RESUMEN
Age-associated B cells (ABCs) are a subset of B cells dependent on the transcription factor T-bet that accumulate prematurely in autoimmune settings. The pathways that regulate ABCs in autoimmunity are largely unknown. SWAP-70 and DEF6 (also known as IBP or SLAT) are the only two members of the SWEF family, a unique family of Rho GTPase-regulatory proteins that control both cytoskeletal dynamics and the activity of the transcription factor IRF4. Notably, DEF6 is a newly identified human risk variant for systemic lupus erythematosus. Here we found that the lupus syndrome that developed in SWEF-deficient mice was accompanied by the accumulation of ABCs that produced autoantibodies after stimulation. ABCs from SWEF-deficient mice exhibited a distinctive transcriptome and a unique chromatin landscape characterized by enrichment for motifs bound by transcription factors of the IRF and AP-1 families and the transcription factor T-bet. Enhanced ABC formation in SWEF-deficient mice was controlled by the cytokine IL-21 and IRF5, whose variants are strongly associated with lupus. The lack of SWEF proteins led to dysregulated activity of IRF5 in response to stimulation with IL-21. These studies thus elucidate a previously unknown signaling pathway that controls ABCs in autoimmunity.
Asunto(s)
Autoinmunidad/inmunología , Subgrupos de Linfocitos B/inmunología , Factores Reguladores del Interferón/inmunología , Lupus Eritematoso Sistémico/inmunología , Animales , Subgrupos de Linfocitos B/patología , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/inmunología , Femenino , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/inmunología , Lupus Eritematoso Sistémico/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Antígenos de Histocompatibilidad Menor/inmunología , Proteínas Nucleares/deficiencia , Proteínas Nucleares/inmunologíaRESUMEN
Crosslinking of FcεRI-bound IgE triggers the release of a large number of biologically active, potentially anaphylactic compounds by mast cells. FcεRI activation ought to be well-controlled to restrict adverse activation. As mast cells are embedded in tissues, adhesion molecules may contribute to limiting premature activation. Here, we report that E-Cadherin serves that purpose. Having confirmed that cultured mast cells express E-Cadherin, a mast-cell-specific E-Cadherin deficiency, Mcpt5-Cre E-Cdhfl/fl mice, was used to analyze mast cell degranulation in vitro and in vivo. Cultured peritoneal mast cells from Mcpt5-Cre E-Cdhfl/fl mice were normal with respect to many parameters but showed much-enhanced degranulation in three independent assays. Soluble E-Cadherin reduced the degranulation of control cells. The release of some newly synthesized inflammatory cytokines was decreased by E-Cadherin deficiency. Compared to controls, Mcpt5-Cre E-Cdhfl/fl mice reacted much stronger to IgE-dependent stimuli, developing anaphylactic shock. We suggest E-Cadherin-mediated tissue interactions restrict mast cell degranulation to prevent their precocious activation.
Asunto(s)
Cadherinas/inmunología , Degranulación de la Célula/inmunología , Mastocitos/inmunología , Animales , Cadherinas/genética , Degranulación de la Célula/genética , Citocinas/genética , Citocinas/inmunología , Inmunoglobulina E/genética , Inmunoglobulina E/inmunología , Inflamación/genética , Inflamación/inmunología , Ratones , Ratones Transgénicos , Receptores de IgE/genética , Receptores de IgE/inmunologíaRESUMEN
F-actin binding and bundling are crucial to a plethora of cell processes, including morphogenesis, migration, adhesion and many others. SWAP-70 was recently described as an in vitro F-actin-binding and -bundling protein. Fluorescence cross-correlation spectroscopy measurements with purified recombinant SWAP-70 confirmed that it forms stable oligomers that facilitate F-actin bundling. However, it remained unclear how SWAP-70 oligomerization and F-actin binding are controlled in living cells. We addressed this by biophysical approaches, including seFRET, FACS-FRET and FLIM-FRET. PIP3-mediated association with the cytoplasmic membrane and non-phosphorylated Y426 are required for SWAP-70 to dimerize and to bind F-actin. The dimerization region was identified near the C terminus where R546 is required for dimerization and, thus, F-actin bundling. The in vitro and in vivo data presented here reveal the functional relationship between the cytoplasm-to-membrane translocation and dimerization of SWAP-70, and F-actin binding and bundling, and demonstrate that SWAP-70 is a finely controlled modulator of membrane-proximal F-actin dynamics.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Actinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas Nucleares/metabolismo , Animales , Estructuras de la Membrana Celular/metabolismo , Células HEK293 , Humanos , Melanoma Experimental , Ratones , Multimerización de ProteínaRESUMEN
Although GM-CSF has been widely used in dendritic cell (DC) research, the mechanisms, factors, and signals regulating steady-state differentiation and maturation of GM-CSF-dependent DCs are insufficiently known. We found that the absence, individually or combined, of the related proteins DEF6 and SWAP-70 strongly enhances differentiation of murine GM-CSF-derived DCs. Contrasting SWAP-70, control through DEF6 does not depend on RHOA activation. DEF6 deficiency leads to expression of the DC-specific transcription factor ZBTB46 and prolonged STAT5 activation in GM-CSF cultures. SWAP-70 and DEF6-mediated restriction of DC differentiation converges mechanistically at the NF-κB pathway. DEF6 acts at early stages of DC differentiation in CD115-cKIT+ myeloid DC progenitors, whereas SWAP-70 acts subsequently. SWAP-70 and DEF6 regulate steady-state DC cytokine expression as well as in vivo accumulation in lymphatic tissue of migratory DCs. Our studies thus elucidate previously unknown roles of two closely related factors with distinct and complementary activities in DC differentiation and steady-state DC function.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Células Dendríticas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas Nucleares/metabolismo , Animales , Tejido Linfoide/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Células Progenitoras Mieloides/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción STAT5/metabolismo , Factores de Transcripción/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Somatic loss of function mutations in cohesin genes are frequently associated with various cancer types, while cohesin disruption in the germline causes cohesinopathies such as Cornelia-de-Lange syndrome (CdLS). Here, we present the discovery of a recurrent heterozygous RAD21 germline aberration at amino acid position 298 (p.P298S/A) identified in three children with lymphoblastic leukemia or lymphoma in a total dataset of 482 pediatric cancer patients. While RAD21 p.P298S/A did not disrupt the formation of the cohesin complex, it altered RAD21 gene expression, DNA damage response and primary patient fibroblasts showed increased G2/M arrest after irradiation and Mitomycin-C treatment. Subsequent single-cell RNA-sequencing analysis of healthy human bone marrow confirmed the upregulation of distinct cohesin gene patterns during hematopoiesis, highlighting the importance of RAD21 expression within proliferating B- and T-cells. Our clinical and functional data therefore suggest that RAD21 germline variants can predispose to childhood lymphoblastic leukemia or lymphoma without displaying a CdLS phenotype.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Linfoma , Leucemia-Linfoma Linfoblástico de Células Precursoras , Apoptosis , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Niño , Proteínas de Unión al ADN/genética , Síndrome de Cornelia de Lange/genética , Puntos de Control de la Fase G2 del Ciclo Celular , Células Germinativas/metabolismo , Humanos , Linfoma/genética , Mutación , Fenotipo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMEN
Signal peptide peptidase (SPP) and the four homologous SPP-like (SPPL) proteases constitute a family of intramembrane aspartyl proteases with selectivity for type II-oriented transmembrane segments. Here, we analyse the physiological function of the orphan protease SPPL2c, previously considered to represent a non-expressed pseudogene. We demonstrate proteolytic activity of SPPL2c towards selected tail-anchored proteins. Despite shared ER localisation, SPPL2c and SPP exhibit distinct, though partially overlapping substrate spectra and inhibitory profiles, and are organised in different high molecular weight complexes. Interestingly, SPPL2c is specifically expressed in murine and human testis where it is primarily localised in spermatids. In mice, SPPL2c deficiency leads to a partial loss of elongated spermatids and reduced motility of mature spermatozoa, but preserved fertility. However, matings of male and female SPPL2c-/- mice exhibit reduced litter sizes. Using proteomics we identify the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2)-regulating protein phospholamban (PLN) as a physiological SPPL2c substrate. Accumulation of PLN correlates with a decrease in intracellular Ca2+ levels in elongated spermatids that likely contribute to the compromised male germ cell differentiation and function of SPPL2c-/- mice.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Membrana Celular/enzimología , Células Germinativas/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Ácido Aspártico Endopeptidasas/química , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Femenino , Células HEK293 , Células HeLa , Homeostasis , Humanos , Masculino , Proteínas de la Membrana/química , Ratones , Especificidad de Órganos , Espermátides/metabolismo , Especificidad por Sustrato , Testículo/enzimologíaRESUMEN
Tudor containing protein 6 (TDRD6) is a male germ line-specific protein essential for chromatoid body (ChB) structure, elongated spermatid development and male fertility. Here we show that in meiotic prophase I spermatocytes TDRD6 interacts with the key protein arginine methyl transferase PRMT5, which supports splicing. TDRD6 also associates with spliceosomal core protein SmB in the absence of RNA and in an arginine methylation dependent manner. In Tdrd6-/- diplotene spermatocytes PRMT5 association with SmB and arginine dimethylation of SmB are much reduced. TDRD6 deficiency impairs the assembly of spliceosomes, which feature 3.5-fold increased levels of U5 snRNPs. In the nucleus, these deficiencies in spliceosome maturation correlate with decreased numbers of SMN-positive bodies and Cajal bodies involved in nuclear snRNP maturation. Transcriptome analysis of TDRD6-deficient diplotene spermatocytes revealed high numbers of splicing defects such as aberrant usage of intron and exons as well as aberrant representation of splice junctions. Together, this study demonstrates a novel function of TDRD6 in spliceosome maturation and mRNA splicing in prophase I spermatocytes.
Asunto(s)
Proteína-Arginina N-Metiltransferasas/metabolismo , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/fisiología , Espermatocitos/metabolismo , Empalmosomas/metabolismo , Animales , Arginina/química , Cromátides/química , Cuerpos Enrollados/metabolismo , Metilación de ADN , Desoxiadenosinas/química , Exones , Ácidos Grasos Insaturados/química , Intrones , Masculino , Metilación , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Dominios Proteicos , Empalme del ARN , ARN Mensajero/metabolismo , Espermatocitos/citología , Tionucleósidos/química , TranscriptomaRESUMEN
[This corrects the article DOI: 10.1371/journal.pgen.1006660.].
RESUMEN
Mast cells (MCs), which are best known for their detrimental role in patients with allergic diseases, act in a diverse array of physiologic and pathologic functions made possible by the plurality of MC types. Their various developmental avenues and distinct sensitivity to (micro-) environmental conditions convey extensive heterogeneity, resulting in diverse functions. We briefly summarize this heterogeneity, elaborate on molecular determinants that allow MCs to communicate with their environment to fulfill their tasks, discuss the protease repertoire stored in secretory lysosomes, and consider different aspects of MC signaling. Furthermore, we describe key MC governance mechanisms (ie, the high-affinity receptor for IgE [FcεRI]), the stem cell factor receptor KIT, the IL-4 system, and both Ca2+- and phosphatase-dependent mechanisms. Finally, we focus on distinct physiologic functions, such as chemotaxis, phagocytosis, host defense, and the regulation of MC functions at the mucosal barriers of the lung, gastrointestinal tract, and skin. A deeper knowledge of the pleiotropic functions of MC mediators, as well as the molecular processes of MC regulation and communication, should enable us to promote beneficial MC traits in physiology and suppress detrimental MC functions in patients with disease.
Asunto(s)
Quimiotaxis/inmunología , Mucosa Intestinal/inmunología , Mastocitos/inmunología , Fagocitosis , Mucosa Respiratoria/inmunología , Transducción de Señal/inmunología , Animales , Calcio/inmunología , Humanos , Interleucina-4/inmunología , Mucosa Intestinal/patología , Lisosomas/inmunología , Lisosomas/patología , Mastocitos/patología , Proteínas Proto-Oncogénicas c-kit/inmunología , Receptores de IgE/inmunología , Mucosa Respiratoria/patologíaRESUMEN
Lipids affect the membrane properties determining essential biological processes. Earlier studies have suggested a role of switch-activated protein 70 (SWAP-70) in lipid raft formation of dendritic cells. We used lipidomics combined with genetic and biochemical assays to analyze the role of SWAP-70 in lipid dynamics. TLR activation using LPS as a ligand represented a pathogenic immunogenic stimulus, physical disruption of cell-cell contacts a tolerogenic stimulus. Physical disruption, but not LPS, caused an increase of phosphatidylcholine ether and cholesteryl esters in CD11c+ immune cells. An increase of ceramide (Cer) was a hallmark for LPS activation. SWAP-70 was required for regulating the increase and localization of Cers in the cell membrane. SWAP-70 controls Cer accumulation through the regulation of pH-dependent acid-sphingomyelinase activity and of RhoA-dependent transport of endosomal contents to the plasma membrane. Poor accumulation of Cers in Swap70-/- cells caused decreased apoptosis. This shows that two different pathways of activation, immunogenic and tolerogenic, induce different changes in the lipid composition of cultured CD11c+ cells, and highlights the important role of SWAP-70 in Cer dynamics in dendritic cells.
Asunto(s)
Antígeno CD11c/inmunología , Ceramidas/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Dendríticas/inmunología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Tolerancia Inmunológica , Lípidos/inmunología , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas Nucleares/metabolismo , Animales , Apoptosis , Línea Celular , Células Cultivadas , Ceramidas/inmunología , Ésteres del Colesterol/genética , Ésteres del Colesterol/inmunología , Medios de Cultivo/química , Citocinas/biosíntesis , Citocinas/inmunología , Proteínas de Unión al ADN/genética , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Lípidos/análisis , Lipopolisacáridos/inmunología , Ratones , Antígenos de Histocompatibilidad Menor/genética , Proteínas Nucleares/genética , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
Mammalian meiocytes feature four meiosis-specific cohesin proteins in addition to ubiquitous ones, but the roles of the individual cohesin complexes are incompletely understood. To decipher the functions of the two meiosis-specific kleisins, REC8 or RAD21L, together with the only meiosis-specific SMC protein SMC1ß, we generated Smc1ß-/-Rec8-/- and Smc1ß-/-Rad21L-/- mouse mutants. Analysis of spermatocyte chromosomes revealed that besides SMC1ß complexes, SMC1α/RAD21 and to a small extent SMC1α/REC8 contribute to chromosome axis length. Removal of SMC1ß and RAD21L almost completely abolishes all chromosome axes. The sex chromosomes do not pair in single or double mutants, and autosomal synapsis is impaired in all mutants. Super resolution microscopy revealed synapsis-associated SYCP1 aberrantly deposited between sister chromatids and on single chromatids in Smc1ß-/-Rad21L-/- cells. All mutants show telomere length reduction and structural disruptions, while wild-type telomeres feature a circular TRF2 structure reminiscent of t-loops. There is no loss of centromeric cohesion in both double mutants at leptonema/early zygonema, indicating that, at least in the mutant backgrounds, an SMC1α/RAD21 complex provides centromeric cohesion at this early stage. Thus, in early prophase I the most prominent roles of the meiosis-specific cohesins are in axis-related features such as axis length, synapsis and telomere integrity rather than centromeric cohesion.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Meiosis/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Espermatogénesis/genética , Animales , Centrómero/genética , Cromátides/genética , Emparejamiento Cromosómico/genética , Segregación Cromosómica/genética , Proteínas de Unión al ADN , Masculino , Mamíferos , Profase Meiótica I/genética , Ratones , Ratones Noqueados , Espermatocitos/crecimiento & desarrollo , Complejo Sinaptonémico/genética , CohesinasRESUMEN
Chromatoid bodies (CBs) are spermiogenesis-specific organelles of largely unknown function. CBs harbor various RNA species, RNA-associated proteins and proteins of the tudor domain family like TDRD6, which is required for a proper CB architecture. Proteome analysis of purified CBs revealed components of the nonsense-mediated mRNA decay (NMD) machinery including UPF1. TDRD6 is essential for UPF1 localization to CBs, for UPF1-UPF2 and UPF1-MVH interactions. Upon removal of TDRD6, the association of several mRNAs with UPF1 and UPF2 is disturbed, and the long 3' UTR-stimulated but not the downstream exon-exon junction triggered pathway of NMD is impaired. Reduced association of the long 3' UTR mRNAs with UPF1 and UPF2 correlates with increased stability and enhanced translational activity. Thus, we identified TDRD6 within CBs as required for mRNA degradation, specifically the extended 3' UTR-triggered NMD pathway, and provide evidence for the requirement of NMD in spermiogenesis. This function depends on TDRD6-promoted assembly of mRNA and decay enzymes in CBs.
Asunto(s)
Proteínas Portadoras/genética , Glutatión/análogos & derivados , Estabilidad del ARN/genética , Ribonucleoproteínas/genética , Espermatogénesis/genética , Regiones no Traducidas 3'/genética , Animales , Células Germinativas/crecimiento & desarrollo , Células Germinativas/metabolismo , Glutatión/genética , Masculino , Ratones , Ratones Noqueados , Dominios Proteicos/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/metabolismo , Espermátides/crecimiento & desarrollo , Espermátides/metabolismoRESUMEN
The cohesin complex is essential for mitosis and meiosis. The specific meiotic roles of individual cohesin proteins are incompletely understood. We report in vivo functions of the only meiosis-specific STAG component of cohesin, STAG3. Newly generated STAG3-deficient mice of both sexes are sterile with meiotic arrest. In these mice, meiotic chromosome architecture is severely disrupted as no bona fide axial elements (AE) form and homologous chromosomes do not synapse. Axial element protein SYCP3 forms dot-like structures, many partially overlapping with centromeres. Asynapsis marker HORMAD1 is diffusely distributed throughout the chromatin, and SYCP1, which normally marks synapsed axes, is largely absent. Centromeric and telomeric sister chromatid cohesion are impaired. Centromere and telomere clustering occurs in the absence of STAG3, and telomere structure is not severely affected. Other cohesin proteins are present, localize throughout the STAG3-devoid chromatin, and form complexes with cohesin SMC1ß. No other deficiency in a single meiosis-specific cohesin causes a phenotype as drastic as STAG3 deficiency. STAG3 emerges as the key STAG cohesin involved in major functions of meiotic cohesin.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromátides/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/genética , Meiosis/genética , Proteínas Nucleares/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Centrómero/genética , Centrómero/fisiología , Cromátides/genética , Proteínas Cromosómicas no Histona/genética , Cromosomas/genética , Femenino , Masculino , Ratones , Proteínas Nucleares/genética , Oocitos/citología , Oocitos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Organismos Libres de Patógenos Específicos , Espermatocitos/citología , Espermatocitos/metabolismo , Complejo Sinaptonémico/genética , Complejo Sinaptonémico/metabolismo , Telómero/genética , Telómero/fisiología , Testículo/citología , Testículo/metabolismo , CohesinasRESUMEN
To determine the site and mechanism of suppression by regulatory T (Treg) cells, we investigated their migration and function in an islet allograft model. Treg cells first migrated from blood to the inflamed allograft where they were essential for the suppression of alloimmunity. This process was dependent on the chemokine receptors CCR2, CCR4, and CCR5 and P- and E-selectin ligands. In the allograft, Treg cells were activated and subsequently migrated to the draining lymph nodes (dLNs) in a CCR2, CCR5, and CCR7 fashion; this movement was essential for optimal suppression. Treg cells inhibited dendritic cell migration in a TGF-beta and IL-10 dependent fashion and suppressed antigen-specific T effector cell migration, accumulation, and proliferation in dLNs and allografts. These results showed that sequential migration from blood to the target tissue and to dLNs is required for Treg cells to differentiate and execute fully their suppressive function.
Asunto(s)
Autoinmunidad/inmunología , Movimiento Celular/inmunología , Ganglios Linfáticos/inmunología , Subgrupos Linfocitarios/inmunología , Linfocitos T Reguladores/inmunología , Animales , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/fisiología , Inflamación , Islotes Pancreáticos/citología , Islotes Pancreáticos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Humans suffer a steep increase in aneuploidies when oocytes age, and deterioration of cohesin was suggested recently as a prominent cause. In the November 15, 2010, issue of Genes & Development, Tachibana-Konwalski and colleagues (pp. 2505-2516) answered a question central to this hypothesis: Can cohesin be reloaded onto mouse oocyte chromosomes long after birth? They found that it cannot, or at least not with an efficiency adequate to rescue cohesin deficiency. With no chance for sufficient replenishment, age-related loss of sister chromatid cohesion seems unavoidable.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Oocitos/metabolismo , Factores de Edad , Aneuploidia , Animales , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , CohesinasRESUMEN
Telomeres are repetitive DNA structures that, together with the shelterin and the CST complex, protect the ends of chromosomes. Telomere shortening is mitigated in stem and cancer cells through the de novo addition of telomeric repeats by telomerase. Telomere elongation requires the delivery of the telomerase complex to telomeres through a not yet fully understood mechanism. Factors promoting telomerase-telomere interaction are expected to directly bind telomeres and physically interact with the telomerase complex. In search for such a factor we carried out a SILAC-based DNA-protein interaction screen and identified HMBOX1, hereafter referred to as homeobox telomere-binding protein 1 (HOT1). HOT1 directly and specifically binds double-stranded telomere repeats, with the in vivo association correlating with binding to actively processed telomeres. Depletion and overexpression experiments classify HOT1 as a positive regulator of telomere length. Furthermore, immunoprecipitation and cell fractionation analyses show that HOT1 associates with the active telomerase complex and promotes chromatin association of telomerase. Collectively, these findings suggest that HOT1 supports telomerase-dependent telomere elongation.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Complejos Multiproteicos/metabolismo , Telomerasa/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Cromatina/genética , Cromatina/metabolismo , Células HeLa , Proteínas de Homeodominio/genética , Humanos , Complejos Multiproteicos/genética , Secuencias Repetitivas de Ácidos Nucleicos/fisiología , Telomerasa/genética , Telómero/genética , Proteínas de Unión a Telómeros/genéticaRESUMEN
Mouse embryo fibroblasts (MEFs) grow slowly after cultivation from animals, however, after an extended period of cultivation, their growth accelerates. We found that SWAP-70 deficient MEFs failed to increase growth rates. They maintain normal growth rates and proliferation cycles for at least 5 years. Complementing SWAP-70 deficiency in one of these MEF clones, MEF1F2, by expressing human SWAP-70 resulted in fast growth of the cells after further cultivation for a long period. The resulting cells show a transformation phenotype, since they grow on top of each other and do not show contact inhibition. This phenotype was reverted when sanguinarine, a putative SWAP-70 inhibitor, was added. Two SWAP-70 expressing clones were examined in detail. Even after cell density became very high their cdc2 and NFκB were still activated suggesting that they do not stop growing. One of the clones formed colonies in soft agar and formed tumors in nude mice. Lately, one more clone became transformed being able to make colonies in soft agar. We maintain 4 human SWAP-70 expressing MEF1F2 cell lines. Three out of 4 clones exhibited transforming phenotypes. The mouse SWAP-70 gene also promoted transformation of MEFs. Taken together our data suggest that SWAP-70 is not a typical oncogene, but is required for spontaneous transformation of MEFs.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas Nucleares/metabolismo , Benzofenantridinas/farmacología , Proteína Quinasa CDC2/metabolismo , Línea Celular , ADN Complementario/genética , Proteínas de Unión al ADN/deficiencia , Fibroblastos/efectos de los fármacos , Factores de Intercambio de Guanina Nucleótido/deficiencia , Humanos , Isoquinolinas/farmacología , FN-kappa B/metabolismo , Proteínas Nucleares/deficiencia , Fenotipo , Factores de TiempoRESUMEN
Cohesin subunit SMC1ß is specific and essential for meiosis. Previous studies showed functions of SMC1ß in determining the axis-loop structure of synaptonemal complexes (SCs), in providing sister chromatid cohesion (SCC) in metaphase I and thereafter, in protecting telomere structure, and in synapsis. However, several central questions remained unanswered and concern roles of SMC1ß in SCC and synapsis and processes related to these two processes. Here we show that SMC1ß substantially supports prophase I SCC at centromeres but not along chromosome arms. Arm cohesion and some of centromeric cohesion in prophase I are provided by non-phosphorylated SMC1α. Besides supporting synapsis of autosomes, SMC1ß is also required for synapsis and silencing of sex chromosomes. In absence of SMC1ß, the silencing factor γH2AX remains associated with asynapsed autosomes and fails to localize to sex chromosomes. Microarray expression studies revealed up-regulated sex chromosome genes and many down-regulated autosomal genes. SMC1ß is further required for non-homologous chromosome associations observed in absence of SPO11 and thus of programmed double-strand breaks. These breaks are properly generated in Smc1ßâ»/â» spermatocytes, but their repair is delayed on asynapsed chromosomes. SMC1α alone cannot support non-homologous associations. Together with previous knowledge, three main functions of SMC1ß have emerged, which have multiple consequences for spermatocyte biology: generation of the loop-axis architecture of SCs, homologous and non-homologous synapsis, and SCC starting in early prophase I.
Asunto(s)
Proteínas de Ciclo Celular/genética , Emparejamiento Cromosómico/genética , Meiosis/genética , Profase Meiótica I/genética , Espermatocitos/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Centrómero/genética , Centrómero/metabolismo , Segregación Cromosómica/genética , Endodesoxirribonucleasas/genética , Masculino , Ratones , Espermatocitos/crecimiento & desarrollo , Complejo Sinaptonémico/genética , Telómero/genética , Telómero/metabolismoRESUMEN
Based on studies in mice and humans, cohesin loss from chromosomes during the period of protracted meiotic arrest appears to play a major role in chromosome segregation errors during female meiosis. In mice, mutations in meiosis-specific cohesin genes cause meiotic disturbances and infertility. However, the more clinically relevant situation, heterozygosity for mutations in these genes, has not been evaluated. We report here evidence from the mouse that partial loss of gene function for either Smc1b or Rec8 causes perturbations in the formation of the synaptonemal complex (SC) and affects both synapsis and recombination between homologs during meiotic prophase. Importantly, these defects increase the frequency of chromosomally abnormal eggs in the adult female. These findings have important implications for humans: they suggest that women who carry mutations or variants that affect cohesin function have an elevated risk of aneuploid pregnancies and may even be at increased risk of transmitting structural chromosome abnormalities.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/genética , Cromosomas , Meiosis/genética , Animales , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Emparejamiento Cromosómico/genética , Cromosomas/genética , Cromosomas/ultraestructura , Femenino , Dosificación de Gen , Humanos , Ratones , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Recombinación Genética , Complejo Sinaptonémico/genética , CohesinasRESUMEN
Four members of the structural maintenance of chromosome (SMC) protein family have essential functions in chromosome condensation (SMC2/4) and sister-chromatid cohesion (SMC1/3). The SMC5/6 complex has been implicated in chromosome replication, DNA repair and chromosome segregation in somatic cells, but its possible functions during mammalian meiosis are unknown. Here, we show in mouse spermatocytes that SMC5 and SMC6 are located at the central region of the synaptonemal complex from zygotene until diplotene. During late diplotene both proteins load to the chromocenters, where they colocalize with DNA Topoisomerase IIα, and then accumulate at the inner domain of the centromeres during the first and second meiotic divisions. Interestingly, SMC6 and DNA Topoisomerase IIα colocalize at stretched strands that join kinetochores during the metaphase II to anaphase II transition, and both are observed on stretched lagging chromosomes at anaphase II following treatment with Etoposide. During mitosis, SMC6 and DNA Topoisomerase IIα colocalize at the centromeres and chromatid axes. Our results are consistent with the participation of SMC5 and SMC6 in homologous chromosome synapsis during prophase I, chromosome and centromere structure during meiosis I and mitosis and, with DNA Topoisomerase IIα, in regulating centromere cohesion during meiosis II.