RESUMEN
Cell-free DNA (cfDNA) (e.g. fetal- or tumor-derived DNA) is DNA found in the blood circulation. It is now widely investigated as a biomarker for prenatal screening, tumor diagnosis, and tumor monitoring as "liquid biopsies". However, the biological and biochemical aspects of cfDNA remain unclear. Although cfDNA is considered to be mainly derived from dead cells, information is scarce as to whether it is apoptotic or necrotic and what kinds of endonucleases or DNases are involved. We induced in vivo hepatocyte necrosis and apoptosis in mice deficient in DNase1L3 (also named DNase γ) and/or caspase-activated DNase (CAD) genes with acetaminophen overdose and anti-Fas antibody treatments. We found that (i) DNase1L3 was the endonuclease responsible for generating cfDNA in acetaminophen-induced hepatocyte necrosis and (ii) CAD and DNase1L3 cooperated in producing cfDNA for anti-Fas mediated hepatocyte apoptosis.
Asunto(s)
Ácidos Nucleicos Libres de Células/genética , Desoxirribonucleasas/genética , Endodesoxirribonucleasas/genética , Necrosis/genética , Receptor fas/genética , Acetaminofén/administración & dosificación , Animales , Anticuerpos Neutralizantes/farmacología , Ácidos Nucleicos Libres de Células/sangre , Desoxirribonucleasas/sangre , Endodesoxirribonucleasas/sangre , Trampas Extracelulares/metabolismo , Regulación de la Expresión Génica , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Necrosis/sangre , Necrosis/inducido químicamente , Necrosis/patología , Transducción de Señal , Receptor fas/antagonistas & inhibidores , Receptor fas/metabolismoRESUMEN
Although chromatin condensation is a well-known hallmark of apoptosis, the generation mechanism has not been clarified. Histone H1, a positively-charged abundant nuclear protein, is located in the linker region of chromatin. There are several Histone H1 subtypes that are encoded by variant genes. Using serial histone H1-deletion mutant cells established from the chicken B-cell leukemia line DT40, we found that apoptotic chromatin condensation was decreased in relation to histone H1 protein level and that the chromatin in nuclei prepared from the live null mutant cells had a high accessibility of DNases and transposase. This indicated that linker histone H1 was the general chromatin condensation factor and that the loss of histone H1 generated open chromatin in both apoptotic and live cells.
Asunto(s)
Apoptosis , Supervivencia Celular , Cromatina/metabolismo , Histonas/metabolismo , Animales , Línea Celular , Pollos , Cromatina/ultraestructura , Eliminación de Gen , Heterocromatina/metabolismo , Heterocromatina/ultraestructura , Histonas/genéticaRESUMEN
Serum endonucleases are essential for degrading the chromatin released from dead cells and preventing autoimmune diseases such as systemic lupus erythematosus. Serum DNase I is known as the major endonuclease, but recently, another endonuclease, DNase γ/DNase I-like 3, gained attention. However, the precise role of each endonuclease, especially that of DNase γ, remains unclear. In this study, we distinguished the activities of DNase γ from those of DNase I in mouse serum and concluded that both cooperated in degrading DNA during necrosis: DNase γ functions as the primary chromatolytic activity, causing internucleosomal DNA fragmentation, and DNase I as the secondary one, causing random DNA digestion for its complete degradation. These results were confirmed by two in vivo experimental mouse models, in which necrosis was induced, acetaminophen-induced hepatic injury and streptozotocin-induced ß-cell necrosis models. We also determined that DNase γ functions as a backup endonuclease for caspase-activated DNase (CAD) in the secondary necrosis phase after γ-ray-induced apoptosis in vivo.
Asunto(s)
Degradación Necrótica del ADN , Desoxirribonucleasas de Localización Especificada Tipo I/sangre , Desoxirribonucleasas/sangre , Endodesoxirribonucleasas/sangre , Animales , Apoptosis , Línea Celular Tumoral , Fragmentación del ADN , Femenino , Humanos , Hígado/metabolismo , Hígado/ultraestructura , Masculino , Ratones , Ratones Noqueados , Complejos Multienzimáticos , Necrosis/sangre , Proteínas de Unión a Poli-ADP-Ribosa , Bazo/metabolismoRESUMEN
DNASE1L3, an enzyme highly expressed in DCs, is functionally important for regulating autoimmune responses to self-DNA and chromatin. Deficiency of DNASE1L3 leads to development of autoimmune diseases in both humans and mice. However, despite the well-established causal relationship between DNASE1L3 and immunity, little is known about the involvement of DNASE1L3 in regulation of antitumor immunity, the foundation of modern antitumor immunotherapy. In this study, we identify DNASE1L3 as a potentially new regulator of antitumor immunity and a tumor suppressor in colon cancer. In humans, DNASE1L3 is downregulated in tumor-infiltrating DCs, and this downregulation is associated with poor patient prognosis and reduced tumor immune cell infiltration in many cancer types. In mice, Dnase1l3 deficiency in the tumor microenvironment enhances tumor formation and growth in several colon cancer models. Notably, the increased tumor formation and growth in Dnase1l3-deficient mice are associated with impaired antitumor immunity, as evidenced by a substantial reduction of cytotoxic T cells and a unique subset of DCs. Consistently, Dnase1l3-deficient DCs directly modulate cytotoxic T cells in vitro. To our knowledge, our study unveils a previously unknown link between DNASE1L3 and antitumor immunity and further suggests that restoration of DNASE1L3 activity may represent a potential therapeutic approach for anticancer therapy.
Asunto(s)
Neoplasias del Colon , Humanos , Ratones , Animales , Neoplasias del Colon/metabolismo , Cromatina/metabolismo , Inmunoterapia , Linfocitos T Citotóxicos , Microambiente Tumoral , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismoRESUMEN
Acetaminophen-induced liver injury in mice is a model system of human acetaminophen overdose and oxidative stress in vivo. The system is technically established, and we usually obtain severe liver damage in the treated mice; however, it is possible that the degree of liver damage is affected by the type of chow fed to mice. Thus, in this experiment, we investigated the effect of different chows on mice by comparing acetaminophen-induced liver damage, liver antioxidant level, and serum amino-acid concentrations. The results showed that differences in chows, even standard ones, affected mouse physiological conditions, with the response to oxidative stress greatly affected.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas , Hepatopatías , Enfermedades de los Roedores , Acetaminofén/toxicidad , Animales , Antioxidantes/uso terapéutico , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Enfermedad Hepática Inducida por Sustancias y Drogas/veterinaria , Glutatión , Hígado/metabolismo , Hepatopatías/tratamiento farmacológico , Hepatopatías/veterinaria , Ratones , Ratones Endogámicos C57BL , Estrés OxidativoRESUMEN
Karyolysis is the complete dissolution of nuclear components of a dying cell. However, the generation mechanism has not been clarified. We studied a necrotic DNA fragmentation factor DNase γ (also known as DNase1L3) and previously found that karyolysis was inhibited in DNase γ deficient (DNase γ-/-) mice. To confirm this, we transiently expressed DNase γ in the liver of DNase γ-/- mice and caused hepatocyte necrosis by acetaminophen overdose. As expected, karyolysis was induced in the necrotic hepatocytes. We also found that the depletion of Kupffer cells from wild type mice reduced the expression and activity of DNase γ in the liver. Thus, we concluded that DNase γ produced from Kupffer cells caused karyolysis of necrotic hepatocytes.
Asunto(s)
Muerte Celular , Fragmentación del ADN , Hepatocitos/patología , Acetaminofén/toxicidad , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas , Endodesoxirribonucleasas , Macrófagos del Hígado/metabolismo , Hígado/enzimología , Hígado/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Oligonucleosomal DNA fragmentation and chromatin condensation are two hallmarks of apoptosis. However, their generation mechanisms are not entirely understood. Histone H1, a positively charged nuclear protein located in the linker region of chromatin, is involved in higher-order chromatin structures and tight chromatin packing. On the basis of the physical and biochemical characteristics of histone H1, we hypothesized that histone H1 plays a role in determining the efficiencies of apoptotic DNA fragmentation and chromatin condensation. Therefore, we examined histone H1 quantity in five human leukemia cell lines and compared the efficiencies. The cell lines were categorized into two groups according to their origins: (i) Ramos and Molt-4 cells of lymphoid origin and (ii) U937, ML-1, and HL60 cells of myeloid origin. Compared to the lymphoid-origin group, the myeloid-origin group had lower levels of histone H1 but more open chromatin. Furthermore, the myeloid-origin group showed marked DNA fragmentation but less chromatin condensation during apoptosis. These results suggested that histone H1 determined chromatin structure and that its quantity affected the efficiencies of DNA fragmentation and chromatin condensation in apoptosis.
Asunto(s)
Apoptosis , Cromatina/genética , Cromatina/metabolismo , Fragmentación del ADN , Histonas/metabolismo , Apoptosis/genética , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Fragmentación del ADN/efectos de la radiación , Humanos , Rayos UltravioletaRESUMEN
Cigarette smoke is a strong and independent risk factor for esophageal cancer, while the consumption of cow's milk has been proposed as a protective factor. The mechanistic role of milk in preventing cancer, however, has not been clarified. We focused our study on acrolein, an abundant unsaturated aldehyde present in cigarette smoke. Acrolein is a highly toxic compound and a putative carcinogen. Using a cell culture system, we found that (1) acrolein caused necrosis in Ramos Burkitt's lymphoma cells, (2) the necrosis was inhibited by preincubation of acrolein with milk, and (3) acrolein formed adducts with milk proteins. These results indicated the protective effects of cow's milk against acrolein-induced cytotoxicity via protein-acrolein adduct formation.
Asunto(s)
Acroleína/antagonistas & inhibidores , Carcinógenos/antagonistas & inhibidores , Proteínas de la Leche/química , Fumar/efectos adversos , Animales , Bovinos , Femenino , Masculino , Leche/químicaRESUMEN
Our previous study suggested that the highly toxic α,ß-unsaturated aldehyde acrolein, a byproduct of oxidative stress, plays a major role in acetaminophen-induced liver injury. In this study, to determine the involvement of acrolein in the liver injury and to identify novel therapeutic options for the liver damage, we examined two putative acrolein scavengers, a thiol compound cysteamine and a hydroxylamine N-benzylhydroxylamine, in cell culture and in mice. Our results showed that cysteamine and N-benzylhydroxylamine effectively prevented the cell toxicity of acrolein in vitro and acetaminophen-induced liver injury in vivo, which suggested that acrolein is involved in the liver damage, and these two drugs can be potential therapeutic options for this condition.
Asunto(s)
Acetaminofén/toxicidad , Acroleína/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Cisteamina/farmacología , Sobredosis de Droga/tratamiento farmacológico , Hidroxilaminas/farmacología , Hígado/efectos de los fármacos , Animales , Línea Celular Tumoral , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Cisteamina/uso terapéutico , Sobredosis de Droga/patología , Humanos , Hidroxilaminas/uso terapéutico , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Platelet and fibrin clots occlude blood vessels in hemostasis and thrombosis. Here we report a noncanonical mechanism for vascular occlusion based on neutrophil extracellular traps (NETs), DNA fibers released by neutrophils during inflammation. We investigated which host factors control NETs in vivo and found that two deoxyribonucleases (DNases), DNase1 and DNase1-like 3, degraded NETs in circulation during sterile neutrophilia and septicemia. In the absence of both DNases, intravascular NETs formed clots that obstructed blood vessels and caused organ damage. Vascular occlusions in patients with severe bacterial infections were associated with a defect to degrade NETs ex vivo and the formation of intravascular NET clots. DNase1 and DNase1-like 3 are independently expressed and thus provide dual host protection against deleterious effects of intravascular NETs.
Asunto(s)
ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Endodesoxirribonucleasas/metabolismo , Trampas Extracelulares/enzimología , Trastornos Hemostáticos/enzimología , Neutrófilos/enzimología , Trombosis/enzimología , Animales , Desoxirribonucleasa I/sangre , Desoxirribonucleasa I/genética , Endodesoxirribonucleasas/sangre , Endodesoxirribonucleasas/genética , Trampas Extracelulares/genética , Factor Estimulante de Colonias de Granulocitos/genética , Factor Estimulante de Colonias de Granulocitos/metabolismo , Hemostasis/genética , Hemostasis/fisiología , Trastornos Hemostáticos/genética , Humanos , Inflamación/sangre , Inflamación/enzimología , Hígado/metabolismo , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Mutantes , Sepsis/sangre , Sepsis/enzimología , Trombosis/genéticaRESUMEN
Although acetaminophen-induced liver injury in mice has been extensively studied as a model of human acute drug-induced hepatitis, the mechanism of liver injury remains unclear. Liver injury is believed to be initiated by metabolic conversion of acetaminophen to the highly reactive intermediate N-acetyl p-benzoquinoneimine, and is aggravated by subsequent oxidative stress via reactive oxygen species (ROS), including hydrogen peroxide (H2O2) and the hydroxyl radical (â¢OH). In this study, we found that a highly toxic unsaturated aldehyde acrolein, a byproduct of oxidative stress, has a major role in acetaminophen-induced liver injury. Acetaminophen administration in mice resulted in liver damage and increased acrolein-protein adduct formation. However, both of them were decreased by treatment with N-acetyl-L-cysteine (NAC) or sodium 2-mercaptoethanesulfonate (MESNA), two known acrolein scavengers. The specificity of NAC and MESNA was confirmed in cell culture, because acrolein toxicity, but not H2O2 or â¢OH toxicity, was inhibited by NAC and MESNA. These results suggest that acrolein may be more strongly correlated with acetaminophen-induced liver injury than ROS, and that acrolein produced by acetaminophen-induced oxidative stress can spread from dying cells at the primary injury site, causing damage to the adjacent cells and aggravating liver injury.
Asunto(s)
Acetaminofén/toxicidad , Acroleína/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Hígado/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Animales , Western Blotting , Técnicas de Cultivo de Célula , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Sobredosis de Droga , Hígado/patología , Mesna/farmacología , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Apoptosis and necrosis, two major forms of cell death, can be distinguished morphologically and biochemically. Internucleosomal DNA fragmentation (INDF) is a biochemical hallmark of apoptosis, and caspase-activated DNase (CAD), also known as DNA fragmentation factor 40 kDa (DFF40), is one of the major effector endonucleases. DNase γ, a Mg(2+)/Ca(2+)-dependent endonuclease, is also known to generate INDF but its role among other apoptosis-associated endonucleases in cell death is unclear. Here we show that (i) INDF occurs even during necrosis in cell lines, primary cells, and in tissues of mice in vivo, and (ii) DNase γ, but not CAD, is the effector endonuclease for INDF in cells undergoing necrosis. These results document a previously unappreciated role for INDF in necrosis and define its molecular basis.
Asunto(s)
Fragmentación del ADN , Endodesoxirribonucleasas/metabolismo , Animales , Desoxirribonucleasas/genética , Desoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Humanos , Ratones , Necrosis , Proteínas de Unión a Poli-ADP-Ribosa , Células U937RESUMEN
High-mobility group box 1 (HMGB1) protein is the major component of the nonhistone nuclear protein group and is involved in nucleosome stabilization and transcription regulation. HMGB1 has recently been focused on as a proinflammatory cytokine associated with various inflammatory diseases and as a target of anti-inflammatory therapy. Mastitis, a serious inflammatory disease of dairy cows, is caused by infection of the mammary gland and has detrimental effects on the quantity and quality of milk. By detecting the presence of HMGB1 in milk, we investigated the correlation between HMGB1 concentration and the severity of bovine mastitis, which was determined using the California Mastitis Test and somatic cell count (SCC). We detected a substantial amount of HMGB1 in mastitic milk but not in the milk from normal cows. We used the Spearman rank correlation coefficient to assess the relationship between HMGB1 concentration and SCC and found a significant correlation (n = 12, r = 0.975). Thus, we confirmed the positive correlation between HMGB1 concentration and SCC in milk, i.e., the severity of mastitis, which suggested that HMGB1 in milk is a new indicator of bovine mastitis.
Asunto(s)
Proteína HMGB1/metabolismo , Mastitis Bovina/metabolismo , Leche/metabolismo , Animales , Western Blotting/veterinaria , Bovinos , Recuento de Células/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Mastitis Bovina/diagnóstico , Mastitis Bovina/microbiología , Leche/microbiología , Estadísticas no ParamétricasRESUMEN
Somatic hypermutation (SHM) diversifies the rearranged immunoglobulin variable (V) region gene in B cells, contributing to affinity maturation of antibodies. It is believed that SHM is generated either by direct replication or by error-prone repair systems resolving V region DNA lesions caused directly or indirectly by cytidine deaminase AID. In accord with a part of these mechanisms, it was reported that SHM is associated with staggered double-strand DNA breaks (DSBs) occurring in the rearranged V regions. However, endonucleases responsible for the DSBs remain elusive. Here we show that DNase gamma, a member of DNase I family endonucleases, contributes to the generation of SHM including point mutation, and nucleotide insertion and deletion in chicken DT40 B cell line. DNase gamma also contributes to the generation of staggered DSBs in the rearranged V region. These results raise a possibility that DNase gamma is involved in the V gene mutation machinery.
Asunto(s)
Linfocitos B/inmunología , Endodesoxirribonucleasas/metabolismo , Genes de Inmunoglobulinas , Región Variable de Inmunoglobulina/genética , Hipermutación Somática de Inmunoglobulina , Animales , Linfocitos B/metabolismo , Línea Celular , Pollos , ADN/inmunología , ADN/metabolismo , Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas/genética , MutaciónRESUMEN
DNA fragmentation is a biochemical hallmark of apoptosis. Several endonucleases, including CAD/DFF40 and endonuclease G, are implicated in DNA fragmentation. DNase gamma has also been considered to be one of the enzymes involved, but its role in relation to CAD/DFF40 in apoptosis has not been fully elucidated. Here, we distinguished between DNase gamma-dependent and CAD/DFF40-dependent DNA fragmentations. We found that DNase gamma activities appeared in the late apoptotic phase and accelerated DNA fragmentation. Thus, even if the apoptotic DNA fragmentation is initiated by CAD/DFF40, DNase gamma is required for the more complete digestion of the genomic DNA in dying cells.
Asunto(s)
Apoptosis/fisiología , Linfoma de Burkitt/genética , Línea Celular Tumoral , Fragmentación del ADN , Endodesoxirribonucleasas/metabolismo , Linfoma de Burkitt/patología , Núcleo Celular/metabolismo , Desoxirribonucleasas/genética , Desoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Inhibidores Enzimáticos/metabolismo , Humanos , Proteínas de Unión a Poli-ADP-Ribosa , Estaurosporina/metabolismoRESUMEN
The internucleosomal cleavage of genomic DNA is a biochemical hallmark of apoptosis. DNase gamma, a Mg2+/Ca2+-dependent endonuclease, has been suggested to be one of the apoptotic endonucleases, but its biochemical characteristic has not been fully elucidated. Here, using recombinant DNase gamma, we showed that DNase gamma is a Mg2+/Ca2+-dependent single-stranded DNA nickase and has a high activity at low ionic strength. Under higher ionic strength, such as physiological buffer conditions, the endonuclease activity of DNase gamma is restricted, but its activity is enhanced in the presence of linker histone H1, which explains DNA cleavage at linker regions of apoptotic nuclei.
Asunto(s)
Cromatina/metabolismo , ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Apoptosis/fisiología , Secuencia de Bases , Línea Celular Tumoral , ADN de Cadena Simple/metabolismo , Desoxirribonucleasa I/metabolismo , Histonas/metabolismo , Histonas/farmacología , Humanos , Datos de Secuencia Molecular , Concentración Osmolar , Especificidad por SustratoRESUMEN
It is suggested that the formation of the switch (S) region RNA-DNA hybrid and the subsequent generation of higher-order chromatin structures including R-loop initiate a class switch recombination of the immunoglobulin gene. The primary factor of this recombination is the S-region derived noncoding RNA. However, the biochemical character of this guanine-rich (G-rich) transcript is poorly understood. The present study was performed to analyze the structure of this G-rich RNA using atomic force microscope (AFM). The in vitro transcribed S-region RNA was spread on a mica plate, air-dried and observed by non-contact mode AFM in air. The G-rich transcripts tend to aggregate on the template DNA and to generate a higher-order RNA-DNA complex. However, the transcripts that incorporated guanine analogues as substitutes for guanine neither aggregated nor generated higher-order structures. Incorporation of guanine analogues in transcribed RNA partially disrupts hydrogen bonds related to guanine, such as Watson-Crick GC-base pair and Hoogsteen bond GG-base pair. Thus, aggregation of S-region RNA and generation of the higher-order RNA-DNA complex are attributed to hydrogen bonds of guanine.
Asunto(s)
ADN/metabolismo , Guanina/química , Región de Cambio de la Inmunoglobulina/genética , Microscopía de Fuerza Atómica/métodos , ARN/química , ARN/metabolismo , Recombinación Genética , ADN/química , ADN/ultraestructura , Cambio de Clase de Inmunoglobulina , Conformación de Ácido Nucleico , ARN/ultraestructuraRESUMEN
Rolling circle amplification (RCA) of plasmid DNA using random hexamers and bacteriophage phi29 DNA polymerase is an increasingly applied technique for amplifying template DNA for DNA sequencing. We analyzed this RCA reaction at a single-molecular level by atomic force microscopy (AFM) and found that multibranched amplified products containing tandem repeats of a circle unit are formed within 1 h. We also used the RCA product of a GFP expression vector for the protein expression in cells, and found that the crude RCA product from one bacterial colony is sufficient for the GFP expression. Thus, the RCA reaction is useful in amplifying DNA for both DNA sequencing and protein expression.
Asunto(s)
Fagos de Bacillus/genética , ADN Circular/genética , Microscopía de Fuerza Atómica/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Plásmidos/genética , Animales , Fagos de Bacillus/enzimología , Células COS , Chlorocebus aethiops , ADN Viral/genética , ADN Polimerasa Dirigida por ADN , Proteínas Fluorescentes Verdes , Cinética , Proteínas Luminiscentes/metabolismo , Análisis de Secuencia de ADN , Secuencias Repetidas en Tándem , Moldes Genéticos , Transfección , Proteínas Virales/metabolismoRESUMEN
A cDNA encoding a rat Smubp-2 has been cloned from a lambdagt11 library by South-Western blot screening using a 50-bp tannic acid responsive element [J. Biol. Chem. 273 (1998) 12499] of the mouse mammary tumor virus (MMTV) promoter region as a probe. The full-length cDNA encodes a protein with a predicted size of 108 kDa. Northern blot analysis revealed that the gene expression of Smubp-2 is comparatively high in testis, moderate in brain, and low in other tissues. The recombinant Smubp-2 protein was expressed as a GST- or Trx-fusion protein in Escherichia coli and purified by affinity column chromatography. Gel mobility shift competition analysis indicated that the recombinant Smubp-2 protein binds to region II (containing the ACTG-motif) in the 50-bp element in the MMTV promoter. A transient transfection assay of the Smubp-2 expression vector with MMTV promoter-containing Luciferase (Luc) reporter plasmids into mouse cells suggested that Smubp-2 is a negative transcription factor. Furthermore, the MMTV promoter activity was suppressed in cells expressing high levels of Smubp-2. Insertion of the 50-bp element upstream of the SV40 promoter negatively responded to the induced expression of Smubp-2. These results suggest that the negative transcriptional effect of Smubp-2 arises from its binding to the 50-bp element located in the MMTV promoter region.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Virus del Tumor Mamario del Ratón/genética , Regiones Promotoras Genéticas/genética , Serina Endopeptidasas/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Western Blotting , Línea Celular Tumoral , ADN Complementario/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Regulación de la Expresión Génica , Taninos Hidrolizables/farmacología , Ratones , Datos de Secuencia Molecular , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Elementos de Respuesta/genética , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Periodic accumulation and degradation of RAG2 (recombination-activating gene 2) protein controls the cell-cycle-dependent V(D)J recombination of lymphocyte antigen receptor genes. Here we show the molecular mechanism of RAG2 degradation. The RAG2 protein is translocated from the nucleus to the cytoplasm and degraded through the ubiquitin/proteasome system. RAG2 translocation is mediated by the Thr-490 phosphorylation of RAG2. Inhibition of this phosphorylation by p27Kip1 stabilizes the RAG2 protein in the nucleus. These results suggest that RAG2 sequestration in the cytoplasm and its subsequent degradation by the ubiquitin/proteasome system upon entering the S phase is an integral part of G0/G1-specific V(D)J recombination.