Polycomb (PcG) proteins are epigenetic modifiers that modulate accessibility of genomic loci by covalent modification of N-terminal histone tails. In mammals, PcG proteins act within at least two functionally and biochemically distinct complexes, named Polycomb repressive complex 1 and 2 (PRC1 and PRC2). Within PRC2, the Ezh2 methyltransferase is able to di- and tri-methylate lysine 27 of histone H3. This mark is recognized by chromodomain-containing PRC1 subunits. Once bound, PRC1 catalyzes monoubiquitination of lysine 119 of histone H2A, which results in permanent, yet reversible, and heritable locus silencing. PcG proteins modulate expression of genes involved in development, lineage specification and cell identity, and their deregulation leads to aberrant differentiation of a number of cellular lineages, including the hematopoietic subsets. Recently, independent studies on a number of PcG mutants have highlighted an essential contribution of the Polycomb system to homeostasis of the lymphoid lineage. Expression of Ring1a and Ring1b, the catalytic subunits of PRC1, is maintained throughout B cell development. Using a conditional gene targeting approach in vivo, we addressed their function in resting naïve B lymphocytes and in B cells participating in a T cell-dependent immune response. Removal of a single subunit did not affect B cell development nor immune responses in mice. Instead, complete ablation of PRC1 function in B cells at early developmental stages resulted in a nearly complete block at the pro- to pre-B cell stage. Moreover, selective PRC1 inactivation in transitional B cells led to aberrant differentiation, with B cells acquiring features of mature cells while simultaneously retaining markers of earlier developmental stages. At the molecular level, loss of PRC1 in resting B cells read out in extensive transcriptional deregulation involving aberrant expression of several lineage determinants. Selective loss of Ring1a and Ring1b in GC B cells caused a significant reduction in numbers and frequency of GC B cells. As a result of the impaired GC reaction, serum titers of antigen-specific, class-switched antibodies were significantly decreased and formation of memory B cells significantly impaired in mutant mice. Instead, mutant GC B cells showed premature onset of terminal differentiation, as assessed by the induction of genes coding for the master regulators of plasma cell differentiation Prdm1, Irf4 and Xbp1. This result was confirmed by performing in vitro LPS stimulation assays, which showed an accelerated appearance of plasmablasts co-expressing Syndecan-1/CD138 and high levels of the Irf4 transcription factor in Ezh2 mutant B cell cultures. Increased apoptosis of PRC1 mutant cells as compared to control was also observed in the context of in vitro B cell activation. Importantly, activation of B cells with the RP105 Toll-Like Receptor agonist, which does not induce expression of Activation Induced Deaminase (AID) normalized the apoptotic rate of PRC1 deficient cells to wild-type levels. Moreover, several target genes of the germinal center master regulator Bcl6 were found up-regulated in PRC1 mutant cells. Together, this suggests that PRC1 may support GC function through at least two mechanisms. On the one hand, it may co-operate in establishing Bcl6 transcriptional program, thereby preventing premature terminal differentiation. On the other, it may participate in the repair of DNA damage caused by AID activity, thereby allowing tolerance of AID genotoxic activity by GC B cells.

THE ROLE OF PRC1 IN B CELL DEVELOPMENT AND ADAPTIVE IMMUNE RESPONSES / F. Alberghini ; supervisor: S. Casola ; cosupervisors: G. Natoli, M. Merkenschlager. Università degli Studi di Milano, 2014 Mar 25. 25. ciclo, Anno Accademico 2013. [10.13130/alberghini-federica_phd2014-03-25].

THE ROLE OF PRC1 IN B CELL DEVELOPMENT AND ADAPTIVE IMMUNE RESPONSES

F. Alberghini
2014

Abstract

Polycomb (PcG) proteins are epigenetic modifiers that modulate accessibility of genomic loci by covalent modification of N-terminal histone tails. In mammals, PcG proteins act within at least two functionally and biochemically distinct complexes, named Polycomb repressive complex 1 and 2 (PRC1 and PRC2). Within PRC2, the Ezh2 methyltransferase is able to di- and tri-methylate lysine 27 of histone H3. This mark is recognized by chromodomain-containing PRC1 subunits. Once bound, PRC1 catalyzes monoubiquitination of lysine 119 of histone H2A, which results in permanent, yet reversible, and heritable locus silencing. PcG proteins modulate expression of genes involved in development, lineage specification and cell identity, and their deregulation leads to aberrant differentiation of a number of cellular lineages, including the hematopoietic subsets. Recently, independent studies on a number of PcG mutants have highlighted an essential contribution of the Polycomb system to homeostasis of the lymphoid lineage. Expression of Ring1a and Ring1b, the catalytic subunits of PRC1, is maintained throughout B cell development. Using a conditional gene targeting approach in vivo, we addressed their function in resting naïve B lymphocytes and in B cells participating in a T cell-dependent immune response. Removal of a single subunit did not affect B cell development nor immune responses in mice. Instead, complete ablation of PRC1 function in B cells at early developmental stages resulted in a nearly complete block at the pro- to pre-B cell stage. Moreover, selective PRC1 inactivation in transitional B cells led to aberrant differentiation, with B cells acquiring features of mature cells while simultaneously retaining markers of earlier developmental stages. At the molecular level, loss of PRC1 in resting B cells read out in extensive transcriptional deregulation involving aberrant expression of several lineage determinants. Selective loss of Ring1a and Ring1b in GC B cells caused a significant reduction in numbers and frequency of GC B cells. As a result of the impaired GC reaction, serum titers of antigen-specific, class-switched antibodies were significantly decreased and formation of memory B cells significantly impaired in mutant mice. Instead, mutant GC B cells showed premature onset of terminal differentiation, as assessed by the induction of genes coding for the master regulators of plasma cell differentiation Prdm1, Irf4 and Xbp1. This result was confirmed by performing in vitro LPS stimulation assays, which showed an accelerated appearance of plasmablasts co-expressing Syndecan-1/CD138 and high levels of the Irf4 transcription factor in Ezh2 mutant B cell cultures. Increased apoptosis of PRC1 mutant cells as compared to control was also observed in the context of in vitro B cell activation. Importantly, activation of B cells with the RP105 Toll-Like Receptor agonist, which does not induce expression of Activation Induced Deaminase (AID) normalized the apoptotic rate of PRC1 deficient cells to wild-type levels. Moreover, several target genes of the germinal center master regulator Bcl6 were found up-regulated in PRC1 mutant cells. Together, this suggests that PRC1 may support GC function through at least two mechanisms. On the one hand, it may co-operate in establishing Bcl6 transcriptional program, thereby preventing premature terminal differentiation. On the other, it may participate in the repair of DNA damage caused by AID activity, thereby allowing tolerance of AID genotoxic activity by GC B cells.
25-mar-2014
Settore BIO/10 - Biochimica
Epigenetics ; Polycomb ; Immune system ; Lymphocyte Development ; Lymphocyte Activation
CASOLA , STEFANO
Doctoral Thesis
THE ROLE OF PRC1 IN B CELL DEVELOPMENT AND ADAPTIVE IMMUNE RESPONSES / F. Alberghini ; supervisor: S. Casola ; cosupervisors: G. Natoli, M. Merkenschlager. Università degli Studi di Milano, 2014 Mar 25. 25. ciclo, Anno Accademico 2013. [10.13130/alberghini-federica_phd2014-03-25].
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