RESUMO
DNA double-strand breaks (DSBs) activate a canonical DNA damage response, including highly conserved cell cycle checkpoint pathways that prevent cells with DSBs from progressing through the cell cycle. In developing B cells, pre-B cell receptor (pre-BCR) signals initiate immunoglobulin light (Igl) chain gene assembly, leading to RAG-mediated DNA DSBs. The pre-BCR also promotes cell cycle entry, which could cause aberrant DSB repair and genome instability in pre-B cells. Here, we show that RAG DSBs inhibit pre-BCR signals through the ATM- and NF-κB2-dependent induction of SPIC, a hematopoietic-specific transcriptional repressor. SPIC inhibits expression of the SYK tyrosine kinase and BLNK adaptor, resulting in suppression of pre-BCR signaling. This regulatory circuit prevents the pre-BCR from inducing additional Igl chain gene rearrangements and driving pre-B cells with RAG DSBs into cycle. We propose that pre-B cells toggle between pre-BCR signals and a RAG DSB-dependent checkpoint to maintain genome stability while iteratively assembling Igl chain genes.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Homeodomínio/metabolismo , Receptores de Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/imunologia , Células Precursoras de Linfócitos B/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Pontos de Checagem do Ciclo Celular/imunologia , Proliferação de Células , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Rearranjo Gênico de Cadeia Leve de Linfócito B , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Subunidade p52 de NF-kappa B/deficiência , Subunidade p52 de NF-kappa B/genética , Subunidade p52 de NF-kappa B/metabolismo , Células Precursoras de Linfócitos B/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/imunologia , Quinase Syk , Transativadores/metabolismo , Quinase Induzida por NF-kappaBRESUMO
Allelic exclusion is enforced through the ability of antigen receptor chains expressed from one allele to signal feedback inhibition of V-to-(D)J recombination on the other allele. To achieve allelic exclusion by such means, only one allele can initiate V-to-(D)J recombination within the time required to signal feedback inhibition. DNA double-strand breaks (DSBs) induced by the RAG endonuclease during V(D)J recombination activate the Ataxia Telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) kinases. We demonstrate that ATM enforces Igκ allelic exclusion, and that RAG DSBs induced during Igκ recombination in primary pre-B cells signal through ATM, but not DNA-PK, to suppress initiation of additional Igκ rearrangements. ATM promotes high-density histone H2AX phosphorylation to create binding sites for MDC1, which functions with H2AX to amplify a subset of ATM-dependent signals. However, neither H2AX nor MDC1 is required for ATM to enforce Igκ allelic exclusion and suppress Igκ rearrangements. Upon activation in response to RAG Igκ cleavage, ATM signals down-regulation of Gadd45α with concomitant repression of the Gadd45α targets Rag1 and Rag2. Our data indicate that ATM kinases activated by RAG DSBs during Igκ recombination transduce transient H2AX/MDC1-independent signals that suppress initiation of further Igκ rearrangements to control Igκ allelic exclusion.