ABSTRACT
Oct-2 and OBF-1 (also called OCA-B or Bob-1) are B cell-specific transcription factors that bind to the conserved octamer site of immunoglobulin promoters, yet their role in immunoglobulin transcription has remained unclear. We generated mice in which the lymphoid compartment was reconstituted with cells that lack both Oct-2 and OBF-1. Even in the absence of these two transcription factors, B cells develop normally to the membrane immunoglobulin M-positive (IgM+) stage and immunoglobulin gene transcription is essentially unaffected. These observations imply that the ubiquitous factor Oct-1 plays a previously unrecognized role in the control of immunoglobulin gene transcription and suggest the existence of another, as yet unidentified, cofactor. In addition, both factors are essential for germinal center formation, although OBF-1 is more important than Oct-2 for IgG production after immunization.
Subject(s)
B-Lymphocytes/cytology , B-Lymphocytes/immunology , DNA-Binding Proteins/deficiency , Genes, Immunoglobulin , Trans-Activators/deficiency , Transcription Factors/deficiency , Animals , B-Lymphocytes/metabolism , Base Sequence , Cell Differentiation , DNA Primers/genetics , DNA-Binding Proteins/genetics , Germinal Center/cytology , Germinal Center/immunology , Immunoglobulin G/biosynthesis , Immunoglobulin M/biosynthesis , Mice , Mice, Knockout , Models, Biological , Octamer Transcription Factor-2 , RNA/genetics , RNA/metabolism , Trans-Activators/genetics , Transcription Factors/genetics , Transcription, GeneticABSTRACT
This study attempted to evaluate and compare the role of various B cell-specific markers for anti-viral immune responses in mouse strains lacking molecules belonging to the B cell receptor (BCR) complex (IgM, Ig alpha and C(kappa)), the co-stimulatory molecules (CD19 and CD22), the protein kinases [Bruton's tyrosine kinase (Btk)] or the transcription factors (OBF-1). These mice were tested in two model infections [vesicular stomatitis virus (VSV) and lymphocytic choriomeningitis virus (LCMV)] using T cell-independent (TI) or T cell-dependent (TD) antigens. All mice controlled an LCMV infection indicating that cytotoxic T cell functions were within normal ranges. In contrast, OBF-1(-/-) mice were partially protected and mb-1(delta c/delta c) mice not at all protected against VSV infection, a virus that is controlled virtually exclusively by neutralizing antibodies. Susceptibility to VSV infection was correlated with structural defects in the spleen: absence of mature B cells and follicles with marginal zone macrophages and absence of germinal centers with follicular dendritic cells correlated with lack or substantial reduction of protective IgM and IgG responses respectively. The lack of kappa light chain did not affect the neutralizing response, indicating that it could easily be replaced by the lambda chain. Absence of the co-stimulatory molecules CD19 and CD22 or of the signaling molecule Btk had modulating effects, but did not increase susceptibility to VSV or LCMV. Our findings suggest that there are crucial molecules for B cell activation at the beginning (BCR complex) and the end (transcription) of the signaling cascade, whereas fine-tuning factors modulating the response in between exhibit considerable functional overlap.
Subject(s)
B-Lymphocytes/immunology , Cell Adhesion Molecules , Lectins , Lymphocytic Choriomeningitis/prevention & control , Lymphocytic choriomeningitis virus/immunology , Rhabdoviridae Infections/prevention & control , Spleen/immunology , Vesicular stomatitis Indiana virus/immunology , Agammaglobulinaemia Tyrosine Kinase , Animals , Antibodies, Viral/blood , Antigens, CD/analysis , Antigens, CD/genetics , Antigens, CD19/genetics , Antigens, Differentiation, B-Lymphocyte/genetics , Antigens, Viral/administration & dosage , CD79 Antigens , DNA-Binding Proteins/genetics , Immunization , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Protein Kinases/genetics , Protein-Tyrosine Kinases/analysis , Receptors, Antigen, B-Cell/analysis , Sialic Acid Binding Ig-like Lectin 2 , Specific Pathogen-Free Organisms , Time Factors , Transcription Factors/geneticsABSTRACT
OBF-1 is a B cell-restricted transcriptional coactivator that is recruited to octamer-containing promoters by interacting with the POU domain of Oct-1 or Oct-2. We have shown earlier that mice lacking OBF-1 were dramatically impaired in their ability to mount humoral immune responses and did not develop germinal centers in the spleen; however, they had a largely normal B cell development in the bone marrow. In this study, we demonstrate that OBF-1-deficient mice also have an early defect in B cell development and show that OBF-1-/- immature B cells are greatly impaired at the transition from the bone marrow to the spleen. In addition, when the OBF-1 mutation is combined to a mutation in the gene encoding Bruton's tyrosine kinase, a striking phenotype is observed. These double-deficient animals lack peripheral B cells and have virtually no serum Igs, thus closely resembling human X chromosome-linked agammaglobulinemia.
Subject(s)
Agammaglobulinemia/genetics , B-Lymphocytes/immunology , Lymphopenia/genetics , Protein-Tyrosine Kinases/deficiency , Trans-Activators/deficiency , Agammaglobulinaemia Tyrosine Kinase , Agammaglobulinemia/enzymology , Agammaglobulinemia/immunology , Agammaglobulinemia/pathology , Animals , B-Lymphocytes/enzymology , B-Lymphocytes/pathology , Cell Differentiation/genetics , Cell Differentiation/immunology , Female , Gene Expression Regulation/immunology , Gene Expression Regulation, Developmental/immunology , Genes, Immunoglobulin , Genetic Linkage/immunology , Humans , Immunoglobulins/biosynthesis , Immunoglobulins/blood , Lymphoid Tissue/pathology , Lymphopenia/enzymology , Lymphopenia/immunology , Lymphopenia/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Knockout , Multigene Family/immunology , Phenotype , Protein-Tyrosine Kinases/genetics , Trans-Activators/genetics , X Chromosome/genetics , X Chromosome/immunologyABSTRACT
The B-lymphocyte-specific transcriptional factor called Oct binding factor (OBF)-1, OCA-B or Bob1 (refs 1-3) is thought to be involved in the transcription of immunoglobulin genes through recruitment to the highly conserved octamer site of immunoglobulin promoters, mediated by either Oct-1 or Oct-2. To define the in vivo role of OBF-1 we have used gene targeting in embryonic stem cells to generate mice lacking the coactivator OBF-1. Such OBF-1-/- mice are born normally, are fertile and seem healthy, and surprisingly, rearrangement and transcription of immunoglobulin genes are largely unaffected. However, mice deficient in OBF-1 have reduced numbers of mature B cells and a severe reduction in the number of recirculating B cells, but otherwise show normal B-cell differentiation. Serum IgA and particularly IgG levels are greatly reduced. If mutant mice are immunized with either a thymus-independent or a thymus-dependent antigen, their immune responses are dramatically weakened. Strikingly, germinal centres completely fail to develop after immunization with thymus-dependent antigen. Our results demonstrate that in vivo OBF-1 is not required for initial transcription of immunoglobulin genes or for B cell development, but instead is essential for the response of B cells to antigens, and is required for the formation of germinal centres.
Subject(s)
B-Lymphocytes/immunology , Germinal Center/cytology , Immunity/physiology , Trans-Activators/physiology , Animals , Antibody Formation/physiology , B-Lymphocytes/cytology , Bone Marrow Cells , Cell Differentiation/genetics , Cell Differentiation/physiology , Gene Targeting , Germinal Center/immunology , Immunoglobulin A/blood , Immunoglobulin G/blood , Mice , Mice, Inbred C57BL , Mutagenesis , Spleen/cytology , Trans-Activators/geneticsABSTRACT
The B cell-specific activity of immunoglobulin (Ig) gene promoters is to a large extent mediated by the conserved octamer motif ATTTGCAT. This requires the DNA binding octamer factors Oct-1 and/or Oct-2, as well as an additional B cell-restricted non-DNA binding cofactor. We recently cloned such a coactivator specific for Oct-1 or Oct-2 from human B cells and called it OBF-1. Here we report the isolation and characterization of the murine homologue. Full-length cDNA clones as well as genomic clones were isolated and the gene structure was determined. The deduced protein sequence shows that the mouse protein has an identical length, is likewise proline rich and shows 89% overall identity to the human protein. The OBF-1 gene is expressed in a very highly B cell-specific manner and is transcribed in cells representative of all stages of B cell differentiation, including the earliest ones. We show that OBF-1 interacts in the absence of DNA with the POU domain of Oct-1 or Oct-2 and also with the general transcription factors TBP and TFIIB. Furthermore, we demonstrate that although OBF-1 efficiently activates promoter octamer sites, it does not activate enhancer octamer sites.