Poor-Quality Vβ Recombination Signal Sequences and the DNA Damage Response ATM Kinase Collaborate to Establish TCRβ Gene Repertoire and Allelic Exclusion

Abstract

The monoallelic expression (allelic exclusion) of diverse lymphocyte Ag receptor genes enables specific immune responses. Allelic exclusion is achieved by asynchronous initiation of V(D)J recombination between alleles and protein encoded by successful rearrangement on the first allele signaling permanent inhibition of V rearrangement on the other allele. The ATM kinase that guides DNA repair and transiently suppresses V(D)J recombination also helps impose allelic exclusion through undetermined mechanisms. At the TCRβ locus, one Vβ gene segment (V31) rearranges only by inversion, whereas all other Vβ segments rearrange by deletion except for rare cases in which they rearrange through inversion following V31 rearrangement. The poor-quality recombination signal sequences (RSSs) of V31 and V2 help establish TCRβ gene repertoire and allelic exclusion by stochastically limiting initiation of Vβ rearrangements before TCRβ protein-signaled permanent silencing of Vβ recombination. We show in this study in mice that ATM functions with these RSSs and the weak V1 RSS to shape TCRβ gene repertoire by restricting their Vβ segments from initiating recombination and hindering aberrant nonfunctional Vβ recombination products, especially during inversional V31 rearrangements. We find that ATM collaborates with the V1 and V2 RSSs to help enforce allelic exclusion by facilitating competition between alleles for initiation and functional completion of rearrangements of these Vβ segments. Our data demonstrate that the fundamental genetic DNA elements that underlie inefficient Vβ recombination cooperate with ATM-mediated rapid DNA damage responses to help establish diversity and allelic exclusion of TCRβ genes.

Figure 1.. The V31 RSS and ATM cooperate to preserve functional V31 rearrangements.

(A) Schematic of the Tcrb locus. Vβ segments are open rectangles. Other gene segments and the β-enhancer (Eβ) are labeled. (B – E) Representative and quantified data of the frequency of V2⁺ or V31⁺ (B and D) or V2⁺V31⁺ (C and E) cells in indicated mice. (F) Quantification of total thymocytes in indicated mice. Data from four experiments, each with at least one mouse of each genotype. Two-way (C) or one-way (E and F) ANOVA with Tukey’s (C, E and F) multiple post-tests. ns = not significant, *p<0.05 **p<0.01 ***p<0.001, ****p<0.0001.

Figure 2.. The V31 RSS and ATM limit formation of nonproductive V31-5’Dβ RSS hybrid joins.

(A - C) Schematic representations of the resolution of recombination intermediates following escape of the 3’Dβ1 RSS from RAG post-cleavage complexes during attempted inversional V31^R rearrangement resulting in a HJ and an intra-locus deletion (A), deletional V2^R rearrangement resulting in a CJ and an assembled TCRβ gene (B), attempted inversional V2^R rearrangement resulting in a HJ and a deletion (C), or attempted deletional V2^R rearrangement resulting in two HJs and an inversion (D). (E) Quantification of indicated HJs in thymocytes of the indicated mice. Data are from three biological replicates. Two-way ANOVA with Tukey’s multiple comparisons test. **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3.. Poor-quality V1 and V2 RSSs and ATM cooperate to enforce TCRβ allelic exclusion.

(A - B) Representative (A) and quantified (B) data of V1⁺ cells in indicated mice. (C) Quantification of total thymocytes in indicated mice. (A - C) Data from five experiments, two with V1^R/+ and WT mice and three with V1^R/R and V1^R/Eb⁻ mice, where at least one mouse of each genotype included in each experiment. (D - G) Representative and quantified data of V1⁺ or V2⁺ (D and F) or V1⁺V2⁺ (E and G) cells in indicated genotypes of mice. (H) Quantification of total thymocytes in indicated mice. Data from four experiments, each with at least one mouse of each genotype. (B, C, F, G, and H) One-way ANOVA with Tukey’s multiple comparisons test. ns = not significant, **p<0.01, ****p<0.0001.

Figure 4.. Poor-quality Vβ RSSs and ATM function together to impose competition between alleles for Vβ recombination.

(A) Quantification of total thymocytes in indicated mice. One-way ANOVA with Tukey’s multiple comparisons test. ns = not significant. (B - E) Representative and quantified data of V1⁺ (B and D) or V2⁺ (D and E) cells in indicated mouse genotypes. (F and G) Quantification of indicated HJs in thymocytes of the indicated mice. (H and I) Representative (H) and quantified (I) data of V31⁺ cells in indicated mouse genotypes. (A – E, H and I) Data from three experiments, each with at least one mouse of each genotype. Unpaired parametric t tests with Welch’s correction. ***p<0.001, ****p<0.0001. (J) Quantification of indicated HJs in thymocytes of the indicated mice. (F, G, and J). Data are from three biological replicates Two-way ANOVA with Sidak’s multiple comparisons test. *p<0.05, ***p<0.001, ****p<0.0001