Identification of E2A target genes in B lymphocyte development by using a gene tagging-based chromatin immunoprecipitation system.
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.
Greenbaum
Stephen
S
Department of Immunology, Duke University Medical Center, Box 3010, Durham, NC 27710, USA.
Zhuang
Yuan
Y
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D002843:Chromatin / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics* / Q000276:immunology; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000172:deficiency / Q000235:genetics* / Q000276:immunology; D014158:Transcription, Genetic
D051792:Basic Helix-Loop-Helix Proteins; D002843:Chromatin; D017931:DNA Primers; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C407909:TCF3 protein, human