Obiettivo Antibody-secreting cells consisting of short-lived proliferating plasmablasts and long-lived quiescent plasma cells are essential for the acute response to infection and long-term protection of the host against pathogens. Only a few regulators (Blimp1, IRF4, XBP1, Aiolos, Ikaros and E-proteins) have been implicated in the transcriptional control of antibody-secreting cells, and their target genes, with the exception of Blimp1 and E-proteins, are still unknown. This proposal aims to systematically identify key players in the development and function of antibody-secreting cells by using the CRISPR/Cas9 and Cre/loxP methods. For this, we improved existing protocols to extend the duration of in vitro plasmablast differentiation and showed that Rosa26(Cas9/+) B cells infected with Blimp1 or Xbp1 sgRNA-expressing retroviruses recapitulated the Blimp1 and Xbp1 mutant phenotypes in this proof-of-principle experiment. Moreover, Cre retrovirus-mediated deletion of Irf4, Ikaros and Aiolos strongly impaired plasmablast differentiation in this optimized system. To discover new regulators of plasma cell differentiation, CRISPR/Cas9-based screens will be performed with pooled sgRNA libraries targeting all known upregulated genes in plasmablasts and plasma cells, followed by individual validation of the best hits. Selected top-ranked genes will be analyzed in vivo by conditional mutagenesis with newly generated, plasma cell-specific Cre lines. Regulated target genes of IRF4, Ikaros, Aiolos, XBP1 and the XBP1-regulated transcription factor Bhlha15 will be identified in plasmablasts by ChIP- and RNA-seq analyses. Target genes with potentially interesting functions will be further characterized by CRISPR/Cas9- or Cre/loxP-mediated mutagenesis. These experiments will provide fundamentally new insight into the molecular mechanisms controlling the development and function of antibody-secreting cells, which are the essential effector cells of humoral immunity. Parole chiave Plasma cell development and function CRISPR Cas9 screen for key regulators conditional Cre-mediated mutagenesis target genes of plasma cell-specific transcription factors transcriptional networks Programma(i) H2020-EU.1.1. - EXCELLENT SCIENCE - European Research Council (ERC) Main Programme Argomento(i) ERC-2016-ADG - ERC Advanced Grant Invito a presentare proposte ERC-2016-ADG Vedi altri progetti per questo bando Meccanismo di finanziamento ERC-ADG - Advanced Grant Istituzione ospitante FORSCHUNGSINSTITUT FUR MOLEKULARE PATHOLOGIE GESELLSCHAFT MBH Contribution nette de l'UE € 2 500 000,00 Indirizzo CAMPUS-VIENNA-BIOCENTER 1 1030 Wien Austria Mostra sulla mappa Regione Ostösterreich Wien Wien Tipo di attività Private for-profit entities (excluding Higher or Secondary Education Establishments) Collegamenti Contatta l’organizzazione Opens in new window Sito web Opens in new window Partecipazione a programmi di R&I dell'UE Opens in new window Rete di collaborazione HORIZON Opens in new window Costo totale € 2 500 000,00 Beneficiari (1) Classifica in ordine alfabetico Classifica per Contributo netto dell'UE Espandi tutto Riduci tutto FORSCHUNGSINSTITUT FUR MOLEKULARE PATHOLOGIE GESELLSCHAFT MBH Austria Contribution nette de l'UE € 2 500 000,00 Indirizzo CAMPUS-VIENNA-BIOCENTER 1 1030 Wien Mostra sulla mappa Regione Ostösterreich Wien Wien Tipo di attività Private for-profit entities (excluding Higher or Secondary Education Establishments) Collegamenti Contatta l’organizzazione Opens in new window Sito web Opens in new window Partecipazione a programmi di R&I dell'UE Opens in new window Rete di collaborazione HORIZON Opens in new window Costo totale € 2 500 000,00
