RESUMEN
By genetically ablating IkappaB kinase (IKK)-mediated activation of the transcription factor NF-kappaB in the B cell lineage and by analyzing a mouse mutant in which immunoglobulin lambda-chain-positive B cells are generated in the absence of rearrangements in the locus encoding immunoglobulin kappa-chain, we define here two distinct, consecutive phases of early B cell development that differ in their dependence on IKK-mediated NF-kappaB signaling. During the first phase, in which NF-kappaB signaling is dispensable, predominantly kappa-chain-positive B cells are generated, which undergo efficient receptor editing. In the second phase, predominantly lambda-chain-positive B cells are generated whose development is ontogenetically timed to occur after rearrangements of the locus encoding kappa-chain. This second phase of development is dependent on NF-kappaB signals, which can be substituted by transgenic expression of the prosurvival factor Bcl-2.
Asunto(s)
Linfocitos B/citología , Cadenas kappa de Inmunoglobulina/metabolismo , Cadenas lambda de Inmunoglobulina/metabolismo , FN-kappa B/metabolismo , Animales , Linfocitos B/metabolismo , Diferenciación Celular , Quinasa I-kappa B/genética , Cadenas kappa de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , FN-kappa B/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2 , Transducción de SeñalRESUMEN
Variable-(diversity)-joining (V(D)J) recombination at loci encoding the immunoglobulin heavy chain (Igh) and immunoglobulin light chain (Igk) takes place sequentially during successive stages in B cell development. Using three-dimensional DNA fluorescence in situ hybridization, here we identify a lineage-specific and stage-specific interchromosomal association between these two loci that marks the transition between Igh and Igk recombination. Colocalization occurred between pericentromerically located alleles in pre-B cells and was mediated by the 3' Igk enhancer. Deletion of this regulatory element prevented association of the Igh and Igk loci, inhibited pericentromeric recruitment and locus 'decontraction' of an Igh allele, and resulted in greater distal rearrangement of the gene encoding the variable heavy-chain region. Our data indicate involvement of the Igk locus and its 3' enhancer in directing the Igh locus to a repressive nuclear subcompartment and inducing the Igh locus to decontract.
Asunto(s)
Elementos de Facilitación Genéticos/inmunología , Reordenamiento Génico de Cadena Pesada de Linfocito B , Genes de las Cadenas Pesadas de las Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Inmunoglobulinas/genética , Células Precursoras de Linfocitos B/inmunología , Región de Flanqueo 3'/genética , Animales , Cromosomas/genética , Cromosomas/metabolismo , Genes de las Cadenas Pesadas de las Inmunoglobulinas/fisiología , Inmunoglobulinas/fisiología , Ratones , Ratones Noqueados , Células Precursoras de Linfocitos B/metabolismo , Recombinación GenéticaRESUMEN
In yeast, increased levels of the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase isozyme, Hmg1p, induce assembly of nuclear-associated ER membranes called karmellae. To identify additional genes involved in karmellae assembly, we screened temperature-sensitive mutants for karmellae assembly defects. Two independently isolated, temperature-sensitive strains that were also defective for karmellae biogenesis carried mutations in VPS16, a gene involved in vacuolar protein sorting. Karmellae biogenesis was defective in all 13 other vacuole biogenesis mutants tested, although the severity of the karmellae assembly defect varied depending on the particular mutation. The hypersensitivity of 14 vacuole biogenesis mutants to tunicamycin was well correlated with pronounced defects in karmellae assembly, suggesting that the karmellae assembly defect reflected alteration of ER structure or function. Consistent with this hypothesis, seven of eight mutations causing defects in secretion also affected karmellae assembly. However, the vacuole biogenesis mutants were able to proliferate their ER in response to Hmg2p, indicating that the mutants did not have a global defect in the process of ER biogenesis.
Asunto(s)
Retículo Endoplásmico/fisiología , Proteínas de la Membrana , Saccharomyces cerevisiae/genética , Vacuolas/fisiología , Antibacterianos/farmacología , Retículo Endoplásmico/ultraestructura , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Genes Fúngicos , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Proteína HMGB2/genética , Proteína HMGB2/metabolismo , Calor , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , Pruebas de Sensibilidad Microbiana , Microscopía Fluorescente , Plásmidos , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Tunicamicina/farmacología , Vacuolas/ultraestructura , Proteínas de Transporte VesicularRESUMEN
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.
Asunto(s)
FN-kappa B/metabolismo , Células Precursoras de Linfocitos B/inmunología , Células Precursoras de Linfocitos B/metabolismo , Edición de ARN , Animales , Secuencia de Bases , Diferenciación Celular , Línea Celular , Cartilla de ADN/genética , ADN Complementario/genética , Reordenamiento Génico de Cadena Ligera de Linfocito B , Proteínas I-kappa B/genética , Factores Reguladores del Interferón/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Inhibidor NF-kappaB alfa , Células Precursoras de Linfocitos B/citología , Receptores de Antígenos de Linfocitos B/genética , Autotolerancia/genéticaRESUMEN
B cell receptor (BCR) signaling plays a critical role in B cell tolerance and activation. Here, we show that mice with B cell-specific ablation of both Cbl and Cbl-b (Cbl-/-Cblb-/-) manifested systemic lupus erythematosus (SLE)-like autoimmune disease. The Cbl double deficiency resulted in a substantial increase in marginal zone (MZ) and B1 B cells. The mutant B cells were not hyperresponsive in terms of proliferation and antibody production upon BCR stimulation; however, B cell anergy to protein antigen appeared to be impaired. Concomitantly, BCR-proximal signaling, including tyrosine phosphorylation of Syk tyrosine kinase, Phospholipase C-gamma2 (PLC-gamma2), and Rho-family GTP-GDP exchange factor Vav, and Ca2+ mobilization were enhanced, whereas tyrosine phosphorylation of adaptor protein BLNK was substantially attenuated in the mutant B cells. These results suggested that the loss of coordination between these pathways was responsible for the impaired B cell tolerance induction. Thus, Cbl proteins control B cell-intrinsic checkpoint of immune tolerance, possibly through coordinating multiple BCR-proximal signaling pathways during anergy induction.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Linfocitos B/enzimología , Linfocitos B/inmunología , Tolerancia Inmunológica/inmunología , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Anticuerpos/inmunología , Antígenos/inmunología , Linfocitos B/citología , Diferenciación Celular , Células Cultivadas , Regulación hacia Abajo , Inmunoglobulinas/inmunología , Inmunoglobulinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lupus Eritematoso Sistémico/enzimología , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Ratones , Ratones Noqueados , Unión Proteica , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-cbl/deficiencia , Proteínas Proto-Oncogénicas c-cbl/genética , Transducción de Señal , Quinasa Syk , Ubiquitina/metabolismoRESUMEN
Increased levels of HMG-CoA reductase induce cell type- and isozyme-specific proliferation of the endoplasmic reticulum. In yeast, the ER proliferations induced by Hmg1p consist of nuclear-associated stacks of smooth ER membranes known as karmellae. To identify genes required for karmellae assembly, we compared the composition of populations of homozygous diploid S. cerevisiae deletion mutants following 20 generations of growth with and without karmellae. Using an initial population of 1,557 deletion mutants, 120 potential mutants were identified as a result of three independent experiments. Each experiment produced a largely non-overlapping set of potential mutants, suggesting that differences in specific growth conditions could be used to maximize the comprehensiveness of similar parallel analysis screens. Only two genes, UBC7 and YAL011W, were identified in all three experiments. Subsequent analysis of individual mutant strains confirmed that each experiment was identifying valid mutations, based on the mutant's sensitivity to elevated HMG-CoA reductase and inability to assemble normal karmellae. The largest class of HMG-CoA reductase-sensitive mutations was a subset of genes that are involved in chromatin structure and transcriptional regulation, suggesting that karmellae assembly requires changes in transcription or that the presence of karmellae may interfere with normal transcriptional regulation.