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

Abstract

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

Figure 1

Eδ promotes rearrangement of a TCRβ minilocus. (A) Schematic representation of TCRβ^PF, Eδ, and its position in Eδ⁺TCRβ^PF. The Vβ14, Dβ1, Jβ1.1, and Jβ1.2 segments and the locations of the BglII (Bg) and BamHI (B) sites and the V, P, and C oligonucleotides used for analysis are shown. (B) Southern blot analysis of thymocyte DNA from Eδ⁺ (Eδ⁺14.2 and 19.1) and Eδ⁻ (Eδ⁻69.1 and 9.2) TCRβ^PF and nontransfected (E) and transfected (ET) ES cells using probe B. The positions of molecular weight markers and bands generated by DJ and VDJ rearrangements are indicated. (C) Southern blotting of VDJ rearrangements in thymocyte DNA from mice containing Eδ⁻ (Eδ⁻ 9.2), Eδ⁺ (Eδ⁺19.1), or iEμ⁺ (iEμ⁺1.163) amplified by PCR with V and C primers and probed with the P oligonucleotide. Threefold serial dilutions of template DNA are shown. RAG2 control PCRs are also shown.

Figure 2

Schematic of F₁ Eα/EδR ES cells and characterization of F₁ Eα/EδR thymocytes. (A) Schematic representation of the TCRα/δ loci in F₁ Eα/EδR cells. (B) FACS analysis of thymocytes isolated from F₁ Eα/EδR and wild-type mice with anti-CD4 and anti-CD8 antibodies. The numbers of thymocytes from representative mice are indicated.

Figure 3

Defective αβ T cell development in EδR/EδR and EαCR/EαCR mice. (A) FACS analysis of thymocytes isolated from EδR/EδR, EαCR/EαCR, EαΔ/EαΔ, and wild-type mice with anti-CD4 and anti-CD8 antibodies. The numbers of thymocytes from representative mice are indicated. (B) FACS analysis of peripheral lymphocytes isolated from the lymph nodes of the same mice with anti-Vα2 and anti-TCRβ antibodies. The percentage of the overall number of cells expressing Vα2⁺ αβ TCR and αβ TCR of other Vαs are indicated.