Developmental regulation of VDJ recombination by the core fragment of the T cell receptor alpha enhancer

Abstract

The role of T cell receptor alpha enhancer (E alpha) cis-acting elements in the developmental regulation of VDJ recombination at the TCR alpha/delta locus was examined in transgenic mice containing variants of a minilocus VDJ recombination substrate. We demonstrate that the 116-bp T alpha 1,2 core enhancer fragment of the 1.4-kb E alpha is sufficient to activate the enhancer-dependent step of minilocus rearrangement, and that within T alpha 1,2, intact binding sites for TCF/LEF and Ets family transcription factors are essential. Although minilocus rearrangement under the control of the 1.4-kb E alpha initiates at fetal day 16.5 and is strictly limited to alpha beta T cells, we find that rearrangement under the control of T alpha 1,2 initiates slightly earlier during ontogeny and occurs in both gamma delta and alpha beta T cells. We conclude that the core fragment of E alpha can establish accessibility to the recombinase in developing thymocytes in vivo in a fashion that is dependent on the binding of TCF/LEF and Ets family transcription factors, but that these and other factors that bind to the E alpha core cannot account for the precise developmental onset of accessibility that is provided by the intact E alpha. Rather, our data suggests a critical role for factors that bind E alpha outside of the core T alpha 1,2 region in establishing the precise developmental onset of TCR alpha rearrangement in vivo.

Figure 6

Tα1,2 minilocus rearrangement in αβ and γδ thymocytes. Genomic DNA templates from sorted αβ and γδ thymocytes and total unfractionated thymocytes (Tot.) of Tα1,2 line T2, T5, and T7 mice (4 wk old), and a no DNA control (−) were amplified by PCR and probed as in Fig. 2.

Figure 2

PCR analysis of Tα1,2 and Tα1,2mTCF minilocus rearrangement. Genomic DNA templates from unfractionated thymocytes of Tα1,2 mice from lines T2, T3, T5, and T7, and of Tα1,2mTCF mice from lines JI, JJ, JK, JL, and JM (all 4 wk old) were amplifed by PCR using the indicated primers. Southern blots were probed with radiolabeled C_δ, V_δ1, or V_δ2 DNA fragments. The positions of 1.2-kb VD and 0.3-kb VDJ rearrangement products are indicated.

Figure 1

Human TCR-δ gene minilocus. (A) Diagram of the three Tα1,2-containing minilocus constructs. Solid boxes, exons, open boxes, protein binding sites. Wild-type and mutant Tα2 sequences are shown. (B) PCR products generated from V_δ1 rearrangements are depicted along with the V_δ1 and J_δ1 primers (arrows) used. Similar products are generated using V_δ2 and J_δ1 primers. Specific PCR products are not generated from unrearranged templates because of the large distances between primers. The primers do not amplify products from the endogenous murine TCR-δ locus.

Figure 3

PCR analysis of E_α, Tα1,2, and Tα1,2mEts minilocus rearrangement. Genomic DNA templates from unfractionated thymocytes of an E_α mouse from line J, of Tα1,2 mice from lines T2 and T5, and of Tα1,2mEts mice from lines JN, JR, and JO (all 4 wk old) were amplified by PCR and probed as in Fig. 2.

Figure 4

Analysis of minilocus rearrangement by genomic Southern blot. PstI plus EcoRI-digested tail and thymus genomic DNA samples from Eα line L, Tα1,2 line T2, Tα1,2mTCF line JI, and Tα1,2mEts line JO mice (all 4 wk old) were analyzed by Southern blot using a radiolabeled 1.0-kb V_δ1 genomic PstI fragment. Positions of the expected 1.0-kb germline, 0.9-kb V_δ1-D_δ3, and 3.2-kb V_δ1-D_δ3-J_δ1 rearranged fragments are indicated.

Figure 5

Time course of Tα1,2 minilocus rearrangement during fetal ontogeny. Genomic DNA samples from Tα1,2 line T2 thymi isolated on days 14.5–17.5 of gestation, as well as from a postnatal (PN) T2 mouse (4 wk old), were amplified by PCR and probed as in Fig. 2.