RESUMO
To produce a diverse antibody repertoire, immunoglobulin heavy-chain (Igh) loci undergo large-scale alterations in structure to facilitate juxtaposition and recombination of spatially separated variable (VH), diversity (DH), and joining (JH) genes. These chromosomal alterations are poorly understood. Uncovering their patterns shows how chromosome dynamics underpins antibody diversity. Using tiled Capture Hi-C, we produce a comprehensive map of chromatin interactions throughout the 2.8-Mb Igh locus in progenitor B cells. We find that the Igh locus folds into semi-rigid subdomains and undergoes flexible looping of the VH genes to its 3' end, reconciling two views of locus organization. Deconvolution of single Igh locus conformations using polymer simulations identifies thousands of different structures. This heterogeneity may underpin the diversity of V(D)J recombination events. All three immunoglobulin loci also participate in a highly specific, developmentally regulated network of interchromosomal interactions with genes encoding B cell-lineage factors. This suggests a model of interchromosomal coordination of B cell development.
Assuntos
Linfócitos B , Imunoglobulinas , Recombinação V(D)J/genética , Genes de Cadeia Pesada de Imunoglobulina/genética , Células Precursoras de Linfócitos BRESUMO
Generation of the primary antibody repertoire requires V(D)J recombination of hundreds of gene segments in the immunoglobulin heavy chain (Igh) locus. The role of interleukin-7 receptor (IL-7R) signaling in Igh recombination has been difficult to partition from its role in B cell survival and proliferation. With a detailed description of the Igh repertoire in murine IL-7Rα-/- bone marrow B cells, we demonstrate that IL-7R signaling profoundly influences VH gene selection during VH-to-DJH recombination. We find skewing toward 3' VH genes during de novo VH-to-DJH recombination more severe than the fetal liver (FL) repertoire and uncover a role for IL-7R signaling in DH-to-JH recombination. Transcriptome and accessibility analyses suggest reduced expression of B lineage transcription factors (TFs) and targets and loss of DH and VH antisense transcription in IL-7Rα-/- B cells. Thus, in addition to its roles in survival and proliferation, IL-7R signaling shapes the Igh repertoire by activating underpinning mechanisms.
Assuntos
Diversidade de Anticorpos/genética , Linfócitos B/metabolismo , Medula Óssea/metabolismo , Genes de Cadeia Pesada de Imunoglobulina , Região Variável de Imunoglobulina/genética , Receptores de Interleucina-7/metabolismo , Transdução de Sinais , Animais , Sequência de Bases , Linhagem da Célula/genética , Cromatina/metabolismo , DNA Intergênico/genética , Feto/metabolismo , Fígado/embriologia , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Motivos de Nucleotídeos/genética , Fator de Transcrição PAX5/metabolismo , Transativadores/metabolismo , Transcrição GênicaRESUMO
The generation of protective humoral immunity after vaccination relies on the productive interaction between antigen-specific B cells and T follicular helper (Tfh) cells. Despite the central role of Tfh cells in vaccine responses, there is currently no validated way to enhance their differentiation in humans. From paired human lymph node and blood samples, we identify a population of circulating Tfh cells that are transcriptionally and clonally similar to germinal center Tfh cells. In a clinical trial of vaccine formulations, circulating Tfh cells were expanded in Tanzanian volunteers when an experimental malaria vaccine was adjuvanted in GLA-SE but not when formulated in Alum. The GLA-SE-formulated peptide was associated with an increase in the extrafollicular antibody response, long-lived antibody production, and the emergence of public TCRß clonotypes in circulating Tfh cells. We demonstrate that altering vaccine adjuvants is a rational approach for enhancing Tfh cells in humans, thereby supporting the long-lived humoral immunity that is required for effective vaccines.
Assuntos
Adjuvantes Imunológicos/farmacologia , Composição de Medicamentos/métodos , Glucosídeos/farmacologia , Lipídeo A/farmacologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Vacinação/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Hidróxido de Alumínio/farmacologia , Anticorpos Antivirais/efeitos dos fármacos , Anticorpos Antivirais/imunologia , Antígenos de Protozoários/imunologia , Linfócitos B/imunologia , Células Cultivadas , Feminino , Centro Germinativo/imunologia , Humanos , Imunidade Humoral/imunologia , Vacinas contra Influenza/imunologia , Linfonodos/imunologia , Vacinas Antimaláricas/imunologia , Masculino , Pessoa de Meia-Idade , Plasmodium falciparum/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Adulto JovemRESUMO
BACKGROUND: Aging is characterized by loss of function of the adaptive immune system, but the underlying causes are poorly understood. To assess the molecular effects of aging on B cell development, we profiled gene expression and chromatin features genome-wide, including histone modifications and chromosome conformation, in bone marrow pro-B and pre-B cells from young and aged mice. RESULTS: Our analysis reveals that the expression levels of most genes are generally preserved in B cell precursors isolated from aged compared with young mice. Nonetheless, age-specific expression changes are observed at numerous genes, including microRNA encoding genes. Importantly, these changes are underpinned by multi-layered alterations in chromatin structure, including chromatin accessibility, histone modifications, long-range promoter interactions, and nuclear compartmentalization. Previous work has shown that differentiation is linked to changes in promoter-regulatory element interactions. We find that aging in B cell precursors is accompanied by rewiring of such interactions. We identify transcriptional downregulation of components of the insulin-like growth factor signaling pathway, in particular downregulation of Irs1 and upregulation of Let-7 microRNA expression, as a signature of the aged phenotype. These changes in expression are associated with specific alterations in H3K27me3 occupancy, suggesting that Polycomb-mediated repression plays a role in precursor B cell aging. CONCLUSIONS: Changes in chromatin and 3D genome organization play an important role in shaping the altered gene expression profile of aged precursor B cells. Components of the insulin-like growth factor signaling pathways are key targets of epigenetic regulation in aging in bone marrow B cell precursors.
Assuntos
Envelhecimento/genética , Linfócitos B/metabolismo , Cromatina/química , Epigênese Genética , Somatomedinas/fisiologia , Transcriptoma , Envelhecimento/imunologia , Animais , Linfócitos B/imunologia , Regulação para Baixo , Genoma , Masculino , Camundongos Endogâmicos C57BL , Transdução de Sinais/genética , Células-Tronco/imunologia , Células-Tronco/metabolismoRESUMO
For high-throughput sequencing and quantification of immunoglobulin repertoires, most methodologies use RNA. However, output varies enormously between recombined genes due to different promoter strengths and differential activation of lymphocyte subsets, precluding quantitation of recombinants on a per-cell basis. To date, DNA-based approaches have used V gene primer cocktails, with substantial inherent biases. Here, we describe VDJ sequencing (VDJ-seq), which accurately quantitates immunoglobulin diversity at the DNA level in an unbiased manner. This is accomplished with a single primer-extension step using biotinylated J gene primers. By addition of unique molecular identifiers (UMIs) before primer extension, we reliably remove duplicate sequences and correct for sequencing and PCR errors. Furthermore, VDJ-seq captures productive and nonproductive VDJ and DJ recombination events on a per-cell basis. Library preparation takes 3 d, with 2 d of sequencing and 1 d of data processing and analysis.
Assuntos
Genes de Imunoglobulinas , Variação Genética , Imunoglobulinas/genética , Análise de Sequência de DNA/métodos , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , CamundongosRESUMO
V(D)J recombination is essential for the generation of diverse antigen receptor (AgR) repertoires. In B cells, immunoglobulin kappa (Igκ) light chain recombination follows immunoglobulin heavy chain (Igh) recombination. We recently developed the DNA-based VDJ-seq assay for the unbiased quantitation of Igh VH and DH repertoires. Integration of VDJ-seq data with genome-wide datasets revealed that two chromatin states at the recombination signal sequence (RSS) of VH genes are highly predictive of recombination in mouse pro-B cells. It is unknown whether local chromatin states contribute to Vκ gene choice during Igκ recombination. Here we adapt VDJ-seq to profile the Igκ VκJκ repertoire and present a comprehensive readout in mouse pre-B cells, revealing highly variable Vκ gene usage. Integration with genome-wide datasets for histone modifications, DNase hypersensitivity, transcription factor binding and germline transcription identified PU.1 binding at the RSS, which was unimportant for Igh, as highly predictive of whether a Vκ gene will recombine or not, suggesting that it plays a binary, all-or-nothing role, priming genes for recombination. Thereafter, the frequency with which these genes recombine was shaped both by the presence and level of enrichment of several other chromatin features, including H3K4 methylation and IKAROS binding. Moreover, in contrast to the Igh locus, the chromatin landscape of the promoter, as well as of the RSS, contributes to Vκ gene recombination. Thus, multiple facets of local chromatin features explain much of the variation in Vκ gene usage. Together, these findings reveal shared and divergent roles for epigenetic features and transcription factors in AgR V(D)J recombination and provide avenues for further investigation of chromatin signatures that may underpin V(D)J-mediated chromosomal translocations.
RESUMO
Variable (V), diversity (D), and joining (J) (V(D)J) recombination is the first determinant of antigen receptor diversity. Understanding how recombination is regulated requires a comprehensive, unbiased readout of V gene usage. We have developed VDJ sequencing (VDJ-seq), a DNA-based next-generation-sequencing technique that quantitatively profiles recombination products. We reveal a 200-fold range of recombination efficiency among recombining V genes in the primary mouse Igh repertoire. We used machine learning to integrate these data with local chromatin profiles to identify combinatorial patterns of epigenetic features that associate with active VH gene recombination. These features localize downstream of VH genes and are excised by recombination, revealing a class of cis-regulatory element that governs recombination, distinct from expression. We detect two mutually exclusive chromatin signatures at these elements, characterized by CTCF/RAD21 and PAX5/IRF4, which segregate with the evolutionary history of associated VH genes. Thus, local chromatin signatures downstream of VH genes provide an essential layer of regulation that determines recombination efficiency.
Assuntos
Cromatina/metabolismo , Recombinação V(D)J/genética , Algoritmos , Animais , Epigênese Genética , Evolução Molecular , Regulação da Expressão Gênica , Loci Gênicos , Proteínas de Homeodomínio/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Camundongos , Receptores de Antígenos , Análise de Sequência de DNA , Transcrição GênicaRESUMO
3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.
Assuntos
Sondas de DNA/química , DNA/química , Imageamento Tridimensional/métodos , Hibridização in Situ Fluorescente/métodos , Compostos Alílicos/química , Animais , Corantes Fluorescentes/química , Camundongos , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/químicaRESUMO
Defective V(D)J rearrangement of immunoglobulin heavy or light chain (IgH or IgL) or class switch recombination (CSR) can initiate chromosomal translocations. The DNA-damage kinase ATM is required for the suppression of chromosomal translocations but ATM regulation is incompletely understood. Here, we show that mice lacking the ATM cofactor ATMIN in B cells (ATMIN(ΔB/ΔB)) have impaired ATM signaling and develop B cell lymphomas. Notably, ATMIN(ΔB/ΔB) cells exhibited defective peripheral V(D)J rearrangement and CSR, resulting in translocations involving the Igh and Igl loci, indicating that ATMIN is required for efficient repair of DNA breaks generated during somatic recombination. Thus, our results identify a role for ATMIN in regulating the maintenance of genomic stability and tumor suppression in B cells.
Assuntos
Linfócitos B/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genes de Cadeia Pesada de Imunoglobulina , Genes de Cadeia Leve de Imunoglobulina , Instabilidade Genômica , Linfoma de Células B/prevenção & controle , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Antígenos CD19/genética , Antígenos CD19/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Linfócitos B/imunologia , Proteínas de Transporte/genética , Células Cultivadas , Quebras de DNA , Regulação Neoplásica da Expressão Gênica , Switching de Imunoglobulina , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Camundongos Nus , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Recombinação Genética , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genéticaRESUMO
B cell development is controlled by a series of checkpoints that ensure that the immunoglobulin (Ig)-encoding genes produce a functional B cell receptor (BCR) and antibodies. As part of this process, recombination-activating gene (Rag) proteins regulate the in-frame assembly of the Ig-encoding genes. The BCR consists of Ig proteins in complex with the immunoreceptor tyrosine-based activation motif (ITAM)-containing Igalpha and Igbeta chains. Whereas the activation of the tyrosine kinases Src and Syk is essential for BCR signaling, the pathways that act downstream of these kinases are incompletely defined. Previous work has revealed a key role for the p110delta isoform of phosphatidylinositol 3-kinase (PI3K) in agonist-induced BCR signaling; however, early B cell development and mature B cell survival, which depend on agonist-independent or "tonic" BCR signaling, are not substantially affected by a deficiency in p110delta. Here, we show that p110alpha, but not p110beta, compensated in the absence of p110delta to promote early B cell development in the bone marrow and B cell survival in the spleen. In the absence of both p110alpha and p110delta activities, pre-BCR signaling failed to suppress the production of Rag proteins and to promote developmental progression of B cell progenitors. Unlike p110delta, however, p110alpha did not contribute to agonist-induced BCR signaling. These studies indicate that either p110alpha or p110delta can mediate tonic signaling from the BCR, but only p110delta can contribute to antigen-dependent activation of B cells.
Assuntos
Linfócitos B/citologia , Fosfatidilinositol 3-Quinases/imunologia , Receptores de Células Precursoras de Linfócitos B/metabolismo , Transdução de Sinais/imunologia , Análise de Variância , Animais , Linfócitos B/imunologia , Western Blotting , Classe I de Fosfatidilinositol 3-Quinases , Primers do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Camundongos , Camundongos Mutantes , Organismos Livres de Patógenos EspecíficosRESUMO
V(D)J recombination in lymphocytes is the cutting and pasting together of antigen receptor genes in cis to generate the enormous variety of coding sequences required to produce diverse antigen receptor proteins. It is the key role of the adaptive immune response, which must potentially combat millions of different foreign antigens. Most antigen receptor loci have evolved to be extremely large and contain multiple individual V, D and J genes. The immunoglobulin heavy chain (Igh) and immunoglobulin kappa light chain (Igk) loci are the largest multigene loci in the mammalian genome and V(D)J recombination is one of the most complicated genetic processes in the nucleus. The challenge for the appropriate lymphocyte is one of macro-management-to make all of the antigen receptor genes in a particular locus available for recombination at the appropriate developmental time-point. Conversely, these large loci must be kept closed in lymphocytes in which they do not normally recombine, to guard against genomic instability generated by the DNA double strand breaks inherent to the V(D)J recombination process. To manage all of these demanding criteria, V(D)J recombination is regulated at numerous levels. It is restricted to lymphocytes since the Rag genes which control the DNA double-strand break step of recombination are only expressed in these cells. Within the lymphocyte lineage, immunoglobulin recombination is restricted to B-lymphocytes and TCR recombination to T-lymphocytes by regulation of locus accessibility, which occurs at multiple levels. Accessibility of recombination signal sequences (RSSs) flanking individual V, D and J genes at the nucleosomal level is the key micro-management mechanism, which is discussed in greater detail in other chapters. This chapter will explore how the antigen receptor loci are regulated as a whole, focussing on the Igh locus as a paradigm for the mechanisms involved. Numerous recent studies have begun to unravel the complex and complementary processes involved in this large-scale locus organisation. We will examine the structure of the Igh locus and the large-scale and higher-order chromatin remodelling processes associated with V(D)J recombination, at the level of the locus itself, its conformational changes and its dynamic localisation within the nucleus.
Assuntos
Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica , Cadeias Pesadas de Imunoglobulinas/genética , Alelos , Animais , Linfócitos B/imunologia , Linfócitos B/fisiologia , Cromatina/metabolismo , Cromatina/ultraestrutura , Rearranjo Gênico , Recombinação Genética , Transcrição GênicaRESUMO
Chromosomal translocation requires formation of paired double-strand DNA breaks (DSBs) on heterologous chromosomes. One of the most well characterized oncogenic translocations juxtaposes c-myc and the immunoglobulin heavy-chain locus (IgH) and is found in Burkitt's lymphomas in humans and plasmacytomas in mice. DNA breaks in IgH leading to c-myc/IgH translocations are created by activation-induced cytidine deaminase (AID) during antibody class switch recombination or somatic hypermutation. However, the source of DNA breaks at c-myc is not known. Here, we provide evidence for the c-myc promoter region being required in targeting AID-mediated DNA damage to produce DSBs in c-myc that lead to c-myc/IgH translocations in primary B lymphocytes. Thus, in addition to producing somatic mutations and DNA breaks in antibody genes, AID is also responsible for the DNA lesions in oncogenes that are required for their translocation.
Assuntos
Citidina Desaminase/metabolismo , Genes de Cadeia Pesada de Imunoglobulina , Genes myc , Translocação Genética , Animais , Linfócitos B/metabolismo , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Quebras de DNA de Cadeia Dupla , Células-Tronco Embrionárias , Humanos , Integrases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Plasmocitoma/genética , Plasmocitoma/metabolismoRESUMO
In healthy mammals, maturation of B cells expressing heavy (H) chain immunoglobulin (Ig) without light (L) chain is prevented by chaperone association of the H chain in the endoplasmic reticulum. Camelids are an exception, expressing homodimeric IgGs, an antibody type that to date has not been found in mice or humans. In camelids, immunization with viral epitopes generates high affinity H chain-only antibodies, which, because of their smaller size, recognize clefts and protrusions not readily distinguished by typical antibodies. Developmental processes leading to H chain antibody expression are unknown. We show that L(-/-) (kappa(-/-)lambda(-/-)-deficient) mice, in which conventional B cell development is blocked at the immature B cell stage, produce diverse H chain-only antibodies in serum. The generation of H chain-only IgG is caused by the loss of constant (C) gamma exon 1, which is accomplished by genomic alterations in C(H)1-circumventing chaperone association. These mutations can be attributed to errors in class switch recombination, which facilitate the generation of H chain-only Ig-secreting plasma cells. Surprisingly, transcripts with a similar deletion can be found in normal mice. Thus, naturally occurring H chain transcripts without C(H)1 (V(H)DJ(H)-hinge-C(H)2-C(H)3) are selected for and lead to the formation of fully functional and diverse H chain-only antibodies in L(-/-) animals.
Assuntos
Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Alelos , Animais , Anticorpos/química , Western Blotting , Linhagem Celular , DNA/metabolismo , Citometria de Fluxo , Genes de Imunoglobulinas , Sistema Imunitário , Hibridização in Situ Fluorescente , Camundongos , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Facioscapulohumeral muscular dystrophy (FSHD) is caused by deletions within the polymorphic DNA tandem array D4Z4. Each D4Z4 repeat unit has an open reading frame (ORF), termed "DUX4," containing two homeobox sequences. Because there has been no evidence of a transcript from the array, these deletions are thought to cause FSHD by a position effect on other genes. Here, we identify D4Z4 homologues in the genomes of rodents, Afrotheria (superorder of elephants and related species), and other species and show that the DUX4 ORF is conserved. Phylogenetic analysis suggests that primate and Afrotherian D4Z4 arrays are orthologous and originated from a retrotransposed copy of an intron-containing DUX gene, DUXC. Reverse-transcriptase polymerase chain reaction and RNA fluorescence and tissue in situ hybridization data indicate transcription of the mouse array. Together with the conservation of the DUX4 ORF for >100 million years, this strongly supports a coding function for D4Z4 and necessitates re-examination of current models of the FSHD disease mechanism.
Assuntos
Evolução Molecular , Distrofia Muscular Facioescapuloumeral/genética , Sequências de Repetição em Tandem , Sequência de Aminoácidos , Animais , Sequência Conservada , Proteínas de Homeodomínio , Humanos , Hibridização in Situ Fluorescente , Mamíferos , Camundongos , Dados de Sequência Molecular , Mutação , Filogenia , Primatas , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Transcription in mammalian nuclei is highly compartmentalized in RNA polymerase II-enriched nuclear foci known as transcription factories. Genes in cis and trans can share the same factory, suggesting that genes migrate to preassembled transcription sites. We used fluorescent in situ hybridization to investigate the dynamics of gene association with transcription factories during immediate early (IE) gene induction in mouse B lymphocytes. Here, we show that induction involves rapid gene relocation to transcription factories. Importantly, we find that the Myc proto-oncogene on Chromosome 15 is preferentially recruited to the same transcription factory as the highly transcribed Igh gene located on Chromosome 12. Myc and Igh are the most frequent translocation partners in plasmacytoma and Burkitt lymphoma. Our results show that transcriptional activation of IE genes involves rapid relocation to preassembled transcription factories. Furthermore, the data imply a direct link between the nonrandom interchromosomal organization of transcribed genes at transcription factories and the incidence of specific chromosomal translocations.
Assuntos
Regulação da Expressão Gênica , Genes Precoces , Genes de Cadeia Pesada de Imunoglobulina , Cadeias Pesadas de Imunoglobulinas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transcrição Gênica , Alelos , Animais , Linfócitos B/citologia , Linfócitos B/fisiologia , Núcleo Celular/metabolismo , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , RNA Polimerase II/metabolismo , Ativação Transcricional , Translocação GenéticaRESUMO
V(D)J recombination is believed to be regulated by alterations in chromatin accessibility to the recombinase machinery, but the mechanisms responsible remain unclear. We previously proposed that antisense intergenic transcription, activated throughout the mouse Igh VH region in pro-B cells, remodels chromatin for VH-to-DJH recombination. Using RNA fluorescence in situ hybridization, we now show that antisense intergenic transcription occurs throughout the Igh DHJH region before D-to-J recombination, indicating that this is a widespread process in V(D)J recombination. Transcription initiates near the Igh intronic enhancer Emu and is abrogated in mice lacking this enhancer, indicating that Emu regulates DH antisense transcription. Emu was recently demonstrated to regulate DH-to-JH recombination of the Igh locus. Together, these data suggest that Emu controls DH-to-JH recombination by activating this form of germ line Igh transcription, thus providing a long-range, processive mechanism by which Emu can regulate chromatin accessibility throughout the DH region. In contrast, Emu deletion has no effect on VH antisense intergenic transcription, which is rarely associated with DH antisense transcription, suggesting differential regulation and separate roles for these processes at sequential stages of V(D)J recombination. These results support a directive role for antisense intergenic transcription in enabling access to the recombination machinery.
Assuntos
DNA Antissenso/genética , DNA Intergênico/genética , Elementos Facilitadores Genéticos/genética , Genes de Cadeia Pesada de Imunoglobulina , Íntrons/genética , Recombinação Genética , Transcrição Gênica , Alelos , Animais , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Proteínas de Homeodomínio/metabolismo , Camundongos , Sítio de Iniciação de TranscriçãoRESUMO
The mechanisms that regulate variable (V) gene selection during the development of the mouse IgH repertoire are not fully understood, due in part to the absence of the complete locus sequence. To better understand these processes, we have assembled the entire 2.5-Mb mouse IgH (Igh) V region sequence of the C57BL/6 strain from public sequences and present the first complete annotated map of the region, including V genes, pseudogenes, repeats, and nonrepetitive intergenic sequences. In so doing, we have discovered a new V gene family, VH16. We have identified clusters of conserved region-specific intergenic sequences and have verified our assembly by genic and intergenic Southern blotting. We have observed that V pseudogenes are not evenly spread throughout the V region, but rather cluster together. The largest J558 family, which spans more than half of the locus, has two strikingly different domains, which suggest points of evolutionary divergence or duplication. The 5' end contains widely spaced J558 genes interspersed with 3609 genes and is pseudogene poor. The 3' end contains closely spaced J558 genes, no 3609 genes, and is pseudogene rich. Each occupies a different branch of the phylogenetic tree. Detailed analysis of 500-bp upstream of all functional genes has revealed several conserved binding sites, general and B cell-specific, as well as key differences between families. This complete and definitive assembly of the mouse Igh V region will facilitate detailed study of promoter function and large-scale mechanisms associated with V(D)J recombination including locus contraction and antisense intergenic transcription.
Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Animais , Sequência de Bases , Sítios de Ligação , Southern Blotting , Camundongos , Camundongos Endogâmicos C57BL , Família Multigênica/genética , Filogenia , Pseudogenes/genética , Análise de Sequência de DNARESUMO
The assembly of Ag receptor genes by V(D)J recombination is regulated by transcriptional promoters and enhancers which control chromatin accessibility at Ig and TCR gene segments to the RAG-1/RAG-2 recombinase complex. Paradoxically, germline deletions of the IgH enhancer (Emu) only modestly reduce D(H)-->J(H) rearrangements when assessed in peripheral B cells. However, deletion of Emu severely impairs recombination of V(H) gene segments, which are located over 100 kb away. We now test two alternative explanations for the minimal effect of Emu deletions on primary D(H)-->J(H) rearrangement: 1) Accessibility at the D(H)J(H) cluster is controlled by a redundant cis-element in the absence of Emu. One candidate for this element lies 5' to D(Q52) (PD(Q52)) and exhibits promoter/enhancer activity in pre-B cells. 2) In contrast to endpoint B cells, D(H)-->J(H) recombination may be significantly impaired in pro-B cells from enhancer-deficient mice. To elucidate the roles of PD(Q52) and Emu in the regulation of IgH locus accessibility, we generated mice with targeted deletions of these elements. We report that the defined PD(Q52) promoter is dispensable for germline transcription and recombination of the D(H)J(H) cluster. In contrast, we demonstrate that Emu directly regulates accessibility of the D(H)J(H) region. These findings reveal a significant role for Emu in the control mechanisms that activate IgH gene assembly and suggest that impaired V(H)-->D(H)J(H) rearrangement in enhancer-deficient cells may be a downstream consequence of the primary block in D(H)-->J(H) recombination.
Assuntos
Elementos Facilitadores Genéticos/genética , Rearranjo Gênico do Linfócito B/genética , Genes de Cadeia Pesada de Imunoglobulina/genética , Íntrons/genética , Recombinação Genética/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Diferenciação Celular , Deleção de Genes , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Mutação/genética , Transcrição Gênica/genéticaRESUMO
Antigen receptor genes undergo variable, diversity and joining (V(D)J) recombination, which requires ordered large-scale chromatin remodeling. Here we show that antisense transcription, both genic and intergenic, occurs extensively in the V region of the immunoglobulin heavy chain locus. RNA fluorescence in situ hybridization demonstrates antisense transcription is strictly developmentally regulated and is initiated during the transition from DJ(H) to VDJ(H) recombination and terminates concomitantly with VDJ(H) recombination. Our data show antisense transcription is specific to the V region and suggest transcripts extend across several genes. We propose that antisense transcription remodels the V region to facilitate V(H)-to-DJ(H) recombination. These findings have wider implications for V(D)J recombination of other antigen receptor loci and developmental regulation of multigene loci.
Assuntos
DNA Antissenso/metabolismo , DNA Intergênico/metabolismo , Rearranjo Gênico/fisiologia , Genes de Imunoglobulinas/fisiologia , Transcrição Gênica/fisiologia , Animais , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos C57BL , RNA/metabolismoRESUMO
The genomic basis of facioscapulohumeral muscular dystrophy (FSHD) is of considerable interest because of the unique nature of the molecular mutation, which is a deletion within a large, complex DNA tandem array (D4Z4). This repeat maps within 30 kb of the 4q telomere. Although D4Z4 repeat units each contain an open reading frame that could encode a homeodomain protein, there is no evidence that the repeat is transcribed, and the underlying disease mechanism probably involves a position effect. A recent study has identified a protein complex bound to D4Z4 that contains YY1 and HMGB2, implicating a role for D4Z4 as a repressor. The 4q telomere has two variants, 4qA and 4qB. Although these alleles are present at almost equal frequencies in the general population, FSHD is associated only with the 4qA allele and never with 4qB. This suggests a functional difference between the telomere variants, either in predisposition to deletions within D4Z4 or in the pathological consequence of the deletion. Comparative mapping studies of the FSHD region in primates, mouse and Fugu rubripes have given insights into the evolutionary history of the D4Z4 repeat and of 4qter, although as yet they have not provided any solutions to the FSHD puzzle.