RESUMO
Developing B lymphocytes undergo clonal expansion following successful immunoglobulin heavy chain gene rearrangement. During this proliferative burst, expression of the Rag genes is transiently repressed to prevent the generation of double-stranded DNA (dsDNA) breaks in cycling large pre-B cells. The Rag genes are then reexpressed in small, resting pre-B cells for immunoglobulin light chain gene rearrangement. We previously identified c-Myb as a repressor of Rag transcription during clonal expansion using Abelson murine leukemia virus-transformed B cells. Nevertheless, the molecular mechanisms by which c-Myb achieved precise spatiotemporal repression of Rag expression remained obscure. Here, we identify two mechanisms by which c-Myb represses Rag transcription. First, c-Myb negatively regulates the expression of the Rag activator Foxo1, an activity dependent on M303 in c-Myb's transactivation domain, and likely the recruitment of corepressors to the Foxo1 locus by c-Myb. Second, c-Myb represses Rag transcription directly by occupying the Erag enhancer and antagonizing Foxo1 binding to a consensus forkhead site in this cis-regulatory element that we show is crucial for Rag expression in Abelson pre-B cell lines. This work provides important mechanistic insight into how spatiotemporal expression of the Rag genes is tightly controlled during B lymphocyte development to prevent mistimed dsDNA breaks and their deleterious consequences.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Proteínas Repressoras/metabolismo , Animais , Sítios de Ligação , Sistemas CRISPR-Cas/genética , Proliferação de Células , Elementos Facilitadores Genéticos/genética , Proteína Forkhead Box O1/metabolismo , Camundongos , Modelos Biológicos , Mutação/genética , Ligação Proteica , Transcrição GênicaRESUMO
V(D)J recombination creates antibody light chain diversity by joining a Vκ gene segment with one of four Jκ segments. Two Jκ germline-transcript (GT) promoters control Vκ-Jκ joining, but the mechanisms that govern Jκ choice are unclear. Here, we show in gene-targeted mice that the proximal GT promoter helps targeting rearrangements to Jκ1 by preventing premature DNA breaks at Jκ2. Consequently, cells lacking the proximal GT promoter show a biased utilization of downstream Jκ segments, resulting in a diminished potential for receptor editing. Surprisingly, the proximal--in contrast to the distal--GT promoter is transcriptionally inactive prior to Igκ recombination, indicating that its role in Jκ choice is independent of classical promoter function. Removal of the proximal GT promoter increases H3K4me3 levels at Jκ segments, suggesting that this promoter could act as a suppressor of recombination by limiting chromatin accessibility to RAG. Our findings identify the first cis-element critical for Jκ choice and demonstrate that ordered Igκ recombination facilitates receptor editing.
Assuntos
Região de Junção de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Regiões Promotoras Genéticas/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Recombinação V(D)J/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Feminino , Citometria de Fluxo , Expressão Gênica/imunologia , Células Germinativas/imunologia , Células Germinativas/metabolismo , Histonas/imunologia , Histonas/metabolismo , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Lisina/imunologia , Lisina/metabolismo , Masculino , Metilação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Receptores de Antígenos de Linfócitos B/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Recombinação V(D)J/genéticaRESUMO
Growth factor independence genes (Gfi1 and Gfi1b) repress recombination activating genes (Rag) transcription in developing B lymphocytes. Because all blood lineages originate from hematopoietic stem cells (HSCs) and different lineage progenitors have been shown to share transcription factor networks prior to cell fate commitment, we hypothesized that GFI family proteins may also play a role in repressing Rag transcription or a global lymphoid transcriptional program in other blood lineages. We tested the level of Rag transcription in various blood cells when Gfi1 and Gfi1b were deleted, and observed an upregulation of Rag expression in plasmacytoid dendritic cells (pDCs). Using microarray analysis, we observed that Gfi1 and Gfi1b do not regulate a lymphoid or pDC-specific transcriptional program. This study establishes a role for Gfi1 and Gfi1b in Rag regulation in a non-B lineage cell type.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Dendríticas/metabolismo , Regulação da Expressão Gênica/genética , Proteínas de Homeodomínio/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Biologia Computacional , Proteínas de Ligação a DNA/genética , Citometria de Fluxo , Humanos , Camundongos , Análise em Microsséries , Proteínas Proto-Oncogênicas/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genéticaRESUMO
The temporal control of RAG (Rag) expression in developing lymphocytes prevents DNA breaks during periods of proliferation that could threaten genomic integrity. In developing B cells, the IL-7R and precursor B cell Ag receptor (pre-BCR) synergize to induce proliferation and the repression of Rag at the protein and mRNA levels for a brief period following successful Ig H chain gene rearrangement. Whereas the mechanism of RAG2 protein downregulation is well defined, little is known about the pathways and transcription factors that mediate transcriptional repression of Rag. Using Abelson murine leukemia virus-transformed B cells to model this stage of development, we identified early B cell factor 1 (Ebf1) as a strong repressor of Rag transcription. Short hairpin RNA-mediated knockdown of either Ebf1 or its downstream target c-Myb was sufficient to induce Rag transcription in these highly proliferative cells. Ebf1 and c-Myb antagonize Rag transcription by negatively regulating the binding of Foxo1 to the Rag locus. Ebf1 accomplishes this through both direct negative regulation of Foxo1 expression and direct positive regulation of Gfi1b expression. Ebf1 expression is driven by the IL-7R downstream effector Stat5, providing a link between the negative regulation of Rag transcription by IL-7 and a novel repressive pathway involving Ebf1 and c-Myb.
Assuntos
Linfócitos B/metabolismo , Proteínas de Homeodomínio/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo , Fator de Transcrição STAT5/genética , Transativadores/metabolismo , Animais , Diferenciação Celular/imunologia , Proliferação de Células , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/metabolismo , Interleucina-7 , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-myb/genética , Interferência de RNA , RNA Interferente Pequeno , Proteínas Repressoras/biossíntese , Transdução de Sinais , Transativadores/genética , Transcrição GênicaRESUMO
Foxo1 is a critical, direct regulator of Rag (recombination activating gene) transcription during B cell development and is thus essential for the generation of a diverse repertoire of antigen receptors. Although Foxo1 regulation has been widely studied in many cell types, pathways regulating Foxo1 in B cells have not been fully elucidated. By screening a panel of Foxo1 mutants, we identified serine 215 on Foxo1 as a novel phosphorylation site that is essential for the activation of Rag transcription. Mutation of S215 strongly attenuated transactivation of Rag but did not affect most other Foxo1 target genes. We show that MK5, a MAPK-activated protein kinase, is a previously unidentified upstream regulator of Foxo1. MK5 was necessary and sufficient to activate Rag transcription in transformed and primary pro-B cells. Together, our experiments show that MK5 positively regulates Rag transcription via phosphorylation of Foxo1 in developing B cells.
Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Genes RAG-1 , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Animais , Benzamidas/farmacologia , Linhagem Celular Transformada , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/genética , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Mesilato de Imatinib , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Piperazinas/farmacologia , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Alinhamento de Sequência , Serina/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
Regulated expression of miRNAs influences development in a wide variety of contexts. We report here that miR290-5p (100049710) and miR292-5p (100049711) are induced at the pre-B stage of murine B cell development and that they influence assembly of the Igκ light chain gene (243469) by contributing to the activation of germline Igκ transcription (κGT). We found that upon forced over-expression of miR290-5p/292-5p in Abelson Murine Leukemia Virus (AMuLV) transformed pro-B cells, two known activators of κGT, E2A (21423) and NF-κB (19697), show increased chromosomal binding to the kappa intronic enhancer. Conversely, knockdown of miR290-5p/292-5p in AMuLV pro-B cells blunts drug-induced activation of κGT. Furthermore, miR290-5p/292-5p knockdown also diminishes κGT activation, but not Rag1/2 (19373, 19374) expression, in an IL-7 dependent primary pro-B cell culture system. In addition, we identified a deficiency in κGT induction in miR290 cluster knockout mice. We hypothesize that increased expression of miR290-5p and miR292-5p contributes to the induction of κGT at the pre-B stage of B cell development through increased binding of NF-κB and E2A to kappa locus regulatory sequences.
Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Loci Gênicos/genética , Cadeias kappa de Imunoglobulina/genética , MicroRNAs/metabolismo , Vírus da Leucemia Murina de Abelson/fisiologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/virologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Benzamidas , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Transformação Celular Viral/efeitos dos fármacos , Transformação Celular Viral/genética , DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Mesilato de Imatinib , Íntrons/genética , Camundongos , MicroRNAs/genética , NF-kappa B/metabolismo , Piperazinas/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
To what extent might the regulation of translation contribute to differentiation programs, or to the molecular pathogenesis of cancer? Pre-B cells transformed with the viral oncogene v-Abl are suspended in an immortalized, cycling state that mimics leukemias with a BCR-ABL1 translocation, such as Chronic Myelogenous Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL). Inhibition of the oncogenic Abl kinase with imatinib reverses transformation, allowing progression to the next stage of B cell development. We employed a genome-wide polysome profiling assay called Gradient Encoding to investigate the extent and potential contribution of translational regulation to transformation and differentiation in v-Abl-transformed pre-B cells. Over half of the significantly translationally regulated genes did not change significantly at the level of mRNA abundance, revealing biology that might have been missed by measuring changes in transcript abundance alone. We found extensive, gene-specific changes in translation affecting genes with known roles in B cell signaling and differentiation, cancerous transformation, and cytoskeletal reorganization potentially affecting adhesion. These results highlight a major role for gene-specific translational regulation in remodeling the gene expression program in differentiation and malignant transformation.
Assuntos
Linfócitos B/metabolismo , Linfócitos B/patologia , Diferenciação Celular/genética , Transformação Celular Viral/genética , Proteínas Oncogênicas v-abl/metabolismo , Biossíntese de Proteínas , Transcriptoma , Linfócitos B/efeitos dos fármacos , Benzamidas , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Transformação Celular Viral/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Oncogênicas v-abl/antagonistas & inibidores , Proteínas Oncogênicas v-abl/genética , Piperazinas/farmacologia , Polirribossomos/efeitos dos fármacos , Polirribossomos/genética , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/patologia , Biossíntese de Proteínas/efeitos dos fármacos , Pirimidinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
Precise regulation of Rag (recombination-activating gene) expression is crucial to prevent genomic instability caused by the generation of Rag-mediated DNA breaks. Although mechanisms of Rag activation have been well characterized, the mechanism by which Rag expression is down-regulated in early B cell development has not been fully elucidated. Using a complementary DNA library screen, we identified the transcriptional repressor Gfi1b as negative regulator of the Rag locus. Expression of Gfi1b causes repression of Rag1 and Rag2 in cell lines and primary mouse cells. Conversely, Gfi1b-deficient cell lines exhibit increased Rag expression, double-strand breaks and recombination, and cell cycle defects. In primary cells, transcription of Gfi1b inversely correlates with Rag transcription, and simultaneous inactivation of Gfi1 and Gfi1b leads to an increase in Rag transcription early in B cell development. In addition, deletion of Gfi1 and Gfi1b in vivo results in a severe block in B cell development. Gfi1b orchestrates Rag repression via a dual mechanism. Direct binding of Gfi1b to a site 5' of the B cell-specific Erag enhancer results in epigenetic changes in the Rag locus, whereas indirect inhibition is achieved through repression of the trans-activator Foxo1. Together, our experiments show that Gfi family members are essential for normal B cell development and play an important role in modulating expression of the V(D)J recombinase.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Diferenciação Celular/genética , Montagem e Desmontagem da Cromatina , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína Forkhead Box O1 , Deleção de Genes , Regulação da Expressão Gênica , Biblioteca Gênica , Marcação de Genes , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Recombinação Genética , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Interleukin 7 (IL-7) promotes pre-B cell survival and proliferation by activating the Pim1 and Akt kinases. These signals must be attenuated to induce G1 cell cycle arrest and expression of the RAG endonuclease, which are both required for IgL chain gene rearrangement. As lost IL-7 signals would limit pre-B cell survival, how cells survive during IgL chain gene rearrangement remains unclear. We show that RAG-induced DNA double-strand breaks (DSBs) generated during IgL chain gene assembly paradoxically promote pre-B cell survival. This occurs through the ATM-dependent induction of Pim2 kinase expression. Similar to Pim1, Pim2 phosphorylates BAD, which antagonizes the pro-apoptotic function of BAX. However, unlike IL-7 induction of Pim1, RAG DSB-mediated induction of Pim2 does not drive proliferation. Rather, Pim2 has antiproliferative functions that prevent the transit of pre-B cells harboring RAG DSBs from G1 into S phase, where these DNA breaks could be aberrantly repaired. Thus, signals from IL-7 and RAG DSBs activate distinct Pim kinase family members that have context-dependent activities in regulating pre-B cell proliferation and survival.
Assuntos
Quebras de DNA de Cadeia Dupla , Células Precursoras de Linfócitos B/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Transposases/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Sobrevivência Celular/genética , Dano ao DNA , Genes RAG-1 , Interleucina-7/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transposases/genética , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Immunoglobulin heavy chain (IgH) variable region exons are assembled from V(H), D and J(H) gene segments in developing B lymphocytes. Within the 2.7-megabase mouse Igh locus, V(D)J recombination is regulated to ensure specific and diverse antibody repertoires. Here we report in mice a key Igh V(D)J recombination regulatory region, termed intergenic control region 1 (IGCR1), which lies between the V(H) and D clusters. Functionally, IGCR1 uses CTCF looping/insulator factor-binding elements and, correspondingly, mediates Igh loops containing distant enhancers. IGCR1 promotes normal B-cell development and balances antibody repertoires by inhibiting transcription and rearrangement of D(H)-proximal V(H) gene segments and promoting rearrangement of distal V(H) segments. IGCR1 maintains ordered and lineage-specific V(H)(D)J(H) recombination by suppressing V(H) joining to D segments not joined to J(H) segments, and V(H) to DJ(H) joins in thymocytes, respectively. IGCR1 is also required for feedback regulation and allelic exclusion of proximal V(H)-to-DJ(H) recombination. Our studies elucidate a long-sought Igh V(D)J recombination control region and indicate a new role for the generally expressed CTCF protein.
Assuntos
DNA Intergênico/genética , Rearranjo Gênico de Cadeia Pesada de Linfócito B/genética , Recombinação Genética/genética , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas Repressoras/metabolismo , Éxons VDJ/genética , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Fator de Ligação a CCCTC , Linhagem da Célula/genética , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , Elementos Facilitadores Genéticos/genética , Retroalimentação Fisiológica , Células Germinativas/metabolismo , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Camundongos , Mutação/genética , Timo/citologia , Transcrição Gênica/genéticaRESUMO
The gene encoding c-ABL, a nonreceptor protein tyrosine kinase, is involved in a chromosomal translocation resulting in expression of a BCR-Abl fusion protein that causes most chronic myelogenous and some acute lymphocytic leukemias (CML and ALL) in humans. The Abelson murine leukemia virus (A-MuLV) expresses an alternative form of c-Abl, v-Abl, that transforms murine pro-B cells, resulting in acute leukemia and providing an experimental model for human disease. Gleevec (STI571) inhibits the Abl kinase and has shown great utility against CML and ALL in humans, although its usefulness is limited by acquired resistance. Since STI571 is active against A-MuLV-transformed cells in vitro, we performed a retroviral cDNA library screen for genes that confer resistance to apoptosis induced by STI571. We found that forced expression of Cdk6 promotes continued cell division and decreased apoptosis of leukemic cells. We then determined that the transcription factor E2A negatively regulates Cdk6 transcription in leukemic pro-B cells and that the v-Abl kinase stimulates Cdk6 expression via an extracellular signal-regulated kinase 1-dependent pathway. Finally, we show that the cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor PD0332991 can act synergistically with STI571 to enhance leukemic cell death, suggesting a potential role for CDK6 inhibitors in the treatment of STI571-resistant CML or ALL.
Assuntos
Antineoplásicos/uso terapêutico , Quinase 6 Dependente de Ciclina/genética , Resistencia a Medicamentos Antineoplásicos/genética , Piperazinas/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Benzamidas , Ciclo Celular , Linhagem Celular Tumoral , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Biblioteca Gênica , Humanos , Mesilato de Imatinib , Piperazinas/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimologia , Piridinas/farmacologia , Transcrição GênicaRESUMO
Compaction and looping of the ~2.5-Mb Igh locus during V(D)J rearrangement is essential to allow all V(H) genes to be brought in proximity with D(H)-J(H) segments to create a diverse antibody repertoire, but the proteins directly responsible for this are unknown. Because CCCTC-binding factor (CTCF) has been demonstrated to be involved in long-range chromosomal interactions, we hypothesized that CTCF may promote the contraction of the Igh locus. ChIP sequencing was performed on pro-B cells, revealing colocalization of CTCF and Rad21 binding at ~60 sites throughout the V(H) region and 2 other sites within the Igh locus. These numerous CTCF/cohesin sites potentially form the bases of the multiloop rosette structures at the Igh locus that compact during Ig heavy chain rearrangement. To test whether CTCF was involved in locus compaction, we used 3D-FISH to measure compaction in pro-B cells transduced with CTCF shRNA retroviruses. Reduction of CTCF binding resulted in a decrease in Igh locus compaction. Long-range interactions within the Igh locus were measured with the chromosomal conformation capture assay, revealing direct interactions between CTCF sites 5' of DFL16 and the 3' regulatory region, and also the intronic enhancer (Eµ), creating a D(H)-J(H)-Eµ-C(H) domain. Knockdown of CTCF also resulted in the increase of antisense transcription throughout the D(H) region and parts of the V(H) locus, suggesting a widespread regulatory role for CTCF. Together, our findings demonstrate that CTCF plays an important role in the 3D structure of the Igh locus and in the regulation of antisense germline transcription and that it contributes to the compaction of the Igh locus.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Proteínas Repressoras/metabolismo , Animais , Sítios de Ligação/genética , Western Blotting , Fator de Ligação a CCCTC , Proteínas de Ciclo Celular/genética , Linhagem Celular , Células Cultivadas , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/genética , DNA Antissenso/genética , Proteínas de Ligação a DNA , Elementos Facilitadores Genéticos/genética , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Interferência de RNA , RNA Antissenso/genética , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , CoesinasAssuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Rearranjo Gênico do Linfócito B/imunologia , Tolerância Imunológica/genética , Modelos Imunológicos , Edição de RNA/imunologia , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Camundongos , Camundongos TransgênicosRESUMO
The allelic exclusion of immunoglobulin (Ig) genes is one of the most evolutionarily conserved features of the adaptive immune system and underlies the monospecificity of B cells. While much has been learned about how Ig allelic exclusion is established during B-cell development, the relevance of monospecificity to B-cell function remains enigmatic. Here, we review the theoretical models that have been proposed to explain the establishment of Ig allelic exclusion and focus on the molecular mechanisms utilized by developing B cells to ensure the monoallelic expression of Ig kappa and Ig lambda light chain genes. We also discuss the physiological consequences of Ig allelic exclusion and speculate on the importance of monospecificity of B cells for immune recognition.
Assuntos
Alelos , Linfócitos B/imunologia , Genes de Imunoglobulinas , Modelos Imunológicos , Animais , Humanos , Cadeias gama de Imunoglobulina/imunologia , Cadeias lambda de Imunoglobulina/imunologiaRESUMO
V(D)J recombination of antigen receptor gene segments in B and T cells is mediated by the lymphoid-specific proteins RAG1 and RAG2. Now, Ji et al. (2010) demonstrate how RAG1 and RAG2 use DNA sequence specificity and modified histones within chromatin to target specific loci for V(D)J recombination at different stages of lymphoid development.
RESUMO
The Abelson Murine Leukemia Virus (A-MuLV) encodes v-Abl, an oncogenic form of the ubiquitous cellular non-receptor tyrosine kinase, c-Abl. A-MuLV specifically transforms murine B cell precursors both in vivo and in vitro. Inhibition of v-Abl by addition of the small molecule inhibitor STI-571 causes these cells to arrest in the G1 phase of the cell cycle prior to undergoing apoptosis. We found that inhibition of v-Abl activity results in upregulation of transcription of the pro-apoptotic TNF-family ligand tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL). Similarly to BCR-Abl-transformed human cells, activation of the transcription factor Foxo3a led to increased TRAIL transcription and induction of a G1 arrest in the absence of v-Abl inhibition, and this effect could be inhibited by the expression of a constitutively active AKT mutant. Multiple pathways act to inhibit FoxO3a activity within Abelson cells. In addition to diminishing transcription factor activity via inhibitory phosphorylation by AKT family members, we found that inhibition of IKKbeta activity results in an increase in the total protein level of FoxO3a. Furthermore overexpression of the p65 subunit of NF-kappaB results in an increase in TRAIL transcription and in apoptosis and deletion of IKKalpha and beta diminishes TRAIL expression and induction. We conclude that in Abelson cells, the inhibition of both NF-kappaB and FoxO3a activity is required for suppression of TRAIL transcription and maintenance of the transformed state.
Assuntos
Linfócitos B/metabolismo , Fatores de Transcrição Forkhead/metabolismo , NF-kappa B/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Vírus da Leucemia Murina de Abelson/fisiologia , Animais , Apoptose/efeitos dos fármacos , Linfócitos B/citologia , Linfócitos B/virologia , Benzamidas , Western Blotting , Linhagem Celular Transformada , Transformação Celular Viral , Citometria de Fluxo , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Fase G1 , Interações Hospedeiro-Patógeno , Quinase I-kappa B/metabolismo , Mesilato de Imatinib , Camundongos , Mutação , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ligante Indutor de Apoptose Relacionado a TNF/genética , Transcrição GênicaRESUMO
Because of the extreme diversity in immunoglobulin genes, tolerance mechanisms are necessary to ensure that B cells do not respond to self-antigens. One such tolerance mechanism is called receptor editing. If the B cell receptor (BCR) on an immature B cell recognizes self-antigen, it is down-regulated from the cell surface, and light chain gene rearrangement continues in an attempt to edit the autoreactive specificity. Analysis of a heterozygous mutant mouse in which the NF-kappaB-dependent IkappaB alpha gene was replaced with a lacZ (beta-gal) reporter complementary DNA (cDNA; IkappaB alpha(+/lacZ)) suggests a potential role for NF-kappaB in receptor editing. Sorted beta-gal(+) pre-B cells showed increased levels of various markers of receptor editing. In IkappaB alpha(+/lacZ) reporter mice expressing either innocuous or self-specific knocked in BCRs, beta-gal was preferentially expressed in pre-B cells from the mice with self-specific BCRs. Retroviral-mediated expression of a cDNA encoding an IkappaB alpha superrepressor in primary bone marrow cultures resulted in diminished germline kappa and rearranged lambda transcripts but similar levels of RAG expression as compared with controls. We found that IRF4 transcripts were up-regulated in beta-gal(+) pre-B cells. Because IRF4 is a target of NF-kappaB and is required for receptor editing, we suggest that NF-kappaB could be acting through IRF4 to regulate receptor editing.