Distinct contracted conformations of the Tcra/Tcrd locus during Tcra and Tcrd recombination

Abstract

Studies have suggested that antigen receptor loci adopt contracted conformations to promote long-distance interactions between gene segments during V(D)J recombination. The Tcra/Tcrd locus is unique because it undergoes highly divergent Tcrd and Tcra recombination programs in CD4(-)CD8(-) double negative (DN) and CD4(+)CD8(+) double positive (DP) thymocytes, respectively. Using three-dimensional fluorescence in situ hybridization, we asked whether these divergent recombination programs are supported by distinct conformational states of the Tcra/Tcrd locus. We found that the 3' portion of the locus is contracted in DN and DP thymocytes but not in B cells. Remarkably, the 5' portion of the locus is contracted in DN thymocytes but is decontracted in DP thymocytes. We propose that the fully contracted conformation in DN thymocytes allows Tcrd rearrangements involving V(delta) gene segments distributed over 1 Mb, whereas the unique 3'-contracted, 5'-decontracted conformation in DP thymocytes biases initial Tcra rearrangements to the most 3' of the available V(alpha) gene segments. This would maintain a large pool of distal 5' V(alpha) gene segments for subsequent rounds of recombination. Thus, distinct contracted conformations of the Tcra/Tcrd locus may facilitate a transition from a Tcrd to a Tcra mode of recombination during thymocyte development.

Figure 1.

Subregion-specific contraction of the Tcra/Tcrd locus during thymocyte development. (A) Organization of Tcra/Tcrd locus and relative positions of four BAC probes (A, B, C, and D). V segments are categorized as proximal, central, central duplication, or distal. Probe B (C57BL/6 origin) hybridizes discontinuously to the central and central duplication regions of the strain 129 Tcra/Tcrd locus. (B) 3D-FISH analysis of strain 129 Tcra/Tcrd locus conformation in splenic B cells and recombinase-deficient DN and DP thymocytes using probes A, B, and D. Each image represents a single z-plane with representative loci. Bars, 1 µm. (C) Scatter plots display pairwise distance measurements between the centers of probe hybridization compiled from use of the A–B–D, A–C–D, and A–B–C probe combinations. Data were accumulated from two to five independent experiments for each cell type (184–592 total alleles) with 78–126 alleles per experiment. Median values are indicated by horizontal lines. **, P < 0.01; ***, P < 0.001; ns, not statistically significant.

Figure 2.

Conformation of central V segments as defined by probe B hybridization. Representative examples of alleles with multiple foci detected by probe B in B cells and DP thymocytes. Tcra/Tcrd alleles were detected using a combination of probes A, B, and C. The top row of images shows probe B hybridization; the bottom row shows a combination of probes A and B. Probe C identified a single focus in each instance (not depicted). Similar results were observed in two independent experiments using the same probe combination. Bars, 1 µm.

Figure 3.

3D models of Tcra/Tcrd locus conformation. Median distances for the three cell types, determined from the entire dataset in Fig. 2, were used to generate 3D models that are aligned so that they share point A and the A–B axis. Two views are shown from different angles.