Memory B cells (Mem B) and long-lived plasma cells (LLPC) ensure the long-term antibody-mediated protection against pathogens. However, the factors that support their differentiation and sustain their exceptional longevity remain largely unknown. We will address this question through the study of the human ICF syndrome (Immunodeficiency, Centromeric instability and Facial abnormalities), a rare genetic disease characterized by a severe decrease in both immunoglobulin titers and Mem B cells numbers. ICF is caused by mutations in DNMT3B (ICF1), a de novo DNA methyltransferase, or ZBTB24 (ICF2), a putative transcription factor. ZBTB24, together with DNMT3B, could play a role in DNA methylation since ICF patients display hypomethylation of juxtacentromeric satellite repeats which causes centromeric instability. As the ICF phenotype is consistent with an alteration of LLPC and Mem B cell compartments, elucidating the link between its genetic basis and the immunodeficiency should be very informative. We will first determine which stage of B cell differentiation is affected by ICF mutations. We will study their impact on in vitro differentiation of human B cells into PC and/or Mem B cells, using both cells from patients and RNA interference. In parallel, two new Zbtb24 mutant mouse strains will be characterized: one with a gene-trapped allele that can be converted into a conditional knockout or a reporter (LacZ) allele, and one with knock-in mutations that reproduce those of an ICF2 patient. We will confirm that both recapitulate the ICF phenotype and Zbtb24 expression will be tracked with the LacZ allele. The lymphoid development of Zbtb24 mutants and their ability to mount memory antibody responses will be analyzed and we will assess if this defect is B-cell intrinsic. At last, we will determine if Dnmt3b deficiency similarly impairs late B-cell differentiation, by lymphoid reconstitution with fetal liver from our mouse strain with hypomorphic mutations found in ICF1 patients. Our second aim is to delineate how Zbtb24 deficiency affects B cells. To identify Zbtb24 direct targets, we will perform transcriptome and methylome analyses on the mutant mice and compare these datasets to those generated earlier from the Dnmt3b deficient mice described above. In addition, Zbtb24 deficiency could alter the heterochromatinization of juxtacentromeric regions. This defect, combined with the spontaneous repression of p53 in germinal center B cells, could lead to the transcription of satellite-derived dsRNA in this cell type and the subsequent triggering of a type-I interferon mediated apoptotic pathway. We will test this hypothesis by breeding our mice with a mouse strain deficient for the type-I IFN receptor. Alternatively, hypomethylation and transcription of satellite repeats could lead to perturbed nuclear organization, and ultimately, to perturbed expression programs associated with B-cell identity and fate. We will characterize transcription of satellite repeats and nuclear organization, especially heterochromatin compartments, in the nuclei of progenitors and B cells from mouse models. Our third aim is to search for Zbtb24 binding partners to understand how it regulates DNA methylation. We will undertake a global yeast double-hybrid screening and we will validate in mammalian cells the interaction with several candidate proteins with known implication in DNA methylation and assembly of heterochromatin. Using loss of function approaches, we will then define whether these factors are implicated in the recruitment of ZBTB24 or DNMT3B or both at heterochromatin regions and target genes identified in Aim 2. In conclusion, our study should refine our knowledge on B-cell differentiation and pathways to DNA methylation. The long-term outputs could be the improvement of the humoral responses of ICF patients, and more generally a better understanding of B-cell memory formation, a key knowledge for the development of efficient vaccines.