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1.
Hereditas ; 160(1): 29, 2023 Jun 22.
Artículo en Inglés | MEDLINE | ID: mdl-37349788

RESUMEN

BACKGROUND: Glioma stem cells (GSCs) are responsible for glioma recurrence and drug resistance, yet the mechanisms underlying their maintenance remains unclear. This study aimed to identify enhancer-controlled genes involved in GSCs maintenance and elucidate the mechanisms underlying their regulation. METHODS: We analyzed RNA-seq data and H3K27ac ChIP-seq data from GSE119776 to identify differentially expressed genes and enhancers, respectively. Gene Ontology analysis was performed for functional enrichment. Transcription factors were predicted using the Toolkit for Cistrome Data Browser. Prognostic analysis and gene expression correlation was conducted using the Chinese Glioma Genome Atlas (CGGA) data. Two GSC cell lines, GSC-A172 and GSC-U138MG, were isolated from A172 and U138MG cell lines. qRT-PCR was used to detect gene transcription levels. ChIP-qPCR was used to detect H3K27ac of enhancers, and binding of E2F4 to target gene enhancers. Western blot was used to analyze protein levels of p-ATR and γH2AX. Sphere formation, limiting dilution and cell growth assays were used to analyze GSCs growth and self-renewal. RESULTS: We found that upregulated genes in GSCs were associated with ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) pathway activation, and that seven enhancer-controlled genes related to ATR pathway activation (LIN9, MCM8, CEP72, POLA1, DBF4, NDE1, and CDKN2C) were identified. Expression of these genes corresponded to poor prognosis in glioma patients. E2F4 was identified as a transcription factor that regulates enhancer-controlled genes related to the ATR pathway activation, with MCM8 having the highest hazard ratio among genes positively correlated with E2F4 expression. E2F4 bound to MCM8 enhancers to promote its transcription. Overexpression of MCM8 partially restored the inhibition of GSCs self-renewal, cell growth, and the ATR pathway activation caused by E2F4 knockdown. CONCLUSION: Our study demonstrated that E2F4-mediated enhancer activation of MCM8 promotes the ATR pathway activation and GSCs characteristics. These findings offer promising targets for the development of new therapies for gliomas.


Asunto(s)
Glioma , Humanos , Glioma/genética , Glioma/metabolismo , Factores de Transcripción/metabolismo , Proliferación Celular/genética , Células Madre Neoplásicas/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Factor de Transcripción E2F4/metabolismo , Proteínas Asociadas a Microtúbulos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo
2.
J Cell Physiol ; 237(6): 2690-2702, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35403704

RESUMEN

E2f4 is a multifunctional transcription factor that is essential for many cellular processes. Although the role of E2f4 during cell cycle progression has been investigated in great detail, less is known about E2f4 during embryonic development. Here, we investigated the role of E2f4 during zebrafish development. Zebrafish e2f4 mutants displayed ectopic otolith formation due to abnormal ciliary beating in the otic vesicle. The beating defects of motile cilia were caused by abnormal expression of ciliary motility genes. The expression of two genes, lrrc23 and ccdc151, were significantly decreased in the absence of E2f4. In addition to that, e2f4 mutants also displayed growth retardation both in the body length and body weight and mostly died at around 6 months old. Although food intake was normal in the mutants, we found that the microvilli of the intestinal epithelia were significantly affected in the mutants. Finally, the intestinal epithelia of e2f4 mutants also displayed reduced cell proliferation, together with an increased level of cell apoptosis. Our data suggested a tissue-specific role of E2f4 during zebrafish development, which is distinct from the traditional views of this protein as a transcription repressor.


Asunto(s)
Factor de Transcripción E2F4/metabolismo , Proteínas de Pez Cebra , Pez Cebra , Animales , Cilios/genética , Cilios/metabolismo , Intestinos , Membrana Otolítica/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
3.
Cerebrovasc Dis ; 51(5): 678-685, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35421860

RESUMEN

BACKGROUND: Recent studies have shown that curcumin can reduce the symptoms of hydrocephalus. However, the underlying mechanisms remain unclear. Our previous studies demonstrated that E2F transcription factor 4 (E2F4) protein plays an important role in hydrocephalus; hence, we hypothesized that E2F4 may involve in curcumin mediated anti-hydrocephalus benefits. METHODS: E2F4 expression and functions in different human tissues and cell lines were determined and analyzed using the all RNA-seq and ChIP-seq sample and signature search database and ChIP-atlas database. Hydrocephalus mouse model was established through stereotactic injection of shE2F4 into frontal cortex. Mice were treated with curcumin, and then hydrocephalus severity, the expression of E2F4, and downstream targets were analyzed. RESULTS: E2F4 was highly expressed in the nervous system, which was downregulated in the bran of hydrocephalus patients. Knockdown E2F4 in mice could mimic the phenotype of human hydrocephalus. Upon curcumin administration, E2F4 expression level was increased, and the hydrocephalus severity score was significantly decreased in mouse model. Mechanistically, curcumin attenuated hydrocephalus through activating E2F4 signaling pathway. CONCLUSION: Curcumin suppresses hydrocephalus progression via activation of E2F4, which could be a target for hydrocephalus treatment.


Asunto(s)
Curcumina , Animales , Línea Celular , Curcumina/farmacología , Factor de Transcripción E2F4/genética , Factor de Transcripción E2F4/metabolismo , Humanos , Ratones
4.
Exp Cell Res ; 401(1): 112521, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33609534

RESUMEN

Oxygen therapy is a common treatment in neonatal intensive care units, but long-term continuous hyperoxia ventilation may induce acute lung injury (ALI). Gasdermin D (GSDMD)-mediated pyroptosis participates in various diseases including ALI, but the role of GSDMD in hyperoxia-induced ALI is yet understood. Here, we showed a significant increase in GSDMD after exposure to high oxygen. To elucidate the molecular mechanisms involved in GSDMD regulation, we identified the core promoter of GSDMD, -98 ~ -12 bp relative to the transcriptional start site (TSS). The results of mutational analysis, overexpression or siRNA interference, EMSA and ChIP demonstrated that E2F4 and TFAP2A positively regulate the transcriptional activity of the GSDMD by binding to its promoter. However, only TFAP2A showed a regulatory effect on the expression of GSDMD. Moreover, TFAP2A was increased in the lung tissues of rats exposed to hyperoxia and showed a strong linear correlation with GSDMD. Our results indicated that TFAP2A positively regulates the GSDMD expression via binding to the promoter region of GSDMD.


Asunto(s)
Lesión Pulmonar Aguda/genética , Factor de Transcripción E2F4/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Oxígeno/efectos adversos , Proteínas de Unión a Fosfato/genética , Factor de Transcripción AP-2/genética , Células A549 , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/patología , Animales , Animales Recién Nacidos , Hipoxia de la Célula/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Unidades de Cuidado Intensivo Neonatal , Oxígeno/uso terapéutico , Regiones Promotoras Genéticas/genética , Piroptosis/genética , Ratas , Sitio de Iniciación de la Transcripción
5.
Nucleic Acids Res ; 48(21): 12085-12101, 2020 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-33166399

RESUMEN

Transcriptional regulation of DNA repair is of outmost importance for the restoration of DNA integrity upon genotoxic stress. Here we report that the potent environmental carcinogen benzo[a]pyrene (B[a]P) activates a cellular DNA damage response resulting in transcriptional repression of mismatch repair (MMR) genes (MSH2, MSH6, EXO1) and of RAD51, the central homologous recombination repair (HR) component, ultimately leading to downregulation of MMR and HR. B[a]P-induced gene repression is caused by abrogated E2F1 signalling. This occurs through proteasomal degradation of E2F1 in G2-arrested cells and downregulation of E2F1 mRNA expression in G1-arrested cells. Repression of E2F1-mediated transcription and silencing of repair genes is further mediated by the p21-dependent E2F4/DREAM complex. Notably, repression of DNA repair is also observed following exposure to the active B[a]P metabolite BPDE and upon ionizing radiation and occurs in response to a p53/p21-triggered, irreversible cell cycle arrest marking the onset of cellular senescence. Overall, our results suggest that repression of MMR and HR is an early event during genotoxic-stress induced senescence. We propose that persistent downregulation of DNA repair might play a role in the maintenance of the senescence phenotype, which is associated with an accumulation of unrepairable DNA lesions.


Asunto(s)
Benzo(a)pireno/toxicidad , Carcinógenos/toxicidad , Senescencia Celular/genética , ADN/genética , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F4/genética , Puntos de Control del Ciclo Celular , Línea Celular Transformada , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , ADN/metabolismo , Daño del ADN , Reparación de la Incompatibilidad de ADN/efectos de los fármacos , Reparación de la Incompatibilidad de ADN/efectos de la radiación , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F4/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/efectos de la radiación , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Rayos gamma , Humanos , Proteínas de Interacción con los Canales Kv/genética , Proteínas de Interacción con los Canales Kv/metabolismo , Células MCF-7 , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Reparación del ADN por Recombinación/efectos de los fármacos , Reparación del ADN por Recombinación/efectos de la radiación , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal
6.
J Clin Lab Anal ; 36(4): e24322, 2022 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-35262965

RESUMEN

BACKGROUND: Cervical cancer is the most common gynecological cancer worldwide and is associated with high morbidity and mortality. Despite improvements in therapeutic strategies, the network regulation mechanism remains unclear and the treatment effect is not satisfactory. Therefore, there is a need to continue studying the mechanism of cervical cancer to explore effective gene targets and precise targeted therapy drugs. METHODS: First, three paired tissues (cancer tissues and noncancerous tissues) from patients with cervical squamous cell carcinoma were collected, grouped, and analyzed by microarray. Second, differentially expressed mRNAs (DEMs) and differentially expressed lncRNAs (DELs) (|fold change| ≥ 2 and p < 0.05) between the two groups were screened. For DEMs, functional annotation and pathway analysis were performed using DAVID. Functional prediction of DELs was then performed and their cis-regulatory and trans-regulatory networks were explored. RESULTS: Function prediction of DELs (both up-regulated and down-regulated) shows that the highest frequency Cellular Component (CC) item is cytosol, the highest frequency Molecular function (MF) item is mitotic cell cycle and the highest frequency Biological Process (BP) item is protein binding. Through cis-regulation analysis of DELs, the cis-regulatory relationship of 96 DELs was predicted. The lncRNA-trans-regulation network analysis suggested that E2F4 may be the core transcription factor in the lncRNA-TF regulatory network in cervical cancer. CONCLUSIONS: The lncRNA-TF regulatory network plays an important role in the occurrence and progression of cervical cancer, and E2F4 may be a critical transcription factor in the regulatory network.


Asunto(s)
MicroARNs , ARN Largo no Codificante , Neoplasias del Cuello Uterino , Factor de Transcripción E2F4/genética , Factor de Transcripción E2F4/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes/genética , Humanos , MicroARNs/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , Factores de Transcripción/genética , Neoplasias del Cuello Uterino/genética
7.
FASEB J ; 34(4): 5578-5589, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32108965

RESUMEN

Most obese patients develop hyperleptinaemia. Leptin, mainly produced by adipocytes, demonstrates a promotional role in liver fibrosis. Hepatic stellate cell (HSC) activation, a key step in liver fibrogenesis, requires global reprogramming of gene expression. The remodeling of DNA methylation is a mechanism of the epigenetic regulation of gene expression. The biosynthesis of S-adenosylmethionine, a principle biological methyl donor, is catalyzed by methionine adenosyltransferase (MAT) such as MATⅡ which has been shown to promote HSC activation in vitro. This study was mainly aimed to determine the effect of leptin on MAT2A expression (the catalytic subunit of MATⅡ) in HSCs. Results showed that MAT2A knockdown reduced leptin-induced HSC activation and liver fibrosis in the leptin-deficient mouse model. Leptin promoted MAT2A expression in HSCs and increased MAT2A promoter activity. The axis of the ß-catenin pathway/E2F-4 mediated the effect of leptin on MAT2A expression. Leptin-induced ß-catenin signaling reduced E2F-4 expression and thus abated E2F-4 binding to MAT2A promoter at a site around -2779 bp, leading to an increase in the MAT2A promoter activity. These data might shed more light on the mechanisms responsible for liver fibrogenesis in obese patients with hyperleptinaemia.


Asunto(s)
Factor de Transcripción E2F4/antagonistas & inhibidores , Regulación de la Expresión Génica/efectos de los fármacos , Células Estrelladas Hepáticas/patología , Leptina/farmacología , Cirrosis Hepática/patología , Metionina Adenosiltransferasa/metabolismo , beta Catenina/metabolismo , Animales , Factor de Transcripción E2F4/metabolismo , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/etiología , Cirrosis Hepática/metabolismo , Metionina Adenosiltransferasa/genética , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/fisiopatología , beta Catenina/genética
8.
FASEB J ; 34(5): 6055-6069, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32239565

RESUMEN

Esophageal cancer represents the eighth most frequently occurring cancer, as well as the sixth most widespread cause of cancer-related deaths. In recent years, accumulating evidence has implicated long non-coding RNAs in the progression of esophageal squamous cell carcinoma (ESCC). The aim of the present study was to investigate the potential involvement and underlying mechanisms of LINC00337 in ESCC. Expression patterns of LINC00337 and targeting protein for Xenopus kinesin-like protein 2 (TPX2) in ESCC tissues and cells were detected using RT-qPCR and immunohistochemical staining. Next, the interactions among LINC00337, E2F4, and TPX2 were identified using chromatin immunoprecipitation, dual-luciferase reporter, and RNA-binding protein immunoprecipitation assays, suggesting that LINC00337 could recruit E2F4 to enhance the transcription of TPX2. Thereafter, the regulatory roles of LINC00337 and TPX2 in ESCC were analyzed by altering the expression of LINC00337 or TPX2 in ESCC cells following treatment with cisplatin (DDP). The levels of autophagy-related proteins Beclin1 and LC3II/LC3I, viability, autophagy, apoptosis, and chemoresistance of ESCC cells to DDP were measured following transfection treatment with different plasmids. Additionally, the role of the LINC00337/E2F4/TPX2 axis was assessed in vivo by measuring tumor formation in nude mice. The results demonstrated that LINC00337 upregulated TPX2, consequently leading to elevated levels of Beclin1 and LC3II/LC3I, promoted cell viability and autophagy, while inhibiting apoptosis and chemosensitivity to DDP in ESCC. In sum, the current study evidenced that the overexpression of LINC00337 could potentially enhance ESCC cell autophagy and chemoresistance to DDP via the upregulation of TPX2 by recruiting E2F4. Thus, LINC00337 may serve as a potential candidate for the treatment of ESCC.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cisplatino/farmacología , Resistencia a Antineoplásicos , Factor de Transcripción E2F4/metabolismo , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/patología , Proteínas Asociadas a Microtúbulos/metabolismo , ARN Largo no Codificante/genética , Anciano , Animales , Antineoplásicos/farmacología , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proteínas de Ciclo Celular/genética , Proliferación Celular , Factor de Transcripción E2F4/genética , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Asociadas a Microtúbulos/genética , Persona de Mediana Edad , Pronóstico , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
9.
Mol Cell ; 50(4): 552-64, 2013 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-23706820

RESUMEN

Cellular stress results in profound changes in RNA and protein synthesis. How cells integrate this intrinsic, p53-centered program with extracellular signals is largely unknown. We demonstrate that TGF-ß1 signaling interferes with the stress response through coordinate transcriptional and translational repression of p53 levels, which reduces p53-activated transcription, and apoptosis in precancerous cells. Mechanistically, E2F-4 binds constitutively to the TP53 gene and induces transcription. TGF-ß1-activated Smads are recruited to a composite Smad/E2F-4 element by an E2F-4/p107 complex that switches to a Smad corepressor, which represses TP53 transcription. TGF-ß1 also causes dissociation of ribosomal protein RPL26 and elongation factor eEF1A from p53 mRNA, thereby reducing p53 mRNA association with polyribosomes and p53 translation. TGF-ß1 signaling is dominant over stress-induced transcription and translation of p53 and prevents stress-imposed downregulation of Smad proteins. Thus, crosstalk between the TGF-ß and p53 pathways defines a major node of regulation in the cellular stress response, enhancing drug resistance.


Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacología , Proteína p53 Supresora de Tumor/genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Secuencia de Bases , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Factor de Transcripción E2F4/genética , Factor de Transcripción E2F4/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Inmunohistoquímica , Glándulas Mamarias Humanas/citología , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteínas Smad/genética , Proteínas Smad/metabolismo , Estrés Fisiológico/genética , Proteína p53 Supresora de Tumor/metabolismo
10.
J Cell Mol Med ; 24(3): 2157-2168, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31943751

RESUMEN

Acute myeloid leukaemia (AML) is an aggressive and mostly incurable haematological malignancy with frequent relapse after an initial response to standard chemotherapy. Therefore, novel therapies are urgently required to improve AML clinical outcome. Here, we aim to study the dysregulation of a particular transcription factor, E2F4, and its role in the progression of AML. In this study, human clinical data from the Gene Expression Profiling Interactive Analysis (GEPIA) revealed that increased E2F4 expression was associated with poor prognosis in AML patients. Moreover, the experimental results showed that E2F4 was aberrantly overexpressed in human AML patients and cell lines. Depletion of E2F4 inhibited the proliferation, induced the differentiation and suppressed the growth of AML cells in a nude mouse model. By contrast, overexpression of E2F4 promoted the proliferation and inhibited the differentiation of AML cells in vitro. Additionally, E2F4 expression not only is positively correlated with EZH2 but also can bind to EZH2. RNA microarray results also showed that E2F4 can regulate MAPK signalling pathway. EZH2 can reverse the inhibitory effect of E2F4 silencing on MAPK signaling pathway. In summary, our data suggest that E2F4 may be a potential therapeutic target for AML therapy.


Asunto(s)
Factor de Transcripción E2F4/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Genes Supresores de Tumor/fisiología , Leucemia Mieloide Aguda/genética , Sistema de Señalización de MAP Quinasas/genética , Transducción de Señal/genética , Animales , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Células Cultivadas , Epigénesis Genética/genética , Perfilación de la Expresión Génica/métodos , Humanos , Masculino , Ratones , Ratones Desnudos , Células THP-1
11.
J Cell Mol Med ; 24(11): 6015-6027, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32314545

RESUMEN

Cervical cancer is one of the major malignancies, the pathophysiology and progression of which remain to be scarcely understood. Long non-coding RNAs (lncRNAs) have been previously implicated in the progression of cervical cancer. Here, the purpose of this study was to identify whether lncRNA heart- and neural crest derivative-expressed 2-antisense RNA 1 (HAND2-AS1) affect the development of cervical cancer through regulation of chromosome 16 open reading frame 74 (C16orf74) by mediating a transcription factor E2F4. RT-qPCR was performed to determine the expression of HAND2-AS1 in cervical cancer cells. Then, cervical cancer cells were treated with HAND2-AS1 or si-E2F4 RNA, or C16orf74, after which the proliferation, colony formation, migration and invasion were detected. Moreover, the binding between HAND2-AS1 and E2F4 or between E2F4 and C16orf74 was explored. Finally, the tumorigenesis of cervical cancer cells was measured in nude mice with altered HAND2-AS1/E2F4/C16orf74 expression. HAND2-AS1 exhibited poor expression in cervical cancer, and HAND2-AS1 overexpression suppressed the proliferation, colony formation, migration and invasion of cervical cancer cells. In addition, HAND2-AS1 was found to recruit transcription factor E2F4 to C16orf74 promoter region and down-regulate C16orf74 expression. Lastly, HAND2-AS1/E2F4/C16orf74 modulated the tumorigenesis of cervical cancer in nude mice. In conclusion, this study provided evidence on the inhibitory effect of HAND2-AS1 on the development of cervical cancer through the suppression of C16orf74 expression by recruiting transcription factor E2F4. This study highlights the potential of lncRNA HAND2-AS1 as a target in the treatment of cervical cancer.


Asunto(s)
Progresión de la Enfermedad , Factor de Transcripción E2F4/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Regiones Promotoras Genéticas , Proteínas/metabolismo , ARN Largo no Codificante/metabolismo , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Adulto , Anciano , Animales , Sitios de Unión , Carcinogénesis/genética , Carcinogénesis/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación hacia Abajo/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Modelos Biológicos , Invasividad Neoplásica , ARN Largo no Codificante/genética
12.
J Biol Chem ; 294(21): 8617-8629, 2019 05 24.
Artículo en Inglés | MEDLINE | ID: mdl-30967472

RESUMEN

We previously reported that the cell cycle-related cyclin-dependent kinase 4-retinoblastoma (RB) transcriptional corepressor pathway is essential for stroke-induced cell death both in vitro and in vivo However, how this signaling pathway induces cell death is unclear. Previously, we found that the cyclin-dependent kinase 4 pathway activates the pro-apoptotic transcriptional co-regulator Cited2 in vitro after DNA damage. In the present study, we report that Cited2 protein expression is also dramatically increased following stroke/ischemic insult. Critically, utilizing conditional knockout mice, we show that Cited2 is required for neuronal cell death, both in culture and in mice after ischemic insult. Importantly, determining the mechanism by which Cited2 levels are regulated, we found that E2F transcription factor (E2F) family members participate in Cited2 regulation. First, E2F1 expression induced Cited2 transcription, and E2F1 deficiency reduced Cited2 expression. Moreover, determining the potential E2F-binding regions on the Cited2 gene regulatory sequence by ChIP analysis, we provide evidence that E2F1/4 proteins bind to this DNA region. A luciferase reporter assay to probe the functional outcomes of this interaction revealed that E2F1 activates and E2F4 inhibits Cited2 transcription. Moreover, we identified the functional binding motif for E2F1 in the Cited2 gene promoter by demonstrating that mutation of this site dramatically reduces E2F1-mediated Cited2 transcription. Finally, E2F1 and E2F4 regulated Cited2 expression in neurons after stroke-related insults. Taken together, these results indicate that the E2F-Cited2 regulatory pathway is critically involved in stroke injury.


Asunto(s)
Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F4/metabolismo , Regulación de la Expresión Génica , Neuronas/metabolismo , Proteínas Represoras/biosíntesis , Accidente Cerebrovascular/metabolismo , Transactivadores/biosíntesis , Secuencias de Aminoácidos , Animales , Muerte Celular , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F4/genética , Ratones , Ratones Transgénicos , Neuronas/patología , Proteínas Represoras/genética , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/patología , Transactivadores/genética
13.
FASEB J ; 33(1): 219-230, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-29995440

RESUMEN

Aurora kinases are critical mitotic serine/threonine kinases and are often implicated in tumorigenesis. Recent studies of the interphase functions for aurora kinase (Aurk)A have considerably expanded our understanding of its role beyond mitosis. To identify the unknown targets of AurkA, we used peptide array-based screening and found E2F4 to be a novel substrate. Phosphorylation of E2F4 by AurkA at Ser75 regulates its DNA binding and subcellular localization. Because E2F4 plays an important role in skeletal muscle differentiation, we attempted to gain insight into E2F4 phosphorylation in this context. We observed that a block in E2F4 phosphorylation retained it better within the nucleus and inhibited muscle differentiation. RNA sequencing analysis revealed a perturbation of the gene network involved in the process of muscle differentiation and mitochondrial biogenesis. Collectively, our findings establish a novel role of AurkA in the process of skeletal muscle differentiation.-Dhanasekaran, K., Bose, A., Rao, V. J., Boopathi, R., Shankar, S. R., Rao, V. K., Swaminathan, A., Vasudevan, M., Taneja, R., Kundu, T. K. Unravelling the role of aurora A beyond centrosomes and spindle assembly: implications in muscle differentiation.


Asunto(s)
Aurora Quinasa A/metabolismo , Diferenciación Celular , Centrosoma/metabolismo , Factor de Transcripción E2F4/metabolismo , Músculo Esquelético/citología , Mioblastos/citología , Huso Acromático/metabolismo , Animales , Aurora Quinasa A/genética , Ciclo Celular , Células Cultivadas , Factor de Transcripción E2F4/genética , Células HEK293 , Humanos , Ratones , Mitosis , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Fosforilación
14.
Nucleic Acids Res ; 46(1): 174-188, 2018 01 09.
Artículo en Inglés | MEDLINE | ID: mdl-29059406

RESUMEN

The Histone 3 lysine 4 methylation (H3K4me3) mark closely correlates with active transcription. E2F-responsive promoters display dynamic changes in H3K4 methylation during the course of cell cycle progression. However, how and when these marks are reset, is not known. Here we show that the retinoblastoma binding protein RBP2/KDM5A, capable of removing tri-methylation marks on H3K4, associates with the E2F4 transcription factor via the pocket protein-p130-in a cell-cycle-stage specific manner. The association of RBP2 with p130 is LxCxE motif dependent. RNAi experiments reveal that p130 recruits RBP2 to E2F-responsive promoters in early G1 phase to bring about H3K4 demethylation and gene repression. A point mutation in LxCxE motif of RBP2 renders it incapable of p130-interaction and hence, repression of E2F-regulated gene promoters. We also examine how RBP2 may be recruited to non-E2F responsive promoters. Our studies provide insight into how the chromatin landscape needs to be adjusted rapidly and periodically during cell-cycle progression, concomitantly with temporal transcription, to bring about expression/repression of specific gene sets.


Asunto(s)
Factores de Transcripción E2F/metabolismo , Histonas/metabolismo , Regiones Promotoras Genéticas/genética , Proteína 2 de Unión a Retinoblastoma/metabolismo , Proteína p130 Similar a la del Retinoblastoma/metabolismo , Proteínas Celulares de Unión al Retinol/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Células Cultivadas , Factor de Transcripción E2F4/metabolismo , Fase G1/genética , Células HeLa , Humanos , Metilación , Ratones , Mutación , Unión Proteica , Interferencia de ARN , Proteína 2 de Unión a Retinoblastoma/genética , Proteína p130 Similar a la del Retinoblastoma/genética , Proteínas Celulares de Unión al Retinol/genética
15.
Dev Biol ; 443(2): 165-172, 2018 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-30218642

RESUMEN

Multiciliated cells (MCCs) differentiate arrays of motile cilia that beat to drive fluid flow over epithelia. Recent studies have established two Geminin family coiled-coil containing nuclear regulatory proteins, Gmnc and Multicilin (Mci), in the specification and differentiation of the MCCs. Both Gmnc and Mci are devoid of a DNA binding domain: they regulate transcription by associating with E2f family transcription factors, notably E2f4 and E2f5. Here, we have studied the relative contribution of these two E2f factors in MCC development using the zebrafish embryo, which differentiates MCCs within kidney tubules and the nose. We found that while E2f4 is fully dispensable, E2f5 is essential for MCCs to form in the kidney tubules. Moreover, using a variety of double mutant combinations we show that E2f5 has a more prominent role in MCC development in the zebrafish than E2f4. This contrasts with current evidence from the mouse, where E2f4 seems to be more important. Thus, distinct combinatorial activities of the E2f4 and E2f5 proteins regulate the specification and differentiation of MCCs in zebrafish and mice.


Asunto(s)
Factor de Transcripción E2F4/metabolismo , Factor de Transcripción E2F5/metabolismo , Pez Cebra/embriología , Animales , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/genética , Cilios/metabolismo , Cilios/fisiología , Factor de Transcripción E2F4/fisiología , Factor de Transcripción E2F5/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Células HEK293 , Humanos , Proteínas Nucleares/metabolismo , Factores de Transcripción , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
16.
J Biol Chem ; 293(41): 15815-15826, 2018 10 12.
Artículo en Inglés | MEDLINE | ID: mdl-30166342

RESUMEN

Heat shock protein 27 (HSP27) protects cells under stress. Here, we demonstrate that HSP27 also promotes cell cycle progression of MRC-5 human lung fibroblast cells. Serum starvation for 24 h induced G1 arrest in these cells, and upon serum refeeding, the cells initiated cell cycle progression accompanied by an increase in HSP27 protein levels. HSP27 levels peaked at 12 h, and transcriptional up-regulation of six G2/M-related genes (CCNA2, CCNB1, CCNB2, CDC25C, CDCA3, and CDK1) peaked at 24-48 h. siRNA-mediated HSP27 silencing in proliferating MRC-5 cells induced G2 arrest coinciding with down-regulation of these six genes. Of note, the promoters of all of these genes have the cell cycle-dependent element and/or the cell cycle gene-homology region. These promoter regions are known to be bound by the E2F family proteins (E2F-1 to E2F-8) and retinoblastoma (RB) family proteins (RB1, p107, and p130), among which E2F-4 and p130 were strongly up-regulated in HSP27-knockdown cells. E2F-4 or p130 knockdown concomitant with the HSP27 knockdown rescued MRC-5 cells from G2 arrest and up-regulated the six cell cycle genes. Moreover, we observed cellular senescence in MRC-5 cells on day 3 after the HSP27 knockdown, as evidenced by increased senescence-associated ß-gal activity and up-regulated inflammatory cytokines. The cellular senescence was also suppressed by the concomitant knockdown of E2F-4/HSP27 or p130/HSP27. Our findings indicate that HSP27 promotes cell cycle progression of MRC-5 cells by suppressing expression of the transcriptional repressors E2F-4 and p130.


Asunto(s)
Ciclo Celular , Factor de Transcripción E2F4/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Proteína p130 Similar a la del Retinoblastoma/metabolismo , Línea Celular Tumoral , Proliferación Celular , Senescencia Celular , Regulación hacia Abajo , Fibroblastos/metabolismo , Fase G2 , Silenciador del Gen , Proteínas de Choque Térmico , Humanos , Pulmón/metabolismo , Chaperonas Moleculares , Oxígeno/química , ARN Interferente Pequeño/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba
17.
Chromosoma ; 127(2): 151-174, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29243212

RESUMEN

To ensure that the genetic material is accurately passed down to daughter cells during mitosis, dividing cells must duplicate their chromosomes and centrosomes once and only once per cell cycle. The same key steps-licensing, duplication, and segregation-control both the chromosome and the centrosome cycle, which must occur in concert to safeguard genome integrity. Aberrations in genome content or centrosome numbers lead to genomic instability and are linked to tumorigenesis. Such aberrations, however, can also be part of the normal life cycle of specific cell types. Multiciliated cells best exemplify the deviation from a normal centrosome cycle. They are post-mitotic cells which massively amplify their centrioles, bypassing the rule for once-per-cell-cycle centriole duplication. Hundreds of centrioles dock to the apical cell surface and generate motile cilia, whose concerted movement ensures fluid flow across epithelia. The early steps that control the generation of multiciliated cells have lately started to be elucidated. Geminin and the vertebrate-specific GemC1 and McIdas are distantly related coiled-coil proteins, initially identified as cell cycle regulators associated with the chromosome cycle. Geminin is required to ensure once-per-cell-cycle genome replication, while McIdas and GemC1 bind to Geminin and are implicated in DNA replication control. Recent findings highlight Geminin family members as early regulators of multiciliogenesis. GemC1 and McIdas specify the multiciliate cell fate by forming complexes with the E2F4/5 transcription factors to switch on a gene expression program leading to centriole amplification and cilia formation. Positive and negative interactions among Geminin family members may link cell cycle control to centriole amplification and multiciliogenesis, acting close to the point of transition from proliferation to differentiation. We review key steps of centrosome duplication and amplification, present the role of Geminin family members in the centrosome and chromosome cycle, and discuss links with disease.


Asunto(s)
Centriolos/metabolismo , Cilios/metabolismo , Geminina/genética , Genoma , Mitosis , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinogénesis/patología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centriolos/ultraestructura , Cilios/ultraestructura , Replicación del ADN , Enanismo/genética , Enanismo/metabolismo , Enanismo/patología , Factor de Transcripción E2F4/genética , Factor de Transcripción E2F4/metabolismo , Factor de Transcripción E2F5/genética , Factor de Transcripción E2F5/metabolismo , Geminina/metabolismo , Regulación de la Expresión Génica , Inestabilidad Genómica , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Transducción de Señal , Factores de Transcripción
18.
J Immunol ; 198(10): 3978-3988, 2017 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-28381640

RESUMEN

B cell development in Justy mutant mice is blocked due to a precursor mRNA splicing defect that depletes the protein GON4-like (GON4L) in B cell progenitors. Genetic and biochemical studies have suggested that GON4L is a transcriptional regulator that coordinates cell division with differentiation, but its role in B cell development is unknown. To understand the function of GON4L, we characterized B cell differentiation, cell cycle control, and mitotic gene expression in GON4L-deficient B cell progenitors from Justy mice. We found that these cells established key aspects of the transcription factor network that guides B cell development and proliferation and rearranged the IgH gene locus. However, despite intact IL-7 signaling, GON4L-deficient pro-B cell stage precursors failed to undergo a characteristic IL-7-dependent proliferative burst. These cells also failed to upregulate genes required for mitotic division, including those encoding the G1/S cyclin D3 and E2F transcription factors and their targets. Additionally, GON4L-deficient B cell progenitors displayed defects in DNA synthesis and passage through the G1/S transition, contained fragmented DNA, and underwent apoptosis. These phenotypes were not suppressed by transgenic expression of prosurvival factors. However, transgenic expression of cyclin D3 or other regulators of the G1/S transition restored pro-B cell development from Justy progenitor cells, suggesting that GON4L acts at the beginning of the cell cycle. Together, our findings indicate that GON4L is essential for cell cycle progression and division during the early stages of B cell development.


Asunto(s)
Mitosis , Proteínas Nucleares/deficiencia , Proteínas Nucleares/fisiología , Células Precursoras de Linfocitos B/fisiología , Animales , Linfocitos B/fisiología , Ciclo Celular , Proteínas de Ciclo Celular , División Celular , Proliferación Celular , Proteínas Co-Represoras , Ciclina D3/genética , Proteínas de Unión al ADN , Factor de Transcripción E2F4/genética , Factor de Transcripción E2F4/metabolismo , Regulación de la Expresión Génica , Interleucina-7/inmunología , Interleucina-7/metabolismo , Ratones , Proteínas Nucleares/genética
19.
Genes Dev ; 25(8): 801-13, 2011 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-21498570

RESUMEN

In the absence of growth signals, cells exit the cell cycle and enter into G0 or quiescence. Alternatively, cells enter senescence in response to inappropriate growth signals such as oncogene expression. The molecular mechanisms required for cell cycle exit into quiescence or senescence are poorly understood. The DREAM (DP, RB [retinoblastoma], E2F, and MuvB) complex represses cell cycle-dependent genes during quiescence. DREAM contains p130, E2F4, DP1, and a stable core complex of five MuvB-like proteins: LIN9, LIN37, LIN52, LIN54, and RBBP4. In mammalian cells, the MuvB core dissociates from p130 upon entry into the cell cycle and binds to BMYB during S phase to activate the transcription of genes expressed late in the cell cycle. We used mass spectroscopic analysis to identify phosphorylation sites that regulate the switch of the MuvB core from BMYB to DREAM. Here we report that DYRK1A can specifically phosphorylate LIN52 on serine residue 28, and that this phosphorylation is required for DREAM assembly. Inhibiting DYRK1A activity or point mutation of LIN52 disrupts DREAM assembly and reduces the ability of cells to enter quiescence or undergo Ras-induced senescence. These data reveal an important role for DYRK1A in the regulation of DREAM activity and entry into quiescence.


Asunto(s)
Senescencia Celular/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Ciclo Celular/genética , Ciclo Celular/fisiología , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Células Cultivadas , Senescencia Celular/genética , Factor de Transcripción E2F4/genética , Factor de Transcripción E2F4/metabolismo , Humanos , Ratones , Células 3T3 NIH , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Proteína 4 de Unión a Retinoblastoma/genética , Proteína 4 de Unión a Retinoblastoma/metabolismo , Proteína p130 Similar a la del Retinoblastoma/genética , Proteína p130 Similar a la del Retinoblastoma/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factor de Transcripción DP1/genética , Factor de Transcripción DP1/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Quinasas DyrK
20.
J Cell Physiol ; 233(12): 9739-9749, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-29987913

RESUMEN

Bone-marrow-derived mesenchymal stem cells (MSCs) have great potential in transplantation medicine due to their multiple advantages. However, the controlled differentiation of MSCs is one of the key aspects of effective clinical transplantation. Growing evidence suggests that the cell cycle plays an important role in regulating differentiation, while p130 and E2F4 are key to cell cycle checkpoints. The aim of the study is to evaluate the effects and mechanism of p130/E2F4 on the multidifferentiation of MSCs. Our data showed that the transduction efficiencies of p130 or E2F4 mediated by lentiviral vectors were 80.3%-84.4%. p130 and E2F4 mRNA expression was significantly higher in MSC-p130 and MSC-E2F4 cells than in MSC normal control (NC) cells. Similar results were also observed for p130 and E2F4 protein expression. After osteogenic or adipogenic differentiation, the G1 phase was significantly delayed in the MSC-p130 and MSC-E2F4 groups compared with that in the MSC-NC group. However, the G1 phase in the MSC-p130 and MSC-E2F4 groups did the opposite after chondrogenic differentiation. Moreover, overexpressing p130 or E2F4 significantly improved osteogenic differentiation while inhibiting adipogenic and chondrogenic differentiation of mouse MSCs (mMSCs). Moreover, overexpressing p130 or E2F4 significantly improved migration but not proliferation of mMSCs. Our data suggest that cell cycle regulation may be involved in p130/E2F4-mediated changes in the multipotential abilities of bone-marrow-derived mMSCs.


Asunto(s)
Diferenciación Celular/genética , Proteína Sustrato Asociada a CrK/genética , Factor de Transcripción E2F4/genética , Células Madre Mesenquimatosas/metabolismo , Adipogénesis/genética , Células de la Médula Ósea/clasificación , Células de la Médula Ósea/metabolismo , Puntos de Control del Ciclo Celular/genética , Movimiento Celular/genética , Regulación del Desarrollo de la Expresión Génica/genética , Vectores Genéticos , Humanos , Lentivirus/genética , Células Madre Mesenquimatosas/citología , Osteogénesis/genética
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