T cell receptor (TCR) α/δ locus enhancer identity and position are critical for the assembly of TCR δ and α variable region genes

T cell receptor (TCR) delta and alpha variable region genes are assembled from germ-line gene segments located in a single chromosomal locus in which TCRdelta segments are situated between TCRalpha segments. The TCRa enhancer (Ealpha) located at the 3' end of the TCRalpha/delta locus functions over a long chromosomal distance to promote TCRa rearrangement and maximal TCRdelta expression; whereas the TCRdelta enhancer (Edelta) is located among the TCRdelta segments and functions with additional element(s) to mediate TCRdelta rearrangement. We used gene-targeted mutation to evaluate whether the identity of Ealpha and the position of Edelta are critical for the developmental stage-specific assembly of TCR 5 and a variable region genes. Specific replacement of Ealpha with Edelta, the core Ealpha element (EalphaC), or the Ig heavy chain intronic enhancer (iEmu), all of which promote accessibility in the context of transgenic V(D)J recombination substrates, did not promote a significant level of TCRa rearrangement beyond that observed in the absence of Ealpha. Therefore, the identity and full complement of Ealpha-binding sites are critical for promoting accessibility within the TCRa locus. In the absence of the endogenous Edelta element, specific replacement of Ealpha with Edelta also did not promote TCRdelta rearrangement. However, deletion of intervening TCRalpha/delta locus sequences to restore the inserted Edelta to its normal chromosomal position relative to 5' sequences rescued TCRdelta rearrangement. Therefore, unlike Ealpha, Edelta lacks ability to function over the large intervening TCRalpha locus and/or Edelta function requires proximity to additional upstream element(s) to promote TCRdelta accessibility.