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PURPOSE: To evaluate the efficacy and safety of percutaneous endovascular biopsy (PEB) in intravascular filling-defect lesions (IVLs) of the great vessels. MATERIAL AND METHODS: We retrospectively reviewed 19 patients (age 65 ± 12 years), 11 men and eight women, who underwent PEB for IVLs, between March 2004 and November 2014. All PEBs were performed for early diagnosis and/or characterization of the IVL, or in case of reasonable doubt about the IVL nature. Pre-intervention imaging work-up included CT, MRI and/or PET-CT. PEBs were obtained with a 7F biopsy forceps device. Clinical profile, procedure technical success and safety, and clinical success were evaluated. RESULTS: PEB was technically successful in all patients (mean of two samples per IVL). No intra- or post-procedural complications were reported. Histopathological analysis provided a diagnosis in all PEBs with a clinical success of 100%. Of the 19 IVLs, 14 were malignant (74%). The most frequent malignant lesion observed was leiomyosarcoma (29%). Benign lesions (26%) included three thrombi (pulmonary artery) and two myxomas. CONCLUSION: PEB is a safe and efficient procedure providing the most effective technique to obtain a tissue sample of high diagnostic quality, which serves to establish early diagnosis in patients with suspected malignant lesions. KEY POINTS: ⢠Intravascular filling-defect lesions are related to both benign conditions and malignant tumours. ⢠Endovascular biopsy is indicated in case of doubt about intravascular lesion nature. ⢠Percutaneous endovascular biopsy is a safe technique. ⢠Endovascular biopsy provides tissue samples leading to correct histopathological analysis. ⢠Percutaneous endovascular biopsy provides early diagnosis of malignant intravascular lesions.
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Neoplasias Vasculares/patología , Anciano , Anciano de 80 o más Años , Biopsia/métodos , Diagnóstico por Imagen/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Radiología Intervencionista/métodos , Reproducibilidad de los Resultados , Estudios Retrospectivos , Neoplasias Vasculares/diagnóstico por imagenRESUMEN
OBJECTIVE: To evaluate the performance of quantitative diffusion-weighted imaging (DWI) correlated with T2 signal in differentiating non-fatty benign from malignant tumors. MATERIAL AND METHODS: A total of 76 patients with a histologically confirmed non-fatty soft tissue tumors (46 benign and 30 malignant) were prospectively included in this ethics committee approved study. All patients signed an informed consent and underwent MRI with DWI with two b values (0 and 600). ADC values from the solid components of these tumors were obtained and were correlated with the lesion's signal intensity on T2-weighted fat-saturated sequences. ADC values were obtained from adjacent normal muscle to allow calculation of tumor/muscle ADC ratios. RESULTS: There were 58 hyperintense and 18 iso or hypointense lesions. All hypointense lesions were benign. The mean ADC values for benign and malignant tumors were 1.47 ± 0.54 × 10(-3) and 1.17 ± 0.38 × 10(-3) mm(2)/s respectively (p < 0.005). The mean ADC ratio in benign iso or hypointense tumors was significantly lower than that of hyperintense ones (0.76 ± 0.21 versus 1.58 ± 0.82 - p < 0.0001). An ADC ratio lower than 0.915 was highly specific for malignancy (96.4 %), whereas an ADC ratio higher than 1.32 was highly sensitive for benign lesions (90 %). CONCLUSION: ADC analysis can be useful in the initial characterization of T2 hyperintense non-fatty soft tissue masses, although this technique alone is not likely to change patient management.
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Imagen de Difusión por Resonancia Magnética/métodos , Interpretación de Imagen Asistida por Computador/métodos , Neoplasias de los Tejidos Blandos/patología , Adulto , Anciano , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Studies have shown that DNA (cytosine-5-)-methyltransferase 1 (DNMT1) is the principal enzyme responsible for maintaining CpG methylation and is required for embryonic development and survival of somatic cells in mice. The role of DNMT1 in human cancer cells, however, remains highly controversial. Using homologous recombination, here we have generated a DNMT1 conditional allele in the human colorectal carcinoma cell line HCT116 in which several exons encoding the catalytic domain are flanked by loxP sites. Cre recombinase-mediated disruption of this allele results in hemimethylation of approximately 20% of CpG-CpG dyads in the genome, coupled with activation of the G2/M checkpoint, leading to arrest in the G2 phase of the cell cycle. Although cells gradually escape from this arrest, they show severe mitotic defects and undergo cell death either during mitosis or after arresting in a tetraploid G1 state. Our results thus show that DNMT1 is required for faithfully maintaining DNA methylation patterns in human cancer cells and is essential for their proliferation and survival.
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Neoplasias Colorrectales/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Mitosis , Alelos , Proliferación Celular , Supervivencia Celular , Neoplasias Colorrectales/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/fisiología , Metilación de ADN , Células HCT116 , Humanos , Modelos Biológicos , Modelos GenéticosRESUMEN
OBJECTIVE: The objective of our study was to assess the impact of two methods of apparent diffusion coefficient (ADC) selection on the diagnostic performance of DWI in the characterization of nonfatty soft-tissue masses. SUBJECTS AND METHODS: Sixty-five histologically confirmed soft-tissue tumors imaged from November 2009 through October 2012 were evaluated. The minimal ADC (ADCmin) and average ADC (ADCavg) values for each tumor were obtained using two ROI-positioning methods: manual and semiautomatic. Two readers correlated the findings to lesion histology and morphology on conventional MRI sequences. RESULTS: The ADCmin values obtained using the manual method presented a better sensitivity with a similar specificity when compared with the ADCmin values obtained using the semiautomatic method (manual vs semiautomatic: 75-83% and 59-63% vs 58-67% and 63% and 63%, respectively). The interobserver agreement for the ADCmin values was similar between the ADC selection methods (intraclass correlation coefficient = 0.81 and 0.87 for manual and semiautomatic methods, respectively). In the subgroup of solid lesions, the ADCavg values obtained using the manual method offered a better sensitivity for benign-malignant differentiation (60-81% vs 60% and 60% for ADCavg and ADCmin, respectively). CONCLUSION: The ADCmin values obtained with manual ROI positioning offered the best diagnostic performance for tumor characterization. The semiautomatic method yielded similar specificity values. For solid masses, the ADCavg values were better correlated with tumor histology than the ADCmin values.
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Imagen de Difusión por Resonancia Magnética/métodos , Neoplasias de los Tejidos Blandos/patología , Adulto , Anciano , Anciano de 80 o más Años , Medios de Contraste , Diagnóstico Diferencial , Femenino , Humanos , Interpretación de Imagen Asistida por Computador , Masculino , Persona de Mediana Edad , Posicionamiento del Paciente , Estudios Prospectivos , Sensibilidad y EspecificidadRESUMEN
Differential DNA methylation of the paternal and maternal alleles regulates the parental origin-specific expression of imprinted genes in mammals. The methylation imprints are established in male and female germ cells during gametogenesis, and the de novo DNA methyltransferase DNMT3A and its cofactor DNMT3L are required in this process. However, the mechanisms underlying locus- and parental-specific targeting of the de novo DNA methylation machinery in germline imprinting are poorly understood. Here we show that amine oxidase (flavin-containing) domain 1 (AOF1), a protein related to the lysine demethylase KDM1 (also known as LSD1), functions as a histone H3 lysine 4 (H3K4) demethylase and is required for de novo DNA methylation of some imprinted genes in oocytes. AOF1, now renamed lysine demethylase 1B (KDM1B) following a new nomenclature, is highly expressed in growing oocytes where genomic imprints are established. Targeted disruption of the gene encoding KDM1B had no effect on mouse development and oogenesis. However, oocytes from KDM1B-deficient females showed a substantial increase in H3K4 methylation and failed to set up the DNA methylation marks at four out of seven imprinted genes examined. Embryos derived from these oocytes showed biallelic expression or biallelic suppression of the affected genes and died before mid-gestation. Our results suggest that demethylation of H3K4 is critical for establishing the DNA methylation imprints during oogenesis.
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Metilación de ADN , Impresión Genómica , Histonas/metabolismo , Madres , Oxidorreductasas N-Desmetilantes/metabolismo , Alelos , Animales , Pérdida del Embrión/genética , Embrión de Mamíferos/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes del Desarrollo/genética , Masculino , Ratones , Células 3T3 NIH , Oocitos/metabolismo , Oxidorreductasas N-Desmetilantes/deficiencia , Oxidorreductasas N-Desmetilantes/genéticaRESUMEN
Alveolar echinococcosis is a rare but life-threatening infection caused by the parasite Echinococcus multilocularis. Its natural history is characterized by a slow parasitic growth over several years. Increased incidence and shorter development delay have been reported in immune-compromised patients. We report the reactivation of aborted lesions within 12 months of lung transplantation leading to a fast-growing aggressive hepatic lesion. Timely identification of alveolar echninococcosis allowed prompt albendazole treatment and radical surgery leading to a favorable outcome 42 months after transplantation. However, close clinical, serological and radiological monitoring is required to rule out relapses in the long term. The pre-existence of aborted self-limited lesions of alveolar echinococcosis and the possibility for their atypical rapid growth in patients undergoing profound immunosuppression should be known by healthcare providers, even if working in non-endemic areas.
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Constitutive ablation of the Yin Yang 1 (YY1) transcription factor in mice results in peri-implantation lethality. In this study, we used homologous recombination to generate knockout mice carrying yy1 alleles expressing various amounts of YY1. Phenotypic analysis of yy1 mutant embryos expressing approximately 75%, approximately 50%, and approximately 25% of the normal complement of YY1 identified a dosage-dependent requirement for YY1 during late embryogenesis. Indeed, reduction of YY1 levels impairs embryonic growth and viability in a dose-dependent manner. Analysis of the corresponding mouse embryonic fibroblast cells also revealed a tight correlation between YY1 dosage and cell proliferation, with a complete ablation of YY1 inducing cytokinesis failure and cell cycle arrest. Consistently, RNA interference-mediated inhibition of YY1 in HeLa cells prevents cytokinesis, causes proliferative arrest, and increases cellular sensitivity to various apoptotic agents. Genome-wide expression profiling identified a plethora of YY1 target genes that have been implicated in cell growth, proliferation, cytokinesis, apoptosis, development, and differentiation, suggesting that YY1 coordinates multiple essential biological processes through a complex transcriptional network. These data not only shed new light on the molecular basis for YY1 developmental roles and cellular functions, but also provide insight into the general mechanisms controlling eukaryotic cell proliferation, apoptosis, and differentiation.
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Apoptosis/genética , Retardo del Crecimiento Fetal/genética , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica , Factor de Transcripción YY1/genética , Factor de Transcripción YY1/fisiología , Animales , Ciclo Celular/genética , Citocinesis/genética , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Inducción Embrionaria/genética , Perfilación de la Expresión Génica , Genes p53/genética , Células HeLa , Humanos , Ratones , Ratones Noqueados , Interferencia de ARNRESUMEN
BACKGROUND: Hepatic arterial embolisation therapy (HAET) is a treatment of liver metastases of gastrointestinal neuroendocrine tumours (GI-NETs). HAET increases circulating vascular endothelial growth factor levels. Everolimus is a treatment in NETs that may have antiangiogenic activity. METHODS: This phase II study was conducted in patients with predominant and progressive liver metastases from GI-NETs. Everolimus was initiated 7-30 days after HAET. The hypothesis was that everolimus after HAET would increase hepatic progression-free survival (hPFS) rate at 24 months from 35% to 50%. RESULTS: Among the 74 patients included, 88% had small-bowel primary tumour, 43% had grade I and 57% grade II tumour, and 51% had extrahepatic metastases. Patients underwent one (n = 19), two (n = 54), or three (n = 1) HAET procedures. hPFS at 24 months was 33% (95% confidence interval [CI], 22.5-43.7); 40 (54%) patients had objective response. Median (95% CI) hPFS, PFS, and overall survival were 19 (14-23), 17 (13-22), and 51 (33-60) months. The most common grade III-IV toxicities (>5%) in patients receiving both HAET and everolimus (n = 67) were elevated liver enzymes (55%), fatigue (18%), diarrhoea (16%), anaemia (12%), hypertriglyceridaemia (7%) and mucositis (6%). CONCLUSIONS: The primary end-point was not reached. This sequence allows high liver response with HAET, and everolimus controls the extrahepatic disease. TRIAL REGISTRATION: NCT01678664 (clinicaltrials.gov).
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Antineoplásicos/uso terapéutico , Embolización Terapéutica , Everolimus/uso terapéutico , Neoplasias Gastrointestinales/patología , Arteria Hepática , Neoplasias Hepáticas/terapia , Tumores Neuroendocrinos/terapia , Adulto , Anciano , Anciano de 80 o más Años , Antibióticos Antineoplásicos/administración & dosificación , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/secundario , Quimioembolización Terapéutica , Doxorrubicina/administración & dosificación , Femenino , Humanos , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Ganglios Linfáticos/patología , Masculino , Persona de Mediana Edad , Tumores Neuroendocrinos/secundario , Neoplasias Peritoneales/tratamiento farmacológico , Neoplasias Peritoneales/secundario , Supervivencia sin Progresión , Estreptozocina/administración & dosificaciónRESUMEN
Transcriptional activation of silent genes can require the erasure of epigenetic marks such as DNA methylation at CpGs (cytosine-guanine dinucleotide). Active demethylation events have been observed, and associated processes are repeatedly suspected to involve DNA glycosylases such as mCpG binding domain protein 4, thymine DNA glycosylase (TDG), Demeter, and repressor of silencing 1. A complete characterization of the molecular mechanisms occurring in metazoan is nonetheless awaited. Here, we report that activation of the endogenous vitronectin gene in P19 cells by the nuclear receptor chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) is observed in parallel with the recruitment of TDG and p68 RNA helicase, two components of a putative demethylation complex. Interestingly, when activated, the vitronectin gene was loaded with DNA methyltransferases 3a and 3b (Dnmt3a/b), and a strand-biased decrease in CpG methylation was detected. Dnmt3a was further found to associate with COUP-TFI and TDG in vivo, and cotransfection experiments demonstrated that Dnmt3a/b can enhance COUP-TFI-mediated activation of a methylated reporter gene. These results suggest that Dnmt3a/b could cooperate with the orphan receptor COUP-TFI to regulate transcription of the vitronectin gene.
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Factor de Transcripción COUP I/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Regulación de la Expresión Génica/fisiología , Animales , Células COS , Chlorocebus aethiops , ADN Metiltransferasa 3A , Ratones , Regiones Promotoras Genéticas , Activación Transcripcional , Vitronectina/biosíntesis , Vitronectina/genética , ADN Metiltransferasa 3BRESUMEN
The prostate apoptosis response-4 (Par-4) protein has been shown to function as an effector of cell death in response to various apoptotic stimuli, and down-regulation of this protein has been suggested to be a key event during tumorigenesis. Several studies suggest an essential function for the COOH-terminal leucine repeats/death domain of Par-4 in mediating apoptosis. We investigated the biological role of this domain in vivo by generating knock-out mice expressing a Par-4 mutant protein lacking the COOH terminus domain. We found that the Par-4 mutant mice are viable and fertile with no overt phenotype, thus excluding an essential role for the COOH terminus domain of Par-4 in embryogenesis and developmental apoptosis. To determine the requirement of Par-4 for apoptosis, we treated primary fibroblasts with various stimuli that trigger mitochondria and membrane receptor cell death pathways. Fibroblasts isolated from Par-4 mutant mice are as sensitive as the wild-type cells to these apoptosis-inducing agents. Similar effects were observed following RNA interference (RNAi)-mediated knockdown of Par-4 in these cells. In contrast, RNAi-mediated depletion of Par-4 in HeLa cells resulted in a significant inhibition of apoptosis induced by various proapoptotic agents. Taken together, our findings provide strong genetic evidence that the proapoptotic function of Par-4 is dependent on the cellular context and raise the possibility that alterations of Par-4 function may occur during carcinogenesis.
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Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/fisiología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Animales , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Calcimicina/farmacología , Doxorrubicina/farmacología , Etopósido/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Células HeLa , Humanos , Ionomicina/farmacología , Ratones , Ratones Noqueados , Estructura Terciaria de Proteína , Interferencia de ARN , Estaurosporina/farmacologíaRESUMEN
PURPOSE: The "oven effect" theory assumes that radiofrequency ablation (RFA) would be more efficient on tumors of cirrhotic livers. The aim of the study was to compare the size and volume of the ablation zone following RFA on tumors of cirrhotic versus healthy livers. METHODS: One hundred and eleven patients who underwent RFA from 2011 to 2013 for the treatment of 140 liver tumors (83 hepatocellular carcinomas developed on a cirrhotic liver, i.e., "cirrhosis group," and 57 tumors developed on a healthy liver, mainly liver metastasis, i.e., "healthy liver group") using the same RFA device were retrospectively selected. The diameter and volume of the ablation zone were compared between groups at the end of the procedure (FU0), at first (FU1) and second follow-up (FU2) performed 1.6 months (± 19 days) and 4.7 months (± 40 days) post-RFA, respectively. RESULTS: No differences in the size or volume of the ablation zone were found between groups at FU0 (36.5 ± 12 mm vs. 34.3 ± 10 mm, p = 0.5; and 28 ± 16 mm3 vs. 26.5 ± 16 mm3, p = 0.6, respectively), FU1, or FU2. Similarly, no differences were found at FU0, FU1, or FU2 in the subgroups of tumors treated using a single radiofrequency application. The mean volume of the ablation zone decreased over time, by 33.3% at FU1 and 48.5% at FU2, without any difference between groups. CONCLUSION: In contradiction to the "oven effect" theory, RFA achieves ablation zones of a comparable size and volume in cirrhotic and healthy livers.
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Carcinoma Hepatocelular/diagnóstico por imagen , Carcinoma Hepatocelular/cirugía , Ablación por Catéter/métodos , Cirrosis Hepática/diagnóstico por imagen , Cirrosis Hepática/cirugía , Neoplasias Hepáticas/diagnóstico por imagen , Neoplasias Hepáticas/cirugía , Carcinoma Hepatocelular/complicaciones , Femenino , Humanos , Hígado/diagnóstico por imagen , Hígado/cirugía , Cirrosis Hepática/complicaciones , Neoplasias Hepáticas/complicaciones , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
Histone acetylation and deacetylation have been implicated in the regulation of gene expression. Molecular studies have shown that histone deacetylases (HDACs) function as transcriptional repressors. However, very little is known about their roles during development in multicellular organisms. We previously demonstrated that inhibition of maternal and zygotic expression of histone deacetylase 1 (HDA-1) causes embryonic lethality in Caenorhabditis elegans. Here, we report the identification of an hda-1 genetic mutant which has also been called a gon-10 mutant (for gonadogenesis defective 10) and show that loss of HDA-1 zygotic expression results in specific postembryonic defects in gonadogenesis and vulval development. We provide evidence that the lag-2 gene, which plays a role in gonadogenesis and vulval development and encodes a Notch ligand, is derepressed in gon-10 animals, suggesting that lag-2 may be a target of HDA-1. Our findings reveal a novel and specific function for the ubiquitously expressed HDA-1 in C. elegans gonadogenesis and place hda-1 in the Notch signaling pathway.
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Caenorhabditis elegans/embriología , Caenorhabditis elegans/enzimología , Gónadas/embriología , Gónadas/enzimología , Histona Desacetilasas/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/genética , Homocigoto , Humanos , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Fenotipo , Mutación Puntual , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vulva/embriología , Vulva/enzimologíaRESUMEN
PURPOSE: To report our experience using a multilevel patient management algorithm to direct transarterial embolization (TAE) in managing spontaneous intramuscular hematoma (SIMH). MATERIALS AND METHODS: From May 2006 to January 2014, twenty-seven patients with SIMH had been referred for TAE to our Radiology department. Clinical status and coagulation characteristics of the patients are analyzed. An algorithm integrating CT findings is suggested to manage SIMH. Patients were classified into three groups: Type I, SIMH with no active bleeding (AB); Type II, SIMH with AB and no muscular fascia rupture (MFR); and Type III, SIMH with MFR and AB. Type II is furthermore subcategorized as IIa, IIb and IIc. Types IIb, IIc and III were considered for TAE. The method of embolization as well as the material been used are described. Continuous variables are presented as mean ± SD. Categorical variables are reported as percentages. Technical success, clinical success, complications and 30-day mortality (d30 M) were analyzed. RESULTS: Two patients (7.5%) had Type IIb, four (15%) Type IIc and 21 (77.5%) presented Type III. The detailed CT and CTA findings, embolization procedure and materials used are described. Technical success was 96% with a complication rate of 4%. Clinical success was 88%. The bleeding-related thirty-day mortality was 15% (all with Type III). CONCLUSION: TAE is a safe and efficient technique to control bleeding that should be considered in selected SIMH as soon as possible. The proposed algorithm integrating CT features provides a comprehensive chart to select patients for TAE. LEVEL OF EVIDENCE: 4.
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Abdomen/irrigación sanguínea , Algoritmos , Embolización Terapéutica/métodos , Hematoma/terapia , Enfermedades Musculares/terapia , Pelvis/irrigación sanguínea , Abdomen/diagnóstico por imagen , Anciano , Anciano de 80 o más Años , Angiografía por Tomografía Computarizada , Femenino , Hematoma/diagnóstico por imagen , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/diagnóstico por imagen , Enfermedades Musculares/diagnóstico por imagen , Pelvis/diagnóstico por imagen , Resultado del TratamientoRESUMEN
Chicken ovalbumin upstream promoter transcription factor I (COUP-TFI) is an orphan member of the nuclear hormone receptor superfamily that comprises key regulators of many biological functions, such as embryonic development, metabolism, homeostasis, and reproduction. Although COUP-TFI can both actively silence gene transcription and antagonize the functions of various other nuclear receptors, the COUP-TFI orphan receptor also acts as a transcriptional activator in certain contexts. Moreover, COUP-TFI has recently been shown to serve as an accessory factor for some ligand-bound nuclear receptors, suggesting that it may modulate, both negatively and positively, a wide range of hormonal responses. In the absence of any identified cognate ligand, the mechanisms involved in the regulation of COUP-TFI activity remain unclear. The elucidation of several putative phosphorylation sites for MAPKs, PKC, and casein kinase II within the sequence of this orphan receptor led us to investigate phosphorylation events regulating the various COUP-TFI functions. After showing that COUP-TFI is phosphorylated in vivo, we provide evidence that in vivo inhibition of either MAPK or PKC signaling pathway leads to a specific and pronounced decrease in COUP-TFI-dependent transcriptional activation of the vitronectin gene promoter. Focusing on the molecular mechanisms underlying the MAPK- and PKC-mediated regulation of COUP-TFI activity, we show that COUP-TFI can be directly targeted by PKC and MAPK. These phosphorylation events differentially modulate COUP-TFI functions: PKC-mediated phosphorylation enhances COUP-TFI affinity for DNA and MAPK-mediated phosphorylation positively regulates the transactivation function of COUP-TFI, possibly through enhancing specific coactivator recruitment. These data provide evidence that COUP-TFI is likely to integrate distinct signaling pathways and raise the possibility that multiple extracellular signals influence biological processes controlled by COUP-TFI.
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Pollos/genética , Proteínas de Unión al ADN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Animales , Secuencia de Bases , Células COS , Factor de Transcripción COUP I , Secuencia de Consenso , ADN/metabolismo , Proteínas de Unión al ADN/química , Ensayo de Cambio de Movilidad Electroforética , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteína Quinasa C/metabolismo , Receptores Citoplasmáticos y Nucleares/química , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Elementos de Respuesta/genética , Factores de Transcripción/química , Transcripción Genética/genética , Vitronectina/genéticaRESUMEN
Histone methylation and DNA methylation cooperatively regulate chromatin structure and gene activity. How these two systems coordinate with each other remains unclear. Here we study the biological function of lysine-specific demethylase 1 (LSD1, also known as KDM1 and AOF2), which has been shown to demethylate histone H3 on lysine 4 (H3K4) and lysine 9 (H3K9). We show that LSD1 is required for gastrulation during mouse embryogenesis. Notably, targeted deletion of the gene encoding LSD1 (namely, Aof2) in embryonic stem (ES) cells induces progressive loss of DNA methylation. This loss correlates with a decrease in DNA methyltransferase 1 (Dnmt1) protein, as a result of reduced Dnmt1 stability. Dnmt1 protein is methylated in vivo, and its methylation is enhanced in the absence of LSD1. Furthermore, Dnmt1 can be methylated by Set7/9 (also known as KMT7) and demethylated by LSD1 in vitro. Our findings suggest that LSD1 demethylates and stabilizes Dnmt1, thus providing a previously unknown mechanistic link between the histone and DNA methylation systems.
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Metilación de ADN , Oxidorreductasas N-Desmetilantes/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Desarrollo Embrionario , Células Madre Embrionarias/citología , Células Madre Embrionarias/enzimología , Histona Demetilasas , Ratones , Oxidorreductasas N-Desmetilantes/deficiencia , Especificidad por SustratoRESUMEN
DNA damage repair is crucial for the maintenance of genome integrity and cancer suppression. We found that loss of the mouse transcription factor YY1 resulted in polyploidy and chromatid aberrations, which are signatures of defects in homologous recombination. Further biochemical analyses identified a YY1 complex comprising components of the evolutionarily conserved INO80 chromatin-remodeling complex. Notably, RNA interference-mediated knockdown of YY1 and INO80 increased cellular sensitivity toward DNA-damaging agents. Functional assays revealed that both YY1 and INO80 are essential in homologous recombination-based DNA repair (HRR), which was further supported by the finding that YY1 preferentially bound a recombination-intermediate structure in vitro. Collectively, these observations reveal a link between YY1 and INO80 and roles for both in HRR, providing new insight into mechanisms that control the cellular response to genotoxic stress.
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ADN Helicasas/fisiología , Reparación del ADN/fisiología , Inestabilidad Genómica , Recombinación Genética , Factor de Transcripción YY1/fisiología , Animales , Células Cultivadas , Aberraciones Cromosómicas , Daño del ADN , Células HeLa , Humanos , Ratones , Ratones Noqueados , Poliploidía , Interferencia de ARNRESUMEN
In C. elegans embryos, a Wnt/MAPK signaling pathway downregulates the TCF/LEF transcription factor POP-1, resulting in a lower nuclear level in signal-responsive cells compared to their sisters. Although the beta-catenin WRM-1 is required for POP-1 downregulation, a direct interaction between these two proteins does not seem to be required, as the beta-catenin-interacting domain of POP-1 is dispensable for both POP-1 downregulation and function in early embryos. We show here that WRM-1 downregulates POP-1 by promoting its phosphorylation by the MAP kinase LIT-1 and subsequent nuclear export via a 14-3-3 protein, PAR-5. In signal-responsive cells, we also detect a concurrent upregulation of nuclear LIT-1 that is dependent on Wnt/MAPK signaling. Our results suggest a model whereby Wnt/MAPK signaling downregulates POP-1 levels in responsive cells, in part by increasing nuclear LIT-1 levels, thereby increasing POP-1 phosphorylation and PAR-5-mediated nuclear export.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Transactivadores/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Proteínas 14-3-3 , Transporte Activo de Núcleo Celular/fisiología , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Diferenciación Celular/fisiología , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/fisiología , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/enzimología , Proteínas del Grupo de Alta Movilidad/genética , Sistema de Señalización de MAP Quinasas/fisiología , Sustancias Macromoleculares , Proteínas de la Membrana/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas , Estructura Terciaria de Proteína/fisiología , beta CateninaRESUMEN
The p300/CBP family of transcriptional coactivators possesses multiple functional domains, including a histone acetyltransferase (HAT) and several activation domains. A number of models have been proposed to account for their roles in transcriptional activation, including interactions with basal transcription machinery and chromatin remodeling. However, individual contributions of these domains to transcriptional activation and their significance in living organisms remain unclear. We addressed the importance of the HAT activity of CBP-1, the worm ortholog of p300/CBP, in Caenorhabditis elegans with three different and complementary approaches. These include allele-specific RNA-mediated interference (RNAi), genetic rescue and the use of a specific chemical inhibitor of the p300/CBP HAT. Our findings demonstrate that HAT activity is of primary importance for CBP-1 to regulate transcription and to promote differentiation during C. elegans embryogenesis.
Asunto(s)
Acetiltransferasas/fisiología , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/fisiología , Diferenciación Celular/fisiología , Proteínas del Helminto/fisiología , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción , Transcripción Genética , Alelos , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Eliminación de Gen , Histona Acetiltransferasas , Mutación , Mutación Puntual , Regiones Promotoras Genéticas , Estructura Terciaria de ProteínaRESUMEN
The enhancement of the human estrogen receptor alpha (hER alpha, NR3A1) activity by the orphan nuclear receptor COUP-TFI is found to depend on the establishment of a tight hER alpha-COUP-TFI complex. Formation of this complex seems to involve dynamic mechanisms different from those allowing hER alpha homodimerization. Although the hER alpha-COUP-TFI complex is present in all cells tested, the transcriptional cooperation between the two nuclear receptors is restricted to cell lines permissive to hER alpha activation function 1 (AF-1). In these cells, the physical interaction between COUP-TFI and hER alpha increases the affinity of hER alpha for ERK2/p42(MAPK), resulting in an enhanced phosphorylation state of the hER alpha Ser118. hER alpha thus acquires a strengthened AF-1 activity due to its hyperphosphorylation. These data indicate an alternative interaction process between nuclear receptors and demonstrate a novel protein intercommunication pathway that modulates hER alpha AF-1.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Procesamiento Proteico-Postraduccional , Receptores de Estrógenos/metabolismo , Factores de Transcripción/metabolismo , Adenocarcinoma/patología , Neoplasias de la Mama/patología , Factor de Transcripción COUP I , ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Receptor alfa de Estrógeno , Femenino , Flavonoides/farmacología , Humanos , Sustancias Macromoleculares , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Fosforilación , Fosfoserina/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Transducción de Señal , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Lymphoid enhancer factor/T-cell factor (LEF/TCF) are transcription factors that mediate the Wnt signaling pathway, and have crucial roles during embryonic development in various organisms. Here we report that acetylation enhances nuclear retention of POP-1, the Caenorhabditis elegans LEF/TCF homolog, through increasing nuclear import and blocking nuclear export. We identify three lysines that are acetylated in vivo, and demonstrate their essential requirement for proper nuclear localization and biological activity of POP-1 during C. elegans embryogenesis. The conservation of these lysines among other LEF/TCF family members suggests that acetylation may be an important, evolutionarily conserved mechanism regulating subcellular distribution of LEF/TCF factors.