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Research



SYNOPSIS
RESEARCH GOALS
CURRENT PROJECTS
ALTERNATIVE SPLICING OF INTRONS AND THE DIVERSIFICATION OF TRANSCRIPTIONAL OUTPUT
CENTROMERIC REPEATS TRANSCRIPTION
DNA METHYLATION AND GENOME MAINTENANCE


THE TEAM


From left to right : Baptiste Bogard (PhD student), Giacomo Grillo (PhD student), Claire Francastel (The Boss),
Florent Hubé (Researcher), Ivana Ivkovic (Ass. engineer), Guillaume Velasco (Ass. professor).



SYNOPSISGo to the Top

The overall interest of our team is on the multiplicity and intricacy of the regulatory networks that control expression and maintenance of mammalian genomes, from local transcriptional and epigenetic control to global genome organization within the nuclear space. With the genomes of complex eukaryotes being pervasively transcribed but mostly made up of large amounts of sequences that do not carry information to make proteins, a challenging task is to document this huge non-coding transcriptional output and understand its functional significance in regulatory networks that govern normal cell fate. We are particularly focused on understanding whether and how perturbations to this non-coding production is a driving force is disease, and which are the mechanisms and factors that maintain their normal biogenesis and hence, genome and cell integrity. Over the past years, we focused on inherently non-coding genomic regions that represent a vast fraction of mammalian genomes, i.e. repeated sequences and introns, as paradigms to address these questions and as unconventional targets of (post)transcriptional and epigenetic perturbations that underpin human diseases.
The strategies of our team are both basic and patient-oriented, based on (i) the development of mouse models, (ii) the establishment of cohorts of patients in collaboration with physicians, (iii) a combination of complementary expertise in the study of gene expression, DNA methylation, non-coding RNA and their associated complexes, RNA splicing and processing and nuclear architecture, and (iv) capitalize on unique cellular models that we developed, including mouse models, ES cells and cells from patients, and the generation of high throughput data. Technical approaches include classical molecular biology, high throughput techniques for epigenome and transcriptome analysis, cellular imaging and in situ assays in the mouse.



RESEARCH GOALSGo to the Top

Our main goals are to:
– document the non-coding transcriptional output from these regions in normal and physiopathological situations
– characterize the biogenesis of the produced transcripts, their processing, epigenetic regulation, sub-cellular localization and associated complexes
– understand if and how their deregulation represents a driving force in disease
– provide an integrated view of pan-genomic epigenetic, transcriptional and splicing defects in the context of pathological defects of the DNA methylation machinery



CURRENT PROJECTSGo to the Top

ALTERNATIVE SPLICING OF INTRONS AND THE DIVERSIFICATION OF TRANSCRIPTIONAL OUTPUTGo to the Top
A paradigm to test the functionality of non-protein-coding genomic regions

teachingIntrons represent almost half of the human genome, although their vast majority is eliminated from eukaryotic transcripts through RNA splicing. Yet, important information is embedded within introns, the most remarkable being the release of different classes of small regulatory ncRNAs directly from splicing. In addition, we showed that their excision or retention, depending on cellular context, contributes to the diversification of the information carried by genes by producing functional RNAs instead of a protein-coding mRNA.
Hence, alternative splicing is a versatile developmental switch that provides plasticity to eukaryotic genomes transcriptional output, increasing not only proteome but also transcriptome diversity.
We explore alternative splicing of introns as a mechanism to fine-tune the production of long and short intron-derived regulatory ncRNAs (that we termed SID) during normal and pathological muscle differentiation where splicing is impaired like in Myotonic Dystrophy type 1, through bioinformatics predictions, high-throughput RNA sequencing and functional validation in model systems.

Related publications:

  • Hubé F, Ulveling D, Sureau A, Forveille S, Francastel C. Short intron-derived ncRNAs. Nucleic Acids Res. 2017 Jan 3;45(8):4768-4781. PMID: 28053119
  • Hubé F, Francastel C. “Pocket-sized RNA-Seq”: a method to capture new mature microRNA produced from a genomic region of interest. Non-coding RNA. 2015 June;1(2):127-138. Paper here
  • Hubé F, Francastel C. Mammalian Introns: When the Junk Generates Molecular Diversity. Int J of Mol Sci. 2015 Feb 20;16(3):4429-4452. PMID: 25710723
  • Ulveling D, Dinger M, Francastel C, Hubé F. Identification of a dinucleotide signature that discriminates coding from non-coding long RNAs. Front Genet. 2014 Sep 9;5:316. PMID: 25250049
  • Ulveling D, Francastel C, Hubé F. When one is better than two: RNA with dual functions. Biochimie. 2011 Apr;93(4):633-44. Review. PMID: 21111023
  • Ulveling D, Francastel C, Hubé F. Identification of potentially new bifunctional RNA based on genome-wide data-mining of alternative splicing events. Biochimie. 2011 Nov;93(11):2024-7. PMID: 21729736
  • Hubé F, Velasco G, Rollin J, Furling D, Francastel C. Steroid receptor RNA activator protein binds to and counteracts SRA RNA-mediated activation of MyoD and muscle differentiation. Nucleic Acids Res. 2011 Jan;39(2):513-25. PMID: 20855289



    CENTROMERIC REPEATS TRANSCRIPTIONGo to the Top
    A paradigm to link transcription of DNA repeats to global molecular and cellular effects

    teachingTandem repeats that underlie centromeric regions have a structural role at the chromosomal level, providing the assembly platforms for the kinetochore and attachment of the mitotic spindle, but also in the functional organization of the nucleus and long-range control of genome expression.
    We characterized transcripts that originate from murine centromeric repeats and showed that they are essential for centromere identity and function, whereas their unscheduled accumulation is causally linked to perturbed nuclear organization and cellular phenotypes. However, we showed that the outcome greatly depends on cellular and genetic contexts. In primary cells, increased transcription of centromeric repeats functions as a sensor of stress promoting cell cycle arrest and safeguard mechanisms; in contrast, in contexts of loss of the p53 checkpoints it leads to chromosomal instability.
    We explore the causal link between aberrant transcription of repetitive sequences and perturbed molecular and cellular programs, ex vivo in various cellular and genotype context and in mouse models. We also question the mechanisms that lead to their deregulated transcription, with special interest on DNA methylation that is tightly linked with maintenance of integrity of these sequences and hence, with maintenance of genome stability. Ultimately, we aim at deciphering the cellular functions and regulatory factors deregulated by their unscheduled accumulation.

    Related publications:

  • Hédouin S, Grillo G, Ivkovic I, Velasco G, Francastel C. CENP-A chromatin disassembly in stressed and senescent murine cells. Sci Rep. 2017 Feb 10;7:42520. doi: 10.1038/srep42520. PMID: 28186195
  • Guillemin C, Francastel C. [Heterochromatin compartments and gene silencing: human hematopoietic differentiation as a model study]. Biol Aujourdhui. 2010;204(3):221-33. Review. French. PMID: 20950566
  • Guillemin C, Maleszewska M, Guais A, Maës J, Rouyez MC, Yacia A, Fichelson S, Goodhardt M, Francastel C. Chromatin modifications in hematopoietic multipotent and committed progenitors are independent of gene subnuclear positioning relative to repressive compartments. Stem Cells. 2009 Jan;27(1):108-15. PMID: 18974210
  • Ferri F, Bouzinba-Segard H, Velasco G, Hubé F, Francastel C. Non-coding murine centromeric transcripts associate with and potentiate Aurora B kinase. Nucleic Acids Res. 2009 Aug;37(15):5071-80. PMID: 19542185



    DNA METHYLATION AND GENOME MAINTENANCEGo to the Top
    When studying a rare disease sheds new light on the field of DNA methylation

    teachingDNA methylation is among the best-studied epigenetic modification in vertebrates and is essential for normal embryonic development. Given its pivotal role in the control of gene expression and key biological processes, it comes as no surprise that perturbed DNA methylation patterns are hallmarks of many human diseases. In this context, the DNA methylation machinery is frequently perturbed although the causal link is sometimes difficult to formally establish. However, inherited monogenic disorders that disrupt components of the epigenetic machinery offer a unique opportunity to learn about (epi)genome maintenance.
    Our interest in the transcription of repeated sequences has motivated our interest in a rare autosomal recessive disease, the ICF syndrome (Immunodeficiency Centromeric instability Facial anomalies), caused by the remarkable loss of DNA methylation at (peri)centromeric repeats that cause chromosomal instability. Mutations in the DNA methyltransferase DNMT3B were the first reported causes of the disease. The study of its etiology has recently fuelled the field with new candidate players, whose role in DNA methylation and maintenance of genome stability had never been suspected before their implication in the disease.
    This raised important questions regarding their function, their direct or indirect role in pathways to DNA methylation, their genomic targets and the impact of their mutations on transcriptome, epigenome and cell fate. We aim at providing a comprehensive and integrated view of the consequences of perturbed DNA methylation in cells from patients and mouse models with important consequences to (i) understand the genotype/phenotype relationship in such a complex monogenic and epigenetic disease, (ii) establish biomarkers to aid diagnosis and prioritize patients for mutation screening and appropriate management, and (iii) in a more basic research orientation, to shed new light on the molecular mechanisms involved in establishment and maintenance of DNA methylation patterns at DNA repeats and unique genes, and on the consequences for long and short, coding and non-coding transcriptional output, with added relevance to other physiopathological contexts.

    Related publications:

  • Gatto S, Gagliardi M, Franzese M, Leppert S, Papa M, Cammisa M, Grillo G, Velasco G, Francastel C, Toubiana S, D’Esposito M, Angelini C, Matarazzo MR. ICF-specific DNMT3B dysfunction interferes with intragenic regulation of mRNA transcription and alternative splicing . Nucleic Acids Res. 2017 Mar 9; 45(10):5739-5756. PMID: 28334849
  • Sagie S, Toubiana S, Hartono S, Katzir H, Tzur-Gilat A, Havazelet S, Francastel C, Velasco G, Chedin F, Selig S. Telomeres in ICF syndrome cells are vulnerable to DNA damage due to elevated DNA:RNA hybrids. Nat Commun. 2017 Jan 24;8:14015. PMID: 28117327
  • Sterlin D, Velasco G, Moshous D, Touzot F, Mahlaoui N, Fischer A, Suarez F, Francastel C, Picard C. Genetic, Cellular and Clinical Features of ICF Syndrome: a French National Survey . J Clin Immunol. 2016 Feb;36(2):149-59. PMID: 26851945
  • Thijssen PE, Ito Y, Grillo G, Wang J, Velasco G, Nitta H, Unoki M, Yoshihara M, Suyama M, Sun Y, Lemmers RJ, de Greef JC, Gennery A, Picco P, Kloeckener-Gruissem B, Güngör T, Reisli I, Picard C, Kebaili K, Roquelaure B, Iwai T, Kondo I, Kubota T, van Ostaijen-Ten Dam MM, van Tol MJ, Weemaes C, Francastel C, van der Maarel SM, Sasaki H. Mutations in CDCA7 and HELLS cause immunodeficiency-centromeric instability-facial anomalies syndrome. Nat Commun. 2015 Jul 28;6:7870. PMID: 26216346
  • Walton E, Francastel C, Velasco G. Dnmt3b prefers germ line genes and centromeric regions: lessons from ICF and cancer and implications for diseases. Biology (Basel). 2014 Sep 5;3(3):578-605. PMID: 25198254
  • Velasco G, Walton EL, Sterlin D, Hédouin S, Nitta H, Yuya I, Fouyssac F, Mégarbané A, Sasaki H, Picard C, Francastel C. Germline genes hypomethylation and expression define a molecular signature in peripheral blood of ICF patients: implications for diagnosis and etiology. Orphanet J Rare Dis. 2014 Apr 17;9(1):56. PMID: 24742017
  • Nitta H, Unoki M, Ichiyanagi K, Kosho T, Shigemura T, Takahashi H, Velasco G, Francastel C, Picard C, Kubota T, Sasaki H. Three novel ZBTB24 mutations identified in Japanese and Cape Verdean type 2 ICF syndrome patients. J Hum Genet. 2013 Jul;58(7):455-60. PMID: 23739126
  • Walton EL, Francastel C, Velasco G. Maintenance of DNA methylation: Dnmt3b joins the dance. Epigenetics. 2011 Nov;6(11):1373-7. PMID: 22048250
  • Velasco G, Hubé F, Rollin J, Neuillet D, Philippe C, Bouzinba-Segard H, Galvani A, Viegas-Péquignot E, Francastel C. Dnmt3b recruitment through E2F6 transcriptional repressor mediates germ-line gene silencing in murine somatic tissues. Proc Natl Acad Sci U S A. 2010 May 18;107(20):9281-6. PMID: 20439742




    FUNDING SOURCESGo to the Top

    teaching


    Claire Francastel on PubMed
    Florent Hubé on PubMed
    Guillaume Velasco on PubMed