RESUMEN
Simian virus 40 (SV40) is a monkey virus associated with several types of human cancers. SV40 is most frequently detected in mesotheliomas, brain and bone tumors and lymphomas, but the mechanism for SV40 tumorigenesis in humans is not clear. SV40 relative Merkel cell polyomavirus (MCPyV) causes Merkel cell carcinoma (MCC) in humans by expressing truncated large tumor antigen (LT) caused by APOBEC cytidine deaminase family enzymes induced mutations. AID (activation-induced cytidine deaminase), a member of the APOBEC family, is the initiator of the antibody diversification process known as somatic hypermutation (SHM) and its aberrant expression and targeting is a frequent source of lymphomagenesis. In this study, we investigated whether AID-induced mutations could cause truncation of SV40 LT. We demonstrate that the SV40 enhancer has strong SHM targeting activity in several cell types and that AID-induced mutations accumulate to SV40 LT in B cells and kidney cells and cause truncated LT expression in B cells. Our results argue that the ability of the SV40 enhancer to target SHM to LT is a potential source of LT truncation events in various cell types that could contribute to carcinogenesis.
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
Merkel cell carcinoma (MCC) is an aggressive skin cancer, which is frequently caused by Merkel cell polyomavirus (MCPyV). Mutations of MCPyV tumor (T) antigens are major pathologic events of virus-positive (MCPyV+) MCCs, but their source is unclear. Activation-induced cytidine deaminase (AID)/APOBEC family cytidine deaminases contribute to antiviral immunity by mutating viral genomes and are potential carcinogenic mutators. We studied the contribution of AID/APOBEC cytidine deaminases to MCPyV large T (LT) truncation events. The MCPyV LT area in MCCs was enriched with cytosine-targeting mutations, and a strong APOBEC3 mutation signature was observed in MCC sequences. AICDA and APOBEC3 expression were detected in the Finnish MCC sample cohort, and LT expression correlated with APOBEC3H and APOBEC3G. Marginal but statistically significant somatic hypermutation targeting activity was detected in the MCPyV regulatory region. Our results suggest that APOBEC3 cytidine deaminases are a plausible cause of the LT truncating mutations in MCPyV+ MCC, while the role of AID in MCC carcinogenesis is unlikely. Significance: We uncover APOBEC3 mutation signature in MCPyV LT that reveals the likely cause of mutations underlying MCPyV+ MCC. We further reveal an expression pattern of APOBECs in a large Finnish MCC sample cohort. Thus, the findings presented here suggest a molecular mechanism underlying an aggressive carcinoma with poor prognosis.
Asunto(s)
Carcinoma de Células de Merkel , Poliomavirus de Células de Merkel , Infecciones por Polyomavirus , Neoplasias Cutáneas , Humanos , Carcinoma de Células de Merkel/genética , Poliomavirus de Células de Merkel/genética , Antígenos Virales de Tumores/genética , Infecciones por Polyomavirus/genética , Neoplasias Cutáneas/genética , Carcinogénesis/genética , Mutación , Citidina , Desaminasas APOBEC/genéticaRESUMEN
The transcription factor Bach2 is required for germinal center formation, somatic hypermutation (SHM), and class-switch recombination (CSR) of immunoglobulins. SHM and CSR are initiated by activation-induced cytidine deaminase (AID) which has potential to induce human B cell lymphoma. To understand the role of Bach2 in AID-mediated immunoglobulin gene diversification processes, we established a BACH2-deficient DT40 B cell line. We show that in addition to allowing SHM, Bach2 drives immunoglobulin gene conversion (GCV), another AID-dependent antibody gene diversification process. We demonstrate that Bach2 promotes GCV by increasing the expression of AID. Importantly, we found that the regulation of AID is independent of Blimp-1 and that BACH2-deficient cells have altered expression of several genes regulating AID expression, stability and function. Furthermore, re-expression of BACH2 or AID in Bach2KO cells restored the SHM and GCV defects. These results demonstrate that Bach2 has a previously unappreciated role in the production of high-affinity antibodies.
