Gene-targeted deletion and replacement mutations of the T-cell receptor beta-chain enhancer: the role of enhancer elements in controlling V(D)J recombination accessibility.
To assess the role of transcriptional enhancers in regulating accessibility of the T-cell receptor beta-chain (TCRbeta) locus, we generated embryonic stem cell lines in which a single allelic copy of the endogenous TCRbeta enhancer (Ebeta) was either deleted or replaced with the immunoglobulin heavy-chain intronic enhancer. We assayed the effects of these mutations on activation of the TCRbeta locus in normal T- and B-lineage cells by RAG-2 (recombination-activating gene 2)-deficient blastocyst complementation. We found that Ebeta is required for rearrangement and germ-line transcription of the TCRbeta locus in T-lineage cells. In the absence of Ebeta, the heavy-chain intronic enhancer partially supported joining region beta-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 (neo(r)). These results demonstrate a critical role for Ebeta in promoting accessibility of the TCRbeta locus and suggest that additional negative elements may cooperate to further modulate this process.
Bories
J C
JC
Unité 93, Institut National de la Santé et de la Recherche Médicale,Hôpital Saint-Louis, Paris, France.
Demengeot
J
J
Davidson
L
L
Alt
F W
FW
D000483:Alleles; D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D001755:Blastocyst; D002478:Cells, Cultured; D002678:Chimera; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D017931:DNA Primers; D004268:DNA-Binding Proteins; D004742:Enhancer Elements, Genetic; D017353:Gene Deletion; D015333:Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; D005816:Genetic Complementation Test; D015698:Genomic Library; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007133:Immunoglobulin Joining Region / Q000096:biosynthesis / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000096:biosynthesis / Q000235:genetics*; D015183:Restriction Mapping; D017384:Sequence Deletion; D013154:Spleen / Q000276:immunology; D013234:Stem Cells; D014158:Transcription, Genetic; D045567:VDJ Recombinases
D017931:DNA Primers; D004268:DNA-Binding Proteins; D007143:Immunoglobulin Heavy Chains; D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D011506:Proteins; C499308:Rag2 protein, mouse; D016693:Receptors, Antigen, T-Cell, alpha-beta; C064655:V(D)J recombination activating protein 2; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases