RESUMEN
To test the hypothesis that DNA polymerase ζ participates in Ig hypermutation, we generated two mouse models of Pol ζ function: a B cell-specific conditional knockout and a knock-in strain with a Pol ζ mutagenesis-enhancing mutation. Pol ζ-deficient B cells had a reduction in mutation frequency at Ig loci in the spleen and in Peyer's patches, whereas knock-in mice with a mutagenic Pol ζ displayed a marked increase in mutation frequency in Peyer's patches, revealing a pattern that was similar to mutations in yeast strains with a homologous mutation in the gene encoding the catalytic subunit of Pol ζ. Combined, these data are best explained by a direct role for DNA polymerase ζ in Ig hypermutation.
Asunto(s)
Hipermutación Somática de Inmunoglobulina/genética , Hipermutación Somática de Inmunoglobulina/inmunología , Animales , Linfocitos B/enzimología , Linfocitos B/inmunología , Linfocitos B/patología , ADN Polimerasa Dirigida por ADN/deficiencia , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/fisiología , Activación Enzimática/genética , Activación Enzimática/inmunología , Técnicas de Sustitución del Gen , Reordenamiento Génico de Cadena Pesada de Linfocito B , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos AnimalesRESUMEN
We recently identified a novel germinal center GTPase, SLIP-GC, that localizes to replication factories in B cells and that, when reduced, induces DNA breaks in lymphoma B cell lines in an activation-induced deaminase (AID)-dependent manner. Herein, we generated mice deficient in SLIP-GC and examined the impact of SLIP-GC deficiency in immunoglobulin hypermutation and class switch recombination, both AID-dependent mechanisms. SLIP-GC-deficient mice experienced a substantial increase in mutations at G:C base pairs at the region downstream of JH4 in the immunoglobulin heavy chain locus. This change was reflected in the overall mutation frequency, and it was associated with an increase in transitions from G:C base pairs, a hallmark of AID-mediated deamination during replication. In addition, G:C transitions at non-immunoglobulin loci also increased in these mice. Given the intracellular localization of SLIP-GC to sites of replicating DNA, these results suggest that SLIP-GC protects replicating DNA from AID-mediated deamination of cytosines in both strands.
Asunto(s)
Citidina Desaminasa/metabolismo , GTP Fosfohidrolasas/biosíntesis , GTP Fosfohidrolasas/genética , Inmunoglobulinas/genética , Hipermutación Somática de Inmunoglobulina/genética , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Citidina Desaminasa/genética , Citosina/química , Análisis Mutacional de ADN , Replicación del ADN , Genotipo , Centro Germinal/metabolismo , Cambio de Clase de Inmunoglobulina , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Genéticos , Ganglios Linfáticos Agregados/metabolismoAsunto(s)
Linfocitos B/metabolismo , Roturas del ADN de Doble Cadena , Reordenamiento Génico de Linfocito B/genética , Genes de Inmunoglobulinas/genética , Translocación Genética/genética , Animales , Linfocitos B/patología , Citidina Desaminasa/metabolismo , Reparación del ADN/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/metabolismo , Genes myc/genética , Cambio de Clase de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/genética , RatonesRESUMEN
The process of allelic exclusion ensures that each B cell expresses a B-cell receptor encoded by only one of its Ig heavy (IgH) and light (IgL) chain alleles. Although its precise mechanism is unknown, recruitment of the nonfunctional IgH allele to centromeric heterochromatin correlates with the establishment of allelic exclusion. Similarly, recruitment in activated splenic B cells correlates with cell division. In the latter, the recruited IgH allele was reported to be transcriptionally silent. However, it is not known whether monoallelic recruitment during establishment of allelic exclusion correlates with transcriptional silencing. To investigate this, we assessed the transcriptional status of both IgH alleles in single primary cells over the course of B-cell development, using RNA fluorescence in situ hybridization. Before allelic exclusion both alleles are transcribed. Thereafter, in pre-BII and subsequent developmental stages both functional and nonfunctional VDJ- and DJ-transcription is observed. Thus, after the establishment of IgH allelic exclusion, monoallelic recruitment to heterochromatin does not silence VDJ- or DJ-transcription, but serves another purpose.