RESUMEN
Glioblastoma (GBM) is a brain tumor that remains largely incurable because of its highly-infiltrative properties. Nuclear factor I (NFI)-type transcription factors regulate genes associated with GBM cell migration and infiltration. We have previously shown that NFI activity depends on the NFI phosphorylation state and that calcineurin phosphatase dephosphorylates and activates NFI. Calcineurin is cleaved and activated by calpain proteases whose activity is, in turn, regulated by an endogenous inhibitor, calpastatin (CAST). The CAST gene is a target of NFI in GBM cells, with differentially phosphorylated NFIs regulating the levels of CAST transcript variants. Here, we uncovered an NFIB-calpain 1-positive feedback loop mediated through CAST and calcineurin. In NFI-hyperphosphorylated GBM cells, NFIB expression decreased the CAST-to-calpain 1 ratio in the cytoplasm. This reduced ratio increased autolysis and activity of cytoplasmic calpain 1. Conversely, in NFI-hypophosphorylated cells, NFIB expression induced differential subcellular compartmentalization of CAST and calpain 1, with CAST localizing primarily to the cytoplasm and calpain 1 to the nucleus. Overall, this altered compartmentalization increased nuclear calpain 1 activity. We also show that nuclear calpain 1, by cleaving and activating calcineurin, induces NFIB dephosphorylation. Of note, knockdown of calpain 1, NFIB, or both increased GBM cell migration and up-regulated the pro-migratory factors fatty acid-binding protein 7 (FABP7) and Ras homolog family member A (RHOA). In summary, our findings reveal bidirectional cross-talk between NFIB and calpain 1 in GBM cells. A physiological consequence of this positive feedback loop appears to be decreased GBM cell migration.
Asunto(s)
Calpaína/metabolismo , Movimiento Celular , Glioblastoma/metabolismo , Glioblastoma/patología , Factores de Transcripción NFI/metabolismo , Línea Celular Tumoral , HumanosRESUMEN
Malignant glioma (MG) is the most lethal primary brain tumor. In addition to having inherent resistance to radiation treatment and chemotherapy, MG cells are highly infiltrative, rendering focal therapies ineffective. Genes involved in MG cell migration and glial cell differentiation are up-regulated by hypophosphorylated nuclear factor I (NFI), which is dephosphorylated by the phosphatase calcineurin in MG cells. Calcineurin is cleaved and thereby activated by calpain proteases, which are, in turn, inhibited by calpastatin (CAST). Here, we show that the CAST gene is a target of NFI and has NFI-binding sites in its intron 3 region. We also found that NFI-mediated regulation of CAST depends on NFI's phosphorylation state. We noted that occupation of CAST intron 3 by hypophosphorylated NFI results in increased activation of an alternative promoter. This activation resulted in higher levels of CAST transcript variants, leading to increased levels of CAST protein that lacks the N-terminal XL domain. CAST was primarily present in the cytoplasm of NFI-hypophosphorylated MG cells, with a predominantly perinuclear immunostaining pattern. NFI knockdown in NFI-hypophosphorylated MG cells increased CAST levels at the plasma membrane. These results suggest that NFI plays an integral role in the regulation of CAST variants and CAST subcellular distribution. Along with the previous findings indicating that NFI activity is regulated by calcineurin, these results provide a foundation for further investigations into the possibility of regulatory cross-talk between NFI and the CAST/calpain/calcineurin signaling pathway in MG cells.
Asunto(s)
Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioma/metabolismo , Mutación , Neurofibromina 1/metabolismo , Fracciones Subcelulares/metabolismo , Sitios de Unión , Movimiento Celular , Glioma/patología , Humanos , Neurofibromina 1/genética , Fosforilación , Regiones Promotoras Genéticas , Células Tumorales CultivadasRESUMEN
Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited treatment options and poor prognosis. There is an urgent need to identify and understand the key factors and signalling pathways driving TNBC tumour progression, relapse, and treatment resistance. In this study, we report that gene copy numbers and expression levels of nuclear factor IB (NFIB), a recently identified oncogene in small cell lung cancer, are preferentially increased in TNBC compared to other breast cancer subtypes. Furthermore, increased levels of NFIB are significantly associated with high tumour grade, poor prognosis, and reduced chemotherapy response. Concurrent TP53 mutations and NFIB overexpression (z-scores > 0) were observed in 77.9% of TNBCs, in contrast to 28.5% in non-TNBCs. Depletion of NFIB in TP53-mutated TNBC cell lines promotes cell death, cell cycle arrest, and enhances sensitivity to docetaxel, a first-line chemotherapeutic drug in breast cancer treatment. Importantly, these alterations in growth properties were accompanied by induction of CDKN1A, the gene encoding p21, a downstream effector of p53. We show that NFIB directly interacts with the CDKN1A promoter in TNBC cells. Furthermore, knockdown of combined p21 and NFIB reverses the docetaxel-induced cell growth inhibition observed upon NFIB knockdown, indicating that NFIB's effect on chemotherapeutic drug response is mediated through p21. Our results indicate that NFIB is an important TNBC factor that drives tumour cell growth and drug resistance, leading to poor clinical outcomes. Thus, targeting NFIB in TP53-mutated TNBC may reverse oncogenic properties associated with mutant p53 by restoring p21 activity. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Mutación , Factores de Transcripción NFI/metabolismo , Transcripción Genética , Neoplasias de la Mama Triple Negativas/metabolismo , Proteína p53 Supresora de Tumor/genética , Antineoplásicos/farmacología , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Docetaxel/farmacología , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Factores de Transcripción NFI/genética , Transducción de Señal , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Glioblastomas (GBMs) are highly aggressive brain tumors with a dismal prognosis. Nuclear factor I (NFI) is a family of transcription factors that controls glial cell differentiation in the developing central nervous system. NFIs have previously been shown to regulate the expression of astrocyte markers such as glial fibrillary acidic protein (GFAP) in both normal brain and GBM cells. We used chromatin immunoprecipitation (ChIP)-on-chip to identify additional NFI targets in GBM cells. Analysis of our ChIP data revealed ~400 putative NFI target genes including an effector of the Notch signaling pathway, HEY1, implicated in the maintenance of neural stem cells. All four NFIs (NFIA, NFIB, NFIC, and NFIX) bind to NFI recognition sites located within 1â¯kb upstream of the HEY1 transcription site. We further showed that NFI negatively regulates HEY1 expression, with knockdown of all four NFIs in GBM cells resulting in increased HEY1 RNA levels. HEY1 knockdown in GBM cells decreased cell proliferation, increased cell migration, and decreased neurosphere formation. Finally, we found a general correlation between elevated levels of HEY1 and expression of the brain neural stem/progenitor cell marker B-FABP in GBM cell lines. Knockdown of HEY1 resulted in an increase in the RNA levels of the GFAP astrocyte differentiation marker. Overall, our data indicate that HEY1 is negatively regulated by NFI family members and is associated with increased proliferation, decreased migration, and increased stem cell properties in GBM cells.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Ciclo Celular/genética , Glioblastoma/patología , Factores de Transcripción NFI/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Inmunoprecipitación de Cromatina/métodos , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Humanos , Factores de Transcripción NFI/genética , Células Madre Neoplásicas/patología , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Human Rap1-interacting factor 1 (RIF1) is an important player in the repair of DNA double strand breaks (DSBs). RIF1 acts downstream of 53BP1, with well-documented roles in class switch recombination in B-cells and inhibition of end resection initiation in BRCA1-defective cells. Here, we report that DEAD Box 1 (DDX1), a RNA helicase also implicated in DSB repair, interacts with RIF1, with co-localization of DDX1 and RIF1 observed throughout interphase. Recruitment of DDX1 to DSBs is dependent on RIF1, with RIF1 depletion abolishing DDX1-mediated facilitation of homologous recombination at DSBs. As previously demonstrated for RIF1, DDX1 is also required for chromatin loading of Bloom syndrome helicase (BLM) to ionizing radiation-induced DSBs, a RIF1-related activity that is independent of 53BP1. We show that DDX1 and RIF1 have different nucleic acid requirements for accumulation at DSBs, with RNA-DNA hybrids required for DDX1 accrual at DSBs, and single-strand RNA required for accumulation of RIF1 at these sites. Our data suggest both convergent and divergent roles for DDX1 and RIF1 in DSB repair, and may help explain why RIF1 depletion does not fully mimic 53BP1 ablation in the restoration of homologous recombination defects in BRCA1-deficient cells.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , RecQ Helicasas/metabolismo , Reparación del ADN por Recombinación , Proteínas de Unión a Telómeros/metabolismo , Proteína BRCA1 , ADN/metabolismo , Humanos , Unión Proteica , ARN/metabolismoRESUMEN
Although RNA and RNA-binding proteins have been linked to double-strand breaks (DSBs), little is known regarding their roles in the cellular response to DSBs and, if any, in the repair process. Here, we provide direct evidence for the presence of RNA-DNA hybrids at DSBs and suggest that binding of RNA to DNA at DSBs may impact repair efficiency. Our data indicate that the RNA-unwinding protein DEAD box 1 (DDX1) is required for efficient DSB repair and cell survival after ionizing radiation (IR), with depletion of DDX1 resulting in reduced DSB repair by homologous recombination (HR). While DDX1 is not essential for end resection, a key step in homology-directed DSB repair, DDX1 is required for maintenance of the single-stranded DNA once generated by end resection. We show that transcription deregulation has a significant effect on DSB repair by HR in DDX1-depleted cells and that RNA-DNA duplexes are elevated at DSBs in DDX1-depleted cells. Based on our combined data, we propose a role for DDX1 in resolving RNA-DNA structures that accumulate at DSBs located at sites of active transcription. Our findings point to a previously uncharacterized requirement for clearing RNA at DSBs for efficient repair by HR.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Reparación del ADN , Recombinación Homóloga , ARN/metabolismo , Línea Celular , Supervivencia Celular/efectos de la radiación , ARN Helicasas DEAD-box/genética , Roturas del ADN de Doble Cadena , Regulación de la Expresión Génica , Células HeLa , Humanos , Transcripción Genética/efectos de la radiaciónRESUMEN
BACKGROUND: AP-2δ is the most divergent member of the Activating Protein-2 (TFAP2) family of transcription factors. AP-2δ is restricted to specific regions of the CNS, including a subset of ganglion cells in the retina. Retinal ganglion cells (RGCs), the only output neurons of the retina, are responsible for transmitting the visual signal to the brain. RESULTS: AP-2δ knockout results in loss of Brn3c (Pou4f3) expression in AP-2δ -positive RGCs. While AP-2δ-/- mice have morphologically normal retinas at birth, there is a significant reduction in retinal ganglion cell numbers by P21, after eye opening. Chromatin immunoprecipitation indicates that Brn3c is a target of AP-2δ in the retina. Using fluorochrome-conjugated cholera toxin subunit B to trace ganglion cell axons from the eye to the major visual pathways in the brain, we found 87 % and 32 % decreases in ipsilateral and contralateral projections, respectively, to the superior colliculus in AP-2δ-/- mice. In agreement with anatomical data, visually evoked responses recorded from the brain confirmed that retinal outputs to the brain are compromised. CONCLUSIONS: AP-2δ is important for the maintenance of ganglion cell numbers in the retina. Loss of AP-2δ alters retinal axonal projections to visual centers of the brain, with ipsilaterial projections to the superior colliculus being the most dramatically affected. Our results have important implications for integration of the visual signal at the superior colliculus.
Asunto(s)
Axones/metabolismo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Colículos Superiores/metabolismo , Factor de Transcripción AP-2/deficiencia , Animales , Apoptosis , Recuento de Células , Inmunoprecipitación de Cromatina , Visión de Colores , Adaptación a la Oscuridad , Potenciales Evocados Visuales/fisiología , Cuerpos Geniculados/citología , Cuerpos Geniculados/metabolismo , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Ratones Endogámicos C57BL , Ratones Noqueados , Colículos Superiores/citología , Núcleo Supraquiasmático/citología , Núcleo Supraquiasmático/metabolismo , Factor de Transcripción AP-2/metabolismo , Factor de Transcripción Brn-3C/metabolismoRESUMEN
Arachidonic (AA) and docosahexaenoic acid (DHA) brain accretion is essential for brain development. The impact of DHA-rich maternal diets on offspring brain fatty acid composition has previously been studied up to the weanling stage; however, there has been no follow-up at later stages. Here, we examine the impact of DHA-rich maternal and weaning diets on brain fatty acid composition at weaning and three weeks post-weaning. We report that DHA supplementation during lactation maintains high DHA levels in the brains of pups even when they are fed a DHA-deficient diet for three weeks after weaning. We show that boosting dietary DHA levels for three weeks after weaning compensates for a maternal DHA-deficient diet during lactation. Finally, our data indicate that brain fatty acid binding protein (FABP7), a marker of neural stem cells, is down-regulated in the brains of six-week pups with a high DHA:AA ratio. We propose that elevated levels of DHA in developing brain accelerate brain maturation relative to DHA-deficient brains.
Asunto(s)
Encéfalo/efectos de los fármacos , Suplementos Dietéticos , Ácidos Docosahexaenoicos/farmacología , Lactancia , Destete , Animales , Ácido Araquidónico/metabolismo , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Regulación hacia Abajo , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos/metabolismo , Femenino , Proteínas del Tejido Nervioso/metabolismo , Embarazo , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
DEAD box 1 (DDX1) is a member of the DEAD box family of RNA helicases which are involved in all aspects of RNA metabolism. DDX1 has been implicated in a variety of biological processes, including 3'-end processing of mRNA, DNA repair, microRNA processing, tRNA maturation and mRNA transport. To study the role of DDX1 during development, we have generated mice carrying a constitutive Ddx1 knock-out allele. Ddx1(+/-) mice have no obvious phenotype and express similar levels of DDX1 as wild-type mice indicating compensation from the intact Ddx1 allele. Heterozygote matings produce no viable Ddx1(-/-) progeny, with Ddx1(-/-) embryos dying prior to embryonic day (E) 3.5. Intriguingly, the number of wild-type progeny is significantly decreased in heterozygote crosses, with two different heterozygote populations identified based on parental genotype: (i) normal Ddx1(+/-) mice which generate the expected number of wild-type progeny and (ii) Ddx1*(/-) mice (with * signifying a non-genetically altered allele) which generate a significantly reduced number of wild-type mice. The transgenerational inheritance of wild-type lethality observed upon crossing Ddx1*(/-) mice is independent of parental sex and occurs in cis through a mechanism that is different from other types of previously reported transgenerational epigenetic inheritance.
Asunto(s)
ARN Helicasas DEAD-box/genética , Alelos , Animales , ARN Helicasas DEAD-box/deficiencia , ARN Helicasas DEAD-box/metabolismo , Metilación de ADN , Femenino , Genotipo , Heterocigoto , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
The AP-2δ transcription factor is restricted to a subset of retinal ganglion cells. Overexpression of AP-2δ in chick retina results in induction of polysialylated neural cell adhesion molecule (PSA-NCAM) accompanied by misrouting and bundling of ganglion cell axons. Two polysialyltransferases, ST8SIA2 and ST8SIA4, are responsible for polysialylation of NCAM. Here, we investigate the mechanism driving the increase in PSA-NCAM observed upon AP-2δ overexpression. We show that ST8SIA2 is induced by AP-2δ overexpression in chick retina. We use chromatin immunoprecipitation and gel shift assays to demonstrate direct interaction between AP-2δ and the ST8SIA2 promoter. We propose that up-regulation of ST8SIA2 upon AP-2δ overexpression in retina increases ectopic polysialylation of NCAM which in turn causes premature bundling of axons and alters axonal response to guidance cues.
Asunto(s)
Proteínas Aviares/genética , Retina/enzimología , Sialiltransferasas/genética , Factor de Transcripción AP-2/fisiología , Animales , Proteínas Aviares/metabolismo , Axones/enzimología , Secuencia de Bases , Sitios de Unión , Embrión de Pollo , Regulación Enzimológica de la Expresión Génica , Regiones Promotoras Genéticas , Unión Proteica , Retina/citología , Células Ganglionares de la Retina/enzimología , Sialiltransferasas/metabolismoRESUMEN
Retinal ganglion cells transmit the visual signal from the retina to the brain. We have previously shown that the activator protein 2 (AP-2)δ (TFAP2D) transcription factor is expressed in one third of ganglion cells in developing retina suggesting a specialized role for these AP-2δ-expressing cells. Here, we address the role of AP-2δ in retina by in ovo electroporation of RCAS/AP-2δ retroviral constructs into the eyes of chick embryos at day 2 of gestation. Ectopic expression of AP-2δ does not affect lineage differentiation in the developing retina. However, immunostaining of retinal tissue with markers associated with axonal growth such as growth-associated protein 43 and polysialic acid-neural cell adhesion molecule (PSA-NCAM) demonstrates axonal misrouting and abnormal axonal bundling. Treatment of AP-2δ-misexpressing retinal cell cultures with endoneuraminidase, an enzyme that removes PSA from NCAM, decreases AP-2δ-induced axonal bundling. Our data suggest a role for AP-2δ in polysialylation of NCAM, with ectopic expression of AP-2δ resulting in premature bundling of emerging axons and misrouting of axons. We propose that expression of AP-2δ in a subset of ganglion cells contributes to the fine-tuning of axonal growth in the developing retina.
Asunto(s)
Axones/fisiología , Coristoma , Regulación del Desarrollo de la Expresión Génica , Molécula L1 de Adhesión de Célula Nerviosa/biosíntesis , Retina/embriología , Retina/metabolismo , Ácidos Siálicos/biosíntesis , Factor de Transcripción AP-2/biosíntesis , Animales , Embrión de Pollo , Pollos , Glicósido Hidrolasas/farmacología , Molécula L1 de Adhesión de Célula Nerviosa/antagonistas & inhibidores , Retina/efectos de los fármacos , Ácidos Siálicos/antagonistas & inhibidores , Factor de Transcripción AP-2/antagonistas & inhibidoresRESUMEN
Brain fatty acid-binding protein (FABP7) and PAX6 are both expressed in radial glial cells and have been implicated in neurogenesis and glial cell differentiation. FABP7 and PAX6 have also been postulated to play a role in malignant glioma cell growth and invasion. Here, we address the role of PAX6 in regulating FABP7 gene expression in malignant glioma cells. We report that PAX6 and FABP7 RNA are generally co-expressed in malignant glioma cell lines, tumors and tumor neurospheres. Using the CAT reporter gene assay, we show that FABP7 promoter activity is upregulated by PAX6. Sequential deletion analysis of the FABP7 promoter, combined with gel shift and supershift assays demonstrate the presence of a PAX6 responsive region located upstream of the FABP7 gene, at -862 to -1033 bp. Inclusion of sequences between -1.2 and -1.8 kb reduced CAT activity, suggesting the presence of a repressor element within this region. While PAX6 overexpression did not induce endogenous FABP7 expression in FABP7-negative cells, knock-down of PAX6 in PAX6-positive malignant glioma cells resulted in reduced FABP7 levels. These data provide the first evidence of direct transactivation of the FABP7 proximal promoter by PAX6 and suggest a synergistic mechanism for PAX6 and other co-factor(s) in regulating FABP7 expression in malignant glioma.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Proteínas Portadoras/genética , Proteínas del Ojo/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Glioma/patología , Proteínas de Homeodominio/metabolismo , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Proteínas Supresoras de Tumor/genética , Línea Celular Tumoral , Proteínas del Ojo/genética , Proteína de Unión a los Ácidos Grasos 7 , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/genética , Humanos , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Proteínas Represoras/genética , Activación Transcripcional , TransfecciónRESUMEN
The Reelin-Disabled-1 (Dab1) signaling pathway plays a key role in the positioning of neurons during brain development. Two alternatively spliced Dab1 isoforms have been identified in chick retina and brain: Dab1-E, expressed at early stages of development, and Dab1-L (commonly referred to as Dab1), expressed at later developmental stages. The well-studied Dab1-L serves as an adaptor protein linking Reelin signal to its downstream effectors; however, nothing is known regarding the role of Dab1-E. Here we show that Dab1-E is primarily expressed in proliferating retinal progenitor cells whereas Dab1-L is found exclusively in differentiated neuronal cells. In contrast to Dab1-L, which is tyrosine phosphorylated upon Reelin stimulation, Dab1-E is not tyrosine phosphorylated and may function independently of Reelin. Knockdown of Dab1-E in chick retina results in a significant reduction in the number of proliferating cells and promotes ganglion cell differentiation. Our results demonstrate a role for Dab1-E in the maintenance of the retinal progenitor pool and determination of cell fate.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Pollos/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Retina/citología , Serina Endopeptidasas/metabolismo , Tirosina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/análisis , Proteínas Adaptadoras Transductoras de Señales/genética , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Moléculas de Adhesión Celular Neuronal/genética , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Embrión de Pollo , Pollos/genética , Proteínas de la Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Mitosis , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Reelina , Retina/embriología , Retina/metabolismo , Retina/ultraestructura , Células Ganglionares de la Retina/citología , Serina Endopeptidasas/genética , Células Madre/citologíaRESUMEN
Glial fibrillary acidic protein (GFAP), an intermediate filament protein normally found in astrocytes, and the radial glial marker brain fatty acid-binding protein (B-FABP; also known as FABP7) are co-expressed in malignant glioma cell lines and tumors. Nuclear factor I (NFI) recognition sites have been identified in the B-FABP and GFAP promoters, and transcription of both genes is believed to be regulated by NFI. Here, we study the role of the different members of the NFI family in regulating endogenous and ectopic B-FABP and GFAP gene transcription in human malignant glioma cells. We show by gel shifts that all four members of the NFI family (NFIA, NFIB, NFIC, and NFIX) bind to B-FABP and GFAP NFI consensus sites. Over-expression of NFIs, in conjunction with mutation analysis of NFI consensus sites using a reporter gene assay, supports a role for all four NFIs in the regulation of the GFAP and B-FABP genes. Knock-down of single or combined NFIs reveals promoter-dependent and promoter-context-dependent interaction patterns and suggests cross talk between the different members of the NFI family. Our data indicate that the NFI family of transcription factors plays a key role in the regulation of both the B-FABP and GFAP genes in malignant glioma cells.
Asunto(s)
Proteínas Portadoras/genética , Regulación Neoplásica de la Expresión Génica , Proteína Ácida Fibrilar de la Glía/genética , Glioma/genética , Factores de Transcripción NFI/metabolismo , Proteínas Supresoras de Tumor/genética , Región de Flanqueo 3'/genética , Secuencia de Bases , Sitios de Unión/genética , Línea Celular Tumoral , Proteína de Unión a los Ácidos Grasos 7 , Técnicas de Silenciamiento del Gen , Humanos , Datos de Secuencia Molecular , Mutación , Factores de Transcripción NFI/genética , Regiones Promotoras Genéticas/genéticaRESUMEN
DEAD box proteins are a family of putative RNA helicases associated with all aspects of cellular metabolism involving the modification of RNA secondary structure. DDX1 is a member of the DEAD box protein family that is overexpressed in a subset of retinoblastoma and neuroblastoma cell lines and tumors. DDX1 is found primarily in the nucleus, where it forms two to four large aggregates called DDX1 bodies. Here, we report a rapid redistribution of DDX1 in cells exposed to ionizing radiation, resulting in the formation of numerous foci that colocalize with gamma-H2AX and phosphorylated ATM foci at sites of DNA double-strand breaks (DSBs). The formation of DDX1 ionizing-radiation-induced foci (IRIF) is dependent on ATM, which was shown to phosphorylate DDX1 both in vitro and in vivo. The treatment of cells with RNase H prevented the formation of DDX1 IRIF, suggesting that DDX1 is recruited to sites of DNA damage containing RNA-DNA structures. We have shown that DDX1 has RNase activity toward single-stranded RNA, as well as ADP-dependent RNA-DNA- and RNA-RNA-unwinding activities. We propose that DDX1 plays an RNA clearance role at DSB sites, thereby facilitating the template-guided repair of transcriptionally active regions of the genome.