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.

Locus folding mechanisms determine modes of antigen receptor gene assembly

“…Any inversional rearrangements between the reoriented RSS and its normal partner(s) must occur by diffusion-based synapsis, not by active RAG scanning. To determine whether Tcrb can employ diffusion-based synapsis for Trbv1 recombination, we first replaced its RSS with a more efficient version (3′Dβ1 RSS) in either the normal ( Wu et al, 2022 ) or inverted orientation (the V1 R or V1 Ri allele, respectively; Fig. 5 A ).…”

“…Wild-type (C57BL/6J) and Rag1 −/− (B6.129S7- Rag1 tm1Mom /J) mice were obtained from Jackson Laboratories. The V1 R/R mice were previously described ( Wu et al, 2022 ). We used the Easi-CRISPR method ( Quadros et al, 2017 ) of CRISPR/Cas9-mediated genomic editing in C57BL/6 zygotes to create mice with a scrambled Trbv1 CBE ( V1C Scr allele), a deletion of the Trbv1 promoter ( V1P KO allele), or an inverted 3′Dβ1RSS replacement of the Trbv1 RSS ( V1 Ri allele).…”

“…The mixture used to electroporate zygotes contained two different ctRNAs (4 μM each; two different crRNAs independently annealed to tracrRNA) and no ssODN. For the V1 Ri/Ri mice, we utilized the same crRNA as in a previous study to replace the Trbv1 RSS with the 3′Dβ1 RSS in its normal orientation ( V1 R ; Wu et al, 2022 ). This crRNA targets the sense strand within the spacer of the Trbv1 RSS.…”