RESUMO
Somatic hypermutation (SHM) and class-switch recombination (CSR) of the Ig gene require both the transcription of the locus and the expression of activation-induced cytidine deaminase (AID). During CSR, AID decreases the amount of topoisomerase I (Top1); this decrease alters the DNA structure and induces cleavage in the S region. Similarly, Top1 is involved in transcription-associated mutation at dinucleotide repeats in yeast and in triplet-repeat contraction in mammals. Here, we report that the AID-induced decrease in Top1 is critical for SHM. Top1 knockdown or haploinsufficiency enhanced SHM, whereas Top1 overexpression down-regulated it. A specific Top1 inhibitor, camptothecin, suppressed SHM, indicating that Top1's activity is required for DNA cleavage. Nonetheless, suppression of transcription abolished SHM, even in cells with Top1 knockdown, suggesting that transcription is critical. These results are consistent with a model proposed for CSR and triplet instability, in which transcription-induced non-B structure formation is enhanced by Top1 reduction and provides the target for irreversible cleavage by Top1. We speculate that the mechanism for transcription-coupled genome instability was adopted to generate immune diversity when AID evolved.
Assuntos
Citidina Desaminase/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Switching de Imunoglobulina/fisiologia , Modelos Biológicos , Hipermutação Somática de Imunoglobulina/fisiologia , Animais , Western Blotting , Camptotecina , Linhagem Celular Tumoral , Clonagem Molecular , DNA Topoisomerases Tipo I/genética , Técnicas de Silenciamento de Genes , Vetores Genéticos/genética , Proteínas de Fluorescência Verde , Haploinsuficiência , Humanos , Camundongos , Camundongos KnockoutRESUMO
Activation-induced cytidine deaminase (AID) is shown to be essential and sufficient to induce two genetic alterations in the Ig loci: class switch recombination (CSR) and somatic hypermutation (SHM). However, it is still unknown how a single-molecule AID differentially regulates CSR and SHM. Here we identified Spt6 as an AID-interacting protein by yeast two-hybrid screening and immunoprecipitation followed by mass spectrometry. Knockdown of Spt6 resulted in severe reduction of CSR in both the endogenous Ig locus in B cells and an artificial substrate in fibroblast cells. Conversely, knockdown of Spt6 did not reduce but slightly enhanced SHM in an artificial substrate in B cells, indicating that Spt6 is required for AID to induce CSR but not SHM. These results suggest that Spt6 is involved in differential regulation of CSR and SHM by AID.
Assuntos
Switching de Imunoglobulina , Hipermutação Somática de Imunoglobulina , Fatores de Transcrição/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sequência de Bases , Linhagem Celular , Citidina Desaminase/química , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Primers do DNA/genética , Técnicas de Silenciamento de Genes , Histonas/metabolismo , Humanos , Camundongos , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
To initiate class switch recombination (CSR) activation-induced cytidine deaminase (AID) induces staggered nick cleavage in the S region, which lies 5' to each Ig constant region gene and is rich in palindromic sequences. Topoisomerase 1 (Top1) controls the supercoiling of DNA by nicking, rotating, and religating one strand of DNA. Curiously, Top1 reduction or AID overexpression causes the genomic instability. Here, we report that the inactivation of Top1 by its specific inhibitor camptothecin drastically blocked both the S region cleavage and CSR, indicating that Top1 is responsible for the S region cleavage in CSR. Surprisingly, AID expression suppressed Top1 mRNA translation and reduced its protein level. In addition, the decrease in the Top1 protein by RNA-mediated knockdown augmented the AID-dependent S region cleavage, as well as CSR. Furthermore, Top1 reduction altered DNA structure of the Smu region. Taken together, AID-induced Top1 reduction alters S region DNA structure probably to non-B form, on which Top1 can introduce nicks but cannot religate, resulting in S region cleavage.
