Similar Datasets
Project description:Activation Induced Deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) in germinal center (GC) B cells through the deamination of deoxycytidine residues (dC) into deoxyuridines (dU) in immunoglobulin (Ig) genes. Although AID has a strong preference for Ig genes, it can also target other genomic regions, giving rise to mutations or chromosomal translocations. Thus, understanding the specificity of AID has major implications for oncogenic transformation. However, approaching AID specificity has proved extremely challenging because AID deamination events occur at low frequencies. Here we have sequenced at very high depth >1500 genomic regions from GC B cells and identified 275 genes targeted by AID, including 30 of the previously known 35 AID targets. This has enabled for the first time to define the molecular features predictive of AID target specificity genome-wide. Furthermore, we identify the most highly mutated hotspot for AID activity described to date. We also find that Base Excision Repair (BER) and Mismatch Repair (MMR) systems, which are responsible for the resolution of AID deaminations, back-up each other to faithfully repair AID-induced lesions. Finally, our data establishes a novel link between AID mutagenic activity and malignant transformation.
2018-01-19 | GSE102944 | GEO
Project description:Activation-induced cytidine deaminase (AID) is required for initiation of Ig class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs) involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP) for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq). We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID.
2015-07-16 | GSE66424 | GEO
Project description:It has been shown that DNA demethylation has a pivotal role in the generation of induced pluripotent stem (iPS) cells. However, the underlying mechanism is still unclear. Previous reports indicated that activation-induced cytidine deaminase (Aid) is involved in DNA demethylation in several developmental processes and cell fusion-mediated reprogramming. Based on the reports, we hypothesized that Aid may be involved in DNA demethylation during the iPS cell generation. In this study, we examined the function of Aid in iPS cell generation using Aid knockout (Aid-/-) mice expressing a GFP reporter under the control of a pluripotent stem cell marker, Nanog. By the introduction of Oct3/4, Sox2, Klf4 and c-Myc, Nanog-GFP positive iPS cells could be generated from the fibroblasts and primary B cells of Aid-/- mice. The Aid-/- iPS cells showed normal proliferation and gave rise to chimeras, indicating their capacity for self-renewal and pluripotency. The comprehensive DNA methylation analysis by MBD-sequening demonstrated that there were only a few differences between Aid+/+ and Aid-/- iPS cells. Aid+/+ and Aid-/- iPS colonies were generated from Aid+/+ and Aid-/- MEFs and picked up mechanically. The clones were passaged four times on feeder cells and two times on gelatin-coated dishes to exclude the contamination of feeder cells. Subsequently, the genome was isolated. Four Aid+/+ iPS cell clones and four Aid-/- iPS cell clones were compared. To confirm the validity of MBD-sequencing, four Aid+/+ iPS cell clones were compared with three ES cell clones or three Aid+/+ MEFs.
2014-04-10 | E-GEOD-52136 | biostudies-arrayexpress
Project description:Activation induced cytidine deaminase (AID) initiates antibody gene diversification by creating U:G mismatches. However, AID is not specific for antibody genes. Off-target lesions can activate oncogenes or cause chromosome translocations. Despite its importance in these key transactions little is known about how AID finds its target sites. To examine AID regulation we performed an shRNA screen. We found that Spt5, a factor associated with paused RNA polymerase II (Pol II) and single stranded DNA (ssDNA), is required for class switch recombination (CSR). Spt5 interacts with AID, it is required for the association of AID and Pol II, and for AID recruitment to switch regions. ChIP-seq experiments reveal that Spt5 co-localizes with AID and stalled Pol II. Further, Spt5 accumulation at sites of Pol II pausing is predictive of AID-induced mutation. Our data suggest that Spt5 acts as an adaptor, which brings AID to Pol II on target DNA. PolII, AID, and Spt5 were immunoprecipitated from in vitro activated B cells. For Spt5 and AID, two different antibodies were used in separate, biological replicates
2010-10-05 | E-GEOD-24178 | biostudies-arrayexpress
Project description:PRDM9, a histone methyltransferase, initiates meiotic recombination by binding DNA at recombination hotspots and directing the position of DNA double-strand breaks (DSB). The DSB repair mechanism suggests that hotspots should eventually self-destruct, yet genome-wide recombination levels remain constant, a conundrum known as the hotspot paradox. To test if PRDM9 drives this evolutionary erosion, we compared activity of the Prdm9Cst allele in two Mus musculus subspecies, M.m. castaneus, in which Prdm9Cst arose, and M.m. domesticus, into which Prdm9Cst was introduced. Comparing these two strains, we find that haplotype differences at hotspots leads to qualitative and quantitative changes in PRDM9 binding and activity. Most variants affecting PRDM9Cst binding arose and were fixed in M.m castaneus, suppressing hotspot activity. Furthermore, M.m castaneus x M.m domesticus F1 hybrids exhibit novel hotspots, representing sites of historic evolutionary erosion. Together these data support a model where haplotype-specific PRDM9 binding directs biased gene conversion at hotspots, ultimately leading to hotspot erosion. Identify position of meiotic H3K4me3 from various sub-species of mice and F1 hybrids from crosses between subspecies. In addition, perform ChIP-seq analysis on the meiosis-specific methyltransferase PRDM9.
2015-01-13 | E-GEOD-60906 | biostudies-arrayexpress
Project description:The activation-induced cytidine deaminase enzyme (AID) is required in germinal center (GC) B cells for somatic hyper-mutation and class switch recombination at the immunoglobulin locus. In GC-B cells, AID is highly expressed, with inherent mutator activity that helps generate antibody diversity. However, AID may also regulate gene expression epigenetically, irrespective of mutator activity, by directly deaminating 5-methylcytosine (5mC) in concert with base excision repair glycosylases to exchange unmethylated cytosine. This pathway promotes gene demethylation, thereby removing epigenetic memory. For example, AID promotes active demethylation of the genome in primordial germ cells. However, the range and mechanism by which AID promotes pluripotency is not known. Different studies have suggested either a requirement or a lack of function for promoting pluripotency in somatic nuclei following fusion with embryonic stem cells (ESC). Here we tested directly whether AID regulates epigenetic memory, by comparing the relative ability of cells lacking AID to reprogram from a differentiated cell type to an induced pluripotent stem cell (iPSC). We show that loss of AID impacts two distinct steps of reprogramming: First, AID-null cells are transiently hyper-responsive to the reprogramming process. Second, although they initiate expression of pluripotency genes, they fail to stabilize the pluripotent state. The genome of AID-null cells remains hypermethylated in reprogramming cells, and hypermethylated genes associated with pluripotency fail to be stably up-regulated. MYC target genes are highly enriched in the set of genes hypermethylated and under-expressed in reprogramming cells lacking AID. Recent studies identified a distinctive late step of reprogramming associated with methylation status. AID appears to regulate this step to stabilize the pluripotent state, removing epigenetic memory to promote expression of secondary pluripotency network genes. Transcriptome sequencing of AID-null tail fibroblasts, wildtype tail fibroblasts, AID-null and wildtype tail fibroblasts reprogrammed for three weeks by ectopic expression of transcription factors Oct4, Sox2, KLf4 and cMyc. Methylation profiling by reduced representation bisulphite seuencing of AID-null tail fibroblasts, wildtype tail fibroblasts, AID-null and wildtype tail fibroblasts reprogrammed for three weeks and AID-null and wildtype clones after three weeks of reprogramming (Picked at two weeks)
2013-05-08 | E-GEOD-46700 | biostudies-arrayexpress
Project description:The early detection of tissue and organ damage associated with autoimmune diseases (AID) has been identified as key to improve long-term survival, but non-invasive biomarkers are lacking. Elevated cell-free DNA (cfDNA) levels have been observed in AID and inflammatory bowel disease (IBD), prompting interest to use cfDNA as a potential non-invasive diagnostic and prognostic biomarker. Despite these known disease-related changes in concentration, it remains impossible to identify AID and IBD patients through cfDNA analysis alone. By using unsupervised clustering on large sets of shallow whole-genome sequencing (sWGS) cfDNA data, we uncover AID- and IBD-specific genome-wide patterns in plasma cfDNA in both the obstetric and general AID and IBD populations. Supervised learning of the genome-wide patterns allows AID prediction with 50% sensitivity at 95% specificity. Importantly, the method can identify pregnant women with AID during routine non-invasive prenatal screening. Since AID pregnancies have an increased risk of severe complications, early recognition or detection of new onset AID can redirect pregnancy management and limit potential adverse events. This method opens up new avenues for screening, diagnosis and monitoring of AID and IBD.
2022-09-14 | E-MTAB-11607 | biostudies-arrayexpress