Roles of Zbtb24 and Lsh in DNA methylation and humoral immune

ED BIO SORBONNE PARIS CITE Proposition de sujet de thèse à l’appui d’une demande de contrat doctoral 2016-2017
Renseignements relatifs à l’Unité de Recherche :
Label et intitulé : INSERM U1151, Institut Necker Enfants Malades
Nom et prénom du Directeur : NASSIF Xavier
Téléphone :
PU/PH Paris Descartes
Télécopie :
Courriel: [email protected]
Renseignements relatifs à l’Equipe :
Nom de l’Equipe d’Accueil : Equipe 12, Développement du Système Immunitaire
Nom et prénom du responsable : REYNAUD Claude-Agnès
Qualité du responsable : DRCE CNRS
Téléphone :
01 72 60 64 48
Télécopie : 01 72 60 64 50
Courriel : [email protected]
Renseignements relatifs au sujet de thèse :
Nom et prénom du Directeur de thèse (HDR) : Weller Sandra
Qualité : CR1INSERM
Téléphone :
01 72 60 64 45
Télécopie : 01 72 60 64 50
Courriel : [email protected]
Titre du sujet proposé :
(En français) Rôles de Zbtb24 et Lsh dans la méthylation de l'ADN et la réponse immunitaire humorale
(En anglais) Roles of Zbtb24 and Lsh in DNA methylation and humoral immune response
Département (cocher le département correspondant au sujet de thèse qui n’est pas obligatoirement le
vôtre) :
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Biologie Cellulaire et moléculaire, Physiologie et Physiopathologie
Immunologie
Développement Génétique Neurobiologie et Vieillissement
Infectiologie, Microbiologie
Summary (5 lines maximum) :
Mutations of Zbtb24 and Lsh cause 1/3 of ICF (Immunodeficiency, Centromeric instability and Facial
anomalies) syndrome cases, characterized by hypogammaglobulinemia, an almost complete absence of
memory B cells and disturbed DNA methylation. The aim of the thesis is to use cellular and mouse
models with Zbtb24 or Lsh loss-of-function to determine : 1) which step of late B cell differentiation is
affected; 2) how Zbtb24 controls DNA methylation; 3) what links DNA methylation to humoral response
Proposition de sujet de thèse à l’appui d’une demande de contrat doctoral 2016-2017
(L’ensemble de cette fiche ne doit pas dépasser 1 page)
Nom, prénom du directeur de l'unité de recherche : NASSIF, Xavier
Numéro de l'unité de recherche (et établissement de rattachement) : INSERM U1151
Nom, prénom du responsable de l'équipe d'accueil (EAD) : REYNAUD Claude-Agnès
Nom, prénom du directeur de thèse : Weller Sandra
Titre du sujet de thèse: Roles of Zbtb24 and Lsh in DNA methylation and humoral immune response
Citer 5 mots clés : Memory B cells, Antibodies, Immunodeficiency, DNA methylation, Knockout
Candidat pressenti :
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OUI
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NON
Contenu scientifique du programme de la thèse (en anglais)
Patients who suffer from the ICF syndrome (Immunodeficiency, Centromeric instability and Facial
anomalies) display simultaneously an abnormal profile of DNA methylation and a primary humoral
immune deficiency characterized by a variable hypo-gammaglobulinemia and a constant and deep
decrease in the number of memory B cells. Loss-of-function of Dnmt3b, Lsh, Zbtb24 and CdcA7 account
for most of ICF cases. The goal of the thesis is to elucidate the mechanistic link between the mutations
that cause the ICF syndrome, and the molecular (DNA methylation) and cellular (immunodeficiency)
phenotypes of these patients. This should improve our knowledge of the physiology of memory B cells
and long-lived plasma cells. Our two aims are :
1) delineate how the different ICF mutations cause a primary antibody deficiency
First of all, cellular and mouse models with Zbtb24 and Lsh loss-of-function (LOF) will be
established. Lsh conditional knockout mice (Lsh cKO), which have been established recently by another
team (K. Muegge, USA), are currenlty bread in our animal facility with mouse strains that express the
Cre recombinase at various stages of B cell development (CD21-Cre and mb1-Cre). They will be
immunized and analyzed by FACS and ELISA in order to identify which steps of early and late B-cell
differentiation are affected by Lsh loss-of-function. To perform similar studies with Zbtb24, we have
already set up a retroviral-based RNAi strategy against Zbtb24 that will be applied to transduce
hematopoietic stem cells and reconstitute a murine B-cell compartment deprived of Zbtb24. In parallel,
Zbtb24 cKO mice will be established thanks to Crispr/Cas9 technology applied to mouse ovocytes. The
consequences of the LOF of both genes will be compared and confronted to the phenotypes of the
corresponding ICF patients (in collaboration with C. Picard, Paris Descartes). As a back-up, if we face
trouble with the various mouse models, the "in vitro" germinal center B-cell culture developped by the
group of D. Kitamura will be used in conjunction with our retroviral-based RNAi strategy..
2) determine how Zbtb24 and the other proteins mutated in ICF patients control DNA methylation and
how DNA methylation control late B-cell differentiation
Putative Zbtb24 physical interactors identified by yeast two-hybrid screens will be validated in
human cells in vitro, by DuoLink and co-immunoprecipitation experiments. The functional consequences
of these interactions on genome-vide DNA methylation and heterochromatin assembly will be explored
thanks to the LOF cellular models described in (1), and other knockout cells already imported in the
laboratory, in collaboration with the team of C. Francastel (Paris Diderot). In this way, we hope to identify
putative Lsh and Zbtb24 targets that may explain the specific defects observed in the B-cell
compartment of ICF patients and of our mouse models. In parallel, we will test the hypothesis that the
immunodeficiency is a consequence of a toxic-IFNα pathway triggered by hypomethylation and
derepression of satellite repeats, by breeding our mutant mice onto and IFNAR-/- background.
Indiquez les cinq meilleures publications récentes de l’équipe :
- Bagnara et al., (2015) A Reassessment of IgM Memory Subsets in Humans. J Immunol. 195(8):3716-24
- Descatoire et al., (2014) Identification of a human splenic marginal zone B cell precursor with
NOTCH2-dependent differentiation properties. J Exp Med. 211(5):987-1000.
- Mahévas et al., (2013) B cell depletion in immune thrombocytopenia reveals splenic long-lived plasma
cells. J Clin Invest. 123(1):432-42.
- Weill et al., (2013) Multiple players in mouse B cell memory. Curr Opin Immunol. 25(3):334-8.
- Weller et al., (2012) IgM+IgD+CD27+ B cells are markedly reduced in IRAK-4-, MyD88-, and TIRAPbut not UNC-93B-deficient patients. Blood. 120(25):4992-5001.