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

Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7871-6. doi: 10.1073/pnas.93.15.7871.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alleles
  • Animals
  • B-Lymphocytes / immunology
  • Base Sequence
  • Blastocyst
  • Cells, Cultured
  • Chimera
  • DNA Nucleotidyltransferases / metabolism
  • DNA Primers
  • DNA-Binding Proteins*
  • Enhancer Elements, Genetic*
  • Gene Deletion*
  • Gene Rearrangement, beta-Chain T-Cell Antigen Receptor*
  • Genetic Complementation Test
  • Genomic Library
  • Immunoglobulin Heavy Chains / genetics
  • Immunoglobulin Joining Region / biosynthesis
  • Immunoglobulin Joining Region / genetics
  • Immunoglobulin Variable Region / biosynthesis
  • Immunoglobulin Variable Region / genetics
  • Mice
  • Molecular Sequence Data
  • 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
  • Transcription, Genetic
  • VDJ Recombinases

Substances

  • DNA Primers
  • DNA-Binding Proteins
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Joining Region
  • Immunoglobulin Variable Region
  • Proteins
  • Rag2 protein, mouse
  • Receptors, Antigen, T-Cell, alpha-beta
  • V(D)J recombination activating protein 2
  • DNA Nucleotidyltransferases
  • VDJ Recombinases