RESUMO
OBF1 is a specific coactivator of the POU family transcription factors OCT1 and OCT2. OBF1 and OCT2 are B cell-specific and indispensable for germinal center (GC) formation, but their mechanism of action is unclear. Here, we show by chromatin immunoprecipitation-sequencing that OBF1 extensively colocalizes with OCT1 and OCT2. We found that these factors also often colocalize with transcription factors of the ETS family. Furthermore, we showed that OBF1, OCT2, and OCT1 bind widely to the promoters or enhancers of genes involved in GC formation in mouse and human GC B cells. Short hairpin RNA knockdown experiments demonstrated that OCT1, OCT2, and OBF1 regulate each other and are essential for proliferation of GC-derived lymphoma cell lines. OBF1 downregulation disrupts the GC transcriptional program: genes involved in GC maintenance, such as BCL6, are downregulated, whereas genes related to exit from the GC program, such as IRF4, are upregulated. Ectopic expression of BCL6 does not restore the proliferation of GC-derived lymphoma cells depleted of OBF1 unless IRF4 is also depleted, indicating that OBF1 controls an essential regulatory node in GC differentiation.
Assuntos
Centro Germinativo/metabolismo , Fator 1 de Transcrição de Octâmero/fisiologia , Fator 2 de Transcrição de Octâmero/uso terapêutico , Transativadores/uso terapêutico , Transcrição Gênica/genética , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Ontologia Genética , Células HEK293 , Humanos , Lipopolissacarídeos/farmacologia , Linfoma não Hodgkin/genética , Linfoma não Hodgkin/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator 1 de Transcrição de Octâmero/deficiência , Fator 1 de Transcrição de Octâmero/genética , Fator 2 de Transcrição de Octâmero/deficiência , Fator 2 de Transcrição de Octâmero/genética , Proteína Proto-Oncogênica c-ets-1/análise , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes/metabolismo , Transativadores/deficiência , Transativadores/genéticaRESUMO
Mice lacking a functional gene for the Oct2 transcriptional activator display several developmental and functional deficiencies in the B lymphocyte lineage. These include defective B cell receptor (BCR) and Toll-like receptor 4 signaling, an absence of B-1 and marginal zone populations, and globally reduced levels of serum immunoglobulin (Ig) in naive and immunized animals. Oct2 was originally identified through its ability to bind to regulatory regions in the Ig loci, but genetic evidence has not supported an essential role for Oct2 in the expression of Ig genes. We describe a new Oct2-mediated role in B cells. Oct2 augments the ability of activated B cells to differentiate to antibody-secreting plasma cells (ASCs) under T cell-dependent conditions through direct regulation of the gene encoding the alpha chain of the interleukin (IL) 5 receptor. Ectopic expression of IL-5Ralpha in oct2-deficient B cells largely restores their ability to differentiate to functional ASCs in vitro but does not correct other phenotypic defects in the mutants, such as the maturation and specialization of peripheral B cells, which must therefore rely on distinct Oct2 target genes. IL-5 augments ASC differentiation in vitro, and we show that IL-5 directly activates the plasma cell differentiation program by enhancing blimp1 expression.
Assuntos
Células Produtoras de Anticorpos/citologia , Linfócitos B/imunologia , Subunidade alfa de Receptor de Interleucina-5/imunologia , Fator 2 de Transcrição de Octâmero/imunologia , Animais , Células Produtoras de Anticorpos/efeitos dos fármacos , Células Produtoras de Anticorpos/imunologia , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Linhagem Celular , Eosinófilos/efeitos dos fármacos , Eosinófilos/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-5/farmacologia , Subunidade alfa de Receptor de Interleucina-5/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 de Transcrição de Octâmero/deficiência , Fator 2 de Transcrição de Octâmero/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Baço/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/imunologiaRESUMO
CD86 signals directly in a B cell to activate PI3K and increase the rate of IgG(1) production, without affecting germline transcription. However, the mechanism by which CD86 activates PI3K in a B cell and the relevance of CD86 stimulation in vivo remains unknown. We show that the addition of CD28/Ig to CD40 ligand/IL-4-activated wild-type, but not CD86- or CD19-deficient, B cells increased the level of phosphorylation for Lyn and CD19, as well as the amount of Lyn, Vav, and PI3K that immunoprecipitated with CD19. Adoptive transfer of CD86-deficient B cells and wild-type CD4(+) T cells into RAG2-deficient mice and immunization with trinitrophenylated keyhole limpet hemocyanin resulted in an IL-4 and germline IgG(1) response equivalent to control mice, but a decrease in serum IgG(1). Thus, our findings suggest that CD86 plays a key role in regulating the level of IgG(1) produced in vitro and in vivo, and that Lyn and CD19 may be the signaling intermediates activated by CD86 proximal to PI3K.