Gene-Targeted Deletion and Replacement Mutations of the T-Cell Receptor β -Chain Enhancer: The Role of Enhancer Elements in Controlling V(D)J Recombination Accessibility

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 93; no. 15; pp. 7871 - 7876
• Main Authors: Bories, Jean-Christophe, Demengeot, Jocelyne, Davidson, Laurie, Alt, Frederick W.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 23.07.1996; National Acad Sciences; National Academy of Sciences
• Subjects: Alleles; Animals; B lymphocytes; B-Lymphocytes - immunology; Base Sequence; Blastocyst; Cells; Cells, Cultured; Chimera; Complementary DNA; DNA; DNA Nucleotidyltransferases - metabolism; DNA Primers; DNA probes; DNA-Binding Proteins; Embryos; Enhancer Elements, Genetic; Gene Deletion; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; Genes; Genetic Complementation Test; Genetic loci; Genomic Library; Germ cells; Immunity (Disease); Immunoglobulin Heavy Chains - genetics; Immunoglobulin Joining Region - biosynthesis; Immunoglobulin Joining Region - genetics; Immunoglobulin Variable Region - biosynthesis; Immunoglobulin Variable Region - genetics; Mice; Molecular Sequence Data; Mutation; Polymerase Chain Reaction; Proteins - genetics; Receptors, Antigen, T-Cell, alpha-beta - biosynthesis; Receptors, Antigen, T-Cell, alpha-beta - genetics; Restriction Mapping; Sequence Deletion; Spleen - immunology; Stem Cells; T lymphocytes; Thymocytes; Transcription, Genetic; VDJ Recombinases
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.15.7871

To assess the role of transcriptional enhancers in regulating accessibility of the T-cell receptor β -chain (TCRβ ) locus, we generated embryonic stem cell lines in which a single allelic copy of the endogenous TCRβ enhancer (Eβ ) was either deleted or replaced with the immunoglobulin heavy-chain intronic enhancer. We assayed the effects of these mutations on activation of the TCRβ locus in normal T- and B-lineage cells by RAG-2 (recombination-activating gene 2)-deficient blastocyst complementation. We found that Eβ is required for rearrangement and germ-line transcription of the TCRβ locus in T-lineage cells. In the absence of Eβ , the heavy-chain intronic enhancer partially supported joining region β -chain rearrangement in T- but not in B-lineage cells. However, ability of the heavy-chain intronic enhancer to induce rearrangements was blocked by linkage to an expressed neomycin-resistance gene (neor). These results demonstrate a critical role for Eβ in promoting accessibility of the TCRβ locus and suggest that additional negative elements may cooperate to further modulate this process.