The assembly of immunoglobulin genes occurs in ordered waves during B cell development. The heavy chain (Igh) locus generally recombines first, and each allele has at most one chance to undergo a productive rearrangement. Subsequently, at the κ (Igk) locus, individual alleles can undergo sequential rounds of rearrangement, permitting different light-heavy chain combinations to be tested until a functional, nonself-reactive immunoglobulin is produced. The two waves of recombination are separated by a checkpoint governed by the pre–B cell receptor (pre–BCR), which enforces allelic exclusion at the Igh locus, triggers proliferation, and promotes Igk rearrangement. This raises the question: how do we mitigate the genomic damage that might occur if DNA cleavage and cell cycle entry were initiated simultaneously? In this issue, Bednarski et al. suggest a solution: an unexpected mechanism by which RAG-induced DNA double-strand breaks (DSBs) suppress pre–BCR signaling.
The authors began by identifying genes that undergo RAG-dependent changes in expression as progenitors progress from the large pre–B stage to the small pre–B stage, at which Igk rearrangement begins. Of particular interest were the genes encoding RELB and p100, components of the transcription factor NF-κB2. Because RELB and p100 are induced by DSBs through the activation of ataxia-telangiectasia mutated (ATM), they seemed well positioned to act as a bridge between RAG and downstream transcriptional targets.
Indeed, many of the genes regulated by RAG-dependent DSBs were targets of NF-κB2. One of these targets encodes the transcriptional repressor SPIC, which had been implicated as a negative regulator of B cell development. Bednarski et al. were able to situate RAG activity at the apex of a signaling cascade that induces SPIC through sequential activation of ATM and NF-κB2. SPIC was found to close the circle, inhibiting expression of the pre–BCR signal mediators SYK and BLNK by antagonizing PU.1 at their corresponding genes. The resulting extinction of pre–BCR signaling enforces cell cycle arrest in G1 while suppressing κ rearrangement.
The ability of RAG-induced DSBs to suppress pre–BCR signaling may explain how κ rearrangement is punctuated in pre–B cells: (1) an initial round of κ rearrangement in small pre–B cells generates DSBs; (2) DSBs, acting through ATM, induce SPIC and turn down pre–BCR signaling, inhibiting κ rearrangement and enforcing G1 arrest; and (3) repair of the first wave of DSBs extinguishes ATM signaling and restores pre–BCR signaling, initiating another round of κ rearrangement. The model, which is attractive in its economy, may inform development of therapies that interrupt pre–BCR signaling in malignancies of pre–B cell origin, particularly acute lymphoblastic leukemias.
Would interruption of the linkage between RAG DSBs and pre–BCR signaling promote genomic instability? Other mechanisms, notably activation of p53 and destruction of RAG-2 at the G1-to-S transition, are in place to segregate DSBs from S phase, but these may not be sufficiently genoprotective in the face of persistent pre–BCR signaling. In this regard, it will be interesting to assess genomic instability in SPIC-null mice, particularly in a p53-deficicient setting.