Controls had to fulfill the following criteria: (1) absence of symptoms or history of asthma or other pulmonary diseases, (2) no symptoms or history of allergy, (3) negative skin prick test results ...to a battery of common aeroallergens,1 and (4) absence of familial history of asthma or allergic diseases. Because both allergy and asthma were ruled out, they could be considered as control subjects. ...although further studies are required to explore the causes for its transcriptional deregulation, it is plausible to suggest that the IL4R might hold translational potential as both a biomarker and a therapeutic target in patients with allergic asthma to HDM.
Mismatch repair of AID-generated dU:G mispairs is critical for class switch recombination (CSR) and somatic hypermutation (SHM) in B cells. The generation of a previously unavailable ...Msh2(-/-)Msh6(-/-) mouse has for the first time allowed us to examine the impact of the complete loss of MutSalpha on lymphomagenesis, CSR and SHM. The onset of T cell lymphomas and the survival of Msh2(-/-)Msh6(-/-) and Msh2(-/-)Msh6(-/-)Msh3(-/-) mice are indistinguishable from Msh2(-/-) mice, suggesting that MSH2 plays the critical role in protecting T cells from malignant transformation, presumably because it is essential for the formation of stable MutSalpha heterodimers that maintain genomic stability. The similar defects on switching in Msh2(-/-), Msh2(-/-)Msh6(-/-) and Msh2(-/-)Msh6(-/-)Msh3(-/-) mice confirm that MutSalpha but not MutSbeta plays an important role in CSR. Analysis of SHM in Msh2(-/-)Msh6(-/-) mice not only confirmed the error-prone role of MutSalpha in the generation of strand biased mutations at A:T bases, but also revealed an error-free role of MutSalpha when repairing some of the dU:G mispairs generated by AID on both DNA strands. We propose a model for the role of MutSalpha at the immunoglobulin locus where the local balance of error-free and error-prone repair has an impact in the spectrum of mutations introduced during Phase 2 of SHM.
Small non-coding RNAs (snRNAs) develop important functions related to epigenetic regulation. YRNAs are snRNAs involved in the initiation of DNA replication and RNA stability that regulate gene ...expression. They have been related to autoimmune, cancer and inflammatory diseases but never before to allergy. In this work we described for the first time in allergic patients the differential expression profile of YRNAs, their regulatory mechanisms and their potential as new diagnostic and therapeutic targets.
From a previous whole RNAseq study in B cells of allergic patients, differential expression profiles of coding and non-coding transcripts were obtained. To select the most differentially expressed non coding transcripts, fold change and p-values were analyzed. A validation of the expression differences detected was developed in an independent cohort of 304 individuals, 208 allergic patients and 96 controls by using qPCR. Potential binding and retrotransponibility capacity were characterized by
structural analysis. Using a novel bioinformatics approach, RNA targets identification, functional enrichment and network analyses were performed.
We found that almost 70% of overexpressed non-coding transcripts in allergic patients corresponded to YRNAs. From the three more differentially overexpressed candidates, increased expression was independently confirmed in the peripheral blood of allergic patients. Structural analysis suggested a protein binding capacity decrease and an increase in retrotransponibility. Studies of RNA targets allowed the identification of sequences related to the immune mechanisms underlying allergy.
Overexpression of YRNAs is observed for the first time in allergic patients. Structural and functional information points to their implication on regulatory mechanisms of the disease.
Abstract The generation of effective antibodies depends upon somatic hypermutation (SHM) and class-switch recombination (CSR) of antibody genes by activation induced cytidine deaminase (AID) and the ...subsequent recruitment of error prone base excision and mismatch repair. While AID initiates and is required for SHM, more than half of the base changes that accumulate in V regions are not due to the direct deamination of dC to dU by AID, but rather arise through the recruitment of the mismatch repair complex (MMR) to the U:G mismatch created by AID and the subsequent perversion of mismatch repair from a high fidelity process to one that is very error prone. In addition, the generation of double-strand breaks (DSBs) is essential during CSR, and the resolution of AID-generated mismatches by MMR to promote such DSBs is critical for the efficiency of the process. While a great deal has been learned about how AID and MMR cause hypermutations and DSBs, it is still unclear how the error prone aspect of these processes is largely restricted to antibody genes. The use of knockout models and mice expressing mismatch repair proteins with separation-of-function point mutations have been decisive in gaining a better understanding of the roles of each of the major MMR proteins and providing further insight into how mutation and repair are coordinated. Here, we review the cascade of MMR factors and repair signals that are diverted from their canonical error free role and hijacked by B cells to promote genetic diversification of the Ig locus. This error prone process involves AID as the inducer of enzymatically-mediated DNA mismatches, and a plethora of downstream MMR factors acting as sensors, adaptors and effectors of a complex and tightly regulated process from much of which is not yet well understood.
The somatic hypermutation of Ig variable regions requires the activity of activation-induced cytidine deaminase (AID) which has previously been shown to preferentially deaminate WRC (W = A/T, R = ...A/G) motif hot spots in in vivo and in vitro assays. We compared mutation profiles of in vitro assays for the 3' flanking intron of VhJ558-Jh4 region to previously reported in vivo profiles for the same region in the Msh2⁻/⁻Ung⁻/⁻ mice that lack base excision and mismatch repair. We found that the in vitro and in vivo mutation profiles were highly correlated for the top (nontranscribed) strand, while for the bottom (transcribed) strand the correlation is far lower. We used an in silico model of AID activity to elucidate the relative importance of motif targeting in vivo. We found that the mutation process entails substantial complexity beyond motif targeting, a large part of which is captured in vitro. To elucidate the contribution of the sequence environment to the observed differences between the top and bottom strands, we analyzed intermutational distances. The bottom strand shows an approximately exponential distribution of distances in vivo and in vitro, as expected from a null model. However, the top strand deviates strongly from this distribution in that mutations approximately 50 nucleotides apart are greatly reduced, again both in vivo and in vitro, illustrating an important strand asymmetry. While we have confirmed that AID targeting of hot and cold spots is a key part of the mutation process, our results suggest that the sequence environment plays an equally important role.
Does antisense make sense of AID targeting? Roa, Sergio; Kuang, Fei Li; Scharff, Matthew D
Proceedings of the National Academy of Sciences - PNAS,
03/2008, Letnik:
105, Številka:
10
Journal Article
Highlights ► The DNA mutator AID catalyzes U:G mismatches recognized by mismatch repair (MMR). ► MMR generally fixes DNA mismatches faithfully, except at the immunoglobulin genes. ► MMR ...differentially promotes A:T mutagenesis at V regions and DSB at S regions. ► The MMR sensor complex MutSα (MSH2/MSH6) promotes both SHM and CSR. ► The MMR adaptor complex MutLα (MLH1/PMS2) has no role in SHM but promotes CSR.
Abstract
DNA damage response pathways rely extensively on nuclease activity to process DNA intermediates. Exonuclease 1 (EXO1) is a pleiotropic evolutionary conserved DNA exonuclease involved in ...various DNA repair pathways, replication, antibody diversification, and meiosis. But, whether EXO1 facilitates these DNA metabolic processes through its enzymatic or scaffolding functions remains unclear. Here, we dissect the contribution of EXO1 enzymatic versus scaffolding activity by comparing Exo1DA/DA mice expressing a proven nuclease-dead mutant form of EXO1 to entirely EXO1-deficient Exo1−/− and EXO1 wild type Exo1+/+ mice. We show that Exo1DA/DA and Exo1–/– mice are compromised in canonical DNA repair processing, suggesting that the EXO1 enzymatic role is important for error-free DNA mismatch and double-strand break repair pathways. However, in non-canonical repair pathways, EXO1 appears to have a more nuanced function. Next-generation sequencing of heavy chain V region in B cells showed the mutation spectra of Exo1DA/DA mice to be intermediate between Exo1+/+ and Exo1–/– mice, suggesting that both catalytic and scaffolding roles of EXO1 are important for somatic hypermutation. Similarly, while overall class switch recombination in Exo1DA/DA and Exo1–/– mice was comparably defective, switch junction analysis suggests that EXO1 might fulfill an additional scaffolding function downstream of class switching. In contrast to Exo1−/− mice that are infertile, meiosis progressed normally in Exo1DA/DA and Exo1+/+ cohorts, indicating that a structural but not the nuclease function of EXO1 is critical for meiosis. However, both Exo1DA/DA and Exo1–/– mice displayed similar mortality and cancer predisposition profiles. Taken together, these data demonstrate that EXO1 has both scaffolding and enzymatic functions in distinct DNA repair processes and suggest a more composite and intricate role for EXO1 in DNA metabolic processes and disease.
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