RESUMEN
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
Antigen receptors (AgRs) expressed on B and T cells provide the adaptive immune system with ability to detect numerous foreign antigens. Epigenetic features of B cell receptor (BCR) and T cell receptor (TCR) genes were previously studied in lymphocytes, but little is known about their epigenetic features in other cells. Here, we explored histone modifications and transcription markers at the BCR and TCR loci in lymphocytes (pro-B, DP T cells, and mature CD4+ T cells), compared to embryonic stem (ES) cells and neurons. In B cells, the BCR loci exhibited active histone modifications and transcriptional markers indicative of active loci. Similar results were observed at the TCR loci in T cells. All loci were largely inactive in neurons. Surprisingly, in ES cells all AgR loci displayed a high degree of active histone modifications and markers of active transcription. Locations of these active histone modifications in ES cells were largely distinct from those in pro-B cells, and co-localized at numerous binding locations for transcription factors Oct4, Sox2, and Nanog. ES and pro-B cells also showed distinct binding patterns for the ubiquitous transcription factor YY1 and chromatin remodeler Brg1. On the contrary, there were many overlapping CCCTC-binding factor (CTCF) binding patterns when comparing ES cells, pro-B cells, and neurons. Our study identifies epigenetic features in ES cells and lymphocytes that may be related to ES cell pluripotency and lymphocyte tissue-specific activation at the AgR loci.
RESUMEN
Activation-induced cytidine deaminase (AID) has been implicated as both a positive and a negative factor in the progression of B cell chronic lymphocytic leukemia (CLL), but the role that it plays in the development and progression of this disease is still unclear. We generated an AID knockout CLL mouse model, AID-/-/Eµ-TCL1, and found that these mice die significantly earlier than their AID-proficient counterparts. AID-deficient CLL cells exhibit a higher ER stress response compared to Eµ-TCL1 controls, particularly through activation of the IRE1/XBP1s pathway. The increased production of secretory IgM in AID-deficient CLL cells contributes to their elevated expression levels of XBP1s, while secretory IgM-deficient CLL cells express less XBP1s. This increase in XBP1s in turn leads AID-deficient CLL cells to exhibit higher levels of B cell receptor signaling, supporting leukemic growth and survival. Further, AID-/-/Eµ-TCL1 CLL cells downregulate the tumor suppressive SMAD1/S1PR2 pathway and have altered homing to non-lymphoid organs. Notably, CLL cells from patients with IgHV-unmutated disease express higher levels of XBP1s mRNA compared to those from patients with IgHV-mutated CLL. Our studies thus reveal novel mechanisms by which the loss of AID leads to worsened CLL and may explain why unmutated CLL is more aggressive than mutated CLL.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Animales , Citidina Desaminasa/genética , Leucemia Linfocítica Crónica de Células B/patología , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas , ARN Mensajero/genética , Receptores de Antígenos de Linfocitos B/genéticaRESUMEN
Immunoglobulin heavy chain (IgH) genes are assembled by two sequential DNA rearrangement events that are initiated by recombination activating gene products (RAG) 1 and 2. Diversity (DH) gene segments rearrange first, followed by variable (VH) gene rearrangements. Here, we provide evidence that each rearrangement step is guided by different rules of engagement between rearranging gene segments. DH gene segments, which recombine by deletion of intervening DNA, must be located within a RAG1/2 scanning domain for efficient recombination. In the absence of intergenic control region 1, a regulatory sequence that delineates the RAG scanning domain on wild-type IgH alleles, VH and DH gene segments can recombine with each other by both deletion and inversion of intervening DNA. We propose that VH gene segments find their targets by distinct mechanisms from those that apply to DH gene segments. These distinctions may underlie differential allelic choice associated with each step of IgH gene assembly.
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
Systemic lupus erythematosus (SLE) is an autoimmune disorder that predominantly affects women and is driven by autoreactive T cell-mediated inflammation. It is known that individuals with multiple X-chromosomes are at increased risk for developing SLE; however, the mechanisms underlying this genetic basis are unclear. Here, we use single cell imaging to determine the epigenetic features of the inactive X (Xi) in developing thymocytes, mature T cell subsets, and T cells from SLE patients and mice. We show that Xist RNA and heterochromatin modifications transiently reappear at the Xi and are missing in mature single positive T cells. Activation of mature T cells restores Xist RNA and heterochromatin marks simultaneously back to the Xi. Notably, X-chromosome inactivation (XCI) maintenance is altered in T cells of SLE patients and late-stage-disease NZB/W F1 female mice, and we show that X-linked genes are abnormally upregulated in SLE patient T cells. SLE T cells also have altered expression of XIST RNA interactome genes, accounting for perturbations of Xi epigenetic features. Thus, abnormal XCI maintenance is a feature of SLE disease, and we propose that Xist RNA localization at the Xi could be an important factor for maintaining dosage compensation of X-linked genes in T cells.
Asunto(s)
Autoinmunidad/genética , Lupus Eritematoso Sistémico/genética , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunología , Inactivación del Cromosoma X/inmunología , Animales , Niño , Conjuntos de Datos como Asunto , Modelos Animales de Enfermedad , Femenino , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/inmunología , Activación de Linfocitos , Masculino , Ratones , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , RNA-Seq , Factores Sexuales , Análisis de la Célula Individual , Bazo/citología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Cromosoma X/genética , Cromosoma X/metabolismoRESUMEN
Activation-induced cytidine deaminase (AID) is a mutator enzyme essential for somatic hypermutation (SHM) and class switch recombination (CSR) during effective adaptive immune responses. Its aberrant expression and activity have been detected in lymphomas, leukemias, and solid tumors. In chronic lymphocytic leukemia (CLL) increased expression of alternatively spliced AID variants has been documented. We used real-time RT-PCR to quantify the expression of AID and its alternatively spliced transcripts (AIDΔE4a, AIDΔE4, AIDivs3, and AIDΔE3E4) in 149 CLL patients and correlated this expression to prognostic markers including recurrent chromosomal aberrations, the presence of complex karyotype, mutation status of the immunoglobulin heavy chain variable gene, and recurrent mutations. We report a previously unappreciated association between higher AID transcript levels and trisomy of chromosome 12. Functional analysis of AID splice variants revealed loss of their activity with respect to SHM, CSR, and induction of double-strand DNA breaks. In silico modeling provided insight into the molecular interactions and structural dynamics of wild-type AID and a shortened AID variant closely resembling AIDΔE4, confirming its loss-of-function phenotype.
Asunto(s)
Empalme Alternativo , Citidina Desaminasa , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B , Modelos Biológicos , Proteínas de Neoplasias , Trisomía , Anciano , Animales , Cromosomas Humanos Par 12/enzimología , Cromosomas Humanos Par 12/genética , Simulación por Computador , Citidina Desaminasa/biosíntesis , Citidina Desaminasa/química , Citidina Desaminasa/genética , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Simulación de Dinámica Molecular , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Trisomía/genética , Trisomía/patologíaRESUMEN
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
In females, the long non-coding RNA Xist drives X-chromosome Inactivation (XCI) to equalize X-linked gene dosage between sexes. Unlike other somatic cells, dynamic regulation of Xist RNA and heterochromatin marks on the inactive X (Xi) in female lymphocytes results in biallelic expression of some X-linked genes, including Tlr7, Cxcr3, and Cd40l, implicated in sex-biased autoimmune diseases. We now find that while Xist RNA is dispersed across the nucleus in NK cells and dendritic cells (DCs) and partially co-localizes with H3K27me3 in bone marrow-derived macrophages, it is virtually absent in plasmacytoid DCs (p-DCs). Moreover, H3K27me3 foci are present in only 10-20% of cells and we observed biallelic expression of Tlr7 in p-DCs from wildtype mice and NZB/W F1 mice. Unlike in humans, mouse p-DCs do not exhibit sex differences with interferon alpha production, and interferon signature gene expression in p-DCs is similar between males and females. Despite the absence of Xist RNA from the Xi, female p-DCs maintain dosage compensation of six immunity-related X-linked genes. Thus, immune cells use diverse mechanisms to maintain XCI which could contribute to sex-linked autoimmune diseases.
Asunto(s)
Células Dendríticas/fisiología , Epigénesis Genética , Variación Genética , Células Asesinas Naturales/fisiología , Macrófagos/fisiología , Inactivación del Cromosoma X/genética , Cromosoma X/genética , Análisis de Varianza , Animales , Núcleo Celular/metabolismo , Compensación de Dosificación (Genética) , Femenino , Regulación de la Expresión Génica , Genes Ligados a X , Heterocromatina/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos NZB , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Bazo/citología , Receptor Toll-Like 7/metabolismoRESUMEN
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
YY1 has been implicated as a master regulator of germinal center B cell development as YY1 binding sites are frequently present in promoters of germinal center-expressed genes. YY1 is known to be important for other stages of B cell development including the pro-B and pre-B cells stages. To determine if YY1 plays a critical role in germinal center development, we evaluated YY1 expression during B cell development, and used a YY1 conditional knock-out approach for deletion of YY1 in germinal center B cells (CRE driven by the immunoglobulin heavy chain γ1 switch region promoter; γ1-CRE). We found that YY1 is most highly expressed in germinal center B cells and is increased 3 fold in splenic B cells activated by treatment with anti-IgM and anti-CD40. In addition, deletion of the yy1 gene by action of γ1-CRE recombinase resulted in significant loss of GC cells in both un-immunized and immunized contexts with corresponding loss of serum IgG1. Our results show a crucial role for YY1 in the germinal center reaction.
Asunto(s)
Linfocitos B/inmunología , Centro Germinal/inmunología , Células Precursoras de Linfocitos B/inmunología , Bazo/inmunología , Factor de Transcripción YY1/genética , Animales , Anticuerpos Antiidiotipos/farmacología , Anticuerpos Monoclonales/farmacología , Linfocitos B/citología , Diferenciación Celular , Técnicas de Inactivación de Genes , Centro Germinal/citología , Inmunoglobulina G/sangre , Inmunoglobulina G/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Integrasas/genética , Integrasas/metabolismo , Activación de Linfocitos , Ratones , Ratones Transgénicos , Células Precursoras de Linfocitos B/citología , Regiones Promotoras Genéticas , Bazo/citología , Factor de Transcripción YY1/deficiencia , Factor de Transcripción YY1/inmunologíaRESUMEN
Females have a greater immunological advantage than men, yet they are more prone to autoimmune disorders. The basis for this sex bias lies in the X chromosome, which contains many immunity-related genes. Female mammals use X chromosome inactivation (XCI) to generate a transcriptionally silent inactive X chromosome (Xi) enriched with heterochromatic modifications and XIST/Xist RNA, which equalizes gene expression between the sexes. Here, we examine the maintenance of XCI in lymphocytes from females in mice and humans. Strikingly, we find that mature naïve T and B cells have dispersed patterns of XIST/Xist RNA, and they lack the typical heterochromatic modifications of the Xi. In vitro activation of lymphocytes triggers the return of XIST/Xist RNA transcripts and some chromatin marks (H3K27me3, ubiquitin-H2A) to the Xi. Single-cell RNA FISH analysis of female T cells revealed that the X-linked immunity genes CD40LG and CXCR3 are biallelically expressed in some cells. Using knockout and knockdown approaches, we find that Xist RNA-binding proteins, YY1 and hnRNPU, are critical for recruitment of XIST/Xist RNA back to the Xi. Furthermore, we examined B cells from patients with systemic lupus erythematosus, an autoimmune disorder with a strong female bias, and observed different XIST RNA localization patterns, evidence of biallelic expression of immunity-related genes, and increased transcription of these genes. We propose that the Xi in female lymphocytes is predisposed to become partially reactivated and to overexpress immunity-related genes, providing the first mechanistic evidence to our knowledge for the enhanced immunity of females and their increased susceptibility for autoimmunity.
Asunto(s)
Linfocitos/metabolismo , Inactivación del Cromosoma X , Animales , Femenino , Humanos , Inmunidad/genética , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Activación de Linfocitos , Masculino , Ratones , ARN Largo no Codificante/genética , ARN Mensajero/genéticaRESUMEN
During B cell development, long-distance DNA interactions are needed for V(D)J somatic rearrangement of the immunoglobulin (Ig) loci to produce functional Ig genes, and for class switch recombination (CSR) needed for antibody maturation. The tissue-specificity and developmental timing of these mechanisms is a subject of active investigation. A small number of factors are implicated in controlling Ig locus long-distance interactions including Pax5, Yin Yang 1 (YY1), EZH2, IKAROS, CTCF, cohesin, and condensin proteins. Here we will focus on the role of YY1 in controlling these mechanisms. YY1 is a multifunctional transcription factor involved in transcriptional activation and repression, X chromosome inactivation, Polycomb Group (PcG) protein DNA recruitment, and recruitment of proteins required for epigenetic modifications (acetylation, deacetylation, methylation, ubiquitination, sumoylation, etc.). YY1 conditional knock-out indicated that YY1 is required for B cell development, at least in part, by controlling long-distance DNA interactions at the immunoglobulin heavy chain and Igκ loci. Our recent data show that YY1 is also required for CSR. The mechanisms implicated in YY1 control of long-distance DNA interactions include controlling non-coding antisense RNA transcripts, recruitment of PcG proteins to DNA, and interaction with complexes involved in long-distance DNA interactions including the cohesin and condensin complexes. Though common rearrangement mechanisms operate at all Ig loci, their distinct temporal activation along with the ubiquitous nature of YY1 poses challenges for determining the specific mechanisms of YY1 function in these processes, and their regulation at the tissue-specific and B cell stage-specific level. The large numbers of post-translational modifications that control YY1 functions are possible candidates for regulation.
RESUMEN
Polycomb Group (PcG) proteins are crucial for epigenetic inheritance of cell identity and are functionally conserved from Drosophila to humans. PcG proteins regulate expression of homeotic genes and are essential for axial body patterning during development. Earlier we showed that transcription factor YY1 functions as a PcG protein. YY1 also physically interacts with YAF2, a homolog of RYBP. Here we characterize the mechanism and physiologic relevance of this interaction. We found phenotypic and biochemical correction of dRYBP mutant flies by mouse YAF2 demonstrating functional conservation across species. Further biochemical analysis revealed that YAF2 bridges interaction between YY1 and the PRC1 complex. ChIP assays in HeLa cells showed that YAF2 is responsible for PcG recruitment to DNA, which is mediated by YY1 DNA binding. Knock-down of YY1 abrogated PcG recruitment, which was not compensated by exogenous YAF2 demonstrating that YY1 DNA binding is a priori necessary for Polycomb assembly on chromatin. Finally, we found that although YAF2 and RYBP regulate a similar number of Polycomb target genes, there are very few genes that are regulated by both implying functional distinction between the two proteins. We present a model of YAF2-dependent and independent PcG DNA recruitment by YY1.
Asunto(s)
Silenciador del Gen , Proteínas Musculares/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Proteínas Represoras/metabolismo , Factor de Transcripción YY1/metabolismo , Animales , Cromatina/metabolismo , ADN/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Células HEK293 , Células HeLa , Humanos , Ratones , Proteínas Musculares/química , Proteínas Musculares/fisiología , Mutación , Fenotipo , Dominios y Motivos de Interacción de Proteínas , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Factor de Transcripción YY1/químicaRESUMEN
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
Activation-induced deaminase (AID) is an enzyme required for class switch recombination (CSR) and somatic hypermutation (SHM), processes that ensure antibody maturation and expression of different immunoglobulin isotypes. AID function is tightly regulated by tissue- and stage-specific expression, nuclear localization, and protein stability. Transcription factor YY1 is crucial for early B cell development, but its function at late B cell stages is unknown. Here, we show that YY1 conditional knockout in activated splenic B cells interferes with CSR. Knockout of YY1 did not affect B cell proliferation, transcription of the AID and IgM genes, or levels of various switch region germ line transcripts. However, we show that YY1 physically interacts with AID and controls the accumulation of nuclear AID, at least in part, by increasing nuclear AID stability. We show for the first time that YY1 plays a novel role in CSR and controls nuclear AID protein levels.
Asunto(s)
Linfocitos B/metabolismo , Citidina Desaminasa/metabolismo , Cambio de Clase de Inmunoglobulina/genética , Factor de Transcripción YY1/metabolismo , Animales , Linfocitos B/inmunología , Línea Celular , Núcleo Celular/enzimología , Citidina Desaminasa/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Bazo/inmunología , Factor de Transcripción YY1/genéticaRESUMEN
Ying Yang 1 (YY1) is a multifunctional Polycomb Group (PcG) transcription factor that binds to multiple enhancer binding sites in the immunoglobulin (Ig) loci and plays vital roles in early B cell development. PcG proteins have important functions in hematopoietic stem cell renewal and YY1 is the only mammalian PcG protein with DNA binding specificity. Conditional knock-out of YY1 in the mouse B cell lineage results in arrest at the pro-B cell stage, and dosage effects have been observed at various YY1 expression levels. To investigate the impact of elevated YY1 expression on hematopoetic development, we utilized a mouse in vivo bone marrow reconstitution system. We found that mouse bone marrow cells expressing elevated levels of YY1 exhibited a selective disadvantage as they progressed from hematopoietic stem/progenitor cells to pro-B, pre-B, immature B and re-circulating B cell stages, but no disadvantage of YY1 over-expression was observed in myeloid lineage cells. Furthermore, mouse bone marrow cells expressing elevated levels of YY1 displayed enrichment for cells with surface markers characteristic of long-term hematopoietic stem cells (HSC). YY1 expression induced apoptosis in mouse B cell lines in vitro, and resulted in down-regulated expression of anti-apoptotic genes Bcl-xl and NFκB2, while no impact was observed in a mouse myeloid line. B cell apoptosis and LT-HSC enrichment induced by YY1 suggest that novel strategies to induce YY1 expression could have beneficial effects in the treatment of B lineage malignancies while preserving normal HSCs.
Asunto(s)
Linfocitos B/fisiología , Diferenciación Celular/genética , Proliferación Celular , Células Madre Hematopoyéticas/fisiología , Células Mieloides/fisiología , Factor de Transcripción YY1/genética , Animales , Linfocitos B/metabolismo , Diferenciación Celular/inmunología , Células Cultivadas , Regulación hacia Abajo/inmunología , Regulación del Desarrollo de la Expresión Génica/fisiología , Células HEK293 , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Mieloides/metabolismo , Proteínas del Grupo Polycomb , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/fisiología , Factores de Tiempo , Regulación hacia Arriba/genética , Regulación hacia Arriba/fisiología , Factor de Transcripción YY1/metabolismo , Factor de Transcripción YY1/fisiologíaRESUMEN
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
Yin Yang 1 (YY1) is a ubiquitously expressed transcription factor that performs numerous functions including transcriptional regulation, cell growth control, apoptosis, large-scale chromosomal dynamics, and X-chromosome inactivation. YY1 clearly is able to control cell functions, including proliferation, by acting as a transcription factor either to activate or repress specific genes. Based on its ability to regulate cell growth control genes, it has been argued that YY1 can function as an oncogene that initiates oncogenesis. Although this is an attractive hypothesis, no reports indicate that YY1 can acutely transform cells in culture or form tumors within animals when overexpressed. Thus, it remains unclear whether YY1 is a "classic" oncogene. However, YY1 controls many diverse cell functions, and these functions may provide clues to its role in oncogenesis. We propose that in many cases YY1 may function in oncogenesis and disease progression through "indirect" effects by virtue of its role in either recruiting Polycomb group proteins to DNA, regulating mutator protein accumulation, controlling large-scale chromosomal dynamics or genomic integrity. Disruption of these functions may causally initiate cancer or may contribute to disease progression. Targeting YY1 functions provides possible avenues for clinical intervention.