Initiation of allelic exclusion by stochastic interaction of Tcrb alleles with repressive nuclear compartments

Abstract

Studies of antigen-receptor loci have linked directed monoallelic association with pericentromeric heterochromatin to the initiation or maintenance of allelic exclusion. Here we provide evidence for a fundamentally different basis for T cell antigen receptor-beta (Tcrb) allelic exclusion. Using three-dimensional immunofluorescence in situ hybridization, we found that germline Tcrb alleles associated stochastically and at high frequency with the nuclear lamina or with pericentromeric heterochromatin in developing thymocytes and that such interactions inhibited variable-to-diversity-joining (V(beta)-to-D(beta)J(beta)) recombination before beta-selection. The introduction of an ectopic enhancer into Tcrb resulted in fewer such interactions and impaired allelic exclusion. We propose that initial V(beta)-to-D(beta)J(beta) recombination events are generally monoallelic in developing thymocytes because of frequent stochastic, rather than directed, interactions of Tcrb alleles with repressive nuclear compartments. Such interactions may be essential for Tcrb allelic exclusion.

Figure 1

Subnuclear localization of Tcrb alleles in Rag2^-/- pro-B cells, Rag2^-/- DN thymocytes, and sorted wild-type DP thymocytes. (a) Tcrb locus, including the relative positions of the BAC clones used to visualize its subnuclear localization. (b) 3D Immuno-FISH, showing both Tcrb alleles in representative nuclei of each of three cell types. Nuclei were hybridized with BAC RP23-75P5 to identify the distal end of the Tcrb locus (green) and a plasmid containing γ-satellite repeats to identify peri-centromeric heterochromatin (red), and were stained with an antibody specific for laminB1 to identify the nuclear lamina (blue). (c) Colocalization of Tcrb alleles with either γ-satellite repeats or laminB1. γ-satellite data, 604 pro-B, 432 DN and 862 DP alleles (two to three slides each); laminB1 data, 578 pro-B, 232 DN and 972 DP alleles (two to three slides each). *, P < 0.05.

Figure 2

Subnuclear localization of Tcra alleles in Rag2^-/- pro-B cells, Rag2^-/- DN thymocytes, and sorted wild-type DP thymocytes. (a) Tcra locus, including the position of the BAC clone used to visualize its subnuclear localization. (b) 3D Immuno-FISH, showing Tcra alleles in representative nuclei of each of the three cell types. Nuclei were hybridized with BAC RP23-304L21 to identify the Tcra locus (green) and a plasmid containing γ-satellite repeats to identify peri-centromeric heterochromatin (red), and were stained with an antibody specific for laminB1 to identify the nuclear lamina (blue). (c) Colocalization of Tcra alleles with either γ-satellite repeats or laminB1. γ-satellite data, 302 pro-B, 52 DN and 178 DP alleles (one slide each); laminB1 data, 302 pro-B, 52 DN and 178 DP alleles (one slide each). *, P < 0.05.

Figure 3

Colocalization of Tcrb, Tcra, and Actb alleles with γ-satellite or laminB1. Alleles colocalizing with γ-satellite alone, laminB1 alone, or simultaneously with γ-satellite and laminB1 were summed. Tcrb data, 424 pro-B, 232 DN and 390 DP alleles (one slide each); Tcra data, 302 pro-B, 52 DN and 178 DP alleles (one slide each); Actb data, 246 pro-B, 232 DN and 162 DP alleles (one slide each). *, P < 0.05.

Figure 4

Distribution of nuclei with zero, one, or two Tcrb alleles colocalized with γ-satellite or laminB1. (a) Colocalization with γ-satellite, in 289 pro-B, 116 DN and 486 DP nuclei (b) Colocalization with laminB1, in 302 pro-B, 116 DN and 431 DP nuclei. (c) Colocalization with either γ-satellite or laminB1, in 289 pro-B, 116 DN and 431 DP nuclei. Data were accumulated from two to three independently prepared slides per cell type.

Figure 5

Subnuclear localization of rearranged and unrearranged Tcrb alleles. (a) 3D Immuno-FISH, showing Tcrb alleles in DP nuclei using distal (75P5, green) and proximal (203H5, red) probes. Peri-centromeric heterochromatin (γ-satellite, blue, upper panels). Nuclear lamina (laminB1, blue, lower panels). (b) Colocalization of rearranged and unrearranged alleles with γ-satellite or laminB1 in DP nuclei. γ-satellite, 145 rearranged and 327 unrearranged alleles (one slide); laminB1, 179 rearranged and 403 unrearranged alleles (two slides). (c) Colocalization of rearranged and unrearranged alleles with γ-satellite or laminB1 in the subset of DP cells with nuclei having monoallelic Tcrb locus association and one rearranged Tcrb allele. γ-satellite, 145 nuclei (one slide); laminB1, 179 nuclei (two slides). (d) 3D Immuno-FISH, showing Tcrb alleles in nuclei of Lat^-/- thymocytes. Probe strategy was identical to (a). (e) Colocalization of rearranged and unrearranged alleles with γ-satellite or laminB1 in the subset of Lat^-/- cells with nuclei having monoallelic Tcrb locus association and one rearranged allele. γ-satellite, 161 nuclei (one slide); laminB1, 54 nuclei (one slide). (f) Colocalization of Tcrb alleles with γ-satellite or laminB1 in Rag2^-/- DN nuclei and in Lat^-/- DN nuclei containing two rearranged alleles. γ-satellite, 232 Rag2^-/- and 36 Lat^-/- alleles (two and one slides, respectively); laminB1, 232 Rag2^-/- and 220 Lat^-/- alleles (two slides each). *, P < 0.05.

Figure 6

Influence of E_α on subnuclear localization and allelic exclusion. (a) 3D Immuno-FISH, showing Tcra alleles in nuclei of E_α^-/- Rag2^-/- Tcrb transgenic thymocytes. Tcra (304L21, green), peri-centromeric heterochromatin (γ-satellite, red), nuclear lamina (laminB1, blue). (b) 3D Immuno-FISH, showing Tcrb alleles in nuclei of EαKI thymocytes. Distal Tcrb (75P5, green), proximal Tcrb (203H5, red), peri-centromeric heterochromatin (γ-satellite, blue, left panel), nuclear lamina (laminB1, blue, right panel). (c) Tcra colocalization with γ-satellite or laminB1 in wild-type DP and E_α^-/-Rag2^-/- Tcrb transgenic thymocytes. WT DP, 178 alleles; E_α^-/-Rag2^-/- Tcrb tg DP, 210 alleles (one slide each). (d) Colocalization of unrearranged Tcrb alleles with γ-satellite or laminB1 in wild-type and EαKI DP thymocytes. γ-satellite, 145 WT and 114 EαKI alleles (one slide each); laminB1, 179 WT and 48 EαKI alleles (one slide each). (e) Colocalization of unrearranged Tcrb alleles with γ-satellite or laminB1 in DN thymocytes of Rag2^-/- and EαKI Rag2^-/- mice. γ-satellite, 145 Rag2^-/- and 128 EαKI Rag2^-/- alleles (two slides each); laminB1, 316 Rag2^-/- and 128 EαKI Rag2^-/- alleles (two slides each). (f) Quantification of V_β-to-D_βJ_β recombination in wild-type and EαKI DP nuclei. WT, 527 nuclei; EαKI 129 nuclei (two slides each). *, P < 0.05.