RESUMO
Somatic hypermutation (SHM), initiated by activation-induced cytidine deaminase (AID), generates mutations in the antibody-coding sequence to allow affinity maturation. Why these mutations intrinsically focus on the three nonconsecutive complementarity-determining regions (CDRs) remains enigmatic. Here, we found that predisposition mutagenesis depends on the single-strand (ss) DNA substrate flexibility determined by the mesoscale sequence surrounding AID deaminase motifs. Mesoscale DNA sequences containing flexible pyrimidine-pyrimidine bases bind effectively to the positively charged surface patches of AID, resulting in preferential deamination activities. The CDR hypermutability is mimicable in in vitro deaminase assays and is evolutionarily conserved among species using SHM as a major diversification strategy. We demonstrated that mesoscale sequence alterations tune the in vivo mutability and promote mutations in an otherwise cold region in mice. Our results show a non-coding role of antibody-coding sequence in directing hypermutation, paving the way for the synthetic design of humanized animal models for optimal antibody discovery and explaining the AID mutagenesis pattern in lymphoma.
Assuntos
Citidina Desaminase , Hipermutação Somática de Imunoglobulina , Animais , Camundongos , Anticorpos/genética , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , DNA/genética , DNA de Cadeia Simples , Mutação , Evolução Molecular , Regiões Determinantes de Complementaridade/genética , Motivos de NucleotídeosRESUMO
Recurrent chromosomal translocations are characteristic features of many types of cancers, especially lymphomas and leukemias. Several basic mechanistic factors are required for the generation of most translocations. First, DNA double-strand breaks (DSBs) must be present simultaneously at the two participating loci. Second, the two broken loci must either be in proximity or be moved into proximity to be joined. Finally, cellular DNA repair pathways must be available to join the two broken loci to complete the translocation. These mechanistic factors can vary in different normal and mutant cells and, as a result, substantially influence the frequency at which particular translocations are generated in a given cell type. Ultimately, however, appearance of recurrent oncogenic translocations in tumors is, in most cases, strongly influenced by selection for the translocated oncogene during the tumorigenesis process. In this review, we discuss in depth the factors and pathways that contribute to the generation of translocations in lymphocytes and other cell types. We also discuss recent findings regarding mechanisms that underlie the appearance of recurrent translocations in tumors.
Assuntos
Linfócitos/metabolismo , Translocação Genética , Animais , Citidina Desaminase/genética , Quebras de DNA de Cadeia Dupla , Rearranjo Gênico do Linfócito B , Humanos , Leucemia/genética , Linfoma/genéticaRESUMO
RAG endonuclease initiates antibody heavy chain variable region exon assembly from V, D, and J segments within a chromosomal V(D)J recombination center (RC) by cleaving between paired gene segments and flanking recombination signal sequences (RSSs). The IGCR1 control region promotes DJH intermediate formation by isolating Ds, JHs, and RCs from upstream VHs in a chromatin loop anchored by CTCF-binding elements (CBEs). How VHs access the DJHRC for VH to DJH rearrangement was unknown. We report that CBEs immediately downstream of frequently rearranged VH-RSSs increase recombination potential of their associated VH far beyond that provided by RSSs alone. This CBE activity becomes particularly striking upon IGCR1 inactivation, which allows RAG, likely via loop extrusion, to linearly scan chromatin far upstream. VH-associated CBEs stabilize interactions of D-proximal VHs first encountered by the DJHRC during linear RAG scanning and thereby promote dominant rearrangement of these VHs by an unanticipated chromatin accessibility-enhancing CBE function.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Proteínas de Homeodomínio/metabolismo , Recombinação V(D)J , Animais , Linhagem Celular , DNA Intergênico/genética , DNA Intergênico/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mutagênese , Sinais Direcionadores de Proteínas , RNA Guia de Cinetoplastídeos/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
While neutralizing antibodies are highly effective against ebolavirus infections, current experimental ebolavirus vaccines primarily elicit species-specific antibody responses. Here, we describe an immunization-elicited macaque antibody (CA45) that clamps the internal fusion loop with the N terminus of the ebolavirus glycoproteins (GPs) and potently neutralizes Ebola, Sudan, Bundibugyo, and Reston viruses. CA45, alone or in combination with an antibody that blocks receptor binding, provided full protection against all pathogenic ebolaviruses in mice, guinea pigs, and ferrets. Analysis of memory B cells from the immunized macaque suggests that elicitation of broadly neutralizing antibodies (bNAbs) for ebolaviruses is possible but difficult, potentially due to the rarity of bNAb clones and their precursors. Unexpectedly, germline-reverted CA45, while exhibiting negligible binding to full-length GP, bound a proteolytically remodeled GP with picomolar affinity, suggesting that engineered ebolavirus vaccines could trigger rare bNAb precursors more robustly. These findings have important implications for developing pan-ebolavirus vaccine and immunotherapeutic cocktails.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Vacinas contra Ebola/imunologia , Doença pelo Vírus Ebola/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Regiões Determinantes de Complementaridade , Reações Cruzadas , Ebolavirus/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/imunologia , Feminino , Furões , Cobaias , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos MolecularesRESUMO
Repair of DNA double-strand breaks (DSBs) by non-homologous end joining is critical for neural development, and brain cells frequently contain somatic genomic variations that might involve DSB intermediates. We now use an unbiased, high-throughput approach to identify genomic regions harboring recurrent DSBs in primary neural stem/progenitor cells (NSPCs). We identify 27 recurrent DSB clusters (RDCs), and remarkably, all occur within gene bodies. Most of these NSPC RDCs were detected only upon mild, aphidicolin-induced replication stress, providing a nucleotide-resolution view of replication-associated genomic fragile sites. The vast majority of RDCs occur in long, transcribed, and late-replicating genes. Moreover, almost 90% of identified RDC-containing genes are involved in synapse function and/or neural cell adhesion, with a substantial fraction also implicated in tumor suppression and/or mental disorders. Our characterization of NSPC RDCs reveals a basis of gene fragility and suggests potential impacts of DNA breaks on neurodevelopment and neural functions.
Assuntos
Quebras de DNA , Células-Tronco Neurais/metabolismo , Animais , Afidicolina/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Encéfalo/citologia , Adesão Celular , Moléculas de Adesão Celular Neuronais/metabolismo , Quebras de DNA/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Proteínas Ligadas por GPI/metabolismo , Genoma , Humanos , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Sinapses , Fatores de Transcrição/metabolismo , Translocação GenéticaRESUMO
The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2(∗)02 segment to undergo normal V(D)J recombination and, thereby, leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies, IGHV1-2(∗)02-rearranging mice, which also express a VRC01-antibody precursor light chain, can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , HIV-1/imunologia , Imunização , Cadeias Pesadas de Imunoglobulinas/imunologia , Células Precursoras de Linfócitos B/imunologia , Animais , Anticorpos Monoclonais/genética , Linfócitos B/imunologia , Anticorpos Amplamente Neutralizantes , Linhagem Celular , Modelos Animais de Doenças , Regulação da Expressão Gênica/imunologia , Anticorpos Anti-HIV , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Concentração Inibidora 50 , Camundongos , Deleção de Sequência , Linfócitos T/imunologiaRESUMO
Vaccine elicitation of broadly neutralizing antibodies (bnAbs) is a key HIV-research goal. The VRC01 class of bnAbs targets the CD4-binding site on the HIV-envelope trimer and requires extensive somatic hypermutation (SHM) to neutralize effectively. Despite substantial progress, vaccine-induced VRC01-class antibodies starting from unmutated precursors have exhibited limited neutralization breadth, particularly against viruses bearing glycan on loop D residue N276 (glycan276), present on most circulating strains. Here, using sequential immunization of immunoglobulin (Ig)-humanized mice expressing diverse unmutated VRC01-class antibody precursors, we elicited serum responses capable of neutralizing viruses bearing glycan276 and isolated multiple lineages of VRC01-class bnAbs, including two with >50% breadth on a 208-strain panel. Crystal structures of representative bnAbs revealed the same mode of recognition as known VRC01-class bnAbs. Structure-function studies further pinpointed key mutations and correlated their induction with specific immunizations. VRC01-class bnAbs can thus be matured by sequential immunization from unmutated ancestors to >50% breadth, and we delineate immunogens and regimens inducing key SHM.
Assuntos
Vacinas contra a AIDS/imunologia , Linfócitos B/imunologia , Anticorpos Amplamente Neutralizantes/metabolismo , Anticorpos Anti-HIV/metabolismo , Infecções por HIV/imunologia , HIV-1/fisiologia , Mutação/genética , Animais , Anticorpos Amplamente Neutralizantes/genética , Modelos Animais de Doenças , Células HEK293 , Anticorpos Anti-HIV/genética , Humanos , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Hipermutação Somática de Imunoglobulina , VacinaçãoRESUMO
RAG initiates antibody V(D)J recombination in developing lymphocytes by generating "on-target" DNA breaks at matched pairs of bona fide recombination signal sequences (RSSs). We employ bait RAG-generated breaks in endogenous or ectopically inserted RSS pairs to identify huge numbers of RAG "off-target" breaks. Such breaks occur at the simple CAC motif that defines the RSS cleavage site and are largely confined within convergent CTCF-binding element (CBE)-flanked loop domains containing bait RSS pairs. Marked orientation dependence of RAG off-target activity within loops spanning up to 2 megabases implies involvement of linear tracking. In this regard, major RAG off-targets in chromosomal translocations occur as convergent RSS pairs at enhancers within a loop. Finally, deletion of a CBE-based IgH locus element disrupts V(D)J recombination domains and, correspondingly, alters RAG on- and off-target distributions within IgH. Our findings reveal how RAG activity is developmentally focused and implicate mechanisms by which chromatin domains harness biological processes within them.
Assuntos
Cromossomos de Mamíferos/metabolismo , Sequências Reguladoras de Ácido Nucleico , Recombinação V(D)J , Animais , Fator de Ligação a CCCTC , Cromossomos de Mamíferos/química , Proteínas de Ligação a DNA/metabolismo , Genes myc , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Homeodomínio/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Linfoma/genética , Camundongos , Motivos de Nucleotídeos , Proteínas Repressoras/metabolismo , Análise de Sequência de DNA , Translocação GenéticaRESUMO
In activated B lymphocytes, AID initiates antibody variable (V) exon somatic hypermutation (SHM) for affinity maturation in germinal centers (GCs) and IgH switch (S) region DNA breaks (DSBs) for class-switch recombination (CSR). To resolve long-standing questions, we have developed an in vivo assay to study AID targeting of passenger sequences replacing a V exon. First, we find AID targets SHM hotspots within V exon and S region passengers at similar frequencies and that the normal SHM process frequently generates deletions, indicating that SHM and CSR employ the same mechanism. Second, AID mutates targets in diverse non-Ig passengers in GC B cells at levels similar to those of V exons, definitively establishing the V exon location as "privileged" for SHM. Finally, Peyer's patch GC B cells generate a reservoir of V exons that are highly mutated before selection for affinity maturation. We discuss the implications of these findings for harnessing antibody diversification mechanisms.
Assuntos
Linfócitos B/metabolismo , Citidina Desaminase/genética , Switching de Imunoglobulina , Hipermutação Somática de Imunoglobulina , Recombinação V(D)J , Animais , Humanos , Camundongos , Mutação , Globinas beta/genéticaRESUMO
In developing B cells, V(D)J recombination assembles exons encoding IgH and Igκ variable regions from hundreds of gene segments clustered across Igh and Igk loci. V, D and J gene segments are flanked by conserved recombination signal sequences (RSSs) that target RAG endonuclease1. RAG orchestrates Igh V(D)J recombination upon capturing a JH-RSS within the JH-RSS-based recombination centre1-3 (RC). JH-RSS orientation programmes RAG to scan upstream D- and VH-containing chromatin that is presented in a linear manner by cohesin-mediated loop extrusion4-7. During Igh scanning, RAG robustly utilizes only D-RSSs or VH-RSSs in convergent (deletional) orientation with JH-RSSs4-7. However, for Vκ-to-Jκ joining, RAG utilizes Vκ-RSSs from deletional- and inversional-oriented clusters8, inconsistent with linear scanning2. Here we characterize the Vκ-to-Jκ joining mechanism. Igk undergoes robust primary and secondary rearrangements9,10, which confounds scanning assays. We therefore engineered cells to undergo only primary Vκ-to-Jκ rearrangements and found that RAG scanning from the primary Jκ-RC terminates just 8 kb upstream within the CTCF-site-based Sis element11. Whereas Sis and the Jκ-RC barely interacted with the Vκ locus, the CTCF-site-based Cer element12 4 kb upstream of Sis interacted with various loop extrusion impediments across the locus. Similar to VH locus inversion7, DJH inversion abrogated VH-to-DJH joining; yet Vκ locus or Jκ inversion allowed robust Vκ-to-Jκ joining. Together, these experiments implicated loop extrusion in bringing Vκ segments near Cer for short-range diffusion-mediated capture by RC-based RAG. To identify key mechanistic elements for diffusional V(D)J recombination in Igk versus Igh, we assayed Vκ-to-JH and D-to-Jκ rearrangements in hybrid Igh-Igk loci generated by targeted chromosomal translocations, and pinpointed remarkably strong Vκ and Jκ RSSs. Indeed, RSS replacements in hybrid or normal Igk and Igh loci confirmed the ability of Igk-RSSs to promote robust diffusional joining compared with Igh-RSSs. We propose that Igk evolved strong RSSs to mediate diffusional Vκ-to-Jκ joining, whereas Igh evolved weaker RSSs requisite for modulating VH joining by RAG-scanning impediments.
Assuntos
Cadeias Pesadas de Imunoglobulinas , Região de Junção de Imunoglobulinas , Região Variável de Imunoglobulina , Cadeias kappa de Imunoglobulina , Recombinação V(D)J , Animais , Feminino , Masculino , Camundongos , Alelos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Cromatina/genética , Cromatina/metabolismo , Cromatina/química , Coesinas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Cadeias Pesadas de Imunoglobulinas/genética , Região de Junção de Imunoglobulinas/genética , Cadeias kappa de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Recombinação V(D)J/genéticaRESUMO
Activation-induced cytidine deaminase (AID) initiates both somatic hypermutation (SHM) for antibody affinity maturation and DNA breakage for antibody class switch recombination (CSR) via transcription-dependent cytidine deamination of single-stranded DNA targets. Though largely specific for immunoglobulin genes, AID also acts on a limited set of off-targets, generating oncogenic translocations and mutations that contribute to B cell lymphoma. How AID is recruited to off-targets has been a long-standing mystery. Based on deep GRO-seq studies of mouse and human B lineage cells activated for CSR or SHM, we report that most robust AID off-target translocations occur within highly focal regions of target genes in which sense and antisense transcription converge. Moreover, we found that such AID-targeting "convergent" transcription arises from antisense transcription that emanates from super-enhancers within sense transcribed gene bodies. Our findings provide an explanation for AID off-targeting to a small subset of mostly lineage-specific genes in activated B cells.
Assuntos
Citidina Desaminase/metabolismo , Elementos Facilitadores Genéticos , Instabilidade Genômica , Transcrição Gênica , Animais , Linfócitos B/metabolismo , Humanos , Switching de Imunoglobulina , Camundongos , Sítio de Iniciação de TranscriçãoRESUMO
Chromosomal translocations involving antigen receptor loci are common in lymphoid malignancies. Translocations require DNA double-strand breaks (DSBs) at two chromosomal sites, their physical juxtaposition, and their fusion by end-joining. Ability of lymphocytes to generate diverse repertoires of antigen receptors and effector antibodies derives from programmed genomic alterations that produce DSBs. We discuss these lymphocyte-specific processes, with a focus on mechanisms that provide requisite DSB target specificity and mechanisms that suppress DSB translocation. We also discuss recent work that provides new insights into DSB repair pathways and the influences of three-dimensional genome organization on physiological processes and cancer genomes.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Instabilidade Genômica , Linfócitos/metabolismo , Recombinação V(D)J , Animais , Humanos , Linfócitos/imunologia , Linfoma/genética , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos T/genética , Translocação GenéticaRESUMO
The DNA damage response (DDR) protein 53BP1 protects DNA ends from excessive resection in G1, and thereby favors repair by nonhomologous end-joining (NHEJ) as opposed to homologous recombination (HR). During S phase, BRCA1 antagonizes 53BP1 to promote HR. The pro-NHEJ and antirecombinase functions of 53BP1 are mediated in part by RIF1, the only known factor that requires 53BP1 phosphorylation for its recruitment to double-strand breaks (DSBs). Here, we show that a 53BP1 phosphomutant, 53BP18A, comprising alanine substitutions of the eight most N-terminal S/TQ phosphorylation sites, mimics 53BP1 deficiency by restoring genome stability in BRCA1-deficient cells yet behaves like wild-type 53BP1 with respect to immunoglobulin class switch recombination (CSR). 53BP18A recruits RIF1 but fails to recruit the DDR protein PTIP to DSBs, and disruption of PTIP phenocopies 53BP18A. We conclude that 53BP1 promotes productive CSR and suppresses mutagenic DNA repair through distinct phosphodependent interactions with RIF1 and PTIP.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Switching de Imunoglobulina , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Animais , Linfócitos B/metabolismo , Proteína BRCA1/metabolismo , Proteínas Cromossômicas não Histona/genética , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Instabilidade Genômica , Camundongos , Mutação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
The B cell antigen receptor (BCR) is composed of a membrane-bound class M, D, G, E or A immunoglobulin for antigen recognition1-3 and a disulfide-linked Igα (also known as CD79A) and Igß (also known as CD79B) heterodimer (Igα/ß) that functions as the signalling entity through intracellular immunoreceptor tyrosine-based activation motifs (ITAMs)4,5. The organizing principle of the BCR remains unknown. Here we report cryo-electron microscopy structures of mouse full-length IgM BCR and its Fab-deleted form. At the ectodomain (ECD), the Igα/ß heterodimer mainly uses Igα to associate with Cµ3 and Cµ4 domains of one heavy chain (µHC) while leaving the other heavy chain (µHC') unbound. The transmembrane domain (TMD) helices of µHC and µHC' interact with those of the Igα/ß heterodimer to form a tight four-helix bundle. The asymmetry at the TMD prevents the recruitment of two Igα/ß heterodimers. Notably, the connecting peptide between the ECD and TMD of µHC intervenes in between those of Igα and Igß to guide TMD assembly through charge complementarity. Weaker but distinct density for the Igß ITAM nestles next to the TMD, suggesting potential autoinhibition of ITAM phosphorylation. Interfacial analyses suggest that all BCR classes utilize a general organizational architecture. Our studies provide a structural platform for understanding B cell signalling and designing rational therapies against BCR-mediated diseases.
Assuntos
Microscopia Crioeletrônica , Receptores de Antígenos de Linfócitos B , Animais , Camundongos , Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/biossíntese , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/ultraestrutura , Transdução de Sinais , Fragmentos Fab das Imunoglobulinas , Domínios Proteicos , FosforilaçãoRESUMO
An important class of HIV-1 broadly neutralizing antibodies, termed the VRC01 class, targets the conserved CD4-binding site (CD4bs) of the envelope glycoprotein (Env). An engineered Env outer domain (OD) eOD-GT8 60-mer nanoparticle has been developed as a priming immunogen for eliciting VRC01-class precursors and is planned for clinical trials. However, a substantial portion of eOD-GT8-elicited antibodies target non-CD4bs epitopes, potentially limiting its efficacy. We introduced N-linked glycans into non-CD4bs surfaces of eOD-GT8 to mask irrelevant epitopes and evaluated these mutants in a mouse model that expressed diverse immunoglobulin heavy chains containing human IGHV1-2∗02, the germline VRC01 VH segment. Compared to the parental eOD-GT8, a mutant with five added glycans stimulated significantly higher proportions of CD4bs-specific serum responses and CD4bs-specific immunoglobulin G+ B cells including VRC01-class precursors. These results demonstrate that glycan masking can limit elicitation of off-target antibodies and focus immune responses to the CD4bs, a major target of HIV-1 vaccine design.
Assuntos
Anticorpos Neutralizantes/imunologia , Sítios de Ligação de Anticorpos/imunologia , Antígenos CD4/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Amplamente Neutralizantes , Linhagem Celular , Feminino , Técnicas de Introdução de Genes , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Polissacarídeos/químicaRESUMO
The extent to which the three-dimensional organization of the genome contributes to chromosomal translocations is an important question in cancer genomics. We generated a high-resolution Hi-C spatial organization map of the G1-arrested mouse pro-B cell genome and used high-throughput genome-wide translocation sequencing to map translocations from target DNA double-strand breaks (DSBs) within it. RAG endonuclease-cleaved antigen-receptor loci are dominant translocation partners for target DSBs regardless of genomic position, reflecting high-frequency DSBs at these loci and their colocalization in a fraction of cells. To directly assess spatial proximity contributions, we normalized genomic DSBs via ionizing radiation. Under these conditions, translocations were highly enriched in cis along single chromosomes containing target DSBs and within other chromosomes and subchromosomal domains in a manner directly related to pre-existing spatial proximity. By combining two high-throughput genomic methods in a genetically tractable system, we provide a new lens for viewing cancer genomes.
Assuntos
Genoma , Neoplasias/genética , Translocação Genética , Animais , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Fase G1 , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Células Precursoras de Linfócitos B/citologia , Receptores de Antígenos/genéticaRESUMO
Extrachromosomal circular DNA elements (eccDNAs) have been described in the literature for several decades, and are known for their broad existence across different species1,2. However, their biogenesis and functions are largely unknown. By developing a new circular DNA enrichment method, here we purified and sequenced full-length eccDNAs with Nanopore sequencing. We found that eccDNAs map across the entire genome in a close to random manner, suggesting a biogenesis mechanism of random ligation of genomic DNA fragments. Consistent with this idea, we found that apoptosis inducers can increase eccDNA generation, which is dependent on apoptotic DNA fragmentation followed by ligation by DNA ligase 3. Importantly, we demonstrated that eccDNAs can function as potent innate immunostimulants in a manner that is independent of eccDNA sequence but dependent on eccDNA circularity and the cytosolic DNA sensor Sting. Collectively, our study not only revealed the origin, biogenesis and immunostimulant function of eccDNAs but also uncovered their sensing pathway and potential clinical implications in immune response.
Assuntos
Apoptose , Fragmentação do DNA , DNA Circular/biossíntese , DNA Circular/imunologia , Imunidade Inata , Animais , Células Cultivadas , Mapeamento Cromossômico , DNA Ligase Dependente de ATP/metabolismo , DNA Circular/genética , DNA Circular/isolamento & purificação , Endodesoxirribonucleases/metabolismo , Regulação da Expressão Gênica , Genoma/genética , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Proteínas de Ligação a Poli-ADP-Ribose/metabolismoRESUMO
RAG endonuclease initiates Igh V(D)J recombination in progenitor B cells by binding a JH-recombination signal sequence (RSS) within a recombination centre (RC) and then linearly scanning upstream chromatin, presented by loop extrusion mediated by cohesin, for convergent D-RSSs1,2. The utilization of convergently oriented RSSs and cryptic RSSs is intrinsic to long-range RAG scanning3. Scanning of RAG from the DJH-RC-RSS to upstream convergent VH-RSSs is impeded by D-proximal CTCF-binding elements (CBEs)2-5. Primary progenitor B cells undergo a mechanistically undefined contraction of the VH locus that is proposed to provide distal VHs access to the DJH-RC6-9. Here we report that an inversion of the entire 2.4-Mb VH locus in mouse primary progenitor B cells abrogates rearrangement of both VH-RSSs and normally convergent cryptic RSSs, even though locus contraction still occurs. In addition, this inversion activated both the utilization of cryptic VH-RSSs that are normally in opposite orientation and RAG scanning beyond the VH locus through several convergent CBE domains to the telomere. Together, these findings imply that broad deregulation of CBE impediments in primary progenitor B cells promotes RAG scanning of the VH locus mediated by loop extrusion. We further found that the expression of wings apart-like protein homologue (WAPL)10, a cohesin-unloading factor, was low in primary progenitor B cells compared with v-Abl-transformed progenitor B cell lines that lacked contraction and RAG scanning of the VH locus. Correspondingly, depletion of WAPL in v-Abl-transformed lines activated both processes, further implicating loop extrusion in the locus contraction mechanism.
Assuntos
Linfócitos B/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteínas de Homeodomínio/metabolismo , Cadeias Pesadas de Imunoglobulinas/genética , Conformação de Ácido Nucleico , Animais , Linfócitos B/citologia , Linfócitos B/enzimologia , Linhagem Celular , Células Cultivadas , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Endonucleases/deficiência , Endonucleases/genética , Pontos de Checagem da Fase G1 do Ciclo Celular , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas/genética , Proteínas/metabolismo , Recombinação V(D)J/genéticaRESUMO
In activated B cells, activation-induced cytidine deaminase (AID) generates programmed DNA lesions required for antibody class switch recombination (CSR), which may also threaten genome integrity. AID dynamically shuttles between cytoplasm and nucleus, and the majority stays in the cytoplasm due to active nuclear export mediated by its C-terminal peptide. In immunodeficient-patient cells expressing mutant AID lacking its C-terminus, a catalytically active AID-delC protein accumulates in the nucleus but nevertheless fails to support CSR. To resolve this apparent paradox, we dissected the function of AID-delC proteins in the CSR process and found that they cannot efficiently target antibody genes. We demonstrate that AID-delC proteins form condensates both in vivo and in vitro, dependent on its N-terminus and on a surface arginine-rich patch. Co-expression of AID-delC and wild-type AID leads to an unbalanced nuclear AID-delC/AID ratio, with AID-delC proteins able to trap wild-type AID in condensates, resulting in a dominant-negative phenotype that could contribute to immunodeficiency. The co-condensation model of mutant and wild-type proteins could be an alternative explanation for the dominant-negative effect in genetic disorders.
Assuntos
Citidina Desaminase , Switching de Imunoglobulina , Linfócitos B , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , DNA/metabolismo , Humanos , Switching de Imunoglobulina/genéticaRESUMO
Monoclonal antibodies (mAbs) are an important class of antiviral therapeutics. MAbs are highly selective, well tolerated, and have long in vivo half-life as well as the capacity to induce immune-mediated virus clearance. Their activities can be further enhanced by integration of their variable fragments (Fvs) into bispecific antibodies (bsAbs), affording simultaneous targeting of multiple epitopes to improve potency and breadth and/or to mitigate against viral escape by a single mutation. Here, we explore a bsAb strategy for generation of pan-ebolavirus and pan-filovirus immunotherapeutics. Filoviruses, including Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV), cause severe hemorrhagic fever. Although there are two FDA-approved mAb therapies for EBOV infection, these do not extend to other filoviruses. Here, we combine Fvs from broad ebolavirus mAbs to generate novel pan-ebolavirus bsAbs that are potently neutralizing, confer protection in mice, and are resistant to viral escape. Moreover, we combine Fvs from pan-ebolavirus mAbs with those of protective MARV mAbs to generate pan-filovirus protective bsAbs. These results provide guidelines for broad antiviral bsAb design and generate new immunotherapeutic candidates.