Mammalian cells have evolved a common DNA damage response (DDR) that sustains cellular function, maintains genomic integrity, and suppresses malignant transformation. In pre-B cells, DNA double strand breaks (DSBs) induced at Igκ loci by the Rag1/Rag2 (RAG) endonuclease engage this DDR to modulate transcription of genes that regulate lymphocyte-specific processes. We previously reported that RAG DSBs induced at one Igκ allele signal through the ATM kinase to feedback inhibit RAG expression and RAG cleavage of the other Igκ allele. Here, we show that DSBs induced by ionizing radiation, etoposide, or bleomycin suppress Rag1 and Rag2 mRNA levels in primary pre-B cells, pro-B cells, and pro-T cells, indicating that inhibition of Rag1 and Rag2 expression is a prevalent DSB response among immature lymphocytes. DSBs induced in pre-B cells signal rapid transcriptional repression of Rag1 and Rag2, causing downregulation of both Rag1 and Rag2 mRNA but only Rag1 protein. This transcriptional inhibition requires the ATM kinase and the NF-κB essential modulator protein, implicating a role for ATM-mediated activation of canonical NF-κB transcription factors. Finally, we demonstrate that DSBs induced in pre-B cells by etoposide or bleomycin inhibit recombination of Igκ loci and a chromosomally integrated substrate. Our data indicate that immature lymphocytes exploit a common DDR signaling pathway to limit DSBs at multiple genomic locations within developmental stages wherein monoallelic antigen receptor locus recombination is enforced. We discuss the implications of our findings for mechanisms that orchestrate the differentiation of mono-specific lymphocytes while suppressing oncogenic antigen receptor locus translocations.

Coordination of V rearrangements between loci on homologous chromosomes is critical for Ig and TCR allelic exclusion. The Ataxia Telangietasia mutated (ATM) protein kinase promotes DNA repair and activates checkpoints to suppress aberrant Ig and TCR rearrangements. In response to RAG cleavage of Igκ loci, ATM inhibits RAG expression and suppresses further Vκ-to-Jκ rearrangements to enforce Igκ allelic exclusion. Since V recombination between alleles is more strictly regulated for TCRβ and IgH loci, we evaluated the ability of ATM to restrict bi-allelic expression and V-to-DJ recombination of TCRβ and IgH genes. We detected greater frequencies of lymphocytes with bi-allelic expression or aberrant V-to-DJ rearrangement of TCRβ or IgH loci in mice lacking ATM. A pre-assembled DJβ complex that decreases the number of TCRβ rearrangements needed for a productive TCRβ gene further increased frequencies of ATM-deficient cells with bi-allelic TCRβ expression. IgH and TCRβ proteins drive proliferation of pro-lymphocytes through Cyclin D3, which also inhibits VH transcription. We show that inactivation of Cyclin D3 leads to increased frequencies of lymphocytes with bi-allelic expression of IgH or TCRβ genes. We also show that Cyclin D3 inactivation cooperates with ATM deficiency to increase the frequencies of cells with bi-allelic TCRβ or IgH expression, while decreasing the frequency of ATM-deficient lymphocytes with aberrant V-to-DJ recombination. Our data demonstrate that core components of the DNA damage response and cell cycle machinery cooperate to help enforce IgH and TCRβ allelic exclusion, and indicate that control of V-to-DJ rearrangements between alleles is important to maintain genomic stability.