ABSTRACT
Chronic infection with Plasmodium falciparum was epidemiologically associated with endemic Burkitt's lymphoma, a mature B cell cancer characterized by chromosome translocation between the c-myc oncogene and Igh, over 50 years ago. Whether infection promotes B cell lymphoma, and if so by which mechanism, remains unknown. To investigate the relationship between parasitic disease and lymphomagenesis, we used Plasmodium chabaudi (Pc) to produce chronic malaria infection in mice. Pc induces prolonged expansion of germinal centers (GCs), unique compartments in which B cells undergo rapid clonal expansion and express activation-induced cytidine deaminase (AID), a DNA mutator. GC B cells elicited during Pc infection suffer widespread DNA damage, leading to chromosome translocations. Although infection does not change the overall rate, it modifies lymphomagenesis to favor mature B cell lymphomas that are AID dependent and show chromosome translocations. Thus, malaria infection favors mature B cell cancers by eliciting protracted AID expression in GC B cells. PAPERCLIP.
Subject(s)
Genomic Instability , Lymphoma, B-Cell/genetics , Malaria/complications , Malaria/genetics , Plasmodium chabaudi/physiology , Animals , B-Lymphocytes/pathology , Chronic Disease , Cytidine Deaminase/metabolism , DNA Replication , Genes, p53 , Germinal Center/parasitology , Malaria/parasitology , Malaria/pathology , Mice , Translocation, GeneticABSTRACT
The barrier to curing HIV-1 is thought to reside primarily in CD4(+) T cells containing silent proviruses. To characterize these latently infected cells, we studied the integration profile of HIV-1 in viremic progressors, individuals receiving antiretroviral therapy, and viremic controllers. Clonally expanded T cells represented the majority of all integrations and increased during therapy. However, none of the 75 expanded T cell clones assayed contained intact virus. In contrast, the cells bearing single integration events decreased in frequency over time on therapy, and the surviving cells were enriched for HIV-1 integration in silent regions of the genome. Finally, there was a strong preference for integration into, or in close proximity to, Alu repeats, which were also enriched in local hotspots for integration. The data indicate that dividing clonally expanded T cells contain defective proviruses and that the replication-competent reservoir is primarily found in CD4(+) T cells that remain relatively quiescent.
Subject(s)
CD4-Positive T-Lymphocytes/virology , HIV Infections/virology , HIV-1/physiology , Virus Integration , Virus Latency , Alu Elements , Clone Cells , Defective Viruses/genetics , Defective Viruses/physiology , HIV Infections/drug therapy , HIV-1/genetics , Humans , Immunologic Memory , Proviruses/physiology , Single-Cell AnalysisABSTRACT
The antibody gene mutator activation-induced cytidine deaminase (AID) promiscuously damages oncogenes, leading to chromosomal translocations and tumorigenesis. Why nonimmunoglobulin loci are susceptible to AID activity is unknown. Here, we study AID-mediated lesions in the context of nuclear architecture and the B cell regulome. We show that AID targets are not randomly distributed across the genome but are predominantly grouped within super-enhancers and regulatory clusters. Unexpectedly, in these domains, AID deaminates active promoters and eRNA(+) enhancers interconnected in some instances over megabases of linear chromatin. Using genome editing, we demonstrate that 3D-linked targets cooperate to recruit AID-mediated breaks. Furthermore, a comparison of hypermutation in mouse B cells, AID-induced kataegis in human lymphomas, and translocations in MEFs reveals that AID damages different genes in different cell types. Yet, in all cases, the targets are predominantly associated with topological complex, highly transcribed super-enhancers, demonstrating that these compartments are key mediators of AID recruitment.
Subject(s)
B-Lymphocytes/metabolism , Carcinogenesis , Cytidine Deaminase/genetics , Enhancer Elements, Genetic , Animals , DNA Damage , Humans , Lymphoma/metabolism , MiceABSTRACT
The Omicron variant of SARS-CoV-2 infected many vaccinated and convalescent individuals1-3. Despite the reduced protection from infection, individuals who received three doses of an mRNA vaccine were highly protected from more serious consequences of infection4. Here we examine the memory B cell repertoire in a longitudinal cohort of individuals receiving three mRNA vaccine doses5,6. We find that the third dose is accompanied by an increase in, and evolution of, receptor-binding domain (RBD)-specific memory B cells. The increase is due to expansion of memory B cell clones that were present after the second dose as well as the emergence of new clones. The antibodies encoded by these cells showed significantly increased potency and breadth when compared with antibodies obtained after the second dose. Notably, the increase in potency was especially evident among newly developing clones of memory cells, which differed from persisting clones in targeting more conserved regions of the RBD. Overall, more than 50% of the analysed neutralizing antibodies in the memory compartment after the third mRNA vaccine dose neutralized the Omicron variant. Thus, individuals receiving three doses of an mRNA vaccine have a diverse memory B cell repertoire that can respond rapidly and produce antibodies capable of clearing even diversified variants such as Omicron. These data help to explain why a third dose of a vaccine that was not specifically designed to protect against variants is effective against variant-induced serious disease.
Subject(s)
COVID-19 Vaccines , COVID-19 , Immunization, Secondary , Memory B Cells , SARS-CoV-2 , mRNA Vaccines , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , COVID-19/prevention & control , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/immunology , Humans , Memory B Cells/immunology , RNA, Messenger/genetics , SARS-CoV-2/genetics , SARS-CoV-2/immunology , mRNA Vaccines/administration & dosage , mRNA Vaccines/immunologyABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection produces B cell responses that continue to evolve for at least a year. During that time, memory B cells express increasingly broad and potent antibodies that are resistant to mutations found in variants of concern1. As a result, vaccination of coronavirus disease 2019 (COVID-19) convalescent individuals with currently available mRNA vaccines produces high levels of plasma neutralizing activity against all variants tested1,2. Here we examine memory B cell evolution five months after vaccination with either Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) mRNA vaccine in a cohort of SARS-CoV-2-naive individuals. Between prime and boost, memory B cells produce antibodies that evolve increased neutralizing activity, but there is no further increase in potency or breadth thereafter. Instead, memory B cells that emerge five months after vaccination of naive individuals express antibodies that are similar to those that dominate the initial response. While individual memory antibodies selected over time by natural infection have greater potency and breadth than antibodies elicited by vaccination, the overall neutralizing potency of plasma is greater following vaccination. These results suggest that boosting vaccinated individuals with currently available mRNA vaccines will increase plasma neutralizing activity but may not produce antibodies with equivalent breadth to those obtained by vaccinating convalescent individuals.
Subject(s)
COVID-19 Vaccines/immunology , Evolution, Molecular , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Synthetic/immunology , mRNA Vaccines/immunology , 2019-nCoV Vaccine mRNA-1273/immunology , Adult , Aged , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antibody Affinity , BNT162 Vaccine/immunology , Cohort Studies , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Epitopes, B-Lymphocyte/immunology , Female , Humans , Male , Memory B Cells/immunology , Middle Aged , Neutralization Tests , Protein Domains/immunology , Spike Glycoprotein, Coronavirus/chemistry , Young AdultABSTRACT
More than one year after its inception, the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains difficult to control despite the availability of several working vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies1,2. Here we report on a cohort of 63 individuals who have recovered from COVID-19 assessed at 1.3, 6.2 and 12 months after SARS-CoV-2 infection, 41% of whom also received mRNA vaccines3,4. In the absence of vaccination, antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable between 6 and 12 months after infection. Vaccination increases all components of the humoral response and, as expected, results in serum neutralizing activities against variants of concern similar to or greater than the neutralizing activity against the original Wuhan Hu-1 strain achieved by vaccination of naive individuals2,5-8. The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in the variants of concern4,9. In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand markedly after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants.
Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/immunology , SARS-CoV-2/immunology , Adult , Aged , Antibodies, Monoclonal/immunology , B-Lymphocytes/immunology , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Female , Humans , Immunologic Memory/immunology , Male , Middle Aged , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Time FactorsABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with the development of variable levels of antibodies with neutralizing activity, which can protect against infection in animal models1,2. Antibody levels decrease with time, but, to our knowledge, the nature and quality of the memory B cells that would be required to produce antibodies upon reinfection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection with SARS-CoV-2. We find that titres of IgM and IgG antibodies against the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 decrease significantly over this time period, with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by fivefold in pseudotype virus assays. By contrast, the number of RBD-specific memory B cells remains unchanged at 6.2 months after infection. Memory B cells display clonal turnover after 6.2 months, and the antibodies that they express have greater somatic hypermutation, resistance to RBD mutations and increased potency, indicative of continued evolution of the humoral response. Immunofluorescence and PCR analyses of intestinal biopsies obtained from asymptomatic individuals at 4 months after the onset of coronavirus disease 2019 (COVID-19) revealed the persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 individuals. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.
Subject(s)
Antibodies, Viral/immunology , COVID-19/immunology , Immunity, Humoral/immunology , SARS-CoV-2/immunology , Adolescent , Adult , Aged , Antibodies, Monoclonal/blood , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/genetics , Antigens, Viral/chemistry , Antigens, Viral/genetics , Antigens, Viral/immunology , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Biopsy , COVID-19/blood , Cohort Studies , Fluorescent Antibody Technique , Humans , Immunity, Humoral/genetics , Immunoglobulin A/immunology , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Immunologic Memory/immunology , Intestines/immunology , Middle Aged , Mutation , Somatic Hypermutation, Immunoglobulin , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Time Factors , Young AdultABSTRACT
Chromosomal rearrangements, including translocations, require formation and joining of DNA double strand breaks (DSBs). These events disrupt the integrity of the genome and are frequently involved in producing leukemias, lymphomas and sarcomas. Despite the importance of these events, current understanding of their genesis is limited. To examine the origins of chromosomal rearrangements we developed Translocation Capture Sequencing (TC-Seq), a method to document chromosomal rearrangements genome-wide, in primary cells. We examined over 180,000 rearrangements obtained from 400 million B lymphocytes, revealing that proximity between DSBs, transcriptional activity and chromosome territories are key determinants of genome rearrangement. Specifically, rearrangements tend to occur in cis and to transcribed genes. Finally, we find that activation-induced cytidine deaminase (AID) induces the rearrangement of many genes found as translocation partners in mature B cell lymphoma.
Subject(s)
B-Lymphocytes/metabolism , Genome , Mutagenesis , Translocation, Genetic , Animals , Cells, Cultured , Cytidine Deaminase/metabolism , Genes, myc , Humans , Immunoglobulin Heavy Chains/genetics , Mice , Neoplasms/genetics , Sequence Analysis, DNA/methods , Spleen/cytologyABSTRACT
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 transactions little is known about how AID finds its targets. We performed an shRNA screen to identify factors required for class switch recombination (CSR) of antibody loci. We found that Spt5, a factor associated with stalled RNA polymerase II (Pol II) and single stranded DNA (ssDNA), is required for CSR. Spt5 interacts with AID, it facilitates association between AID and Pol II, and AID recruitment to its Ig and non-Ig targets. ChIP-seq experiments reveal that Spt5 colocalizes with AID and stalled Pol II. Further, Spt5 accumulation at sites of Pol II stalling is predictive of AID-induced mutation. We propose that AID is targeted to sites of Pol II stalling in part via its association with Spt5.
Subject(s)
B-Lymphocytes/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Cytidine Deaminase/metabolism , Immunoglobulin Class Switching , RNA Polymerase II/metabolism , Transcriptional Elongation Factors/metabolism , Animals , Cell Line , Cell Line, Tumor , Fibroblasts/metabolism , Humans , Immunoglobulins/genetics , MiceABSTRACT
Deficiencies in factors that regulate the DNA damage response enhance the incidence of malignancy by destabilizing the genome. However, the precise influence of the DNA damage response on regulation of cancer-associated rearrangements is not well defined. Here we examine the genome-wide impact of tumor protein P53-binding protein 1 (53BP1) deficiency in lymphoma and translocation. While both activation-induced cytidine deaminase (AID) and 53BP1 have been associated with cancer in humans, neither AID overexpression nor loss of 53BP1 is sufficient to produce malignancy. However, the combination of 53BP1 deficiency and AID deregulation results in B cell lymphoma. Deep sequencing of the genome of 53BP1(-/-) cancer cells and translocation capture sequencing (TC-Seq) of primary 53BP1(-/-) B cells revealed that their chromosomal rearrangements differ from those found in wild-type cells in that they show increased DNA end resection. Moreover, loss of 53BP1 alters the translocatome by increasing rearrangements to intergenic regions.
Subject(s)
Cell Transformation, Neoplastic/genetics , Chromosomal Proteins, Non-Histone/physiology , Cytidine Deaminase/physiology , DNA-Binding Proteins/physiology , Gene Rearrangement , Lymphoma, B-Cell/metabolism , Animals , Cells, Cultured , Chromosomal Proteins, Non-Histone/deficiency , Chromosomal Proteins, Non-Histone/genetics , Chromosomes, Mammalian/genetics , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Epigenesis, Genetic , Genes, Tumor Suppressor , Genome-Wide Association Study , Lymphoma, B-Cell/genetics , Mice , Mice, Knockout , Mutation , Sequence Analysis, DNA , Transcription, Genetic , Translocation, Genetic , Tumor Suppressor p53-Binding Protein 1ABSTRACT
Combination antiretroviral therapy controls but does not cure HIV-1 infection because a small fraction of cells harbor latent viruses that can produce rebound viremia when therapy is interrupted. The circulating latent virus reservoir has been documented by a variety of methods, most prominently by viral outgrowth assays (VOAs) in which CD4+ T cells are activated to produce virus in vitro, or more recently by amplifying proviral near full-length (NFL) sequences from DNA. Analysis of samples obtained in clinical studies in which individuals underwent analytical treatment interruption (ATI), showed little if any overlap between circulating latent viruses obtained from outgrowth cultures and rebound viruses from plasma. To determine whether intact proviruses amplified from DNA are more closely related to rebound viruses than those obtained from VOAs, we assayed 12 individuals who underwent ATI after infusion of a combination of two monoclonal anti-HIV-1 antibodies. A total of 435 intact proviruses obtained by NFL sequencing were compared with 650 latent viruses from VOAs and 246 plasma rebound viruses. Although, intact NFL and outgrowth culture sequences showed similar levels of stability and diversity with 39% overlap, the size of the reservoir estimated from NFL sequencing was larger than and did not correlate with VOAs. Finally, intact proviruses documented by NFL sequencing showed no sequence overlap with rebound viruses; however, they appear to contribute to recombinant viruses found in plasma during rebound.
Subject(s)
CD4-Positive T-Lymphocytes/virology , HIV Infections/virology , HIV-1/physiology , Proviruses/physiology , Anti-HIV Agents/administration & dosage , Antibodies, Monoclonal, Humanized , Antibodies, Neutralizing/administration & dosage , Broadly Neutralizing Antibodies , HIV Antibodies/administration & dosage , HIV Infections/drug therapy , HIV-1/classification , HIV-1/genetics , HIV-1/growth & development , Humans , Phylogeny , Proviruses/classification , Proviruses/genetics , Proviruses/growth & development , Virus Latency , Virus ReplicationABSTRACT
HIV-1-infected individuals harbor a latent reservoir of infected CD4+ T cells that is not eradicated by antiretroviral therapy (ART). This reservoir presents the greatest barrier to an HIV-1 cure and has remained difficult to characterize, in part, because the vast majority of integrated sequences are defective and incapable of reactivation. To characterize the replication-competent reservoir, we have combined two techniques, quantitative viral outgrowth and qualitative sequence analysis of clonal outgrowth viruses. Leukapheresis samples from four fully ART-suppressed, chronically infected individuals were assayed at two time points separated by a 4- to 6-mo interval. Overall, 54% of the viruses emerging from the latent reservoir showed gp160 env sequences that were identical to at least one other virus. Moreover, 43% of the env sequences from viruses emerging from the reservoir were part of identical groups at the two time points. Groups of identical expanded sequences made up 54% of proviral DNA, and, as might be expected, the sequences of replication-competent viruses in the active reservoir showed limited overlap with integrated proviral DNA, most of which is known to represent defective viruses. Finally, there was an inverse correlation between proviral DNA clone size and the probability of reactivation, suggesting that replication-competent viruses are less likely to be found among highly expanded provirus-containing cell clones.
ABSTRACT
Recurrent chromosomal translocations underlie both haematopoietic and solid tumours. Their origin has been ascribed to selection of random rearrangements, targeted DNA damage, or frequent nuclear interactions between translocation partners; however, the relative contribution of each of these elements has not been measured directly or on a large scale. Here we examine the role of nuclear architecture and frequency of DNA damage in the genesis of chromosomal translocations by measuring these parameters simultaneously in cultured mouse B lymphocytes. In the absence of recurrent DNA damage, translocations between Igh or Myc and all other genes are directly related to their contact frequency. Conversely, translocations associated with recurrent site-directed DNA damage are proportional to the rate of DNA break formation, as measured by replication protein A accumulation at the site of damage. Thus, non-targeted rearrangements reflect nuclear organization whereas DNA break formation governs the location and frequency of recurrent translocations, including those driving B-cell malignancies.
Subject(s)
B-Lymphocytes/metabolism , B-Lymphocytes/pathology , DNA Damage/genetics , Translocation, Genetic/genetics , Animals , B-Lymphocytes/cytology , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cells, Cultured , Chromosome Positioning , Chromosomes, Mammalian/genetics , Chromosomes, Mammalian/metabolism , Cytidine Deaminase/deficiency , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , DNA Breaks, Double-Stranded , Genes, myc/genetics , Genome/genetics , Immunoglobulin Heavy Chains/genetics , Mice , Replication Protein A/metabolismABSTRACT
MicroRNAs (miRNAs) are small noncoding RNAs that regulate vast networks of genes that share miRNA target sequences. To examine the physiologic effects of an individual miRNA-mRNA interaction in vivo, we generated mice that carry a mutation in the putative microRNA-155 (miR-155) binding site in the 3'-untranslated region of activation-induced cytidine deaminase (AID), designated Aicda(155) mice. AID is required for immunoglobulin gene diversification in B lymphocytes, but it also promotes chromosomal translocations. Aicda(155) caused an increase in steady-state Aicda mRNA and protein amounts by increasing the half-life of the mRNA, resulting in a high degree of Myc-Igh translocations. A similar but more pronounced translocation phenotype was also found in miR-155-deficient mice. Our experiments indicate that miR-155 can act as a tumor suppressor by reducing potentially oncogenic translocations generated by AID.
Subject(s)
B-Lymphocytes/enzymology , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , Genes, myc , Immunoglobulin Heavy Chains/genetics , MicroRNAs/metabolism , Translocation, Genetic , 3' Untranslated Regions , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Genes, Immunoglobulin , Immunoglobulin Class Switching , Lipopolysaccharides/immunology , Mice , Mice, Mutant Strains , MicroRNAs/genetics , Mutation , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , Somatic Hypermutation, ImmunoglobulinABSTRACT
The DNA double-strand break (DSB) repair protein DNA-PKcs and the signal transducer ATM are both activated by DNA breaks and phosphorylate similar substrates in vitro, yet appear to have distinct functions in vivo. Here, we show that ATM and DNA-PKcs have overlapping functions in lymphocytes. Ablation of both kinase activities in cells undergoing immunoglobulin class switch recombination leads to a compound defect in switching and a synergistic increase in chromosomal fragmentation, DNA insertions, and translocations due to aberrant processing of DSBs. These abnormalities are attributed to a compound deficiency in phosphorylation of key proteins required for DNA repair, class switching, and cell death. Notably, both kinases are required for normal levels of p53 phosphorylation in B and T cells and p53-dependent apoptosis. Our experiments reveal a DNA-PKcs-dependent pathway that regulates DNA repair and activation of p53 in the absence of ATM.
Subject(s)
Apoptosis/physiology , Cell Cycle Proteins/metabolism , DNA Breaks, Double-Stranded , DNA Repair , DNA-Activated Protein Kinase/metabolism , DNA-Binding Proteins/metabolism , Lymphocytes/physiology , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Ataxia Telangiectasia Mutated Proteins , Base Sequence , Cell Cycle Proteins/genetics , Cells, Cultured , DNA-Activated Protein Kinase/genetics , DNA-Binding Proteins/genetics , Fibroblasts/cytology , Fibroblasts/physiology , Genomic Instability , Immunoglobulin Class Switching , Lymphocytes/cytology , Mice , Mice, Knockout , Molecular Sequence Data , Nuclear Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Thymus Gland/cytology , Tripartite Motif-Containing Protein 28 , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/geneticsABSTRACT
Activation-induced cytidine deaminase (AID) initiates class switch recombination (CSR) and somatic hypermutation (SHM) by deaminating cytosine residues in immunoglobulin genes (Igh, Igκ, and Igλ). At a lower frequency, AID also causes collateral DNA damage at non-Ig loci, including genes that are rearranged or mutated in B-cell lymphoma. Precisely how AID is recruited to these off-target sites is not entirely understood. To gain further insight into how AID selects its targets, we compared AID-mediated translocations in two different cell types, B cells and mouse embryonic fibroblasts (MEFs). AID targets a distinct set of hotspots in the two cell types. In both cases, hotspots are concentrated in highly transcribed but stalled genes. However, transcription alone is insufficient to recruit AID activity. Comparison of genes similarly transcribed in B cells and MEFs but targeted in only one of the two cell types reveals a common set of epigenetic features associated with AID recruitment in both cells. AID target genes are enriched in chromatin modifications associated with active enhancers (such as H3K27Ac) and marks of active transcription (such as H3K36me3) in both fibroblasts and B cells, indicating that these features are universal mediators of AID recruitment.
Subject(s)
B-Lymphocytes/enzymology , Cytidine Deaminase , Embryo, Mammalian/enzymology , Epigenesis, Genetic , Gene Targeting , Transcription, Genetic/physiology , Animals , B-Lymphocytes/cytology , Cell Line , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , Embryo, Mammalian/cytology , Fibroblasts/cytology , Fibroblasts/enzymology , Histones/genetics , Histones/metabolism , Immunoglobulins/biosynthesis , Immunoglobulins/genetics , Mice , Mice, KnockoutABSTRACT
MOTIVATION: The detection of genomic regions unusually rich in a given pattern is an important undertaking in the analysis of next-generation sequencing data. Recent studies of chromosomal translocations in activated B lymphocytes have identified regions that are frequently translocated to c-myc oncogene. A quantitative method for the identification of translocation hotspots was crucial to this study. Here we improve this analysis by using a simple probabilistic model and the framework provided by scan statistics to define the number and location of translocation breakpoint hotspots. A key feature of our method is that it provides a global chromosome-wide nominal control level to clustering, as opposed to previous methods based on local criteria. While being motivated by a specific application, the detection of unusual clusters is a widespread problem in bioinformatics. We expect our method to be useful in the analysis of data from other experimental approaches such as of ChIP-seq and 4C-seq. RESULTS: The analysis of translocations from B lymphocytes with the method described here reveals the presence of longer hotspots when compared with those defined previously. Further, we show that the hotspot size changes substantially in the absence of DNA repair protein 53BP1. When 53BP1 deficiency is combined with overexpression of activation-induced cytidine deaminase, the hotspot length increases even further. These changes are not detected by previous methods that use local significance criteria for clustering. Our method is also able to identify several exclusive translocation hotspots located in genes of known tumor supressors. AVAILABILITY AND IMPLEMENTATION: The detection of translocation hotspots is done with hot_scan, a program implemented in R and Perl. Source code and documentation are freely available for download at https://github.com/itojal/hot_scan.
Subject(s)
Biometry/methods , Genomics/methods , Translocation, Genetic/genetics , B-Lymphocytes/metabolism , Chromosome Breakpoints , Cluster Analysis , Cytidine Deaminase/genetics , DNA Repair , High-Throughput Nucleotide Sequencing , Models, StatisticalABSTRACT
Classical nonhomologous DNA end-joining (C-NHEJ), which is a major DNA double-strand break (DSB) repair pathway in mammalian cells, plays a dominant role in joining DSBs during Ig heavy chain (IgH) class switch recombination (CSR) in activated B lymphocytes. However, in B cells deficient for one or more requisite C-NHEJ factors, such as DNA ligase 4 (Lig4) or XRCC4, end-joining during CSR occurs by a distinct alternative end-joining (A-EJ) pathway. A-EJ also has been implicated in joining DSBs found in oncogenic chromosomal translocations. DNA ligase 3 (Lig3) and its cofactor XRCC1 are widely considered to be requisite A-EJ factors, based on biochemical studies or extrachromosomal substrate end-joining studies. However, potential roles for these factors in A-EJ of endogenous chromosomal DSBs have not been tested. Here, we report that Xrcc1 inactivation via conditional gene-targeted deletion in WT or XRCC4-deficient primary B cells does not have an impact on either CSR or IgH/c-myc translocations in activated B lymphocytes. Indeed, homozygous deletion of Xrcc1 does not impair A-EJ of I-SceI-induced DSBs in XRCC4-deficient pro-B-cell lines. Correspondingly, substantial depletion of Lig3 in Lig4-deficient primary B cells or B-cell lines does not impair A-EJ of CSR-mediated DSBs or formation of IgH/c-myc translocations. Our findings firmly demonstrate that XRCC1 is not a requisite factor for A-EJ of chromosomal DSBs and raise the possibility that DNA ligase 1 (Lig1) may contribute more to A-EJ than previously considered.
Subject(s)
DNA Repair , DNA-Binding Proteins/physiology , Animals , B-Lymphocytes/cytology , Cell Lineage , Mice , Translocation, Genetic , X-ray Repair Cross Complementing Protein 1ABSTRACT
HIV-1 anti-retroviral therapy is highly effective but fails to eliminate a reservoir of latent proviruses leading to a requirement for life-long treatment. How the site of integration of authentic intact latent proviruses might impact their own or neighboring gene expression or reservoir dynamics is poorly understood. Here we report on proviral and neighboring gene transcription at sites of intact latent HIV-1 integration in cultured T cells obtained directly from people living with HIV, as well as engineered primary T cells and cell lines. Proviral gene expression was correlated to the level of endogenous gene expression under resting but not activated conditions. Notably, latent proviral promoters were 10010,000X less active than in productively infected cells and had little or no measurable impact on neighboring gene expression under resting or activated conditions. Thus, the site of integration has a dominant effect on the transcriptional activity of intact HIV-1 proviruses in the latent reservoir thereby influencing cytopathic effects and proviral immune evasion.
ABSTRACT
HIV-1 antiretroviral therapy is highly effective but fails to eliminate a reservoir of latent proviruses, leading to a requirement for life-long treatment. How the site of integration of authentic intact latent proviruses might impact their own or neighboring gene expression or reservoir dynamics is poorly understood. Here, we report on proviral and neighboring gene transcription at sites of intact latent HIV-1 integration in cultured T cells obtained directly from people living with HIV, as well as engineered primary T cells and cell lines. Proviral gene expression was correlated to the level of endogenous gene expression under resting but not activated conditions. Notably, latent proviral promoters were 100-10,000× less active than in productively infected cells and had little or no measurable impact on neighboring gene expression under resting or activated conditions. Thus, the site of integration has a dominant effect on the transcriptional activity of intact HIV-1 proviruses in the latent reservoir, thereby influencing cytopathic effects and proviral immune evasion.