. Inactivation of nuclear histone deacetylases by EP300 disrupts the MiCEE complex in idiopathic pulmonary fibrosis. Nature communications 10, 2229, doi:10.1038/s41467-019-10066-7 (2019).
12 Dobersch, S. et al. Positioning of nucleosomes containing gamma-H2AX precedes active DNA demethylation and transcription initiation. Nature communications 12, 1072, doi:10.1038/s41467-021-21227-y (2021).
13 Cordero, J. et al. 3D genome organization during TGFB-induced transcription requires nuclear microRNA and G-quadruplexes. bioRxiv, 2023.2012.2022.573061, doi:10.1101/2023.12.22.573061 (2023).
14 Karlic, R., Chung, H. R., Lasserre, J., Vlahovicek, K. & Vingron, M. Histone modification levels are predictive for gene expression. Proceedings of the National Academy of Sciences of the United States of America 107, 2926-2931, doi:10.1073/pnas.0909344107 (2010).
15 Gohlke, J. et al. DNA methylation mediated control of gene expression is critical for development of crown gall tumors. PLoS genetics 9, e1003267, doi:10.1371/journal.pgen.1003267 (2013).
16 Georgakopoulos-Soares, I., Morganella, S., Jain, N., Hemberg, M. & Nik-Zainal, S. Noncanonical secondary structures arising from non-B DNA motifs are determinants of mutagenesis. Genome research 28, 1264-1271, doi:10.1101/gr.231688.117 (2018).
17 Papantonis, A. & Cook, P. R. Genome architecture and the role of transcription. Current opinion in cell biology 22, 271-276, doi:10.1016/j.ceb.2010.03.004 (2010).
18 Jerkovic, I. & Cavalli, G. Understanding 3D genome organization by multidisciplinary methods. Nature reviews. Molecular cell biology 22, 511-528, doi:10.1038/s41580-021-00362-w (2021).
19 Cremer, T. & Cremer, M. Chromosome territories. Cold Spring Harbor perspectives in biology 2, a003889, doi:10.1101/cshperspect.a003889 (2010).
20 Rubio, K., Dobersch, S. & Barreto, G. Functional interactions between scaffold proteins, noncoding RNAs, and genome loci induce liquid-liquid phase separation as organizing principle for 3-dimensional nuclear architecture: implications in cancer. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, fj201802715R, doi:10.1096/fj.201802715R (2019).
21 Pederson, T. The spatial organization of the genome in mammalian cells. Current opinion in genetics & development 14, 203-209, doi:10.1016/j.gde.2004.02.008 (2004).
22 Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nature reviews. Molecular cell biology 18, 285-298, doi:10.1038/nrm.2017.7 (2017).
23 Wang, J. et al. A Molecular Grammar Governing the Driving Forces for Phase Separation of Prion-like RNA Binding Proteins. Cell 174, 688-699 e616, doi:10.1016/j.cell.2018.06.006 (2018).
24 Shin, Y. & Brangwynne, C. P. Liquid phase condensation in cell physiology and disease. Science 357, doi:10.1126/science.aaf4382 (2017).
25 Rhoads, S. N., Monahan, Z. T., Yee, D. S. & Shewmaker, F. P. The Role of Post-Translational Modifications on Prion-Like Aggregation and Liquid-Phase Separation of FUS. International journal of molecular sciences 19, doi:10.3390/ijms19030886 (2018).
26 Varshney, D., Spiegel, J., Zyner, K., Tannahill, D. & Balasubramanian, S. The regulation and functions of DNA and RNA G-quadruplexes. Nature reviews. Molecular cell biology 21, 459-474, doi:10.1038/s41580-020-0236-x (2020).
27 Henderson, E., Hardin, C. C., Walk, S. K., Tinoco, I., Jr. & Blackburn, E. H. Telomeric DNA oligonucleotides form novel intramolecular structures containing guanine-guanine base pairs. Cell 51, 899-908, doi:10.1016/0092-8674(87)90577-0 (1987).
28 Hansel-Hertsch, R. et al. G-quadruplex structures mark human regulatory chromatin. Nature genetics 48, 1267-1272, doi:10.1038/ng.3662 (2016).
29 Zheng, K. W. et al. Detection of genomic G-quadruplexes in living cells using a small artificial protein. Nucleic acids research 48, 11706-11720, doi:10.1093/nar/gkaa841 (2020).
30 Renciuk, D. et al. G-quadruplex formation in the Oct4 promoter positively regulates Oct4 expression. Biochim Biophys Acta Gene Regul Mech 1860, 175-183, doi:10.1016/j.bbagrm.2016.11.002 (2017).
31 Sato, K., Hendrikx, A. G. M. & Knipscheer, P. RNA transcripts suppress G-quadruplex structures through G-loop formation. bioRxiv, 2023.2003.2009.531892, doi:10.1101/2023.03.09.531892 (2023).
32 Cui, W., Yoneda, R., Ueda, N. & Kurokawa, R. Arginine methylation of translocated in liposarcoma (TLS) inhibits its binding to long noncoding RNA, abrogating TLS-mediated repression of CBP/p300 activity. The Journal of biological chemistry 293, 10937-10948, doi:10.1074/jbc.RA117.000598 (2018).
33 Dovey, O. M., Foster, C. T. & Cowley, S. M. Histone deacetylase 1 (HDAC1), but not HDAC2, controls embryonic stem cell differentiation. Proceedings of the National Academy of Sciences of the United States of America 107, 8242-8247, doi:10.1073/pnas.1000478107 (2010).
34 De Magis, A. et al. DNA damage and genome instability by G-quadruplex ligands are mediated by R loops in human cancer cells. Proceedings of the National Academy of Sciences of the United States of America 116, 816-825, doi:10.1073/pnas.1810409116 (2019).
35 Lyu, J., Shao, R., Kwong Yung, P. Y. & Elsasser, S. J. Genome-wide mapping of G-quadruplex structures with CUT&Tag. Nucleic acids research 50, e13, doi:10.1093/nar/gkab1073 (2022).
36 Rubio, K., Castillo-Negrete, R. & Barreto, G. Non-coding RNAs and nuclear architecture during epithelial-mesenchymal transition in lung cancer and idiopathic pulmonary fibrosis. Cell Signal 70, 109593, doi:10.1016/j.cellsig.2020.109593 (2020).