ABSTRACT
The transcription factors encoded by the E2A gene have been shown to play essential roles in the initiation and progression of lymphocyte development. However, there is still a lack of comprehensive understanding of E2A downstream genes in B-cell development. We previously developed a gene tagging-based chromatin immunoprecipitation (ChIP) system to directly evaluate E2A target genes in B-cell development. Here, we have improved this ChIP strategy and used it in conjunction with microarray analysis on E2A-deficient pre-B-cell lines to determine E2A target genes in lymphocyte development. Both microarray data and ChIP studies confirmed that E2A directly controls IgH gene expression. The microarray assay also revealed genes that were significantly up-regulated after E2A disruption. ChIP analysis showed that E2A was most likely to be directly involved in repression of some of these target genes such as Nfil3 and FGFR2. An inducible E2A reconstitution system further demonstrated that E2A-mediated repression of Nfil3 and FGFR2 was reversible. Collectively, these findings indicate that E2A is a positive regulator for one set of genes and a negative regulator for another set of genes in developing B lymphocytes.
Subject(s)
B-Lymphocytes/metabolism , DNA-Binding Proteins/physiology , Gene Expression Regulation , Transcription Factors/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors , Cell Differentiation , Cell Line , Cell Line, Transformed , Cell Line, Tumor , Cell Lineage , Cell Proliferation , Chromatin/metabolism , DNA/chemistry , DNA-Binding Proteins/metabolism , Flow Cytometry , Gene Deletion , Green Fluorescent Proteins/metabolism , Humans , Immunoglobulin Heavy Chains/chemistry , Immunoprecipitation , Mice , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/metabolismABSTRACT
Although expression of the RAG1 and RAG2 genes is essential for lymphocyte development, the mechanisms responsible for the lymphoid- and developmental stage-specific regulation of these genes are poorly understood. We have identified a novel, evolutionarily conserved transcriptional enhancer in the RAG locus, called Erag, which was essential for the expression of a chromosomal reporter gene driven by either RAG promoter. Targeted deletion of Erag in the mouse germline results in a partial block in B cell development associated with deficient V(D)J recombination, whereas T cell development appears unaffected. We found that E2A transcription factors bind to Erag in vivo and can transactivate Erag-dependent reporter constructs in cotransfected cell lines. These findings lead us to conclude that RAG transcription is regulated by distinct elements in developing B and T cells and that Erag is required for optimal levels of RAG expression in early B cell precursors but not in T cells.
Subject(s)
B-Lymphocytes/metabolism , DNA-Binding Proteins/genetics , Enhancer Elements, Genetic/physiology , Gene Expression Regulation , Genes, RAG-1 , Animals , B-Lymphocytes/cytology , Base Sequence , Basic Helix-Loop-Helix Transcription Factors , Cell Differentiation , Cell Line , DNA Nucleotidyltransferases/physiology , DNA-Binding Proteins/metabolism , Humans , Mice , Molecular Sequence Data , Nuclear Proteins , Promoter Regions, Genetic , Receptors, Antigen, B-Cell/physiology , Recombinases , Transcription Factors/metabolism , TransfectionABSTRACT
The transcription factors encoded by the E2A gene are known to be essential for B lymphocyte development, and ectopic expression or gene inactivation studies have revealed several potential lineage-specific E2A target genes. However, it remains unknown whether these target genes are directly regulated by E2A at the transcriptional level. We therefore generated mice carrying an affinity-tagged E2A knock-in allele to provide a system for the direct elucidation of E2A target genes based on E2A binding to target regulatory regions. Abelson-transformed pre-B cell lines derived from these mice were used in chromatin immunoprecipitation experiments to identify regulatory sequences bound by E2A in the context of an early B lymphocyte environment. Significant E2A binding was detected at the promoters and enhancers of several essential B-lineage genes, including the Igkappa intronic and 3' enhancers, lambda5 and VpreB surrogate light chain promoters, the EBF locus promoter region, and the mb-1 (Igalpha) promoter. Low levels of E2A binding were observed at several other lymphoid-restricted regulatory regions including the Ig heavy chain (IgH) intronic enhancer, the IgH 3' enhancers hs3b/hs4, the RAG-2 enhancer, and the 5' regions of the B29 and TdT loci. An E2A target gene, the predicted butyrophilin-like gene NG9 (BTL-II), was also identified by using a chromatin immunoprecipitation-based cloning strategy. In summary, our studies have provided evidence that E2A is directly involved in the transcriptional regulation of a number of early B-lineage genes.
Subject(s)
B-Lymphocytes/immunology , Chromatin/genetics , DNA-Binding Proteins/genetics , Transcription Factors/genetics , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors , Chromatin/immunology , Cloning, Molecular , DNA Primers , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/immunology , Humans , Mice , Mice, Knockout , Polymerase Chain Reaction , Promoter Regions, Genetic , Recombinant Fusion Proteins/immunology , Regulatory Sequences, Nucleic Acid , Transcription Factors/deficiency , Transcription Factors/immunology , Transcription, GeneticABSTRACT
Lymphocytes develop from hematopoietic stem cells through a series of highly regulated differentiation events in the bone marrow and thymus. A number of transcription factors are known to collaborate in controlling the timing and specificity of gene expression required for these developmental processes to occur. The basic helix-loop-helix (bHLH) proteins encoded by the E2A gene have been shown to play particularly important roles in the initiation and progression of lymphocyte differentiation. Gene targeting experiments in mice have demonstrated a requirement for E2A proteins at the onset of B lymphocyte development. More recent studies have broadened our view on the function of E2A proteins at multiple stages of lymphopoiesis and in the regulation of lymphoid-specific gene expression. Here we review the mammalian E2A proteins and the accumulated evidence demonstrating central roles for E2A throughout early B and T lymphocyte development. We also speculate on the direction of future research on the mechanisms underlying the lineage and stage-specific functions of E2A in lymphopoiesis.
