Immature Lymphocytes Inhibit Rag1 and Rag2 Transcription and V(D)J Recombination in Response to DNA Double-Strand Breaks

Abstract

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.

V(D)J Recombination Exploits DNA Damage Responses to Promote Immunity

“…Collectively, these observations are consistent with RAG cleavage of one Igκ allele transiently blocking initiation of V(D)J recombination at both Igκ alleles via distinct ATM-dependent signaling pathways that repress Rag1 and Rag2 transcription (Figure 3). Additionally it has been shown that IR downregulates Rag1 and Rag2 transcripts in pro-B and pro-T cells and ATM-deficiency increases bi-allelic V recombination and allelic inclusion for Igh and Tcrb loci [47, 50]. These findings are consistent with a RAG DSB-induced feedback inhibition coordinating initiation of V(D)J recombination between alleles at Igh and Tcrb loci via ATM-dependent repression of RAG expression.…”

“…Thus, this suggested that RAG Igκ DSBs signal ATM-dependent loss of Gadd45a, leading to transcriptional repression of RAG1 expression. However, it was shown that constitutive expression of Gadd45a in primary pre-B cells does not antagonize DSB-induced repression of RAG1 expression [47]. Moreover, it was also found that IR downregulates Rag1 and Rag2 transcripts via mechanisms dependent on Nemo [47], consistent with a role for repression of Rag1 and Rag2 transcription by the p50 NFκB protein [48].…”

“…However, it was shown that constitutive expression of Gadd45a in primary pre-B cells does not antagonize DSB-induced repression of RAG1 expression [47]. Moreover, it was also found that IR downregulates Rag1 and Rag2 transcripts via mechanisms dependent on Nemo [47], consistent with a role for repression of Rag1 and Rag2 transcription by the p50 NFκB protein [48]. Furthermore, another study showed that DSBs induced by IR or genotoxic drugs downregulate Rag1 and Rag2 transcripts concomitant with ATM-dependent loss of FOXO1 transcription factor binding to the Rag1/Rag2 locus Erag transcriptional enhancer [49].…”

“…Furthermore, another study showed that DSBs induced by IR or genotoxic drugs downregulate Rag1 and Rag2 transcripts concomitant with ATM-dependent loss of FOXO1 transcription factor binding to the Rag1/Rag2 locus Erag transcriptional enhancer [49]. Notably, the loss of RAG1 expression in response to DSBs induced by genotoxic drugs correlates with inhibition of V(D)J recombination within retroviral substrates and at Igκ loci [47, 49]. Collectively, these observations are consistent with RAG cleavage of one Igκ allele transiently blocking initiation of V(D)J recombination at both Igκ alleles via distinct ATM-dependent signaling pathways that repress Rag1 and Rag2 transcription (Figure 3).…”

“…In the latter context, it was demonstrated that RAG Igκ DSBs trigger G1 arrest by inhibiting pre-BCR proliferative signals and by transcriptional activation of the Pim2 kinase, which halts cell cycle through undefined mechanisms [27, 51]. Finally, the findings of that DSBs caused by genotoxic agents downregulate RAG expression and suppress initiation of V(D)J recombination imply that this lymphocyte-specific DSB response is important to maintain genomic integrity during AgR gene assembly [47, 49]. …”