RESUMEN
Somatic hypermutation (SHM) drives the genetic diversity of Ig genes in activated B cells and supports the generation of Abs with increased affinity for Ag. SHM is targeted to Ig genes by their enhancers (diversification activators [DIVACs]), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase II (Pol II) and Pol II occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol II or production of full-length transcripts, indicating accumulation of stalled Pol II. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of ssDNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol II stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.
Asunto(s)
Proteínas Aviares/metabolismo , Subgrupos de Linfocitos B/inmunología , Linfoma de Burkitt/inmunología , Elementos de Facilitación Genéticos/genética , Inmunoglobulinas/metabolismo , ARN Polimerasa II/metabolismo , Animales , Diversidad de Anticuerpos , Proteínas Aviares/genética , Linfoma de Burkitt/genética , Pollos , Citidina Desaminasa/genética , Humanos , Inmunoglobulinas/genética , Activación de Linfocitos , Mutagénesis Sitio-Dirigida , Mutación/genética , ARN Polimerasa II/genética , Hipermutación Somática de Inmunoglobulina , Transcripción GenéticaRESUMEN
Somatic hypermutation (SH) generates point mutations within rearranged immunoglobulin (Ig) genes of activated B cells, providing genetic diversity for the affinity maturation of antibodies. SH requires the activation-induced cytidine deaminase (AID) protein and transcription of the mutation target sequence, but how the Ig gene specificity of mutations is achieved has remained elusive. We show here using a sensitive and carefully controlled assay that the Ig enhancers strongly activate SH in neighboring genes even though their stimulation of transcription is negligible. Mutations in certain E-box, NFκB, MEF2, or Ets family binding sites--known to be important for the transcriptional role of Ig enhancers--impair or abolish the activity. Full activation of SH typically requires a combination of multiple Ig enhancer and enhancer-like elements. The mechanism is evolutionarily conserved, as mammalian Ig lambda and Ig heavy chain intron enhancers efficiently stimulate hypermutation in chicken cells. Our results demonstrate a novel regulatory function for Ig enhancers, indicating that they either recruit AID or alter the accessibility of the nearby transcription units.
Asunto(s)
Citidina Desaminasa/genética , Elementos de Facilitación Genéticos/genética , Activación de Linfocitos/genética , Hipermutación Somática de Inmunoglobulina/genética , Animales , Anticuerpos/genética , Anticuerpos/inmunología , Linfocitos B/inmunología , Sitios de Unión/genética , Línea Celular , Pollos , Elementos E-Box/genética , Técnicas de Inactivación de Genes , Proteínas Fluorescentes Verdes/genética , Humanos , Cadenas kappa de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/inmunología , Cadenas lambda de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/inmunología , Activación de Linfocitos/inmunología , Factores de Transcripción MEF2/genética , Ratones , Mutación/genética , FN-kappa B/genética , Alineación de Secuencia , Transcripción Genética , Uracil-ADN Glicosidasa/genéticaRESUMEN
Secondary B cell repertoire diversification occurs by somatic hypermutation (SHM) in germinal centers following Ag stimulation. In SHM, activation-induced cytidine deaminase mutates the V region of the Ig genes to increase the affinity of Abs. Although SHM acts primarily at Ig loci, low levels of off-target mutation can result in oncogenic DNA damage, illustrating the importance of understanding SHM targeting mechanisms. A candidate targeting motif is the E box, a short DNA sequence (CANNTG) found abundantly in the genome and in many SHM target genes. Using a reporter assay in chicken DT40 B cells, we previously identified a 1928-bp portion of the chicken IgL locus capable of supporting robust SHM. In this article, we demonstrate that mutation of all 20 E boxes in this fragment reduces SHM targeting activity by 90%, and that mutation of subsets of E boxes reveals a functional hierarchy in which E boxes within "core" targeting regions are of greatest importance. Strikingly, when the sequence and spacing of the 20 E boxes are preserved but surrounding sequences are altered, SHM targeting activity is eliminated. Hence, although E boxes are vital SHM targeting elements, their function is completely dependent on their surrounding sequence context. These results suggest an intimate cooperation between E boxes and other sequence motifs in SHM targeting to Ig loci and perhaps also in restricting mistargeting to certain non-Ig loci.
Asunto(s)
Linfocitos B/metabolismo , Elementos E-Box/genética , Hipermutación Somática de Inmunoglobulina/genética , Animales , Sitios de Unión , Células Cultivadas , Pollos , Citidina Desaminasa/fisiología , ADN Recombinante/genética , Elementos de Facilitación Genéticos/genética , Genes de las Cadenas Ligeras de las Inmunoglobulinas/genética , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Región Variable de Inmunoglobulina/genética , Mutación , Unión Proteica , Factor de Transcripción 3/metabolismo , Transfección , TransgenesRESUMEN
Somatic hypermutation (SHM) diversifies the V region of Ig genes and underlies the process of affinity maturation, in which B lymphocytes producing high-affinity Abs are generated and selected. SHM is triggered in activated B cells by deamination of deoxycytosine residues mediated by activation-induced deaminase (AID). Whereas mistargeting of SHM and AID results in mutations and DNA damage in many non-Ig genes, they act preferentially at Ig loci. The mechanisms responsible for preferential targeting of SHM and AID activity to Ig loci are poorly understood. Using an assay involving an SHM reporter cassette inserted into the Ig L chain locus (IgL) of chicken DT40 B cells, we have identified a 1.9-kb DIVAC (diversification activator) element derived from chicken IgL that supports high levels of AID-dependent mutation activity. Systematic deletion analysis reveals that targeting activity is spread throughout much of the sequence and identifies two core regions that are particularly critical for function: a 200-bp region within the IgL enhancer, and a 350-bp 3' element. Chromatin immunoprecipitation experiments demonstrate that whereas DIVAC does not alter levels of several epigenetic marks in the mutation cassette, it does increase levels of serine-5 phosphorylated RNA polymerase II in the mutation target region, consistent with an effect on transcriptional elongation/pausing. We propose that multiple, dispersed DNA elements collaborate to recruit and activate the mutational machinery at Ig gene variable regions during SHM.