ABSTRACT
PU.1(+/-)Spi-B(-/-) mice exhibit reduced numbers of immature and mature B lymphocytes, which exhibit severe defects in response to BCR-mediated stimulation and poor survival. We found that expression of c-rel, a member of the Rel/NF-kappa B family, is dramatically reduced in PU.1(+/-)Spi-B(-/-) splenic B cells. Analysis of the murine c-rel promoter identified three PU.1/Spi-B binding sites critical for c-rel promoter activity. Furthermore, reintroduction of Rel protein restored wild-type B cell numbers to mice reconstituted with PU.1(+/-)Spi-B(-/-) bone marrow. These findings are the first to demonstrate that a member of the Rel/NF-kappa B family is directly regulated by Ets proteins and dissect the molecular basis for the function of two Ets factors, PU.1 and Spi-B, in promoting B lymphocyte survival.
Subject(s)
B-Lymphocytes/immunology , DNA-Binding Proteins/physiology , Proto-Oncogene Proteins c-rel/genetics , Proto-Oncogene Proteins/physiology , Trans-Activators/physiology , Transcription Factors/physiology , Animals , Binding Sites , Bone Marrow Cells/immunology , Bone Marrow Transplantation , Cell Line , Cell Survival , Cells, Cultured , DNA-Binding Proteins/genetics , Down-Regulation , Gene Expression Regulation , Mice , Mice, Knockout , Promoter Regions, Genetic , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-rel/biosynthesis , RNA, Messenger/biosynthesis , Spleen/immunology , Trans-Activators/genetics , Transcription Factors/genetics , TransfectionABSTRACT
Previously it was shown that the Ets proteins, PU.1 and Spi-B, exhibit functional redundancy in B lymphocytes. To investigate the possibility that PU.1 or Spi-B or both share overlapping roles with Ets-1 or Elf-1, PU.1(+/-)Ets-1(-/-), PU.1(+/-)Elf-1(-/-), and Spi-B(-/-)Ets-1(-/-) animals were generated. No blood cell defects were observed in these animals except those previously reported for Ets-1(-/-) mice. Therefore, no genetic overlap was detected between PU.1 or Spi-B with Ets-1 or Elf-1. In contrast, the results confirmed functional redundancy for PU.1 and Spi-B in that PU.1(+/-)Spi-B(-/-) bone marrow progenitors yielded smaller colonies in methylcellulose cultures than did wild-type, PU.1(+/-) or Spi-B(-/-) progenitors. In addition, PU.1(+/-)Spi-B(+/+), PU.1(+/-)Spi-B(+/-), and PU.1(+/-) Spi-B(-/-) mice displayed extramedullary splenic hematopoiesis. In summary, PU.1 and Spi-B regulate common target genes required for proliferation of hematopoietic progenitors or their committed descendants, whereas Ets-1 or Elf-1 do not appear to regulate shared target genes with PU.1 or Spi-B.
Subject(s)
DNA-Binding Proteins/immunology , Proto-Oncogene Proteins/immunology , Trans-Activators/immunology , Transcription Factors/immunology , Animals , DNA-Binding Proteins/genetics , Gene Expression Regulation/immunology , Hematopoietic Stem Cells/immunology , Mice , Nuclear Proteins , Proto-Oncogene Protein c-ets-1 , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-ets , Trans-Activators/genetics , Transcription Factors/geneticsABSTRACT
To investigate the in vivo functions of PU.1 and Spi-B, two highly related Ets transcription factors, we previously generated PU. 1(+/+)Spi-B(-/-) and PU.1(+/-)Spi-B(-/-) mice and demonstrated a significant decrease in B-cell receptor (BCR) signaling in mutants. Major components of BCR signaling appear to be expressed at normal levels in these mice, implying that PU.1 and Spi-B cooperate in the transcription of additional target genes important for antigen receptor signaling. We used subtractive hybridization to identify novel in vivo PU.1/Spi-B target genes and determined that the expression of a heptahelical receptor, P2Y10, is dramatically reduced in PU.1(+/-)Spi-B(-/-) B-cells. Further analysis shows that P2Y10 expression is restricted to lymphoid cells and parallels that of Spi-B in B-lymphocytes. Lastly, the P2Y10 promoter contains a PU. 1/Spi-B binding site functionally required for efficient transcription in B-cells. Thus, P2Y10 is likely to be a direct in vivo transcriptional target for PU.1 and Spi-B and provides a unique model to explore transcriptional regulation by this Ets factor subfamily. Furthermore, P2Y10 suggests an intriguing connection between heterotrimeric G-proteins and BCR signaling.
Subject(s)
DNA-Binding Proteins/physiology , Proto-Oncogene Proteins/physiology , Receptors, Antigen, B-Cell/genetics , Trans-Activators/physiology , Transcription Factors/physiology , Transcription, Genetic , 3T3 Cells , Amino Acid Sequence , Animals , B-Lymphocytes/metabolism , Binding Sites , Binding, Competitive , Blotting, Northern , Cell Lineage , DNA/genetics , DNA/metabolism , DNA, Complementary/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression , Mice , Mice, Knockout , Molecular Sequence Data , Promoter Regions, Genetic , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , RNA, Messenger/metabolism , Receptors, Antigen, B-Cell/metabolism , Sequence Homology, Amino Acid , Tissue Distribution , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Cells, CulturedABSTRACT
PU.1 and Spi-B have previously been implicated in the regulation of genes encoding B cell receptor (BCR) signaling components. Spi-B-/- B lymphocytes respond poorly to BCR stimulation; PU.1-/- mice, however, lack B cells, precluding an analysis of BCR responses. We now show that PU.1+/- Spi-B-/- B cells exhibit more extensive defects than Spi-B-/- B cells, indicating that both PU.1 and Spi-B are required for normal BCR signaling. Strikingly, BCR cross-linking results in substantially reduced protein tyrosine phosphorylation in mutant B cells. Further analysis shows that Igalpha is phosphorylated and syk is recruited and becomes phosphorylated but that BLNK and PLCgamma phosphorylation are defective in mutant cells. Our data support the existence of a novel component coupling syk to downstream targets.
Subject(s)
DNA-Binding Proteins/physiology , Receptors, Antigen, B-Cell/physiology , Signal Transduction/immunology , Trans-Activators/physiology , Transcription Factors/physiology , Animals , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Calcium Signaling/immunology , Cell Lineage/genetics , Cell Lineage/immunology , Crosses, Genetic , DNA-Binding Proteins/genetics , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/physiology , Interferon Regulatory Factors , Mice , Mice, Knockout , Phosphorylation , Receptors, Antigen, B-Cell/genetics , Receptors, Antigen, B-Cell/immunology , Receptors, Antigen, B-Cell/metabolism , Trans-Activators/genetics , Transcription Factors/genetics , Tyrosine/metabolismABSTRACT
Spi-B is a hematopoietic-specific Ets family transcription factor closely related to PU.1. Previous gene targeting experiments have shown that PU.1 is essential for the production of both lymphocytes and monocytes. We have now generated mice with a null mutation at the Spi-B locus. Unlike PU.1 mutant mice, Spi-B-/- mice are viable, fertile and possess mature B and T lymphocytes. However, Spi-B-/- mice exhibit severe abnormalities in B cell function and selective T cell-dependent humoral immune responses. First, although Spi-B-/- splenic B cells respond normally to lipopolysaccharide stimulation in vitro, these B cells proliferate poorly and die in response to B cell receptor (surface IgM) cross-linking. Secondly, Spi-B-/- mice display abnormal T-dependent antigenic responses in vivo and produce low levels of antigen-specific IgG1, IgG2a and IgG2b after immunization. Finally, Spi-B-/- mice show a dramatic defect in germinal center formation and maintenance. In contrast to wild-type animals, germinal centers in Spi-B-/- mice are smaller and short-lived with significantly increased numbers of apoptotic B cells. Taken together, these results demonstrate that Spi-B is essential for antigen-dependent expansion of B cells, T-dependent immune responses and maturation of normal germinal centers in vivo.
Subject(s)
B-Lymphocytes/immunology , DNA-Binding Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Receptors, Antigen, B-Cell/immunology , Transcription Factors/metabolism , Animals , Bone Marrow/growth & development , DNA-Binding Proteins/genetics , Germinal Center/immunology , Immunoglobulin M/immunology , Lipopolysaccharides/immunology , Lymphocyte Activation/genetics , Lymphoid Tissue/growth & development , Mice , Mice, Mutant Strains , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-ets , T-Lymphocytes/immunology , Trans-Activators/metabolism , Transcription Factors/geneticsABSTRACT
We have identified a novel zinc-finger protein whose mRNA is expressed at high levels in the epidermal layer of the skin and in epithelial cells in the tongue, palate, esophagus, stomach, and colon of newborn mice. Expression in epithelial cells is first detected at the time of their differentiation during embryonic development. In addition, during early embryonic development there is expression in mesenchymal cells of the skeletal primordia and the metanephric kidney which is later down-regulated. The expression pattern suggests that the protein could be involved in terminal differentiation of several epithelial cell types and could also be involved in early differentiation of the skeleton and kidney. The carboxyl terminus of the protein contains three zinc fingers with a high degree of homology to erythroid krüppel-like factor and binds to DNA fragments containing CACCC motifs. The amino-terminal portion of the protein is proline and serine-rich and can function as a transcriptional activator. The chromosomal location of the gene was mapped using mouse interspecific backcrosses and was shown to localize to mouse chromosome 4 and to cosegregate with the thioredoxin gene.