RESUMEN
Immunoglobulin class switch recombination (CSR) occurs in activated B cells with increased mitochondrial mass and membrane potential. Transcription factor Yin Yang 1 (YY1) is critical for CSR and for formation of the DNA loops involved in this process. We therefore sought to determine if YY1 knockout impacts mitochondrial gene expression and mitochondrial function in murine splenic B cells, providing a potential mechanism for regulating CSR. We identified numerous genes in splenic B cells differentially regulated when cells are induced to undergo CSR. YY1 conditional knockout caused differential expression of 1129 genes, with 59 being mitochondrial-related genes. ChIP-seq analyses showed YY1 was directly bound to nearly half of these mitochondrial-related genes. Surprisingly, at the time when YY1 knockout dramatically reduces DNA loop formation and CSR, mitochondrial mass and membrane potential were not significantly impacted, nor was there a significant change in mitochondrial oxygen consumption, extracellular acidification rate, or mitochondrial complex I or IV activities. Our results indicate that YY1 regulates numerous mitochondrial-related genes in splenic B cells, but this does not account for the impact of YY1 on CSR or long-distance DNA loop formation.
Asunto(s)
Linfocitos B/inmunología , ADN Mitocondrial/inmunología , Genes Mitocondriales/inmunología , Cambio de Clase de Inmunoglobulina , Bazo/inmunología , Factor de Transcripción YY1/inmunología , Animales , Linfocitos B/citología , ADN Mitocondrial/genética , Ratones , Ratones Noqueados , Bazo/citología , Factor de Transcripción YY1/genéticaRESUMEN
X-chromosome inactivation (XCI) in female lymphocytes is uniquely regulated, as the inactive X (Xi) chromosome lacks localized Xist RNA and heterochromatin modifications. Epigenetic profiling reveals that Xist RNA is lost from the Xi at the pro-B cell stage and that additional heterochromatic modifications are gradually lost during B cell development. Activation of mature B cells restores Xist RNA and heterochromatin to the Xi in a dynamic two-step process that differs in timing and pattern, depending on the method of B cell stimulation. Finally, we find that DNA binding domain of YY1 is necessary for XCI in activated B cells, as ex-vivo YY1 deletion results in loss of Xi heterochromatin marks and up-regulation of X-linked genes. Ectopic expression of the YY1 zinc finger domain is sufficient to restore Xist RNA localization during B cell activation. Together, our results indicate that Xist RNA localization is critical for maintaining XCI in female lymphocytes, and that chromatin changes on the Xi during B cell development and the dynamic nature of YY1-dependent XCI maintenance in mature B cells predisposes X-linked immunity genes to reactivation.
Asunto(s)
Silenciador del Gen , Activación de Linfocitos/genética , Células Precursoras de Linfocitos B/metabolismo , ARN Largo no Codificante/genética , Inactivación del Cromosoma X/genética , Factor de Transcripción YY1/metabolismo , Animales , Epigénesis Genética , Femenino , Eliminación de Gen , Genes Ligados a X , Heterocromatina/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , ARN Largo no Codificante/aislamiento & purificación , Análisis de Secuencia de ARN , Bazo/citología , Regulación hacia Arriba , Cromosoma X/genética , Factor de Transcripción YY1/genéticaRESUMEN
Activation-induced cytidine deminase (AID) is crucial for controlling the immunoglobulin (Ig) diversification processes of somatic hypermutation (SHM) and class switch recombination (CSR). AID initiates these processes by deamination of cytosine, ultimately resulting in mutations or double strand DNA breaks needed for SHM and CSR. Levels of AID control mutation rates, and off-target non-Ig gene mutations can contribute to lymphomagenesis. Therefore, factors that control AID levels in the nucleus can regulate SHM and CSR, and may contribute to disease. We previously showed that transcription factor YY1 can regulate the level of AID in the nucleus and Ig CSR. Therefore, we hypothesized that conditional knock-out of YY1 would lead to reduction in AID localization at the Ig locus, and reduced AID-mediated mutations. Using mice that overexpress AID (IgκAID yy1f/f ) or that express normal AID levels (yy1f/f ), we found that conditional knock-out of YY1 results in reduced AID nuclear levels, reduced localization of AID to the Sµ switch region, and reduced AID-mediated mutations. We find that the mechanism of YY1 control of AID nuclear accumulation is likely due to YY1-AID physical interaction which blocks AID ubiquitination.
Asunto(s)
Linfocitos B/fisiología , Mutagénesis/genética , Factor de Transcripción YY1/genética , Animales , Citidina Desaminasa/metabolismo , Roturas del ADN de Doble Cadena , Femenino , Cambio de Clase de Inmunoglobulina/genética , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutación/genética , Unión Proteica , Hipermutación Somática de Inmunoglobulina/genética , UbiquitinaciónRESUMEN
Conditional knock-out (KO) of Polycomb Group (PcG) protein YY1 results in pro-B cell arrest and reduced immunoglobulin locus contraction needed for distal variable gene rearrangement. The mechanisms that control these crucial functions are unknown. We deleted the 25 amino-acid YY1 REPO domain necessary for YY1 PcG function, and used this mutant (YY1ΔREPO), to transduce bone marrow from YY1 conditional KO mice. While wild-type YY1 rescued B-cell development, YY1ΔREPO failed to rescue the B-cell lineage yielding reduced numbers of B lineage cells. Although the IgH rearrangement pattern was normal, there was a selective impact at the Igκ locus that showed a dramatic skewing of the expressed Igκ repertoire. We found that the REPO domain interacts with proteins from the condensin and cohesin complexes, and that YY1, EZH2 and condensin proteins co-localize at numerous sites across the Ig kappa locus. Knock-down of a condensin subunit protein or YY1 reduced rearrangement of Igκ Vκ genes suggesting a direct role for YY1-condensin complexes in Igκ locus structure and rearrangement.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Linfocitos B/fisiología , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Cadenas kappa de Inmunoglobulina/metabolismo , Complejos Multiproteicos/metabolismo , Mapeo de Interacción de Proteínas , Factor de Transcripción YY1/metabolismo , Adenosina Trifosfatasas/genética , Animales , Linfocitos B/inmunología , Diferenciación Celular , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Proteína Potenciadora del Homólogo Zeste 2 , Técnicas de Silenciamiento del Gen , Ratones , Ratones Noqueados , Complejos Multiproteicos/genética , Complejo Represivo Polycomb 2/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Eliminación de Secuencia , Transducción Genética , Factor de Transcripción YY1/genéticaRESUMEN
Stage-specific rearrangement of Ig H and L chain genes poses an enigma because both processes use the same recombinatorial machinery, but the H chain locus is accessible at the pro-B cell stage, whereas the L chain loci become accessible at the pre-B cell stage. Transcription factor STAT5 is a positive-acting factor for rearrangement of distal V(H) genes, but attenuation of IL-7 signaling and loss of activated STAT5 at the pre-B cell stage corresponds with Igκ locus accessibility and rearrangement, suggesting that STAT5 plays an inhibitory role at this locus. Indeed, loss of IL-7 signaling correlates with increased activity at the Igκ intron enhancer. However, the κE3' enhancer must also be regulated as this enhancer plays a role in Igκ rearrangement. We show in this study that STAT5 can repress κE3' enhancer activity. We find that STAT5 binds to a site that overlaps the κE3' PU.1 binding site. We observed reciprocal binding by STAT5 and PU.1 to the κE3' enhancer in primary bone marrow cells, STAT5 and PU.1 retrovirally transduced pro-B cell lines, or embryonic stem cells induced to differentiate into B lineage cells. Binding by STAT5 corresponded with low occupancy of other enhancer binding proteins, whereas PU.1 binding corresponded with recruitment of IRF4 and E2A to the κE3' enhancer. We also find that IRF4 expression can override the repressive activity of STAT5. We propose a novel PU.1/STAT5 displacement model during B cell development, and this, coupled with increased IRF4 and E2A activity, regulates κE3' enhancer function.
Asunto(s)
Diferenciación Celular/inmunología , Proteínas de Unión al ADN/fisiología , Elementos de Facilitación Genéticos/inmunología , Cadenas kappa de Inmunoglobulina/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Animales , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Unión Competitiva/genética , Unión Competitiva/inmunología , Diferenciación Celular/genética , Línea Celular Tumoral , Células Cultivadas , Técnicas de Cocultivo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos/genética , Cadenas kappa de Inmunoglobulina/metabolismo , Cadenas kappa de Inmunoglobulina/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Moleculares , Células 3T3 NIH , Unión Proteica/genética , Unión Proteica/inmunología , Proteínas Proto-Oncogénicas/fisiología , Factor de Transcripción STAT5/fisiología , Transactivadores/fisiologíaRESUMEN
During B cell development, cells progress through multiple developmental stages with the pro-B cell stage defining commitment to the B cell lineage. YY1 is a ubiquitous transcription factor that is capable of both activation and repression functions. We find here that knockout of YY1 at the pro-B cell stage eliminates B lineage commitment. YY1 knockout pro-B cells can generate T lineage cells in vitro using the OP9- DL4 feeder system, as well as in vivo after injection into sub-lethally irradiated Rag1 -/- mice. These T lineage-like cells lose their B lineage transcript profile and gain a T cell lineage profile. Single cell-RNA-seq experiments showed that as YY1 knockout pro-B cells transition into T lineage cells, various cell clusters adopt transcript profiles representing a multiplicity of hematopoietic lineages indicating unusual lineage plasticity. Given the ubiquitous nature of YY1 and its dual activation and repression functions, YY1 likely regulates commitment in multiple cell lineages.
RESUMEN
The presumed involvement of paired box gene 5 (PAX5) in B-lymphomagenesis is based largely on the discovery of Pax5-specific translocations and somatic hypermutations in non-Hodgkin lymphomas. Yet mechanistically, the contribution of Pax5 to neoplastic growth remains undeciphered. Here we used 2 Myc-induced mouse B lymphoma cell lines, Myc5-M5 and Myc5-M12, which spontaneously silence Pax5. Reconstitution of these cells with Pax5-tamoxifen receptor fusion protein (Pax5ER(TAM)) increased neoplastic growth in a hormone-dependent manner. Conversely, expression of dominant-negative Pax5 in murine lymphomas and Pax5 knockdown in human lymphomas negatively affected cell expansion. Expression profiling revealed that Pax5 was required to maintain mRNA levels of several crucial components of B cell receptor (BCR) signaling, including CD79a, a protein with the immunoreceptor tyrosine-based activation motif (ITAM). In contrast, expression of 2 known ITAM antagonists, CD22 and PIR-B, was suppressed. The key role of BCR/ITAM signaling in Pax5-dependent lymphomagenesis was corroborated in Syk, an ITAM-associated tyrosine kinase. Moreover, we observed consistent expression of phosphorylated BLNK, an activated BCR adaptor protein, in human B cell lymphomas. Thus, stimulation of neoplastic growth by Pax5 occurs through BCR and is sensitive to genetic and pharmacological inhibitors of this pathway.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfoma/metabolismo , Linfoma/patología , Factor de Transcripción PAX5/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Activación de Linfocitos/inmunología , Linfoma/genética , Linfoma/inmunología , Ratones , Trasplante de Neoplasias , Factor de Transcripción PAX5/genética , Transducción de SeñalRESUMEN
In mice, zygotic or pro-B-cell-specific knock-out of the Pax5 gene allows differentiation of pro-B-cells into all hematopoietic lineages. We previously generated and characterized a murine B-cell lymphoma, dubbed Myc5, whose cells spontaneously lose Pax5 expression when cultured in vitro, but regain it when re-injected into syngeneic mice. In cultured Myc5 cells, the loss of Pax5 correlates with the acquisition of myeloid markers, such as CD11b and F4/80. Here, we sought to determine whether these cells are truly B-macrophage-restricted or, like Pax5-null progenitors, can give rise to additional hematopoietic lineages. In vitro differentiation assays with various cytokines showed that Myc5 cells do not differentiate into NK cells, dendritic cells, neutrophils, or osteoclasts. At the same time, in the presence of macrophage colony-stimulating factor (M-CSF), they readily phagocytose latex beads and provide T-cell help. Both phenomena are indicative of the bona fide macrophage phenotype. Conversely, enforced Pax5 re-expression in macrophage-like Myc5 cells led to down-regulation of the M-CSF receptor and re-acquisition of some B-cell surface markers (e.g., CD79a) and lineage-specific transcription factors (e.g., IRF4 and Blimp). Retrovirally encoded Pax5 also restored expression of several master B-cell differentiation proteins, such as the IL-7 receptor and transcription factor E2A. In contrast, levels of EBF were unaffected by Pax5 suggesting that EBF acts exclusively upstream of Pax5 and might contribute to Pax5 expression. Indeed, transduction with an EBF-encoding retrovirus partly reactivated endogenous Pax5. Our data reveal the complex relationship between B-cell-specific transcription factors and suggest the existence of numerous feedback mechanisms.
Asunto(s)
Linfocitos B/citología , Epigénesis Genética , Linfoma de Células B/genética , Macrófagos/citología , Factor de Transcripción PAX5/genética , Animales , Biomarcadores/análisis , Diferenciación Celular/genética , Linaje de la Célula/genética , Células Madre Hematopoyéticas/citología , Ratones , Fagocitosis/genética , Linfocitos T/fisiología , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Somatic rearrangement of the Ig genes during B cell development is believed to be controlled, at least in part, by accessibility of the loci to the recombinational machinery. Accessibility is poorly understood, but appears to be controlled by a combination of histone posttranslational modifications, large scale Ig locus contractions, and changes in intranuclear localization of the loci. These changes are regulated by developmental stage-specific as well as tissue-specific mechanisms. We previously isolated a murine B cell lymphoma line, Myc5, that can oscillate between the B cell and macrophage lineages depending upon growth conditions. This line provides an opportunity to study tissue-specific regulation of epigenetic mechanisms operating on the Ig loci. We found that when Myc5 cells are induced to differentiate from B cells into macrophages, expression of macrophage-specific transcripts was induced (M-CSFR, F4/80, and CD14), whereas B cell-specific transcripts decreased dramatically (mb-1, E47, IRF4, Pax5, and Igkappa). Loss of Igkappa transcription was associated with reduced Igkappa locus contraction, as well as increased association with heterochromatin protein-1 and association of the Igkappa locus with the nuclear periphery. Surprisingly, however, we found that histone modifications at the Igkappa locus remained largely unchanged whether the cells were grown in vivo as B cells, or in vitro as macrophages. These results mechanistically uncouple histone modifications at the Igkappa locus from changes in locus contraction and intranuclear localization.
Asunto(s)
Linfocitos B/inmunología , Epigénesis Genética , Histonas/metabolismo , Cadenas kappa de Inmunoglobulina/genética , Región de Control de Posición/genética , Macrófagos/inmunología , Alelos , Animales , Linfocitos B/ultraestructura , Diferenciación Celular , Línea Celular Tumoral , Núcleo Celular/química , Inmunoprecipitación de Cromatina , ADN/análisis , Hibridación Fluorescente in Situ , Macrófagos/ultraestructura , Ratones , Transcripción GenéticaRESUMEN
A recombinant rabies virus (RV) carrying two identical glycoprotein (G) genes (SPBNGA-GA) was constructed and used to determine the effect of RV G overexpression on cell viability and immunity. Immunoprecipitation analysis and flow cytometry showed that tissue culture cells infected with SPBNGA-GA produced, on average, twice as much RV G as cells infected with RV carrying only a single RV G gene (SPBNGA). The overexpression of RV G in SPBNGA-GA-infected NA cells was paralleled by a significant increase in caspase 3 activity followed by a marked decrease in mitochondrial respiration, neither of which was observed in SPBNGA-infected cells. Furthermore, fluorescence staining and confocal microscopy revealed an increased extent of apoptosis and markedly reduced neurofilament and F actin in SPBNGA-GA-infected primary neuron cultures compared with neuronal cells infected with SPBNGA, supporting the concept that RV G or motifs of the RV G gene trigger the apoptosis cascade. Mice immunized with SPBNGA-GA showed substantially higher antibody titers against the RV G and against the nucleoprotein than SPBNGA-immunized mice, suggesting that the speed or extent of apoptosis directly determines the magnitude of the antibody response.