IMCBio International PhD Program IMCBio PhD Call 2025

Skeletal muscle cells present a fascinating challenge in cell biology due to their syncytial structure - thousands of nuclei sharing a single cytoplasm. To manage this complexity, these cells create distinct functional domains within themselves. Although numerous inherited and acquired muscle pathologies exist, the mechanisms underlying most of these disorders remain poorly understood. This is largely because we lack the tools to study molecular events in specific muscle domains, even though many diseases originate from these regions. To overcome this limitation, our team has developed a set of innovative tools enabling domain-specific genetic manipulation within muscle cells. The recruited student will apply these tools to investigate pathological mechanisms in severe congenital dystrophies and advance gene therapy by targeting the precise muscle regions involved. Additionally, our team continues to discover new muscle domains and myonuclear subtypes linked to various processes such as aging, exercise, and cancer, utilizing single-nucleus transcriptome technology. With our unique domain-specific tools, we can now explore these regions in unprecedented detail.

Keywords: Skeletal muscle, muscle domains, mouse genetics, muscle dystrophy and myopathy, gene therapy, disease mechanism

Relevant publications:

- Kim, M*., Franke, V*., Brandt, B., Lowenstein, E.D., Schowel, V., Spuler S., Akaline, A., and Birchmeier, C. (2020) Single-nucleus transcriptomics reveals functional compartmentalization in syncytial skeletal muscle cells. Nature Communications. 11, 6375. *, co-first authors.

- Bavat Bornstein, Lia Heinemann-Yerushalm, iSharon Krief, Ruth Adler, Bareket Dassa, Dena Leshkowitz, Minchul Kim, Guy Bewick, Robert W Banks, and Elazar Zelzer (2023). Molecular characterization of the intact mouse muscle spindle using a multi-omics approach. eLIFE. Feb 6, https://doi.org/10.7554/eLife.81843

- Cristofer Calvo*, Coalesco Smith*, Taejeong Song, Sakthivel Sadayappan, Douglas P. Millay# and Minchul Kim#. Loss of Ufsp1 is compatible with embryogenesis and causes subtle structural changes of the neuromuscular junction. (Under review)

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

Cell motion is involved in a variety of phenomena during development. Its origins are diverse and they often result from self-organisation of tissues. Specifically, groups of cells can undergo rotation and the mechanisms at play can be addressed and understood by studies at the Interfaces between Physics and Biology.

These dynamics of directed motion can be reproduced in vitro with controlled conditions in 2D and in 3D with organoids. In this context, the team has established that tug-of-war between cell polarities pilot the onset of rotation in 2D rings (Ref. 1) and in cell doublets in 3D (Ref. 2). The acto-myosin and microtubule machinery together with adhesion complexes play a key role in regulating motions and in setting cell shapes (Ref. 3). Cells compete with each other and this can result in a global rotation. However the minimal protein machinery involved in the process needs to be determined and characterised. The PhD will consist in triggering rotation in stem cells by the expression of relevant proteins and their dosages guided by theoretical principles. It will involve stem cell biology, microfabrication, quantitative biology, theoretical physics, single cell sequencing and spatial transcriptomics. The project will be associated with international collaborators.

Keywords: organoids, quantitative biology, cytoskeleton, physical biology, single cell sequencing

Relevant publications:

- S. Lo Vecchio, O. Pertz, M. Szopos, L. Navoret, **  D. Riveline**  (2024). Spontaneous rotations in epithelia as an interplay between cell polarity and boundaries. BioRxiv, Nature Physics 20:322.

- Linjie Lu, Tristan Guyomar, Quentin Vagne, Rémi Berthoz, Alejandro Torres-Sánchez, Michèle Lieb, Cecilie Martin-Lemaitre, Kobus van Unen, Alf Honigmann, Olivier Pertz, Daniel Riveline,** Guillaume Salbreux,** (2024) Polarity-driven three-dimensional spontaneous rotation of a cell doublet, BioRxiv, Nature Physics 20:1194.

- Markus Mukenhirn, Chen-Ho Wang, Tristan Guyomar, Matthew J. Bovyn, Michael F. Staddon, Riccardo Maraspini, Linjie Lu, Cecilie Martin-Lemaitre, Masaki Sano, Tetsuya Hiraiwa, Daniel Riveline,** Alf Honigmann,** (2024) Tight junctions regulate lumen morphology via hydrostatic pressure and junctional tension, BioRxiv, Developmental Cell 59:1–16.

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

The inflammasome is an intracellular multiprotein complex that senses sterile tissue damage and infectious agents to initiate innate immune responses. Distinct inflammasomes containing specific sensing molecules exist. The NLRP3 inflammasome is unique as it detects a broad range of cellular stress signals but a primary and converging sensing mechanism initiating inflammasome assembly remains ill-defined. We found that NLRP3 binds altered endomembranes as a result of disruption of inter-organelle contact sites in response to danger signals. However, little is known about this fundamentally new mechanism of pattern recognition linking organelle spatial organization and innate immunity. The organelle-generated signals sensed by NLRP3 and the mechanisms underlying membrane recruitment and activation of the inflammasome remain largely unexplored and thus will be subject of this proposal. The major limit hampering their identification is the difficulty to disentangle the complex cell response to the variety of stimuli leading to NLRP3 activation. We aim to push this limit through an unprecedented combination of approaches ranging from in vitro reconstitution studies with isolated organelles and artificial liposomes and proteo-lipidomics, to cryo-FIB and cryo-ET imaging, molecular modelling, and in vivo testing of the physiological relevance of in vitro findings. This project will lay the foundation for how altered endomembranes serve as danger-associated molecular patterns to trigger innate immune responses.

Keywords: Inflammasome, NLRP3, endosome, PI4P, innate immunity, macrophages, inflammation

Relevant publications:

- KCNN4 links Piezo-dependent mechanotransduction to NLRP3 inflammasome activation. Li R, et al. Science Immunolog. 2023

-  Distinct changes in endosomal composition promote NLRP3 inflammasome activation. Zhang Z, et al. Nat Immunol. 2023

- Protein kinase D at the Golgi controls NLRP3 inflammasome activation. Zhang Z, et al. J. Exp. Med. 2017

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

During the course of immune responses B cells diversify their immunoglobulin (Ig) genes through somatic hypermutation (SHM) and class switch recombination (CSR), increasing antibody affinity and changing antibody isotypes expressed. SHM alters the variable regions of Ig heavy (IgH) and light (IgL) chain genes, creating clones with mutated receptors that are selected based on antigen affinity. CSR combines a variable region with a different constant region, changing the antibody isotype (e.g., IgM to IgG, IgE, or IgA) while preserving antigen specificity.

Both processes are initiated by Activation Induced Cytidine Deaminase (AID), which deaminates cytosines in DNA, leading to lesions recognized by Uracil DNA glycosylase (Ung). These lesions trigger mutations in SHM and DNA breaks in CSR. However, the mechanisms of AID-induced DNA damage and error-prone repair remain unclear.

In a CRISPR/Cas9 knockout screen for genes associated with CSR, we identified Fam72a as a key regulator of the balance between error-prone and error-free DNA repair and antibody diversification through CSR and SHM (Rogier et al., Nature 2021). Fam72a negatively regulates the protein levels of Ung2, the nuclear form of Ung. Based on these findings, we will conduct further CRISPR/Cas9 knockout screens targeting genes interacting with Fam72a and/or Ung2, followed by mechanistic studies. Additionally, conditional knockout mouse models will be developed to explore the role of selected genes in SHM and CSR in vivo.

Keywords: Antibody diversification, Somatic Hypermutation, Class Switch recombination, programmed DNA Damage/Repair, CRISPR/Cas9 screening, Genome Editing, AID, Fam72a, Uracil DNA glycosylase

Relevant publications:

- Rogier, M. et al. Fam72a enforces error-prone DNA repair during antibody diversification. Nature 600, 329, doi:10.1038/s41586- 021-04093-y (2021).

- Yilmaz, D. et al. Activation of homologous recombination in G1 preserves centromeric integrity. Nature, doi:10.1038/s41586-021- 04200-z (2021).

- Amoretti-Villa, R., Rogier, M., Robert, I., Heyer, V. & Reina-San-Martin, B. A novel regulatory region controls IgH locus transcription and switch recombination to a subset of isotypes. Cell Mol Immunol 16, 887-889 (2019).

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

Cancer cells differ from normal cells displaying high level of proliferative capacity. That creates cancer cell vulnerabilities, which can be targeted therapeutically. One of the cellular machineries which are “hijacked” by cancer cells are the nuclear pore complexes (NPCs). NPCs constitute the sole communication gates between the nucleus and the cytoplasm and ensure cellular function and survival. Nucleoporins (Nups) are the building protein units of the NPCs and represent an “achilles heel” of cancer cells. Cancer cells increase their NPC number to adapt to proliferative demand and depletion of Nups required for NPC assembly can selectively kill cancer cells and reduce tumor growth, providing a strong proof of principle for targeting Nups in cancer disease. However, the molecular mechanisms on how rapid spatial assembly of the NPCs can be achieved in cancer cells and how exactly misregulation of Nups homeostasis can cause cancer disease remain largely unknown. Our published findings identified a novel mechanism on how spatial assembly and increased biogenesis of NPCs can be achieved in cancer cells. We plan to characterize the role of newly identified factors in Nups homeostasis in relevant cellular cancer models. Inhibition of these factors should selectively reduce viability of cancer cells. This research may create unprecedented therapeutic perspectives for cancer patients.

Keywords: cancer, nuclear pore complexes, nucleoporins, nucleocytoplasmic transport, cancer-specific vulnerabilities

Relevant publications:

- Krupina K., Kleiss C., Metzger T., Fournane S., Schmucker S., Hofmann K., Fischer B., Paul N., Porter I.M., Raffelsberger W., Poch O., Swedlow J.R., Brino L. and Sumara I. (2016) Ubiquitin receptor protein UBASH3B drives Aurora B recruitment to mitotic microtubules. Developmental Cell. Jan 11, 36 (1): 63–78.

- Agote-Aran A., Schmucker S., Jerabkova K., Boyer I.J., Berto A., Pacini L., Ronchi P., Kleiss C., Guerard L., Schwab Y., Moine H., Mandel J-L., Jacquemont S., Bagni C. and Sumara I. (2020) Spatial control of nucleoporin condensation by fragile X-related proteins. EMBO Journal. Oct 15; 39 (20): e104467

- Pangou E., Bielska O., Guerber L., Schmucker S., Agote-Arán A., Ye T., Liao Y., Puig-Gamez M., Grandgirard E., Kleiss C., Liu Y., Compe E., Zhang Z., Aebersold R., Ricci R. and Sumara I. (2021) A PKD-MFF signaling axis couples mitochondrial fission to mitotic progression. Cell Reports. May 18; 35 (7): 109129

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

Actin, a self-assembling biopolymer, plays a crucial role via the formation of dynamic networks that determine cellular shape and mechanical properties. De novo mutations in cytoskeletal actin (ACTB and ACTG1 genes) lead to a wide range of rare diseases, termed non-muscle actinopathies (NMA). Neurological impairment is the most frequent feature across the whole NMA spectrum. The mechanisms linking precisely actin disfunction to these neurological symptoms, the extent of their severity as well as their progression remain so far unknown. Together with collaborators, the Reymann team aims to understand the molecular to functional consequences of these cytoplasmic actin variants using the model organism C. elegans.

Nine human substitutions, chosen to span a large range of severity in patients, were successfully recapitulated in C. elegans actin-coding gene using CRISPR/Cas9 editing. The main objective of the proposed PhD project is to identify the cellular and molecular mechanisms at play associated with the observed neurodevelopment disorders. We will specifically probe neurogenesis, neuronal migration and neuronal maturation including axonal growth capacities. One of the advantages, is that C. elegans invariance in lineage, cell fate acquisition and body plan is enabling to detect subtle developmental defects due to our ability to identify single neurons. We will notably rely on the very advantageous NeuroPAL transgene, including 41 neuronal fate markers in the form of fluorescent reporters, that enables to unambiguously identify each individual neuron.

Keywords: actin, C. elegans, neurodevelopmental disorders, rare disease

Relevant publications:

- Classification of human actin pathological variants using C. elegans CRISPR-generated models. Hecquet T., Arbogast N., Suhner D., Goetz A., Amann G., Yürekli S., Marangoni F., N. Greve J., Di Donato N., Reymann AC, bioRxiv 2024.07.22.604239; doi: https://doi.org/10.1101/2024.07.22.604239

- The kinesin Kif21b regulates radial migration of cortical projection neurons through a non-canonical function on actin cytoskeleton. José Alvarez Rivera, Laure Asselin, Peggy Tilly, Roxane Benoit, Claire Batisse, Ludovic Richert, Julien Batisse, Bastien Morlet, Florian Levet, Noémie Schwaller, Yves Mély, Marc Ruff, Anne-Cécile Reymann, Juliette Godin, Cell Reports, July 2023. DOI: 10.1016/j.celrep.2023.112744

- Rapid assembly of a polar network architecture drives efficient actomyosin contractility. Costache V, Prigent Garcia S, Plancke C, Li J, Begnaud S, Kumar Suman S, Reymann AC, Kim T, Robin F., Cell Reports, May 2022. DOI: 10.1016/j.celrep.2022.110868

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

Multiple sclerosis (MS) is an autoimmune disease affecting over 3 million people worldwide. Myelin is the major target of autoimmune reactions leading gradually to its loss, loss of oligodendrocytes, the only myelin-producing cells, and as a consequence neuronal degeneration and death. Whereas present treatments stabilize autoimmune causes of disease, failure of regenerative remyelination reflecting a differentiation block of oligodendrocyte progenitor cells (OPCs) is a major unmet issue. Our previous work revealed that retinoid X receptors (RXR) are positive modulators of OPC differentiation, but the mechanisms involved remain unknown. These proteins act as ligand-controlled transcription factors which translate signaling of vitamin A5 into transcriptional events in the cell. We propose to use unbiased proteomic approaches to determine RXR-associated protein complex(s) driving OPC differentiation whereas using bulk and single-cell RNAseq analyses identify transcriptional programs underlying RXR control of OPC differentiation. To this end we will use highly reproducible in vitro models of OPC differentiation. The relevance of obtained data for regenerative oligodendrogenesis during aging and after myelin lesion will be next investigated in mouse models used for research into MS, but also RXR-relevant genetic mouse models including newly generated mouse line designed for tracing cell fate of RXR-expressing cells. This study should allow revealing the molecular identities of individual cells contributing to regeneration to understand the mechanisms of this process and define new therapeutic targets. Finally, we will test whether enhancing the RXR signaling pathway by new RXR ligands or targeting its downstream targets may promote regenerative process in mouse models of demyelinating diseases.

Keywords: multiple sclerosis, neurodegeneration, regenerative medicine, oligodendrocytes, retinoid receptors

Relevant publications:

- Krzyżosiak, A., Podleśny-Drabiniok, A., Vaz, B., Alvarez, R., Rühl, R., de Lera, A.R., and Krężel, W. Vitamin A5/X controls stress-adaptation and prevents depressive-like behaviors in a mouse model of chronic stress. Neurobiology of stress. 2021, 15, 100375.

- Baldassarro VA, Krężel W, Fernández M, Schuhbaur B, Giardino L, Calzà L. The role of nuclear receptors in the differentiation of oligodendrocyte precursor cells derived from fetal and adult neural stem cells. Stem Cell Res. 2019, 37:101443

- Huang JK, Jarjour AA, Nait Oumesmar B, Kerninon C, Williams A, Krezel W, Kagechika H, Bauer J, Zhao C, Baron-Van Evercooren A, Chambon P, Ffrench-Constant C, Franklin RJM. Retinoid X receptor gamma signaling accelerates CNS remyelination. Nat Neurosci. 2011, 14(1):45-53.

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

Several tissues in C. elegans are syncytia, large polynucleated cells. The goal of this project is to understand how functional compartmentalization is implemented within such cells, focusing in particular on the larger syncytium, called Hyp7.  Hyp7 is formed during embryogenesis by the fusion of 23 cells, and lines the ventral and dorsal sides of the worm along an antero-posterior axis. In addition, during larval development, several cells will join this syncytium by fusion, which will then contain 139 nuclei in the adult. This tissue is characterized by the homogeneous expression of markers, visualized in all the nuclei by the use of fluorescent reporters. One of the most commonly used markers of this tissue is dpy-7.

A study of the role of the homeobox-domain transcription factor ceh-14 showed that this factor is expressed in Hyp7. Surprisingly, however, this study suggested that ceh-14 is not homogeneous present but limited to a few nuclei within Hyp7. We have confirmed these observations, and our preliminary data suggest that this differential expression already exists at the ceh-14 messenger level. This suggested that, like in myofibers, some nuclei can exhibit a specialised expression profile within a single cell sharing the same cytoplasm. The ability of specific nuclei to express markers unique to them raises questions about the organization and compartmentalization within a large polynuclear cell, its origin and role. Thus, the proposed project will address the following questions:

A- Which nuclei exactly within hyp7 express unique markers?

B- How do regionalised messengers remain localized in a specific region of this giant cell? In particular, are there structures within Hyp7 that limit the diffusion along the antero-posterior axis of molecules (messenger RNA and proteins)? Indeed, such structures, formed by membrane invaginations have been observed in the syncytium formed by the worm germ line.

C- What happens when additional cell nuclei that are forced to fuse to Hyp7? Is the expression of their own markers lost after fusion in these additional nuclei?

D- Is the function of Hyp7 or of neighboring tissues affected if these markers normally expressed in a few nuclei are forced to be expressed in all?

Several experimental approaches will be implemented: single nuclei RNAseq to evaluate the number of differentially expressed genes in a syncytium, forced cell fusions using the EFF-1 fusogene, genetic aproaches, transgenesis, high-end microscopy on live animals and in situ hybridization on single RNA molecule (smFISH).  This work should allow to elucidate the mechanisms allowing differential expression and compartmentalisation of information, and eventually lead to a better understanding of the functioning of large polynucleated cells. In the long term, it will allow to understand the possible regionalization of their interaction with neighboring tissues, with important implications for human muscle function.

Keywords: Syncytium, regionalized expression, functional compartimentalisation, snRNAseq, C. elegans

Relevant publications:

- Daniele T., Cury J., Morin M.C., Ahier A., Isaia D. & Jarriault S. (2024) Essential and dual effects of Notch activity on a natural transdifferentiation event. Nat. Communications, in press and BioRxiv, doi: https://doi.org/10.1101/2024.09.11.612396

- Riva C., Gally C., Hajduskova M., Suman S.K., Ahier A. & Jarriault S. (2021) A natural transdifferentiation event involving mitosis is empowered by integrating signaling inputs with conserved plasticity factors. Cell Rep. 40(12):111365. doi: 10.1016/j.celrep.2022.111365

- Sequential histone-modifying activities determine the robustness of transdifferentiation. Zuryn S., Ahier A., Portoso M., Redhouse White E., Margueron R., Morin M.C. & Jarriault S. Science 345(6198):826-829.  (2014).  Cited « Recommended » by Faculty 1000.

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

In this project, you will delve into the complex interplay between the cell cycle and cell differentiation regulation in human induced pluripotent stem cells (hiPSCs). By focusing on computational modeling, this thesis aims to uncover how changes in the cell cycle influence cell fate decisions during differentiation. The research builds on cutting-edge approaches in machine learning and biophysical modelling to decode gene regulatory mechanisms underlying the interplay between cell cycle and cell differentiation.

You will work on two main objectives:

Aim 1: Developing context-dependent computational models to explore how cell cycle dynamics interact with cell differentiation. By leveraging RNA velocity techniques and variational autoencoders, we will model how differentiation trajectories unfold and how these processes are regulated at the transcriptional level.

Aim 2: Using single-cell multiomics data, you will study how chromatin accessibility and mRNA metabolism are dynamically regulated during cell cycle and differentiation transitions. This will involve integrating chromatin state changes with RNA-seq data to identify key regulatory mechanisms.

Ultimately, this research will provide crucial insights into how cell cycle and differentiation are linked, aiding in the development of novel differentiation protocols that are crucial for cell therapy and regenerative medicine.

Required skills

• Strong programming skills (Python, R, or similar).

• Interest in learining machine learning and applying it to biological data.

• Interest in perfom single-cell sequencing experiments.

• Ability to work independently and in a multidisciplinary team.

Acquired expertise during the internship

• Advanced machine learning techniques (e.g., variational autoencoders, RNA velocity).

• Hands-on experience with single-cell omics data analysis.

• Deep understanding of cell cycle and differentiation dynamics.

• Development of computational models to predict gene regulatory networks.

• Critical thinking and scientific communication

Keywords:

Relevant publications:

- Single-Cell multiomics reveals the oscillatory dynamics of mRNA metabolism during the cell cycle. M. K. Nariya, D. Santiago-Algarra, O. Tassy, M. Cerciat, T. Ye, A. Riba and N. Molina. bioRxiv 2024 (Under review in Cell Systems).

- Gene-specific RNA homeostasis revealed by perturbation of coactivator complexes.
F. Forouzanfar, F. Plassard, A. Furst, D. F. Moreno, K. A. Oliveira, L. Tora, N. Molina and M. Mendoza. bioRxiv 2024 (Under revision in Science Advances).

- Mammalian promoters are characterised by low occupancy and high turnover of RNA polymerase II K. Chatsirisupachai, C. J. I. Moene, R. Kleinendorst, E. Kreibich, N. Molina, A. Krebs bioRxiv 2024 (Under revision in Molecular Systems Biology).

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

RNA polymerase II (Pol II) transcription initiation is regulated by the sequential assembly of protein complexes called General Transcription Factors (GTFs) at promoters. The first GTF to bind is TFIID. Interestingly, while the canonical TFIID complex is essential in many cells, alternative mechanisms are associated with specific cellular contexts. One of the main axes of the team’s research is to explore the variability in protein complexes linked to Pol II transcription initiation during differentiation and development.

During folliculogenesis, active transcription in growing oocytes facilitates the accumulation of mRNA and proteins, which are essential for acquiring fertilization competence and initiating embryonic development. In mice, our previous research identified a first transition in the transcription machinery, where Pol II transcription initiation occurs without functional TFIID, relying instead on an oocyte-specific non-TFIID complex. After oocyte growth, transcription stops and only resumes after fertilization and early development, during zygotic genome activation. The transcription initiation machinery involved in this second transition remains unknown.

This PhD proposal aims to identify the protein complexes driving Pol II transcription initiation during these transitions, focusing on GTFs and transcription co-activators such as SAGA and ATAC, using the mouse as a model. The study will involve two key approaches: (1) analyzing the expression profiles of nuclear transcription complex subunits via immunofluorescence in growing oocytes and early embryos, and (2) assessing the functional significance of these complexes using siRNA electroporation in these stages.

Keywords: RNA polymerase II, transcription initiation, mouse, growing oocytes, pre-implantation embryos, genomics, transcriptomics

Relevant publications:

- V. Hisler, P. Bardot, D. Detilleux, A. Bernardini, M. Stierle, E. Garcia Sanchez, C. Richard, L. Hadj Arab, C. Ehrhard, B. Morlet, Y. Hadzhiev, M. Jung, S. Le Gras, L. Négroni, F. Müller, L. Tora and S.D. Vincent, RNA polymerase II transcription in holo-TFIID depleted mouse embryonic stem cells (2024), Cell Reports, 43(10):114791, 10.1016/j.celrep.2024.114791

- V. Fischer, V. Hisler, E. Scheer, E. Lata, B. Morlet, D. Plassard, D. Helmlinger, D. Devys, L. Tora and S.D. Vincent, SUPT3H-less SAGA coactivator can assemble and function without significantly perturbing RNA polymerase II transcription in mammalian cells (2022), Nucleic Acids Res., 14(50):7972-7990, 10.1093/nar/gkac637

- C. Yu, N. Cvetesic, V. Hisler, K. Gupta, T. Ye, E. Gazdag, L. Negroni, I. Berger, P. Hajkova, B. Lenhard, F. Müller and S.D. Vincent & L. Tora, TBPL2/TFIIA complex overhauls oocyte transcriptome during oocyte growth (2020), Nature Commun, 11:6439, 10.1038/s41467-020-20239-4 

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

Cellular senescence and the Senescence-Associated Secretory Phenotype (SASP) play dual roles in tumorigenesis. Cellular senescence, a state of stable cell-cycle arrest, can act as a tumor-suppressive mechanism by halting the proliferation of damaged or transformed cells. However, senescent cells also secrete a complex mixture of pro-inflammatory cytokines, growth factors, and proteases (SASP), which can paradoxically promote tumor growth, immune evasion, and metastasis by modifying the tumor microenvironment. Understanding the interplay between senescence, SASP, and cancer progression offers opportunities to design novel therapies that either enhance the tumor-suppressive effects or mitigate the pro-tumorigenic consequences of senescence, leading to more effective cancer treatments. Recent and ongoing work in our lab has identified that senescent cells can instruct developmental fate and reprogramming, which we believe could have significant impact on cancer cell fate and their behavior in tumors. The PhD project will explore how senescent cells can promote developmental reprogramming and influence tumor progression. This will involve performing single cell sequencing (RNA, ATAC) to identify which genes are activated by senescent cells in different cancer models following therapy, and to explore how they confer diverse fates on the tumor. These genes will then be functionally inhibited in culture, and in vivo in tumors, to further interrogate the mechanisms by which therapy-induced senescence promotes tumor progression and recurrence. Techniques will include sequencing approaches, cell culture, molecular biology approaches and working with mouse models of cancer.

Keywords: Senescence, cancer, therapy, reprogramming, plasticity, aging, mouse models

Relevant publications:

- Durik, M., Sampaio Gonçalves, D., Spiegelhalter, C., Messaddeq, N., Keyes, W.M. Senescent cells deposit intracellular contents through adhesion-dependent fragmentation (2023) BioRxiv doi: https://doi.org/10.1101/2023.01.11.523642

- Ritschka, B., Knauer-Meyer, T., Sampaio-Conçalves, D., Mas, A., Plassat, J.L., Durik, M., Jacobs, H., Pedone, E., Di Vicino, U., Cosma, M.P. Keyes, W.M. (2020) The senotherapeutic drug ABT-737 disrupts aberrant p21 expression to restore liver regeneration in adult mice. Genes & Development, 34(7-8):489-494. 

- Ritschka, B., Storer, M., Mas, A., Heinzmann, F., Ortells, M.C., Morton, J.P., Sansom, O.J., Zender, L. and Keyes, W.M. (2017) The senescence-associated secretory phenotype induces cellular plasticity and tissue regeneration Genes & Development, 31(2):172-183 

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

Prostate cancer is the second most commonly diagnosed neoplasia in men worldwide and one of the major causes of cancer-related deaths. Advances in the past decade have revealed that tumours comprise a very heterogenous coevolving cellular ecosystem. Prostate tumours are considered as “cold tumours“, as their microenvironment is highly immunosuppressive. Current immunotherapies include Sipuleucel-T and pembrolizumab, and are approved for only a limited subset of patients.

To characterise the cascade of molecular and cellular events involved in prostate tumour progression, we generated genetically-engineered Pten(i)pe-/- mice in which Pten is selectively inactivated in prostatic epithelial cells at adulthood. We have shown that such mice develop prostatic intraepithelial neoplasia within a few months and adenocarcinoma at later stage, as seen in human prostate cancer. Moreover, our single-cell RNA-sequencing analyses revealed a high prostatic tumoral cell heterogeneity, and a complex tumour microenvironment, composed of various cancer-associated fibroblasts, tumour-infiltrating immune cells and endothelial cells.

The goal of the project is to determine the dynamic of the various infiltrating immune cell populations during tumour progression and the communications between the various cell populations. Moreover, the impact of stimulation or inhibition of relevant immune cells will be determined. Finally, the infiltrating immune cells will be determined in human prostate tumours, and their profile will be correlated with disease outcome.

Thus, the project will open new avenues to boost the antitumor immunity and develop biomarkers to improve patient stratification and the selection of immunotherapies for prostate cancer.

Keywords: prostate cancer, mouse models, tumour-infiltrating immune cells, single-cell transcriptomics

Relevant publications:

- M. A. Abu el Maaty, E. Grelet, C. Keime, A.-I. Rerra, J. Gantzer, C. Emprou, J. Terzic, R. Lutzing, J.-M. Bornert, G. Laverny, D. Metzger (2021). Single-cell analyses unravel cell type–specific responses to a vitamin D analog in prostatic precancerous lesions. Sci. Adv. 7, eabg5982. PMID: 34330705.

- Mohamed A. Abu el Maaty, Julie Terzic, Céline Keime, Daniela Rovito, Régis Lutzing, Darya Yanushko, Maxime Parisotto, Elise Grelet, Izzie Jacques Namer, Véronique Lindner, Gilles Laverny and Daniel Metzger (2022). Hypoxia-mediated stabilization of HIF1A in prostatic intraepithelial neoplasia promotes cell plasticity and malignant progression. Science advances 8, eabo2295. PMID: 35867798.

- Julie Terzic, Mohamed A. Abu el Maaty, Régis Lutzing, Alexandre Vincent, Rana El Bizri, Matthieu Jung, Céline Keime and Daniel Metzger (2023). Hypoxia-inducible factor 1A inhibition overcomes castration resistance of prostate tumors. EMBO Molecular Medicine. e17209. https://doi.org/10.15252/emmm.202217209. PMID: 37070472.  

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

Malignant melanoma is responsible for 70% of skin cancer deaths in Western countries. The 5-year survival rate is only 16% for distant stage disease, demonstrating that metastasis is responsible for patient mortality. Treatment options for patients with metastatic melanoma have evolved considerably over the past decade. Combination therapies with inhibitors targeting BRAF (i.e., Vemurafenib and Dabrafenib) and MEK kinases (i.e., Trametinib) have emerged and show high efficacy but are limited by the development of resistance and subsequent progression. The proposed PhD project represents a pioneering endeavor to delve into the intricate molecular landscape of melanoma, focusing on the phenomenon of transcriptional addiction within melanoma cells. With the relentless rise in melanoma incidence worldwide, understanding the molecular underpinnings of this aggressive skin cancer is paramount. This research seeks to unravel the intricacies of transcriptional addiction, a phenomenon wherein cancer cells become heavily reliant on specific transcriptional programs for their survival and proliferation. By employing cutting-edge genomic and bioinformatic approaches, the project aims to identify key transcription factors and regulatory elements that are crucial for the sustained growth and survival of melanoma cells. This investigation will not only shed light on the fundamental biology of melanoma but also holds potential implications for the development of targeted therapeutic interventions. Furthermore, the project's comprehensive approach involves the integration of high-throughput sequencing data, functional genomics, and advanced computational analyses to construct a detailed map of the transcriptional networks driving melanoma progression. Ultimately, the outcomes of this research endeavor have the potential to pave the way for novel therapeutic strategies, providing a deeper understanding of the molecular dependencies that fuel melanoma and offering new avenues for precision medicine in the battle against this formidable malignancy.

Keywords: Melanoma, Transcription addiction, new drugs, TFIIH

Relevant publications:

- Berico, P., Nogaret, M., Cigrang, M., Lallement, A., Vand-Rajabpour, F., Flores-Yanke, A., Gambi, G., Davidson, G., Seno, L., Obid, J., Bujamin H., V, Stephanie Le Gras, Gabrielle Mengus, Tao Ye, Carlos Fernandez Cordero, Mélanie Dalmasso, Emmanuel Compe, Corine Bertolotto, Eva Hernando, Irwin Davidson, Coin. F*. 2023. Super-enhancer-driven expression of BAHCC1 promotes melanoma cell proliferation and genome stability. Cell Reports, Nov 2;42(11):113363. DOI: 10.1016/j.celrep.2023.113363

- Sandoz, J., Cigrang, M., Zachayus, A., Catez, P., Donnio L.M., Elly, C., Nieminuszczy, J., Berico, P., Braun, C., Alekseev, S., Egly, J.M., Niedzwiedz W., Mari-Giglia, G., Compe, E., and Coin, F*. 2023. Active mRNA degradation by EXD2 nuclease elicits recovery of transcription after genotoxic stress. Nature Communications, Jan 20;14(1):341. doi: 10.1038/s41467-023-35922-5.

- Sandoz, J., Nagy, Z., Catez, P., Caliskan, G., Geny, S., Renaud, J.B., Concordet, J.P., Poterszman, A., Tora, L., Egly, J.M., Le May, N., Coin, F*., 2019. Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression. Nature Communications 10, 1288. https://doi.org/10.1038/s41467-019-09270-2

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

Dysregulated immune responses at skin lead to inflammation involved in the development of allergies, autoimmune disorders, cutaneous infections, and cancers. The team is interested in deciphering dendritic cell (DC) -T cell axes implicated in different inflammatory pathologies. DC have specialized subsets whose local development and functions are shaped by tissue microenvironmental cues. In homeostatic and inflammatory conditions, specific DC subsets migrate into draining lymph nodes to present antigen and prime proinflammatory or tolerogenic responses. The latter can generate regulatory T cells (Treg), which are vital to the preservation of immune tolerance and suppression of exacerbated immune responses to foreign antigens, but underlying mechanisms stay poorly understood. The project will focus on a newly identified tolerogenic DC subset, to 1) delineate its function in driving Treg responses and in interacting with proinflammatory axes in steady state and inflammatory contexts particularly autoimmunity and cancer; 2) determine its upstream activation signals; and 3) investigate its tissue localization and dynamics in tissue and tissue-draining lymph nodes. The study will use mouse genetic tools, immunological approaches (­­flow cytometry, cell sorting, adoptive cell transfer, in vitro cell differentiation and cell co-culture), and molecular biology tools particularly single cell RNA-sequencing and bioinformatic analyses. Results are expected to reveal novel tolerogenic mechanisms, and to identify potential therapeutic targets for inflammatory skin diseases and cancer.

Keywords: Immune response; inflammation; dendritic cell; T cell; tolerance; allergy; autoimmune; cancer; skin; mouse models

Relevant publications:

- Yao, W., German B., Chraa, D., Braud, A., Hugel, C., Meyer, P., Davidson, G., Laurette, P., Mengus, G., Flatter, E., Marschall, P., Segaud, J., Guivarch, M., Hener, P., Birling, M, Lipsker, D., Davison, I. and Li, M. Keratinocyte-derived cytokine TSLP promotes growth and metastasis of melanoma by regulating the tumor-associated immune microenvironment. (2022) JCI Insight. DOI: 10.1172/jci.insight.161438. PMID: 36107619.

- Segaud. J., Yao, W., Marschall, P., Daubeuf, F. Lehalle, C., German B., Meyer, P., Hener, P., Hugel, C., Flatter, E., Guivarch, M., Clauss, L., Martin, S., Oulad-Abdelghani, M and Li, M. Context-dependent role of TSLP and IL-1β in skin allergic sensitization and atopic march. (2022) Nat. Commun. DOI: 10.1038/s41467-022-32196-1. PMID: 36050303.

- Marschall, P, Wei, R., Segaud, J., Yao, W., Hener, P., German Falcon, B., Meyer, P., Hugel, C., Silva, G., Braun, R., Kaplan, D. and Li, M. (2020) Dual function of Langerhans cells in skin TSLP-promoted Tfh cell differentiation in mouse atopic dermatitis. J. Allergy Clinic. Immunol. S0091-6749(20)31408-1. doi: 10.1016/j.jaci.2020.10.006. PMID: 33068561.

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

Chromatin, the substrate for crucial processes such as transcription, is highly spatially organized yet dynamic. Work by us and others demonstrated a close link between chromatin architecture and function, such as apparent proximity between distal regulatory elements and genes, and their organization into self-associating topologically associated domains (TADs). An emerging view is that “hubs” of regulatory regions form microenvironments, perhaps as phase-separated condensates, whereby regulatory factors are rapidly exchanged within a high local concentration. However, almost nothing is known about the dynamic biophysical properties of such hubs, nor how loci are recruited to them. The ability of genomic regions to explore their nuclear environment depends on their inherent mobility, a potentially important but as yet uncharacterized regulatory feature. We recently uncovered a surprising dependence of chromatin diffusive parameters on their genomic context and cellular state. By systematically measuring and manipulating chromatin dynamics throughout the genome during differentiation we will ask important questions:

1) What genomic contexts and functional mechanisms define local chromatin diffusive properties and how can they be manipulated?

2) How do chromatin dynamics influence transcriptional regulation and how are they modulated by distal regulatory elements?

Keywords: Transcription; chromatin diffusion; live microscopy; ANCHOR; chromosome conformation

Relevant publications:

- Platania et al., 2024 (in press; Science Advances; bioRxiv: https://www.biorxiv.org/content/10.1101/2023.04.25.538222v1) Transcription processes compete with loop extrusion to homogenize promoter and enhancer dynamics

- Taylor et al., 2022 (Genes and Development; doi: 10.1101/gad.349489.122.) Transcriptional regulation and chromatin architecture maintenance are decoupled functions at the Sox2 locus

- Oliveira et al., 2021 (Nature Communications; doi: 10.1038/s41467-021-26466-7.) Precise measurements of chromatin diffusion dynamics by modeling using Gaussian processes

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

Multicellular organisms establish and maintain different transcriptional states in disparate cell types through complex and specific regulation of gene expression. This regulation is mediated by the cooperative binding of transcription factors to regulatory elements through the recognition of specific DNA sequence motifs. Additionally, the physical access of transcription factors to DNA can be modulated by epigenetic regulation, such as nucleosome positioning and DNA methylation. Failure to maintain this tight regulation of gene expression results in developmental defects and various diseases including cancer.

The identification of transcription factor binding sites remains limited experimentally especially in patient samples or single cells. Our team is currently developing deep learning models of transcription factor binding to better understand how their misregulation is implicated in cancer. During this PhD project, the successful candidate will (1) analyze and integrate different genomic data types (e.g. ChIP-seq, ATAC-seq, RNA-seq), (2) apply and develop deep learning models to predict transcription factor binding from bulk or single-cells samples in healthy and pathological conditions and (3) interpret the models to better understand the sequence features underlying specific transcription factor binding sites.

To ensure the success of the project, our group provides a strong expertise in computational biology as well as the opportunity to perform experimental validations of our predictions.

Keywords: bioinformatics, deep learning, transcription factors, genomics, single-cell, cancer

Relevant publications:

- Balaramane D*, Spill YG*, Weber M#, Bardet AF#. MethyLasso: a segmentation approach to analyze DNA methylation patterns and identify differentially methylation regions from whole-genome datasets. Nucleic Acid Research (2024) gkae880

- Detilleux D*, Spill YG*, Balaramane D, Weber M#, Bardet AF#. Pan-cancer predictions of transcription factors mediating aberrant DNA methylation. Epigenetics & Chromatin (2022) 15:10

- Leporcq C, Spill Y, Balaramane D, Toussaint C, Weber M, Bardet AF#. TFmotifView: a webserver for the visualization of transcription factor motifs in genomic regions. Nucleic Acid Research (2020) 48:W208-W217

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

Hematopoietic stem cells (HSCs) give rise to all blood and immune cells, such as lymphocytes, monocytes and erythrocytes. The mechanisms that drive an HSC to differentiate into the different blood lineages remain only partially understood, but include the orchestrated activities of essential transcription factors that define cell fate choice and identity. The Ikaros family of transcription factors is central to hematopoiesis, and its loss of function is implicated in a variety of human leukemias and immunodeficiencies. Our team aims to understand how Ikaros family proteins establish gene expression programs in HSCs and developing lymphocytes. We apply this knowledge to address how mutations in these factors lead to disease, using in vivo and in vitro models, and a wide range of cellular, molecular and biochemical techniques.

Keywords: Hematopoietic stem cell, leukemia, transcriptional regulation, lymphocyte development

Relevant publications:

- Simand C, Keime C, Cayé A, Arfeuille C, Passet M, Kim R, Cavé H, Clappier E, Kastner P, Chan S, Heizmann B. Ikaros deficiency is associated with aggressive BCR-ABL1 B-cell precursor acute lymphoblastic leukemia independent of the lineage and developmental origin. Haematologica. 2022 Jan 1;107(1):316-320.

- Cova G, Taroni C, Deau MC, Cai Q, Mittelheisser V, Philipps M, Jung M, Cerciat M, Le Gras S, Thibault-Carpentier C, Jost B, Carlsson L, Thornton AM, Shevach EM, Kirstetter P, Kastner P, Chan S. Helios represses megakaryocyte priming in hematopoietic stem and progenitor cells. J Exp Med. 2021 Oct 4;218(10):e20202317.

- Bernardi C, Maurer G, Ye T, Marchal P, Jost B, Wissler M, Maurer U, Kastner P, Chan S, Charvet C. CD4+ T cells require Ikaros to inhibit their differentiation toward a pathogenic cell fate. Proc Natl Acad Sci U S A. 2021 Apr 27;118(17):e2023172118.

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

Translocation Renal Cell Carcinoma (tRCC) is a rare kidney cancer predominantly afflicting children, adolescents, and young adults. TRCC are characterized by translocations affecting TFE3 transcription factor. In the metastatic setting, there are no treatments approved to date, therefore, there is an urgent need to understand molecular underpinnings of these tumors.  Our team developed several murine models expressing specific fusion proteins (NONO-TFE3, TFE3 and ASPSCR1-TFE3) in the kidneys. The project encompasses four key objectives aiming to (1) characterize murine tRCC models, focusing on tumor kinetics, early and late oncogenic steps and microenvironment, (2) compare oncogenic program microenvironments in murine and human tRCC, (3) analyze the convergence between activated pathways in murine model and cultured cell lines and (4) identify and validate therapeutic targets, laying the foundation for novel treatment options in this setting. The project performed will be at the frontiers between different disciplines including molecular biology, bioinformatics and genetics. Cutting-edge techniques involving single-cell RNAseq and spatial transcriptomics will be used. Findings will inform the exploration of personalized treatment strategies and will provide a foundation for improved patient tRCC, addressing critical gaps in understanding and treatment.

Keywords: renal cell carcinomas, mice models, spatial transcriptomics, single-cell RNAseq, MiT family transcription factors

Relevant publications:

- TFE3 fusion proteins drive oxidative metabolism, ferroptosis resistance and general RNA synthesis in translocation renal cell carcinoma. Helleux A, Davidson G, Lallement A, Haller A, Michel I, Fadloun A, Thibault-Carpentier C, Su X, Lindner V, Tricard T, Lang H, Tannir N, Davidson I, Malouf GG (https://www.biorxiv.org/content/10.1101/2024.10.24.620047v1)

- Davidson G, Helleux A, Vano YA, Lindner V, Fattori A, Cerciat M, Elaidi RT, Verkarre V, Sun C, Chevreau C, Bennamoun M, Lang H, Tricard T, Fridman WH, Sautes-Fridman C, Su X, Plassard D, Keime C, Thibault-Carpentier C, Barthelemy P, Oudard S, Davidson I, Malouf GG. Roles of mesenchymal-like tumour cells and myofibroblastic cancer-associated fibroblasts in progression and therapeutic response of clear-cell renal cell carcinoma. Cancer Research, 2023 Sep 1;83(17):2952-2969. 

- Vokshi BH*, Davidson G*, Tawanaie Pour Sedehi N, Helleux A, Rippinger M, Haller A, Gantzer J, Thouvenin J, Baltzinger P, Bouarich R, Manriquez V, Zaidi S, Rao P, Msaouel P, Su X, Lang H, Tricard T, Lindner V, Surdez D, Kurtz JE, Bourdeaut F, Tannir NM, Davidson I and Malouf GG. SMARCB1 regulates a TFCP2L1-MYC transcriptional switch promoting renal medullary carcinoma transformation and ferroptosis resistance. Nat Commun. 2023 May 26;14(1):3034.

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

This project aims to identify the epigenetic mechanisms that control the diversification of macrophages using the model of Drosophila melanogaster. Macrophages are innate immune cells known for phagocytosing debris and pathogens, acting as first responders during embryonic development, and continuing to serve important roles in various tissues into adulthood. Their responses in diseases, such as neurodegenerative disorders and cancers, can either help or worsen symptoms, depending on their behaviour, which is influenced by their environmental conditioning and previous immune challenges. Studies have shown that macrophages can retain a "memory" of immune challenges, affecting their future responses.

Previous work by the laboratory has characterized macrophages at different developmental stages, showing their diversification, which includes chromatin remodelling. The project will examine how the epigenetic landscape of macrophages changes from the embryonic to larval stages using single-cell ATACseq data and will characterize the molecular mechanisms behind the development of macrophage subpopulations, focusing on the impact of a key transcription factor, Gcm, and its interactions with non-coding RNA (ncRNA) on the chromatin remodelling.

The findings of this study will provide deeper insights into the molecular mechanisms regulating macrophage diversification, will lead to future researches on macrophage behaviour in pathological conditions like infections and cancer as well as on the role of ncRNA in the mammalian macrophages.

Keywords: Drosophila, macrophage, non coding RNA, high throughput sequencing

Relevant publications:

- Sakr, R., P. B. Cattenoz, A. Pavlidaki, L. Ciapponi, M. Marzullo et al., 2022 Novel cell- and stage-specific transcriptional signatures defining Drosophila neurons, glia and hemocytes. bioRxiv: 2022.2006.2030.498263.

- Pavlidaki, A., R. Panic, S. Monticelli, C. Riet, Y. Yuasa et al., 2022 An anti-inflammatory transcriptional cascade conserved from flies to humans. Cell Rep 41: 111506.

- Cattenoz, P. B., A. Popkova, T. D. Southall, G. Aiello, A. H. Brand et al., 2016 Functional Conservation of the Glide/Gcm Regulatory Network Controlling Glia, Hemocyte, and Tendon Cell Differentiation in Drosophila. Genetics 202: 191-219.

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

Lysosomes are acidic organelles containing a variety of hydrolases that are essential for the breakdown of cargo delivered by the endocytic and autophagic pathways. They go through a number of dynamic processes (acidification, fusion, tubulation, fission…) that adapt their function to meet cellular demands. Alterations in lysosome dynamics and function contribute to many diseases, ranging from neurodegeneration to cancer, highlighting the importance of lysosomes to human health. However, little is known about the molecular mechanisms regulating lysosome dynamic events, which limits our comprehension of how disease results from their dysfunction. In recent years, physical associations between organelles called membrane contact sites have emerged as critical regulators of organelle dynamics, notably by promoting the remodeling of the membrane lipid composition of organelles. This project proposes to decipher how contacts between the endoplasmic reticulum (ER) and lysosomes orchestrate lysosome dynamics. We will more particularly investigate the role of lipid transfer at ER-lysosome membrane contact sites in the regulation of lysosome dynamics. We will use various cell models, such as human induced pluripotent stem cells, and a combination of super-resolution live-cell imaging, lipidomic, and proteomic techniques. This project will pave the way for understanding how lysosome dynamics are regulated at a fundamental level and how their dysfunction contributes to disease.

Keywords: Lysosome, Membrane contact sites, Organelle dynamics, Super-resolution live imaging

Relevant publications:

- Wilhelm LP, Wendling C, Védie B, Kobayashi T, Chenard MP, Tomasetto C, et al. STARD3 mediates endoplasmic reticulum-to-endosome cholesterol transport at membrane contact sites. EMBO J. 2017 May 15;36(10):1412–33.

- Di Mattia T, Martinet A, Ikhlef S, McEwen AG, Nominé Y, Wendling C, et al. FFAT motif phosphorylation controls formation and lipid transfer function of inter-organelle contacts. EMBO J. 2020 Dec 1;39(23):e104369.

- Boutry M, DiGiovanni LF, Demers N, Fountain A, Mamand S, Botelho RJ, et al. Arf1-PI4KIIIβ positive vesicles regulate PI(3)P signaling to facilitate lysosomal tubule fission. J Cell Biol. 2023 Sep 4;222(9):e202205128.

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

The proposed research project investigates the interplay between epigenetic alterations and inflammation in human diseases using a conditional knock-out (cKO) mouse model. Depleting the histone variant H2A.Z in epidermal stem cells induces a strong inflammatory phenotype. RNA-seq and immunofluorescence reveal activation of interferon-related pathways and cytosolic DNA sensors (cGAS/STING, AIM-2, and Toll-like receptors) in H2A.Z-depleted epidermis. Further studies will examine genome-wide self-DNA patterns in keratinocytes, hypothesizing these fragments originate near H2A.Z nucleosomes. Sources of self-DNA, such as genome alterations, R-loop accumulation, mitotic defects, micronuclei, stalled replication forks, retroelement de-repression, and mitochondrial changes, will be analyzed. The role of the cGAS/STING pathway will be studied by isolating native cGAS complexes from cytosol and nucleus. Preliminary data suggest nucleosome-bound cGAS associates with a specific chromatin remodeling factor. Genomic mapping of cGAS, its associated factors, and H2A.Z will use CUT&RUN/CUT&TAG. Structural characterization of the cGAS nuclear complex and cGAS-nucleosomal complex will employ cryo-EM. These findings aim to clarify the epigenetic crosstalk between skin renewal and innate immunity triggered by H2A.Z ablation, paving the way for specific inhibitors targeting the cGAS/STING pathway.

Keywords: Chromatin, epigenetic, inflammation, innate immunity, cGAS/STING.

Relevant publications: 

- Belotti E, Lacoste N, Iftikhar A, Simonet T, Papin C, Osseni A, Streichenberger N, Mari PO, Girard E, Graies M, Giglia-Mari G, Dimitrov S, Hamiche A*, Schaeffer L. H2A.Z is involved in premature aging and DSB repair initiation in muscle fibers. Nucleic Acids Res. 2024 Apr 12;52(6):3031-3049.

- Fontaine E, Papin C, Martinez G, Le Gras S, Nahed RA, Héry P, Buchou T, Ouararhni K, Favier B, Gautier T, Sabir JSM, Gerard M, Bednar J, Arnoult C, Dimitrov S, Hamiche A. Dual role of histone variant H3.3B in spermatogenesis: positive regulation of piRNA transcription and implication in X-chromosome inactivation. Nucleic Acids Res. 2022.

- Ibrahim A, Papin C, Mohideen-Abdul K, Le Gras S, Stoll I, Bronner C, Dimitrov S, Klaholz BP, Hamiche A. MeCP2 is a long-range chromatin organizer that protects CA repeats from nucleosome invasion. Science. 25 Jun 2021: Vol. 372, Issue 6549, eabd5581, DOI: 10.1126/science.abd5581.

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

Dyneins are a large family of motor proteins that are involved in the minus end directed transport along microtubules. Dyneins fulfil important biological tasks and their versatile cargos include mRNA, whole organelles like mitochondria and nuclei as well as cilia components. Dyneins also carry out crucial functions during mitosis. Defects in dynein function have been linked to a variety of diseases including neurodevelopmental disorders, infertility and chronic lung infections.

Dyneins are multi-protein complexes with a molecular weight of around 1.4 MDa. In all dynein isoforms a    ̴3500 amino-acid motor domain within this complex is responsible for the movement along microtubules. ATP hydrolysis in its hexameric AAA+ ring drives the remodelling of the dynein linker domain, which creates the force for the movement along the microtubule. In this project, we will investigate the role of conserved structural elements in the AAA+ ring during linker remodeling. The student will clone dynein motor domain mutants, express them in insect cells, purify the constructs and characterize these constructs by high-resolution cryoEM. Preliminary data demonstrating the feasibility of the project is already available.

Keywords: Motor protein, dynein, cytoskeleton, AAA+ protein, cryoEM

Relevant publications:

- Remodelling of Rea1 linker domain drives the removal of assembly factors from pre-ribosomal particles, 2024, Nature Communications, accepted for publication

- Fagiewicz R., Crucifix C., Klos T., Deville C., Kieffer B., Nominé Y., Busselez J., Rossolillo P., Schimdt H. In vitro characterization of the full-length human dynein-1 cargo adaptor BicD2, 2022, Structure

- Schimdt H., Zalyte R., Urnavicius L., Carter A. P., Structure of human cytoplasmic dynein-2 primed for its power stroke, 2015, Nature

This project is self-funded by the team proposing the project.

Type 2 DNA topoisomerases (Topo2) control DNA topology during replication, transcription and chromosome segregation. Essential for cell proliferation, these enzymes serve as targets for various classes of antineoplastic agents used in current cancer therapies. Resistance or non-response to cancer treatments highlights the need for personalized therapeutic strategies and a deeper understanding of their mechanisms of action.

We obtained high resolution cryoEM reconstruction of the human Topo2a isoform, in complex with DNA and anti-cancer molecules (Vanden Broeck et al, Nat. Comm 2021). Recent results from our team on the homologous bacterial enzyme showed that forming nucleoprotein complexes requires the use of more physiologically-relevant DNA substrates to capture functional conformational states (Vayssieres et al, Science 2024; Vanden Broeck et al, Nat. Comm 2019). The Topo2 are part of large complexes associated with chromatin factors and regulated by post-translational modifications (Bedez et al, Sci. Rep. 2018; Lotz et al., Can. Drug. Resist. 2020). This thesis proposal aims at providing functional and structural knowledge on the enzymatic mechanisms and on cellular complexes of Topo2 in the context of chromatin. Our team has established cellular models and international collaborations on complementary technologies, to investigate the molecular architecture of Topo2 cellular complexes, as well as the interplay between drug resistance and Topo2 post-translational modifications.

Keywords: DNA topoisomerase, chromatin, Structural biology, cancer

Relevant publications:

- Vayssières M, Marechal N., Yun L., Lopez Duran  B., Murugasamy NK, Fogg  JM, Zechiedrich L., Nadal M., Lamour V. (2024). Structural basis of DNA crossover capture by Escherichia coli DNA gyrase. Science,384(6692):227-232. doi: 10.1126/science.adl5899.

- Vanden Broeck A, Lotz C, Drillien R, Bedez C, Lamour V (2021). Structural basis for the allosteric regulation of Human Topoisomerase 2α. Nat. Commun. 12, 2962. https://doi.org/10.1038/s41467-021-23136-6.

- Vanden Broeck A, Lotz C, Ortiz J, Lamour V. (2019) Cryo-EM structure of the complete E. coli DNA gyrase nucleoprotein complex. Nat. Commun., 10, 4935. https://doi.org/10.1038/s41467-019-12914-y.

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

Mycobacterium tuberculosis (Mtb) and Helicobacter pylori (Hp) cause widespread bacterial infections worldwide but antibiotic resistance is sharply rising. For example, the US CDC lists drug-resistant tuberculosis as a serious threat to human health.

Transcription of DNA by RNA polymerase (RNAP) is a fundamental yet incompletely understood process. Rho-dependent transcription termination is essential and unique to bacteria and therefore an attractive pharmacological target. It relies on the RNA helicase Rho, which moves along the nascent RNA to dissociate RNAP. Rho activity ensures correctly transcribed RNA, recycles RNAP, and prevents erroneous expression of regions downstream of active genes. However, Rho and RNAP are atypical in Mtb and Hp but their activities have never been studied thoroughly.

In collaboration with labs across France, we combine genetics, high-throughput RNA sequencing, biochemistry, and cryo-electron microscopy (cryo-EM) to elucidate the atypical transcription termination mechanism in Mtb and Hp. As a PhD student you will work in an international environment and learn how to isolate, purify, and assemble functional protein-RNA complexes. You will characterize transcription termination complexes of Mtb or Hp and develop biochemical assays for Hp transcription. These efforts will enable you to do a detailed structural analysis by cryo-EM and elucidate the mechanism of Rho-dependent transcription termination in bacterial pathogens.

Our multidisciplinary approach will shed light on the mode of action of Rho-dependent transcription termination - a prerequisite to develop new and effective therapeutic strategies.

The host lab is experienced in the structural characterization of supramolecular complexes with profound roles in bacterial gene expression (see e.g. Guo et al., Mol Cell 2018; Adelkareem et al., Mol Cell 2019; Webster et al., Science 2020; Zhu et al., Nat Comms 2022; Dey et al., Mol Cell 2019; Webster et al., Science 2024).

keywords: RNA polymerase, molecular machines, gene expression, cryo-electron microscopy, structural biology, transcription termination

Relevant publications:

- Webster MW, Chauvier A, Rahil H, Graziadei A, Charles K, Miropolskaya N, Takacs M, Saint-André C, Rappsilber J, Walter NG, Weixlbaumer A (2024). Molecular basis of mRNA delivery to the bacterial ribosome. Science. 386(6725)

- Dey S, Batisse C, Shukla J, Webster MW, Takacs M, Saint-André C, and Weixlbaumer A (2022). Structural insights into RNA-mediated transcription regulation in bacteria.  Mol Cell 82(20), 3885–3900.

- Webster MW, Takacs M, Zhu C, Vidmar V, Eduljee AD, Abdelkareem M,  Weixlbaumer A (2020). Structural basis of transcription-translation coupling and collision in bacteria. Science 369(6509), 1355-1359

This project is self-funded by the team proposing the project.

Human gene expression involves three steps: (i) RNA polymerase II (Pol II) transcribes DNA to pre-mRNA; (ii) pre-mRNA processing yields mature mRNA; and (iii) ribosomes translate mature mRNA to protein. Each step is tightly regulated and misregulation causes severe diseases. Human pre-mRNAs contain coding and non-coding segments, called exons and introns respectively. The spliceosome removes introns and ligates exons to produce mature mRNAs. Although largely studied individually, splicing occurs co-transcriptionally. Pol II interacts with the spliceosome, stimulates spliceosome assembly and splicing rates, and plays decisive roles in alternative splicing. Thus, a complete picture requires studying supramolecular assemblies of Pol II and spliceosome intermediates on the nascent pre-mRNA. In collaboration with the lab of Clément CHARENTON and Sarah CIANFERANI, we will use biochemistry, mass-spectrometry, and single particle cryo-EM to address this phenomenon and improve our mechanistic understanding of the coupling between transcription and splicing in humans.

As a PhD student you will work in an international environment and learn how to isolate, purify, and assemble functional transcription complexes coupled to the splicing machinery. You will then characterize them biochemically and obtain structural information using crosslinking coupled to mass spectrometry in the CIANFERANI lab.

Our work will unravel how synergism and crosstalk of Pol II and the splicing machinery enables faithful constitutive or alternative splicing and how this cooperation regulates the conversion of genotype to phenotype.

The host lab is experienced in the structural characterization of supramolecular complexes with profound roles in bacterial gene expression (see e.g. Guo et al., Mol Cell 2018; Adelkareem et al., Mol Cell 2019; Webster et al., Science 2020; Zhu et al., Nat Comms 2022; Dey et al., Mol Cell 2019; Webster et al., Science 2024).

Keywords: RNA polymerase, transcription, splicing, molecular machines, gene expression, cryo-electron microscopy, structural biology

Relevant publications:

- Webster MW, Chauvier A, Rahil H, Graziadei A, Charles K, Miropolskaya N, Takacs M, Saint-André C, Rappsilber J, Walter NG, Weixlbaumer A (2024). Molecular basis of mRNA delivery to the bacterial ribosome. Science. 386(6725)

- Dey S, Batisse C, Shukla J, Webster MW, Takacs M, Saint-André C, and Weixlbaumer A (2022). Structural insights into RNA-mediated transcription regulation in bacteria.  Mol Cell 82(20), 3885–3900.

- Webster MW, Takacs M, Zhu C, Vidmar V, Eduljee AD, Abdelkareem M,  Weixlbaumer A (2020). Structural basis of transcription-translation coupling and collision in bacteria. Science 369(6509), 1355-1359

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

The molecular interactions between histone and non-histone proteins organize eukaryotic DNA into chromatin, a nucleoprotein complex essential for regulating gene expression. Active genes are located in dispersed, loosely compacted euchromatin, while inactive genomic regions form dense heterochromatin. Although the epigenetic markers and protein interactions defining these chromatin states are well characterized, the mechanisms linking chromatin architecture to functional activity remain poorly understood.

Our team has demonstrated the effectiveness of cryo-electron tomography (cryo-ET) for visualizing nucleosomes and DNA linkers in situ [1]. We also developed Template Learning, a novel approach for automated nucleosome annotation in cryo-ET data [2]. To investigate functionally relevant chromatin architectures, these tools must target euchromatic and heterochromatic domains precisely and selectively. This is now achievable using Repli-Histo labeling [3], a method developed by Prof. Maeshima's group at NIG, Japan. In this project, Repli-Histo will be combined with cryo-fluorescence localization of specific chromatin domains, followed by high-resolution molecular imaging using cryo-ET. We will analyze the nucleosome spatial distribution, chain geometries, and conformational states of nucleosomes characteristic of euchromatin and heterochromatin. This structural information will be integrated with complementary data on nucleosome mobility, provided by Prof. Maeshima’s group.

Our goal is to provide a deeper understanding of the structural mechanisms underlying epigenetic regulation. By linking chromatin architecture and nucleosome dynamics to gene expression and genome organization, this research has the potential to advance fundamental biology and inform the development of epigenetic therapies.

Keywords: Chromatin, Nucleosome, Epigenetics, Cryo-electron tomography

Relevant publications:

- Harastani, M., Patra, G., Kervrann, Ch., Eltsov, M., (2024) Template Learning: Deep Learning with Domain Randomization for Particle Picking in Cryo-Electron Tomography. bioRxiv 2024.03.20.585905; doi: https://doi.org/10.1101/2024.03.20.585905

- Fatmaoui F., Carrivain P., Grewe D., Jakob B., Victor JM., LeforestierA., Eltsov M. Cryo-electron tomography and deep learning denoising reveal native chromatin landscapes of interphase nuclei. bioRxiv 2022.08.16.502515; doi: 10.1101/2022.08.16.502515

- Eltsov M, Grewe D, Lemercier N, Frangakis A, Livolant F, Leforestier A. Nucleosome conformational variability in solution and in interphase nuclei evidenced by cryo-electron microscopy of vitreous sections. Nucleic Acids Res. 2018 Sep 28;46(17):9189-9200. doi: 10.1093/nar/gky670. PMID: 30053160; PMCID: PMC6158616.

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

Tip-growing cells are characterized by a central, typically large-scale protrusion. Their specific shape enables them to explore their environment, transport different sorts of cargo over large distances, or effectively take up nutrients due to their increased surface area. To establish and maintain these functionalities, tip-growing cells heavily depend on the actin cytoskeleton to shape their protrusions, efficiently transport vesicles within them, and define their specific subdomains. Based on fluorescence-microscopy data, the different architectures, e.g., bundles or branched networks, in which actin filaments organize to fulfill this function can be inferred. However, the true ultrastructure of those assemblies, including the distribution of the actin organizers, like bundlers and cross-linkers, orchestrating them, remains unknown. Cryo-electron tomography, enabled by sample thinning through focused ion beam milling, will allow us to study actin filaments all throughout the main protrusions of tip-rowing cells, revealing their organization on the ultrastructural level. These organization patterns will be quantitatively analyzed by employing highly automated processing pipelines, whose development will be a central aspect of the advertised PhD project, yielding deep insights into how their specific architectures prescribe their respective cell biological functions. We further aim to identify the positions and derive the in-situ structures of the most common associated actin organizers to elucidate how their distribution and individual shapes contribute to the overall geometry of the actin assembly in which they are integrated.

Keywords: cytoskeleton, transport, in-situ structural biology, cryo-electron tomography, ultrastructure

Relevant publications:

- F. Fäßler, M. G. Javoor, J. Datler, H. Döring, F. W. Hofer, G. Dimchev, V.-V. Hodirnau, J. Faix, K., Rottner, F. K. M. Schur (2023): ArpC5 isoforms regulate Arp2/3 complex–dependent protrusion through differential Ena/VASP positioning. Science Advances. 9, eadd6495

- W. J. Nicolas, F. Fäßler, P. Dutka, F. K. M. Schur, G. Jensen, E. Meyerowitz (2022): Cryo-electron tomography of the onion cell wall shows bimodally oriented cellulose fibers and reticulated homogalacturonan networks. Current Biology. 32, 2375-2389.e6

- G. Dimchev, B. Amiri, F. Fäßler, M. Falcke, F. K. M. Schur (2021): Computational toolbox for ultrastructural quantitative analysis of filament networks in cryo-ET data. Journal of Structural Biology. 213, 107808

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

Our group investigates how peculiar genetic mutations, namely microsatellite expansions, which are DNA sequences typically composed of more than 50 repeats of 2 to 6 nucleotides-long DNA motifs (for example (CGG)100x, (CAG)29x, etc.) cause muscle and/or neurodegenerative diseases. Importantly these mutations are located in genomic regions annotated as “noncoding” (5’ and 3’UTRs, introns, lncRNAs, etc.). We notably focus on Neuronal Intranuclear Inclusion Disease (NIID), OcculoPharyngoDistal Myopathy (OPDM) and Amyotrophic Lateral Sclerosis (ALS), which affects specifically motor neurons and that is the 3rd most common neurodegenerative disease worldwide.

Our work shows that despite being localized in “non-coding” sequences, these microsatellites repeat expansions are nonetheless translated into novel and toxic proteins (Sellier et al., Neuron 2017, Boivin et al., EMBO Journal 2020, Boivin et al., Neuron 2021, etc.).

Thus, the PHD candidate will continue and deepen this work and investigate how microsatellites repeat expansions causing neuromuscular and neurodegenerative diseases are potentially translated into novel and toxic proteins using a wide range of molecular and cellular approaches, as well as develop novel animal models expressing these mutations.

Overall, this proposal will help to better understand the cause of muscle and neuronal dysfunctions to define therapeutic strategies for these devastating diseases.

This work will take place at Institute of Genetics and Biology Molecular and Cellular (IGBMC, http://www.igbmc.fr/) a large public research laboratory comprising 40 research groups and 12 technological platforms, including all instrument, expertise and common services essential to the present project.

Techniques and approaches employed during this PHD will comprise the following: Clonage, RT-qPCR, cell culture and cell transfection, western blotting, immunoprecipitation, immunofluorescence, FACS, fluorescence microscopy, AAV injection, mouse locomotor phenotyping, histology, IHC, etc.

Keywords: human genetic diseases, muscle and neuronal diseases, non-coding sequences

Relevant publications:

- Charlet N. An unexpected polyglycine route to spinocerebellar ataxia. Nature Genetics. 2024 Jun;56(6):1039-1041.

- Boivin et al., Translation of GGC repeat expansions into a toxic polyglycine protein in NIID defines a novel class of human genetic disorders. Neuron. 2021; 109(11):1825.

- Boivin et al. C9ORF72 haploinsufficiency synergizes DPR proteins toxicity, a double hit mechanism that can be prevented by drugs activating autophagy. EMBO J. 2020; 39(4).

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

The intricate cognitive functions of the cerebral cortex stem from a diverse network of neurons and glial cells, all generated from a uniform pool of progenitors during corticogenesis. This project seeks to elucidate how these progenitors sequentially produce distinct neuronal and glial progeny over time, shaping brain development.

While transcriptional programs have been recognized as key drivers of neuronal diversity, emerging evidence indicates that certain neuronal mRNAs are present in progenitor cells long before their corresponding proteins appear. This observation suggests that translation mechanisms may play a pivotal role in defining cell identity, alongside transcription.

We propose that dynamic regulation of tRNA pools significantly influences translation during cortical patterning, affecting progenitor competency. Our preliminary findings indicate that tRNA availability varies across cell types and developmental stages in the mouse cortex. Through cutting-edge cell labelling and sequencing techniques (tRNA sequencing, (quantitative measurement of translation efficiency), the PhD candidate will investigate whether fluctuations in tRNA abundance correlate with the progenitor's capacity to produce diverse daughter cells and determine whether tRNA abundance correlates with a change in translational programs.

By uncovering these mechanisms, this project not only advances our understanding of neural diversity but also has potential implications for neurodevelopmental disorders.

Keywords: brain development, neuronal diversity, translation, omic analysis, transfer RNA

Relevant publications:

- Asselin L, et al. Mutations in the KIF21B kinesin gene cause neurodevelopmental disorders through imbalanced canonical motor activity. .Nature Communication (2020) 11: 2441

- Ramos-Morales E et al. The structure of the mouse ADAT2/ADAT3 complex reveals the molecular basis for mammalian tRNA wobble adenosine-to-inosine deamination. Nucleic Acids Res (2021) 49: 6529-6548

- Rivera Alvarez J et al. The kinesin Kif21b regulates radial migration of cortical projection neurons through a non-canonical function on actin cytoskeleton. Cell reports (2023) 42, 112744

Financial support of this subject is allocated by the Research Cluster / LabEx INRT based on applicant's merit.

Neuromuscular diseases are rare genetic diseases characterized by muscle weakness originating from structural and functional alterations in the peripheral nerves, the neuromuscular junction, or the skeletal muscle fibers. There is currently no curative treatment for the vast majority of these diseases and the understanding of the pathological mechanisms remains limited. Our team previously identified the genetic basis of several neuromuscular diseases and characterized corresponding cellular and mouse models.

The PhD student will establish novel cellular systems to mimic in vitro the cellular and molecular alterations observed in patients. In addition, he/she will analyze and characterize these alterations in vivo in mouse models already available in the lab. Methods used will combine cell culture, photonic and electronic microscopies, immunofluorescence, histology, several omics approaches, western blotting, quantitative PCR, and motor and sensory phenotyping. Overall, the student will reveal yet unknown mechanisms leading to the development of neuromuscular diseases, with a focus on peripheral neuropathy. In parallel, he/she will also establish the effect of a gene modulation approach in the cellular and animal models to validate a novel therapeutic strategy, using an already identified target and viral vectors and oligonucleotides to mediate gene modulation.

The PhD student will manage the different aspects of his/her project with regular inputs from the team, be responsible for the choice of strategies and presentation of data, and manage the collaborations with technical platforms and external collaborators. Overall, this PhD project should lead to a better understanding of peripheral neuropathies and the validation of a therapeutic strategy that could be developed further to patients. Indeed, our previous work on other neuromuscular diseases led to the development of 3 clinical trials and the creation of a start-up.

Keywords: neuropathy, therapy, genetic disease, omics, virus vector, drug, mouse, cell, imaging

Relevant publications:

- Djeddi S, Reiss D, Menuet A, Freismuth S, de Carvalho Neves J, Djerroud S, Massana-Muñoz X, Sosson AS, Kretz C, Raffelsberger W, Keime C, Dorchies OM, Thompson J, Laporte J. Multi-omics comparisons of different forms of centronuclear myopathies and the effects of several therapeutic strategies. Mol Ther. 2021 Aug 4;29(8):2514-2534. doi: 10.1016/j.ymthe.2021.04.033. (S Djeddi = previous PhD student) 

- Lionello VM, Nicot AS, Sartori M, Kretz C, Kessler P, Buono S, Djerroud S, Messaddeq N, Koebel P, Prokic I, Herault Y, Romero NB, Laporte J*, Cowling BS*. Amphiphysin 2 (BIN1) modulation rescues MTM1 centronuclear myopathy and prevents focal adhesion defects. Science Transl Med. 2019 Mar 20;11(484). doi: 10.1126/scitranslmed.aav1866. (V Lionello = previous PhD student) 

- Gómez-Oca R, Edelweiss E, Djeddi S, Gerbier M, Massana-Muñoz X, Oulad-Abdelghani M, Crucifix C, Spiegelhalter C, Messaddeq N, Poussin-Courmontagne P, Koebel P, Cowling BS^, Laporte J^. Differential impact of ubiquitous and muscle dynamin 2 isoforms in muscle physiology and centronuclear myopathy. Nature Commun. 2022 Nov 11;13(1):6849. doi: 10.1038/s41467-022-34490-4. (R Gomez-Oca = previous PhD student) 

This project is self-funded by the team proposing the project.

Stormorken syndrome (STRMK) is a multi-systemic disorder characterized by small stature, muscle weakness, thrombocytopenia, and spleen anomalies. Our team previously uncovered the genetic causes of the diseases and demonstrated that the mutations in the Ca2+sensor STIM1 and the Ca2+ channel ORAI1 involve a gain-of-function and result in excessive extracellular Ca2+ influx. We also generated and characterized the Stim1R304W/+ mouse model carrying the most common STRMK mutation and recapitulating the human disease.

There is currently no treatment for STRMK. However, Ca2+ entry is amenable to manipulation. We recently provided the proof-of-concept that the genetic downregulation or inhibition of the Ca2+ channel ORAI1 improves the multi-systemic STRMK phenotype in mice (Silva-Rojas et al., 2022 and 2024). We now want to make the next step and establish a practical and translational method to modulate ORAI1 expression and function with the aim to prevent and revert STRMK. The global project is divided into following work packages:

1) Based on thorough test series in the cellular model, we identified two shRNA strongly decreasing ORAI1 expression. The PhD student will produce AAV particles containing both most efficient shRNAs and systemically downregulate ORAI1 in Stim1R304W/+ mice before and after disease onset to assess their capacity to prevent and reverse STRMK. The PhD student quantify muscle force, contraction, relaxation, and fatigue, examine muscle, skin and spleen morphology, and investigate ER stress, Ca2+ balance, bone architecture, and platelet numbers.

2) As an alternative therapeutic strategy, we aim to attenuate ORAI1 activity. We initiated a high-throughput drug screen of more than 1300 small compounds to identify molecules able to decrease ORAI1 permeability and normalize Ca2+ balance, and subsequent test series in the cell model revealed the efficacy of 3 molecules. The PhD student will generate chemical analogues of the molecules, perform additional cellular tests, and administer both most effective molecules to Stim1R304W/+ mice before and after disease onset.  

Taken together, the multi-disciplinary PhD project bears an important bench-to-bedside potential and is expected to greatly accelerate the development of an innovative and practical treatment of STRMK. It may also be of medical interest for other Ca2+-related disorders affecting skeletal muscle, platelets, bones, or spleen.

Keywords: Calcium, Stormorken syndrome, cellular model, mouse model, therapy

relevant publications:

- Pathophysiological effects of overactive STIM1 on murine muscle function and structure. Silva-Rojas R, Charles AL, Djeddi S, Geny B, Laporte J, Böhm J. Cells. 2021 Jul 8;10(7):1730. doi: 10.3390/cells10071730.

- Silencing of the Ca2+ channel ORAI1 improves the multi-systemic phenotype of tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) in mice. Silva-Rojas R, Pérez-Guàrdia L, Lafabrie E, Moulaert D, Laporte J*, Böhm J*. (equal contribution). Int J Mol Sci. 2022 Jun 23;23(13):6968. doi: 10.3390/ijms23136968.

- ORAI1 inhibition as an efficient preclinical therapy for tubular aggregate myopathy and Stormorken syndrome. Silva-Rojas R, Pérez-Guàrdia L, Simon A, Djeddi S, Treves S, Ribes A, Silva-Hernández L, Tard C, Laporte J*, Böhm J*. (equal contribution)- JCI Insight. 2024 Mar 5;9(6):e174866. doi: 10.1172/jci.insight.174866.

This project is self-funded by the team proposing the project.

The chromatin remodeler CHD1L is an enzyme implicated in chromatin remodeling and DNA relaxation process and is known to facilitate DNA repair and pluripotency in stem cells. CHD1L has been extensively studied in the context of cancer but little is known on its role during brain development. The deletion and duplication of the 1q21.1 region (1.35 Mb; 8 genes) containing CHD1L have been found in individuals with variable phenotypes including autism, schizophrenia, cardiac defects and micro/macrocephaly. Through the study of this chromosomal region in cellular and animal models, we found that CHD1L dosage changes modulates the brain size by controlling the proliferation of the neuronal progenitors. Here we propose to follow-up our observations to investigate further the role of CHD1L during neurogenesis. The PhD student will i) identify the transcriptional targets and partners of CHD1L in IPSC derived neurons and glial cells, ii) determine transcriptomic profiles in the context of normal and mutant conditions to identify tissue-specific gene regulation networks in cerebral organoids, iii) Study Excitatory/Inhibitory balance by performing electrophysiological experiments on normal and mutant human iPSC-derived neurons and glial cells.

Keywords: Organoids, Chromatin and gene regulation, iPSC, autism, SC-RNAseq, SN-multiome, electrophysiology

Relevant publications:

- Pahlevan Kakhki M, et al. Nat Commun. 15(1) :6419 (2024)

- Hayot G, et al. Life Sci Alliance, Nov 14;6(1)e202201456 (2022)

- Loviglio MN*, Arbogast T*, et al. Am J Hum Genet. 101 (4):564-577 (2017)

Dengue and Zika viruses cause approximately 400 million new infections each year, and this number is increasing due to climate change. These viruses are transmitted to humans by Aedes mosquitoes and possess the unique ability to replicate in both the insect vector and the mammalian host. The viral RNA polymerase, NS5, is essential for viral genome replication and has additional functions. NS5 is predominantly localized to the nucleus in both mosquito and human cells, even though viral RNA replication is believed to occur in the cytoplasm. In humans, nuclear NS5 has been shown to sequester the antiviral factor STAT2 in the nucleus, although this is likely not its only function. Nuclear NS5 also interacts with the spliceosome, a complex molecular machine responsible for pre-mRNA splicing, influencing gene expression in mammalian cells. The role of nuclear NS5 in mosquitoes remains largely unknown. The main objective of this project is to characterize the function of nuclear NS5 in mosquito and human cells. We will employ cellular and molecular approaches combined with biochemistry and structural biology in both mosquitoes and human systems to elucidate the molecular functions of NS5 during dengue and Zika virus infections.

Keywords: Dengue virus, Zika virus, NS5, Structural biology

Relevant publications:

- Wilkinson, M.E.*†, Charenton, C.*†, Nagai, K.†, 2020. RNA Splicing by the Spliceosome. Annual Review in Biochemistry 89, 359–388.

- Charenton, C.*†, Wilkinson, M.E.*†, Nagai, K.*, 2019. Mechanism of 5′ splice site transfer for human spliceosome activation. Science 364, 362–367.

- Charenton, C., Taverniti, V., Gaudon-Plesse, C., Back, R., Séraphin, B., Graille, M., 2016. Structure of the active form of Dcp1-Dcp2 decapping enzyme bound to m(7)GDP and its Edc3 activator. Nature Structural & Molecular Biology 23, 982–986.

- Mosquito vector competence for dengue is modulated by insect-specific viruses. Olmo RP, Todjro YMH, Aguiar ERGR, de Almeida JPP, Ferreira FV, Armache JN, de Faria IJS, Ferreira AGA, Amadou SCG, Silva ATS, de Souza KPR, Vilela APP, Babarit A, Tan CH, Diallo M, Gaye A, Paupy C, Obame-Nkoghe J, Visser TM, Koenraadt CJM, Wongsokarijo MA, Cruz ALC, Prieto MT, Parra MCP, Nogueira ML, Avelino-Silva V, Mota RN, Borges MAZ, Drumond BP, Kroon EG, Recker M, Sedda L, Marois E, Imler JL, Marques JT*. Nat Microbiol. 2023 Jan;8(1):135-149. doi: 10.1038/s41564-022-01289-4. PMID: 36604511

- Invading viral DNA triggers dsRNA synthesis by RNA polymerase II to activate antiviral RNA interference in Drosophila. de Faria IJS, Aguiar ERGR, Olmo RP, Alves da Silva J, Daeffler L, Carthew RW, Imler JL, Marques JT*. Cell Rep. 2022 Jun 21;39(12):110976. doi: 10.1016/j.celrep.2022.110976. PMID: 35732126.

- Control of dengue virus in the midgut of Aedes aegypti by ectopic expression of the dsRNA-binding protein Loqs2. Olmo RP, Ferreira AGA, Izidoro-Toledo TC, Aguiar ERGR, de Faria IJS, de Souza KPR, Osório KP, Kuhn L, Hammann P, de Andrade EG, Todjro YM, Rocha MN, Leite THJF, Amadou SCG, Armache JN, Paro S, de Oliveira CD, Carvalho FD, Moreira LA, Marois E, Imler JL, Marques JT*. Nat Microbiol. 2018 3(12):1385-1393. doi: 10.1038/s41564-018-0268-6. PMID: 33923055

Financial support of this subject is allocated by the IMCBio Graduate School based on applicant's merit.

Mitochondria are essential organelles in eukaryotes. While their primary function is to synthesize ATP through oxidative phosphorylation, mitochondria are involved in numerous other metabolic pathways and are capable of adapting to the needs of the cell and responding to various stresses. Mitochondria are not static; they are constantly moving and changing shape. Mitochondrial dynamics generally refer to mitochondrial fusion, fission, mitophagy, and mitochondrial trafficking. Dysfunction of these dynamics can lead to dysfunction of mitochondrial functions, and a number of pathologies are associated with them.

Although mitochondrial dynamics is a fundamental cellular process, this mechanism is still largely unknown in higher plants. Most of knowledge is limited to a few proteins that have been identified as being involved in mitochondrial fission.

The aim of this work is to study mitochondrial fission in the model plant Arabidopsis thaliana. To this end, global interactome approaches (co-immunoprecipitation and proximity labeling with Turbo ID, combined with mass spectrometry) will be developed. This work should allow to establish the relationships between the different proteins already known and to identify new partners. The second part of the thesis will be devoted to the validation of the candidates thus identified.

This work should lead to a better understanding of the mitochondrial fission process. It should also be a springboard to address the fine-tuning balance between fission and fusion and, more broadly, mitochondrial dynamics in plants.

Keywords: Mitochondria, fission, plant, co-immunoprecipitation, TurboID, mass spectrometry, confocal microscopy

Relevant publications:

- Extensive import of nucleus-encoded tRNAs into chloroplasts of the photosynthetic lycophyte, Selaginella kraussiana, Berrissou et al, PNAS (2024) 121(46) : e2412221121.

- FRIENDLY is an RNA binding protein associated with cytosolic ribosomes at the mitochondrial surface. Hemono et al, Plant J.  (2022), 12:309-321

- The interactome of CLUH reveals its association to SPAG5 and its co-translational proximity to mitochondrial proteins. Hemono M et al, BMC Biol. (2022) 20:13

Financial support of this subject is guaranteed by the Research Cluster / LabEx MitoCross

Mitochondria are endosymbiotic organelles that originated two billion years ago, transforming eukaryotic evolution. They are central to bioenergetics, providing ATP to eukaryote cells. Chlamydomonas reinhardtii is a unicellular green alga. Its mitochondrial genome combines features of land plants with an architecture resembling mammalian mitochondrion, making it a unique model. Our laboratory could determine the structure of the Chlamydomonas mitoribosome, identifying proteins potentially aiding the reconstitution of fragmented ribosomal RNAs that make the algae mitoribosome. Furthermore, our work showed that Chlamydomonas mitochondrial mRNAs initiate translation at the AUG codon without leader sequences, a process whose mechanisms remains unknown.

The proposed work aims at characterizing the translation initiation process in Chlamydomonas mitochondria. Building on strong preliminary data, this multidisciplinary project will use a combination of genetic, next-generation sequencing, biochemical, and structural approaches (i.e., immunoprecipitation, Ribo-Seq, Cryo-EM) to identify without a priori novel factors involved in algae mitochondrial translation initiation, identify their interplay with universal translation initiation factors, and determine their mode of action. Understanding how mRNA and tRNA are recruited to the mitoribosome and how translation is initiated will contribute to unravel the diversity of translation machineries across eukaryotes.

Keywords: Chlamydomonas, algae, mitochondria, translational machinery, RNA, transcriptomics, proteomics

Relevant publications:

- Waltz, F., Salinas-Giegé, T., Englmeier, R. et al. (2021) How to build a ribosome from RNA fragments in Chlamydomonas mitochondria. Nat Commun 12, 7176. https://doi.org/10.1038/s41467-021-27200-z

- Salinas-Giegé, T., Cavaiuolo, M., Cognat, V. et al. (2017) Polycytidylation of mitochondrial mRNAs in Chlamydomonas reinhardtii, Nucleic Acids Research, Volume 45, Issue 22, Pages 12963–12973, https://doi.org/10.1093/nar/gkx903

- Salinas, T., Duby, F., Larosa, V. et al. (2012) Co-Evolution of mitochondrial tRNA import and codon usage determines translational efficiency in the green alga Chlamydomonas. PLoS Genet, 8(9):e1002946, https://doi.org/10.1371/journal.pgen.1002946

Financial support of this subject is guaranteed by the Research Cluster / LabEx MitoCross

The goal of the PhD is to fill a knowledge gap in chloroplast RNA quality control (QC). Transcriptomics data suggest that the full chloroplast genome is transcribed, which, coupled to inefficient termination, leads to a highly complex primary transcriptome. Moreover, it is now clear that chloroplast also possess abundant non coding RNAs (ncRNAs) that can form RNA duplexes with genic transcripts (dsRNA) although the amplitude and biological meaning are unknown. The chloroplast therefore relies on RNA quality control (QC) to sort through this initial transcript pool for those segments to be preserved and in some cases subdivided, while the remainder is rapidly degraded. It is performed by an assortment of ribonucleases (RNases) described to have low sequence specificity. One poorly understood aspect of chloroplast gene expression and regulation is the functions of asRNAs and more globally double-stranded RNA-related processes. We have recently showed that at least two ribonucleases, PNPase and RNaseJ, have a major role in maintaining the sense/antisense RNA homeostasis. One important role is to eliminate deleterious antisense RNA that could harm translation. The project aims to (1) identify the full set of RNA-RNA duplexes in various RNases mutants and environmental stress conditions and (2) identify and initiate the characterization of new proteins factors involved in dsRNA metabolism.

Methods: In the frame of an ANR project, our team has recently developed a complete analytical pipeline allowing the systematic monitoring of sense, antisense and double stranded RNA in the chloroplast. A combination of Illumina and Nanopore sequencing associated to the DiffSegR bioinformatics tool will be used to identify the different transcripts isoforms in the ribonucleases mutants and under stress conditions. The dsRNA metabolism will be studied thanks to the specific dsRNA antibody J2. We recently used it to develop a dsRIP-Seq assay and the abundance and localization of dsRNA in vivo will be monitored by immunolocalisation in confocal microscopy. Finally, proteins co-purifying with dsRNA will be identified though MS and promising candidates will be characterized using classical reverse genetic approaches.

Keywords: Chloroplast, Arabidopsis, RNA quality control, RNases, RNA-Seq, Nanopore, double stranded RNA

Relevant publications:

- Delannoy E, Liehrmann A, Castandet B. The use of nanopore sequencing to analyze the chloroplast transcriptome bioinformatics analyses and virtual RNA blots. Methods Mol Biol. 2024; 2776:259-267.

- Liehrmann A*, Delannoy E, Launay-Avon A, Gilbault E, Loudet O, Castandet B*, Rigaill G*. DiffSegR: An RNA-Seq data driven method for differential expression analysis using changepoint detection. NAR Genom and Bioinform, 2023, Nov 6; 5(4):lqad098. *co corresponding authors

- MacIntosh GC and Castandet B. Organellar and secretory ribonucleases, major players in RNA homeostasis. Plant Physiology. 2020; 183(4):1438-1452.

Mitochondrial diseases are a clinically heterogeneous group of disorders caused by mitochondrial dysfunctions. They may be caused by mutations of either the nuclear or the mitochondrial genomes and are mostly incurable. In the scope of the present project we are considering two frequent situations: (1) mutations within a single tRNA lead to several disorders and (2) mutations convert a sense codon into a non-sense one leading to premature termination (PTC). In both, we propose that the targeting of a single tRNA into mitochondria will allow counteracting the molecular defects at the origin of the diseases. In the first situation, the therapeutic agent is a replacement tRNA, whose role is to switch the ratio mutant tRNA/wild-type tRNA below the fateful threshold where the disease is triggered. In the second, it is a suppressor tRNA, able to decode a nonsense codon, to read-through the PTC and thus restore the synthesis of a full-length protein. Basing on recent discoveries, we propose to model gene therapy in cultured immortalized and human patients' cells. The design of therapeutic tRNAs will be rationalized according to our knowledge in tRNA identity and codon/anticodon recognition rules. Proof of concept series of experiments will assess for the efficacy of model cells' transfection with the therapeutic tRNA. Finally, rescue of phenotypes in cybrids and patient-derived cells upon tRNA transfection will demonstrate a therapeutic potential of tRNA PTC suppressor or replacement methodologies.

Keywords: Mitochondrial diseases / Mitochondrial DNA mutations / suppressor tRNA / gene therapy modeling

Relevant publications:

- D. Jeandard, A. Smirnova, A. M. Fasemore, L. Coudray, N. Entelis, K. U. Forstner, I. Tarassov, A. Smirnov, CoLoC-seq probes the global topology of organelle transcriptomes. Nucleic acids research 51, e16 (2023); published online EpubFeb 22 (10.1093/nar/gkac1183).

- K. Aure, G. Fayet, I. Chicherin, B. Rucheton, S. Filaut, A. M. Heckel, J. Eichler, F. Caillon, Y. Pereon, N. Entelis, I. Tarassov, A. Lombes, Homoplasmic mitochondrial tRNA(Pro) mutation causing exercise-induced muscle swelling and fatigue. Neurology. Genetics 6, e480 (2020); published online EpubAug (10.1212/NXG.0000000000000480).

- O. A. Kolesnikova, N. S. Entelis, H. Mireau, T. D. Fox, R. P. Martin, I. A. Tarassov, Suppression of mutations in mitochondrial DNA by tRNAs imported from the cytoplasm. Science 289, 1931-1933 (2000); published online EpubSep 15 (10.1126/science.289.5486.1931).

Financial support of this subject is guaranteed by the Cluster / LabEx MitoCross

Elucidating the causes of the awesome phenotypic diversity observed in natural populations is a major challenge in biology. To date quantitative genetics has mainly been focused on the identification of the nuclear determinants of complex trait, leaving mitochondrial DNA aside. The mitochondrial genome has been considered to study specific mitochondrial traits, but its contribution to common complex traits has been largely ignored. There is a growing number of evidence that genetic variations of the mitochondrion (considered alone or through interactions with nuclear variations) account for much of the heritability of complex traits. In this context, we would like to identify mitochondrial genetic determinants of multiple complex traits (including molecular traits like gene expression levels). Moreover, we will characterize the genetic architecture of the interaction between the mitochondrial and nuclear genomes, with respect with those traits. Beyond the description of mitochondrial genetic associations, we want to uncover the molecular mechanisms underlying them, i.e. what are the genes/proteins involved and how are they contribute together to a phenotype. To achieve this, we will develop genetic and genomic approaches (experimental and computational) in the budding yeast Saccharomyces cerevisiae. Reaching a system comprehension of the genetic and molecular mechanisms underlying the nuclear-mitochondrial interactions requires a deep understanding of both molecular and computational biology. Therefore, we will work at the interface between genetics, genomics and computational biology.

Keywords: genetics, population genomics, phenotypic variation, mitochondrion, yeast

Relevant publications:

- Pan-transcriptome reveals a large accessory genome contribution to gene expression variation in yeast. Caudal É, Loegler V, Dutreux F, Vakirlis N, Teyssonnière É, Caradec C, Friedrich A, Hou J, Schacherer J. Nat Genet., 2024 Jun;56(6):1278-1287.

- Telomere-to-telomere assemblies of 142 strains characterize the genome structural landscape in Saccharomyces cerevisiae. O'Donnell S, Yue JX, Saada OA, Agier N, Caradec C, Cokelaer T, De Chiara M, Delmas S, Dutreux F, Fournier T, Friedrich A, Kornobis E, Li J, Miao Z, Tattini L, Schacherer J*, Liti G*, Fischer G*. Nat Genet., 2023, 55(8):1390-1399.

- Genome evolution across 1,011 Saccharomyces cerevisiae isolates. Peter J, De Chiara M, Friedrich A, Yue JX, Pflieger D, Bergström A, Sigwalt A, Barre B, Freel K, Llored A, Cruaud C, Labadie K, Aury JM, Istace B, Lebrigand K, Barbry P, Engelen S, Lemainque A, Wincker P, Liti G, Schacherer J Nature, 2018, 7701:339-344

Financial support of this subject is guaranteed by the Research Cluster / LabEx MitoCross

Prostate cancer (PCa) is the second most commonly diagnosed cancer worldwide, the fifth cause of cancer-related mortality. As a slow-growing disease, PCa takes decades to elicit clinical symptoms. Moreover, chemical or surgical androgen deprivation are the main treatments offered to patients facing advanced PCa. Unfortunately, the disease progresses to castration-resistant PCa for which chemotherapies are poorly effective. It is therefore crucial to identify new molecular players in PCa to develop better and more precise treatments. PCa cells exhibit a unique metabolic reprogramming, drastically affecting various aspects of lipid metabolism from lipid biosynthesis and storage, fatty acids (FA) b-oxidation, to membrane remodeling, and supports cancer growth, survival, metastasis and therapy resistance. This project proposes to identify the physical and functional interplays between organelles involved in lipid metabolism (lipid droplets, mitochondria and peroxisomes), and how they contribute to PCa development, growth and metastasis. To do so, we will establish at the transcriptome and proteome levels components involved in FA metabolism and organelles contacts by multi-omics approaches (RNAseq, Proteomics). We will also characterize LD-mitochondria-peroxisomes dynamics and contacts by microscopy (TEM, confocal) using patient-derived tissues grown in 2D and 3D (organoids) as model. Best candidates will serve as proxies to design new therapeutic drugs targeting organelles contact sites and FA metabolism using genetically-modified mice and patient-derived organoids. 

Keywords: organoids, lipid droplet, mitochondria, peroxisomes, fatty acid, prostate cancer, organelles, contact sites, specialized medicine

Relevant publications:

- M. A. Abu el Maaty, E. Grelet, C. Keime, A.-I. Rerra, J. Gantzer, C. Emprou, J. Terzic, R. Lutzing, J.-M. Bornert, G. Laverny, D. Metzger (2021). Single-cell analyses unravel cell type–specific responses to a vitamin D analog in prostatic precancerous lesions. Sci. Adv. 7, eabg5982. PMID: 34330705.

- Mohamed A. Abu el Maaty, Julie Terzic, Céline Keime, Daniela Rovito, Régis Lutzing, Darya Yanushko, Maxime Parisotto, Elise Grelet, Izzie Jacques Namer, Véronique Lindner, Gilles Laverny and Daniel Metzger (2022). Hypoxia-mediated stabilization of HIF1A in prostatic intraepithelial neoplasia promotes cell plasticity and malignant progression. Science advances 8, eabo2295. PMID: 35867798.

- Julie Terzic, Mohamed A. Abu el Maaty, Régis Lutzing, Alexandre Vincent, Rana El Bizri, Matthieu Jung, Céline Keime and Daniel Metzger (2023). Hypoxia-inducible factor 1A inhibition overcomes castration resistance of prostate tumors. EMBO Molecular Medicine. e17209. https://doi.org/10.15252/emmm.202217209. PMID: 37070472.  

Financial support of this subject is allocated by the IMCBio Graduate School based on applicant's merit.

In the model plant Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing. AGO1 is also an important regulator of antiviral defense, as its mutation enhances susceptibility to different RNA viruses. Under normal conditions, AGO1 associates to the rough endoplasmic reticulum to conduct miRNA-mediated translational repression, mRNA cleavage, and biogenesis of phased siRNAs. It has recently been shown that under heat stress AGO1 protein accumulates in cytosolic condensates where it colocalizes with components of stress granules. AGO1 contains a prion-like domain in its poorly characterized N-terminal Poly-Q domain, which is sufficient to undergo phase separation. These recent results raise a number of important questions. Do other abiotic or even biotic stresses have similar effects on AGO1 subcellular localization. Do AGO1 condensates obtained under different stresses have a similar (protein and RNA) composition? Will the deletion of the entire N-terminal Poly-Q domain of AGO1 be sufficient to block phase separation? Does AGO1 localization in condensates requires post-translational modifications? Given the importance of post-transcriptional gene silencing in plant development, stress responses, and antiviral defense, it will also be essential to determine to which extent the ability of AGO1 to phase separate will affect these different functions. The PhD work will employ different approaches and methods to answer these questions. These include the generation of AGO1 mutations impairing its phase separation in vivo, identifying AGO1 interactors by proximity labeling (TurboID) coupled with mass spectrometry, performing transcriptome and small RNA sequencing to unravel the physiological and molecular functions of AGO1 under stress.

Keywords: RNA silencing, ARGONAUTE1, phase separation, stress responses, post-translational modifications, Arabidopsis

Relevant publications:

- Michaeli S, Clavel M, Lechner E, Viotti C, Wu J, Dubois M, Hacquard T, Derrien B, Izquierdo E, Lecorbeiller M, Bouteiller N, De Cilia J, Ziegler-Graff V, Vaucheret H, Galili G and Genschik P (2019) The viral F-box protein P0 induces an ER-derived autophagy degradation pathway for the clearance of membrane-bound AGO1. Proc Natl Acad Sci USA 116: 22872-22883.

- Hacquard T, Clavel M, Baldrich P, Lechner E, Pérez-Salamó I, Schepetilnikov M, Derrien B, Dubois M, Hammann P, Kuhn L, Brun D, Bouteiller N, Baumberger N, Vaucheret H, Meyers B and Genschik (2022) The Arabidopsis F-box protein FBW2 targets AGO1 for degradation to avoid spurious loading of illegitimate small RNA. Cell Rep. 39: 110671.

- Blagojevic A, Baldrich P, Schiaffini M, Lechner E, Baumberger N, Hammann P, Elmayan T, Garcia D, Vaucheret H, Meyers BC, Genschik P (2024) Heat stress promotes Arabidopsis AGO1 phase separation and association with stress granule components. iScience 27(3):109151.

Financial support of this subject is guaranteed by the Research Cluster / LabEx NetRNA.

Polyadenylation is crucial for the stability and translation of eukaryotic mRNAs. The recent advent of Nanopore sequencing has revealed an unsuspected complexity in mRNA poly(A) tail distribution profiles. Our latest gene-to-gene analyses showed strong disparities in poly(A) tail profiles between different ontological classes of mRNAs in Arabidopsis, but also for mRNAs belonging to the same ontological class. This new layer of post-transcriptional gene regulation is essentially unexplored.

The objective of this thesis project is the functional characterization of genetic determinants conferring specific poly(A) tail distributions. One such determinant has already been identified by the team and will be further characterized by the student. Machine learning tools will also be deployed to identify new genetic determinants. Once identified, the mechanisms underlying their function will be elucidated. Affinity purification and mass spectrometry analyses will allow the identification of trans factors recognizing the genetic determinants. Interfering with the expression of such trans factors, as well as mutating the genetic determinants, will then be used to study their impact on mRNA stability, translation and localization.

This project will allow major advances in understanding connections between gene expression and regulation of mRNA poly(A) tail sizes. The student will acquire theoretical and practical knowledge about mRNA metabolism, reverse genetics, mass spectrometry and Nanopore sequencing, and will develop associated bioinformatic skills.

Keywords: Polyadenylation; mRNA; RNA degradation; translation; Nanopore sequencing; mass spectrometry

Relevant publications:

- Giraudo P, Simonnot Q, Pflieger D, Peter J, Gagliardi D, Zuber H (2024) Nano3'RACE: A Method to Analyze Poly(A) Tail Length and Nucleotide Additions at the 3' Extremity of Selected mRNAs Using Nanopore Sequencing. Methods in Molecular Biology. 2723: 233-252, doi: 10.1007/978-1-0716-3481-3_14

- Joly AC, Garcia S, Hily J-M, Koechler S, Demangeat G, Garcia D, Vigne E, Lemaire O, Zuber H, Gagliardi D (2023) An extensive survey of phytoviral RNA 3' uridylation identifies extreme variations and virus-specific patterns. Plant Physiology 193(1):271-290, doi: 10.1093/plphys/kiad278

- Scheer H, de Almeida C, Ferrier E, Simonnot Q, Poirier L, Pflieger D, Sement FM, Koechler S, Piermaria C, Krawczyk P, Mroczek S, Chicher J, Kuhn L, Dziembowski A, Hammann P, Zuber H and Gagliardi D (2021) The TUTase URT1 connects decapping activators and prevents the accumulation of excessively deadenylated mRNAs to avoid siRNA biogenesis. Nature Communications. 12: 1298. doi: 10.1038/s41467-021-21382-2

Financial support of this subject is guaranteed by the Research Cluster / LabEx NetRNA.

RNA viruses establish membrane-bound viral factories in host cells to replicate their genomes, relying on a complex interplay of viral and host-encoded proteins. This process generates double-stranded RNA (dsRNA) intermediates, key elements in antiviral defense. By focusing on dsRNA in healthy and virus-infected Arabidopsis, we have identified a small set of host proteins that may function as either pro- or antiviral factors. Among these, one highly conserved protein stands out. Despite its evolutionary conservation in plants, its function remains unknown.

Our recent biochemical and structural studies, employing site-directed mutagenesis, X-ray crystallography and Cryo-EM, reveal that this protein has exceptional dsRNA-binding properties, potentially linked to plant innate immunity. This PhD project seeks to unravel the biological role of this protein under normal conditions and during viral infections. Using CRISPR-Cas9-generated mutants, phenotypic screens will be conducted under various environmental conditions, alongside testing viruses with or without silencing suppression activity. RNA-seq and small RNA profiling will be used to investigate changes in gene expression and small RNA pathways.

To explore molecular mechanisms, the project will integrate advanced techniques, super resolution microscopy, co-immunoprecipitation, and purification of native dsRNA-protein complexes for structural studies. This interdisciplinary project offers a unique opportunity to delve into plant immunity and antiviral defense using state-of-the-art molecular, genetic, and structural biology approaches.

Keywords: RNA virus replication, Host-virus interactions, dsRNA-binding proteins, plant virology

Relevant publications:

- Incarbone, M., Clavel, M., Monsion, B., Kuhn, L., Scheer, H., Vantard, E., Poignavent, V., Dunoyer, P., Genschik, P., and Ritzenthaler, C. (2021). Immunocapture of dsRNA-bound proteins provides insight into Tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector. Plant Cell 33, 3402-3420. https://www.ncbi.nlm.nih.gov/pubmed/34436604

- Incarbone, M., Scheer, H., Hily, J.M., Kuhn, L., Erhardt, M., Dunoyer, P., Altenbach, D., and Ritzenthaler, C. (2020). Characterization of a DCL2-Insensitive Tomato Bushy Stunt Virus Isolate Infecting Arabidopsis thaliana. Viruses 12, 1121. https://www.ncbi.nlm.nih.gov/pubmed/33023227

- Monsion, B., Incarbone, M., Hleibieh, K., Poignavent, V., Ghannam, A., Dunoyer, P., Daeffler, L., Tilsner, J., and Ritzenthaler, C. (2018). Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein. Front Plant Sci 9, 70. https://www.ncbi.nlm.nih.gov/pubmed/29449856

Financial support of this subject is guaranteed by the Research Cluster / LabEx NetRNA

Eukaryotes use RNA polymerase II (Pol II) to synthesize mRNAs from genes, allowing their translation into proteins for cellular functions. However, genomes also contain transposable elements (TEs) that can cause mutations when activated. Animals balance the need for gene transcription by Pol II against the risk of deleterious TE mobility by synthesizing small RNAs (piRNAs) that target and silence TEs. In plants, a specialized enzyme, RNA polymerase IV (Pol IV), transcribes TEs to produce small interfering RNAs (siRNAs) that guide DNA methylation to repress TEs (Ferrafiat et al. 2019; Rymen et al. 2020). Pol IV evolved to specifically interact with SNF2-like CLSY proteins rather than general transcription factors (Felgines et al. 2024), supporting its roles in TE silencing, reproduction and pathogen tolerance. Despite these recent findings, how the CLSYs facilitate Pol IV transcription at specific loci remains mysterious. This project aims to determine how structural features of Pol IV and its CLSY recruitment factors permit noncoding RNA transcription in silent chromatin. Using the model plant Nicotiana benthamiana, the student will: (1) Characterize CLSY-Pol IV complexes via functional genetics and complementation (CRISPR/Cas9, genomics); (2) Analyze Pol IV subunits of distinct parental origins in the allotetraploid N. benthamiana (phylogenetics, IP-MS); and (3) purify Pol IV complexes bound to nucleosomes to obtain high-res structures of its novel recruitment machinery (cryo-EM). This project will generate basic knowledge about the structure and function of plant RNA polymerases, provide a deeper understanding of epigenetic regulation, and could help breeders engineer desirable traits linked to Pol IV function, such as enhanced reproductive output and tolerance to viral infection.

Keywords: RNA polymerase structure-function, immunoprecipitation-mass spectrometry (IP-MS), noncoding RNA, small RNAs, transposable elements, epigenetics

Relevant publications:

- Felgines L, Rymen B, Martins LM, et al. Blevins T (2024 in press). CLSY docking to Pol IV requires a conserved domain critical for small RNA biogenesis and transposon silencing. Nat Commun. 10.1038/s41467-024-54268-0 / bioRxiv (10.1101/2023.12.26.573199v1)

- Rymen B, Ferrafiat L, Blevins T (2020). Non-coding RNA polymerases that silence transposable elements and reprogram gene expression in plants. Transcription 11(3-4): 172-191. (10.1080/21541264.2020.1825906)

- Ferrafiat L, Pflieger D, Singh J, et al., Blevins T (2019). The NRPD1 N-terminus contains a Pol IV-specific motif that is critical for genome surveillance in Arabidopsis. Nucleic Acids Res. 47(17): 9037-9052. (10.1093/nar/gkz618)

- Dey S, Batisse C, Shukla J, et al., Weixlbaumer A (2022). Structural insights into RNA-mediated transcription regulation in bacteria. Mol Cell 82(20): 3885-3900.e10. (10.1016/j.molcel.2022.09.020).

- Zhu C, Guo X, Dumas P, et al., Weixlbaumer A (2022). Transcription factors modulate RNA polymerase conformational equilibrium. Nat Commun. 13(1): 1546. (10.1038/s41467-022-29148-0).

- Abdelkareem M, Saint-André C, Takacs M, et al., Weixlbaumer A (2019). Structural Basis of Transcription: RNA Polymerase Backtracking and Its Reactivation. Mol Cell 75(2): 298-309.e4. (10.1016/j.molcel.2019.04.029).

Financial support of this subject is allocated by the IMCBio Graduate School based on applicant's merit.

Liver cancer, particularly hepatocellular carcinoma (HCC), is a global health challenge with high mortality rates and limited therapeutic success. While immunotherapies have shown promise, their efficacy remains hindered by low tumor remission rates. Recent evidence highlights the critical role of gut microbiota in shaping inflammation, antitumor immunity, and therapeutic outcomes in cancers. Our team has identified Claudin-1 as a key driver and potential target in liver cancer progression. This PhD project aims to get inside the mechanisms linking gut microbiota and Claudin-1-driven liver disease biology.       

To decipher how microbiota influences liver cancer and immunotherapy efficacy, we aim to develop innovative microbiota-based therapeutic strategies. The findings from this project could revolutionize liver cancer treatment and have important implications for improving therapeutic outcomes across various Claudin-1-associated cancers.

Join us to be at the forefront of pioneering research in cancer biology and precision medicine and contribute to transforming the landscape of liver cancer treatment.

Keywords: microbiota, microbiome, hepatocarcinoma, cholangiocarcinoma, gut-liver-axis, liver-diseases, liver-tumor microenvironment

Relevant publications:

- Roehlen et. al. A monoclonal antibody targeting nonjunctional claudin-1 inhibits fibrosis in patient-derived models by modulating cell plasticity. Science Translational Medicine. 2022 PMID: 36542691

- Almeida et al. Gut microbiota from patients with mild COVID-19 cause alterations in mice that resemble post-COVID syndrome. Gut Microbes 2023. PMID: 3766831

- Rungue et al. NLRP6-associated host microbiota composition impacts in the intestinal barrier to systemic dissemination of Brucella abortus. PLoS Neglected Tropical Disease. 2021 PMID: 33617596

Financial support of this subject is guaranteed by the Research Cluster / LabEx HepSYS

BACKGROUND:
Hepatitis D is by far the most severe form of chronic viral hepatitis, leading to liver fibrosis, liver failure, hepatocellular carcinoma, and death. Hepatitis D is caused by co-infection Hepatitis D is caused by co-infection with hepatitis B virus (HBV) and hepatitis D virus (HDV). Up to 20 million individuals worldwide are infected with HDV including about 250.000 patients in the European Union. Hepatitis D is a major health burden in distinct regions in Central Asia and Africa but also a particular problem in some European countries such as Romania. Western and Central European countries have high HDV prevalence in immigrant populations, so an adequate cascade of care is a challenge. There is still very limited knowledge about the pathophysiology of the disease and the interactions between host and virus, the latter being the reason for large interindividual variability during HDV infection. We previous demonstrated for Hepatitis C that chronic viral injury dysregulated DNA repair pathways and leaves an epigenetic viral footprint in the genome of the host, which associates with a pro-oncogenic transcriptional signature that persists after viral cure (Lupberger et al. Gastroenterology 2019, Hamdane et al. Gastroenterology 2019, Jühling et al. Gut 2020). This viral footprint highlighted novel biomarkers to identify patients at risk post cure and therapeutic targets to attenuate liver disease progression and cancer risk.
In pilot experiments, we identified HDV-specific transcriptional signature in the livers of infected human liver chimeric mice and profiled posttranslational histone modification linked to transcriptional activation/enhancer activity (H3K27ac, H3K27me1) as well as transcriptional repression (H3K4me3). As the HDV-infected mice show signs of liver fibrosis and perturbed hepatic DNA repair, we hypothesize that these pathways exert a key role in the development of liver fibrosis and cancer.

AIMS:
Embedded in a translational research program for the clinical development of HDV-targeting therapies (HORIZON-HLTH-2021-DISEASE program D-SOLVE) and supervised by two leading teams in the field of chronic liver disease, epigenetics, and DNA repair of the Laboratoire d’Excellence initiative (LabEx HepSYS and LabEx INRT), the PhD thesis aims to identify the role and molecular mechanism of the epigenetic viral imprint during chronic HDV infection and its impact on DNA repair and cancer risk.

APPROACH:
In a first axis, the candidate will unravel the molecular mechanism of HDV-induced histone modifications (H3K27ac, H3K27me1) and study the impact of viral protein expression in vitro on the histone marks associated with the viral footprint in vivo. Therefore, the candidate will perturb candidate regulators of the respective histone marks and predicted candidate transcription factors in the context of viral protein expression and HBV/HDV infection in vitro. Moreover, the candidate will perturb the expression of 2-3 identified candidate drivers highlighted by the viral epigenetic footprint and study their impact on viral infection, pro-fibrogenic and pro-carcinogenic signaling, cell growth, and apoptosis. The candidate will study HBV/HDV infection and viral protein expression using cell co-culture models mimicking the liver microenvironment and assess the activation status of co-cultured hepatocytes with human stellate cells (HSC; extracellular matrix deposition) and liver-resident macrophages (Kupffer cells, KC), which are key actors in the building up of excessive extracellular matrix during fibrosis. Finally, the candidate will study
the effect of the approved HDV drug bulevirtide (BLV) on the persistence of the HDV viral footprint. Therefore, the viral footprint will be studied in livers of mice infected with HDV/HBV treated for 12 weeks with BLV. Liver samples from this already scheduled experiment will be available at the end of May 2024.
In a second axis, the candidate will study the impact of chronic HDV/HBV-infection on the observed dysregulation of DNA repair components. DNA repair and in particular the transcription factor II H had been previously implicated in HBV infection (Schreiner et al. Viruses 2017, Quadri et al., PNAS 1996, Groisman et al., Carcinogenesis 1999) and higher-order chromatin structure (Sandoz et al. Nat. Comm. 2019), however the effects of HDV on this process is unknown. Therefore, the candidate will validate the observed impact of HBV/HDV infection on DNA repair pathways in the livers of chronically infected animals (already available). Accumulation of γH2AX or 53BP1 foci will be measured using IHC. DNA repair will be measured in vitro using cell extract of mouse livers. The candidate will study the role of TFIIH in the regulation of HDV-transcriptional pattern. Potential interactions between HDV proteins and TFIIH subunits will be analyzed using recombinant expression technology. Impact of HDV proteins on TFIIH enzymatic activities (helicase, ATPase, kinase) will be measured in vitro.
Importantly, key technologies and protocols are already established and available in the teams of both supervisors. The animal experiment will be already concluded at the start of the thesis project. All liver samples of HDV/HVB-infected animals and patients are therefore available. The experimental work will be conducted at LabEx HepSYS (axis 1) and LabEx INRT (axis 2). The candidate will be supervised by Dr. Joachim Lupberger (LabEx HepSYS, thesis director) and Dr. Frederic Coin (LabEx INRT, thesis co-director) including bi-monthly joint meetings between the candidate and both supervisors.

IMPACT:
This project is addressing an important unmet medical need of novel antiviral concepts and compounds to tackle the rising HDV epidemic and viral pathogenesis. The research program and the embedded thesis project will have a major impact for HDV-infected patients with advanced liver disease and HCC by advancing the knowledge of HDV-associated liver complications, novel preventive strategies to attenuate liver complications in these patients, and novel biomarkers for liver disease and BLV-treatment response.

Keywords: chronic hepatitis delta, virus, fibrosis, cancer, epigenetics

Relevant publications:

- Mukherji A and Jühling F, et al. An atlas of the human liver diurnal transcriptome and its perturbation by hepatitis C virus infection. Nat Commun. 2024;15(1):7486. doi: 10.1038/s41467-024-51698-8. PMID: 39209804

- Jühling F, et al. Targeting clinical epigenetic reprogramming for chemoprevention of metabolic and viral hepatocellular carcinoma. Gut. 2021;70(1):157-169. PMID: 32217639

- Lupberger J, et. al. Combined Analysis of Metabolomes, Proteomes, and Transcriptomes of Hepatitis C Virus-Infected Cells and Liver to Identify Pathways Associated With Disease Development. Gastroenterology. 2019;157(2):537-551.e9. PMID: 30978357

Financial support of this subject is allocated by the IMCBio Graduate School based on applicant's merit.