Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 37
Filtrar
1.
Genes Dev ; 33(13-14): 828-843, 2019 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-31171701

RESUMEN

Adenovirus transformed cells have a dedifferentiated phenotype. Eliminating E1A in transformed human embryonic kidney cells derepressed ∼2600 genes, generating a gene expression profile closely resembling mesenchymal stem cells (MSCs). This was associated with a dramatic change in cell morphology from one with scant cytoplasm and a globular nucleus to one with increased cytoplasm, extensive actin stress fibers, and actomyosin-dependent flattening against the substratum. E1A-induced hypoacetylation at histone H3 Lys27 and Lys18 (H3K27/18) was reversed. Most of the increase in H3K27/18ac was in enhancers near TEAD transcription factors bound by Hippo signaling-regulated coactivators YAP and TAZ. E1A causes YAP/TAZ cytoplasmic sequestration. After eliminating E1A, YAP/TAZ were transported into nuclei, where they associated with poised enhancers with DNA-bound TEAD4 and H3K4me1. This activation of YAP/TAZ required RHO family GTPase signaling and caused histone acetylation by p300/CBP, chromatin remodeling, and cohesin loading to establish MSC-associated enhancers and then superenhancers. Consistent results were also observed in primary rat embryo kidney cells, human fibroblasts, and human respiratory tract epithelial cells. These results together with earlier studies suggest that YAP/TAZ function in a developmental checkpoint controlled by signaling from the actin cytoskeleton that prevents differentiation of a progenitor cell until it is in the correct cellular and tissue environment.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas E1A de Adenovirus/metabolismo , Diferenciación Celular/genética , Silenciador del Gen , Péptidos y Proteínas de Señalización Intracelular/genética , Fosfoproteínas/genética , Citoesqueleto de Actina/metabolismo , Adenoviridae , Animales , Células Cultivadas , Células HEK293 , Humanos , Ratas , Transducción de Señal , Transactivadores , Factores de Transcripción , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAP
2.
Nucleic Acids Res ; 52(16): 9481-9500, 2024 Sep 09.
Artículo en Inglés | MEDLINE | ID: mdl-39011896

RESUMEN

Alu retrotransposons, which form the largest family of mobile DNA elements in the human genome, have recently come to attention as a potential source of regulatory novelties, most notably by participating in enhancer function. Even though Alu transcription by RNA polymerase III is subjected to tight epigenetic silencing, their expression has long been known to increase in response to various types of stress, including viral infection. Here we show that, in primary human fibroblasts, adenovirus small e1a triggered derepression of hundreds of individual Alus by promoting TFIIIB recruitment by Alu-bound TFIIIC. Epigenome profiling revealed an e1a-induced decrease of H3K27 acetylation and increase of H3K4 monomethylation at derepressed Alus, making them resemble poised enhancers. The enhancer nature of e1a-targeted Alus was confirmed by the enrichment, in their upstream regions, of the EP300/CBP acetyltransferase, EP400 chromatin remodeler and YAP1 and FOS transcription factors. The physical interaction of e1a with EP400 was critical for Alu derepression, which was abrogated upon EP400 ablation. Our data suggest that e1a targets a subset of enhancer Alus whose transcriptional activation, which requires EP400 and is mediated by the e1a-EP400 interaction, may participate in the manipulation of enhancer activity by adenoviruses.


Asunto(s)
Proteínas E1A de Adenovirus , Elementos Alu , ADN Helicasas , Proteínas de Unión al ADN , Elementos de Facilitación Genéticos , Factor de Transcripción AP-1 , Factores de Transcripción , Humanos , Elementos Alu/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1A de Adenovirus/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , ADN Helicasas/metabolismo , ADN Helicasas/genética , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción AP-1/genética , Ensamble y Desensamble de Cromatina , Proteínas Señalizadoras YAP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Activación Transcripcional , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Células Cultivadas , Fibroblastos/metabolismo , Histonas/metabolismo , Proteínas Nucleares , Factores de Transcripción TFIII
3.
J Virol ; 97(12): e0099323, 2023 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-37962355

RESUMEN

IMPORTANCE: Inactivation of EP300/CREBB paralogous cellular lysine acetyltransferases (KATs) during the early phase of infection is a consistent feature of DNA viruses. The cell responds by stabilizing transcription factor IRF3 which activates transcription of scores of interferon-stimulated genes (ISGs), inhibiting viral replication. Human respiratory adenoviruses counter this by assembling a CUL4-based ubiquitin ligase complex that polyubiquitinylates RUVBL1 and 2 inducing their proteasomal degradation. This inhibits accumulation of active IRF3 and the expression of anti-viral ISGs, allowing replication of the respiratory HAdVs in the face of inhibition of EP300/CBEBBP KAT activity by the N-terminal region of E1A.


Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas , Proteínas E1A de Adenovirus , Proteínas Portadoras , ADN Helicasas , Inmunidad Innata , Complejo de la Endopetidasa Proteasomal , Estrés Fisiológico , Humanos , Proteínas E1A de Adenovirus/metabolismo , Adenovirus Humanos/enzimología , Adenovirus Humanos/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Cullin/metabolismo , ADN Helicasas/metabolismo , Interferones/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Estructura Cuaternaria de Proteína , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitinación , Replicación Viral
4.
J Virol ; 92(18)2018 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-29976669

RESUMEN

How histone acetylation promotes transcription is not clearly understood. Here, we confirm an interaction between p300 and the adenovirus 2 large E1A activation domain (AD) and map the interacting regions in E1A by observing colocalization at an integrated lacO array of fusions of LacI-mCherry to E1A fragments with YFP-p300. Viruses with mutations in E1A subdomains were constructed and analyzed for kinetics of early viral RNA expression and association of acetylated H3K9, K18, K27, TBP, and RNA polymerase II (Pol II) across the viral genome. The results indicate that this E1A interaction with p300 is required for H3K18 and H3K27 acetylation at the E2early, E3, and E4 promoters and is required for TBP and Pol II association with the E2early promoter. In contrast, H3K18/27 acetylation was not required for TBP and Pol II association with the E3 and E4 promoters but was required for E4 transcription at a step subsequent to Pol II preinitiation complex assembly.IMPORTANCE Despite a wealth of data associating promoter and enhancer region histone N-terminal tail lysine acetylation with transcriptional activity, there are relatively few examples of studies that establish causation between these histone posttranslational modifications and transcription. While hypoacetylation of histone H3 lysines 18 and 27 is associated with repression, the step(s) in the overall process of transcription that is blocked at a hypoacetylated promoter is not clearly established in most instances. Studies presented here confirm that the adenovirus 2 large E1A protein activation domain interacts with p300, as reported previously (P. Pelka, J. N. G. Ablack, J. Torchia, A. S. Turnell, R. J. A. Grand, J. S. Mymryk, Nucleic Acids Res 37:1095-1106, 2009, https://doi.org/10.1093/nar/gkn1057), and that the resulting acetylation of H3K18/27 affects varied steps in transcription at different viral promoters.


Asunto(s)
Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Histonas/metabolismo , Regiones Promotoras Genéticas , Transcripción Genética , Acetilación , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Proteína p300 Asociada a E1A/genética , Proteína p300 Asociada a E1A/metabolismo , Regulación de la Expresión Génica , Humanos , ARN Polimerasa II/metabolismo , Activación Transcripcional
5.
Genome Res ; 22(7): 1212-21, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22499665

RESUMEN

Adenovirus small e1a oncoprotein causes ~70% reduction in cellular levels of histone H3 lysine 18 acetylation (H3K18ac). It is unclear, however, where this dramatic reduction occurs genome-wide. ChIP-sequencing revealed that by 24 h after expression, e1a erases 95% of H3K18ac peaks in normal, contact-inhibited fibroblasts and replaces them with one-third as many at new genomic locations. The H3K18ac peaks at promoters and intergenic regions of genes with fibroblast-related functions are eliminated after infection, and new H3K18ac peaks are established at promoters of highly induced genes that regulate cell cycling and at new putative enhancers. Strikingly, the regions bound by the retinoblastoma family of proteins in contact-inhibited fibroblasts gain new peaks of H3K18ac in the e1a-expressing cells, including 55% of RB1-bound loci. In contrast, over half of H3K9ac peaks are similarly distributed before and after infection, independently of RB1. The strategic redistribution of H3K18ac by e1a highlights the importance of this modification for transcriptional activation and cellular transformation as well as functional differences between the RB-family member proteins.


Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Adenovirus Humanos/genética , Epigénesis Genética , Genoma Humano , Histonas/metabolismo , Acetilación , Proteínas E1A de Adenovirus/genética , Adenovirus Humanos/metabolismo , Adenovirus Humanos/patogenicidad , Ciclo Celular , Transformación Celular Viral , Células Cultivadas , Inmunoprecipitación de Cromatina , Fibroblastos/metabolismo , Fibroblastos/virología , Regulación Viral de la Expresión Génica , Histonas/genética , Humanos , Pulmón/metabolismo , Pulmón/patología , Pulmón/virología , Anotación de Secuencia Molecular/métodos , Nucleosomas/genética , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Factores de Tiempo , Activación Transcripcional
6.
Nat Rev Genet ; 10(5): 290-4, 2009 05.
Artículo en Inglés | MEDLINE | ID: mdl-19290008

RESUMEN

The cancerous cellular state is associated with multiple epigenetic alterations, but elucidating the precise order of such alterations during tumorigenic progression and their contributions to the transformed phenotype remains a significant challenge in cancer biology. Here we discuss recent findings on how viral oncoproteins exploit specific epigenetic processes to coerce normal cells to replicate when they should remain quiescent - a hallmark of cancer. These findings may highlight roles of epigenetic processes in normal biology and shed light on epigenetic events occurring along the path of non-viral neoplastic transformation.


Asunto(s)
Transformación Celular Viral/genética , Epigénesis Genética/genética , Genoma , Proteínas Oncogénicas Virales/metabolismo , Animales , Humanos , Neoplasias/genética , Neoplasias/metabolismo
7.
Proc Natl Acad Sci U S A ; 109(51): 20913-8, 2012 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-23213214

RESUMEN

Although aberrant protein aggregation has been conclusively linked to dozens of devastating amyloid diseases, scientists remain puzzled about the molecular features that render amyloid fibrils or small oligomers toxic. Here, we report a previously unobserved type of amyloid fibril that tests as cytotoxic: one in which the strands of the contributing ß-sheets are out of register. In all amyloid fibrils previously characterized at the molecular level, only in-register ß-sheets have been observed, in which each strand makes its full complement of hydrogen bonds with the strands above and below it in the fibril. In out-of-register sheets, strands are sheared relative to one another, leaving dangling hydrogen bonds. Based on this finding, we designed out-of-register ß-sheet amyloid mimics, which form both cylindrin-like oligomers and fibrils, and these mimics are cytotoxic. Structural and energetic considerations suggest that out-of-register fibrils can readily convert to toxic cylindrins. We propose that out-of-register ß-sheets and their related cylindrins are part of a toxic amyloid pathway, which is distinct from the more energetically favored in-register amyloid pathway.


Asunto(s)
Amiloide/química , Rojo Congo/farmacología , Cristalografía por Rayos X/métodos , Colorantes Fluorescentes/farmacología , Humanos , Enlace de Hidrógeno , Microscopía Electrónica de Transmisión/métodos , Modelos Moleculares , Conformación Molecular , Péptidos/química , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas/química , Termodinámica , Difracción de Rayos X , Microglobulina beta-2/química
9.
J Virol ; 84(23): 12210-25, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20861261

RESUMEN

Oncogenic transformation by adenovirus E1A and E1B-55K requires E1B-55K inhibition of p53 activity to prevent E1A-induced apoptosis. During viral infection, E1B-55K and E4orf6 substitute for the substrate-binding subunits of the host cell cullin 5 class of ubiquitin ligases, resulting in p53 polyubiquitinylation and proteasomal degradation. Here we show that E1B-55K alone also functions as an E3 SUMO1-p53 ligase. Fluorescence microscopy studies showed that E1B-55K alone, in the absence of other viral proteins, causes p53 to colocalize with E1B-55K in promyelocytic leukemia (PML) nuclear bodies, nuclear domains with a high concentration of sumoylated proteins. Photobleaching experiments with live cells revealed that E1B-55K tethering of p53 in PML nuclear bodies decreases the in vivo nuclear mobility of p53 nearly 2 orders of magnitude. E1B-55K-induced p53 sumoylation contributes to maximal inhibition of p53 function since mutation of the major p53 sumoylation site decreases E1B-55K-induced p53 sumoylation, tethering in PML nuclear bodies, and E1B-55K inhibition of p53 activity. Mutation of the E1B-55K sumoylation site greatly inhibits E1B-55K association with PML nuclear bodies and the p53 nuclear export to cytoplasmic aggresomes observed in E1A-E1B-transformed cells. Purified E1B-55K and p53 form high-molecular-weight complexes potentially through the formation of a network of E1B-55K dimers bound to the N termini of p53 tetramers. In support of this model, a p53 mutation that prevents tetramer formation greatly reduces E1B-55K-induced tethering in PML nuclear bodies and p53 nuclear export. These data indicate that E1B-55K's association with PML nuclear bodies inactivates p53 by first sequestering it in PML nuclear bodies and then greatly facilitating its nuclear export.


Asunto(s)
Proteínas E1B de Adenovirus/metabolismo , Transformación Celular Neoplásica/metabolismo , Cuerpos de Inclusión Intranucleares/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Proteína SUMO-1/metabolismo , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Proteínas E1B de Adenovirus/genética , Línea Celular Tumoral , Dimerización , Humanos , Microscopía Fluorescente , Modelos Biológicos , Mutación/genética , Fotoblanqueo , Proteína de la Leucemia Promielocítica , Sumoilación
10.
Elife ; 102021 03 11.
Artículo en Inglés | MEDLINE | ID: mdl-33704060

RESUMEN

Regulation of RNA polymerase II (Pol2) elongation in the promoter-proximal region is an important and ubiquitous control point for gene expression in metazoans. We report that transcription of the adenovirus 5 E4 region is regulated during the release of paused Pol2 into productive elongation by recruitment of the super-elongation complex, dependent on promoter H3K18/27 acetylation by CBP/p300. We also establish that this is a general transcriptional regulatory mechanism that applies to ~7% of expressed protein-coding genes in primary human airway epithelial cells. We observed that a homeostatic mechanism maintains promoter, but not enhancer, H3K18/27ac in response to extensive inhibition of CBP/p300 acetyl transferase activity by the highly specific small molecule inhibitor A-485. Further, our results suggest a function for BRD4 association at enhancers in regulating paused Pol2 release at nearby promoters. Taken together, our results uncover the processes regulating transcriptional elongation by promoter region histone H3 acetylation and homeostatic maintenance of promoter, but not enhancer, H3K18/27ac in response to inhibition of CBP/p300 acetyl transferase activity.


Asunto(s)
Regulación de la Expresión Génica , Histonas/metabolismo , Homeostasis , Factores de Transcripción p300-CBP/genética , Acetilación , Línea Celular , Humanos , Factores de Transcripción p300-CBP/metabolismo
11.
J Virol ; 83(7): 3249-57, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19158239

RESUMEN

To make a safe, long-lasting gene delivery vehicle, we developed a hybrid vector that leverages the relative strengths of adenovirus and Epstein-Barr virus (EBV). A fully gene-deleted helper-dependent adenovirus (HDAd) is used as the delivery vehicle for its scalability and high transduction efficiency. Upon delivery, a portion of the HDAd vector is recombined to form a circular plasmid. This episome includes two elements from EBV: an EBV nuclear antigen 1 (EBNA1) expression cassette and an EBNA1 binding region. Along with a human replication origin, these elements provide considerable genetic stability to the episome in replicating cells while avoiding insertional mutagenesis. Here, we demonstrate that this hybrid approach is highly efficient at delivering EBV episomes to target cells in vivo. We achieved nearly 100% transduction of hepatocytes after a single intravenous injection in mice. This is a substantial improvement over the transduction efficiency of previously available physical and viral methods. Bioluminescent imaging of vector-transduced mice demonstrated that luciferase transgene expression from the hybrid was robust and compared well to a traditional HDAd vector. Quantitative PCR analysis confirmed that the EBV episome was stable at approximately 30 copies per cell for up to 50 weeks and that it remained circular and extrachromosomal. Approaches for adapting the HDAd-EBV hybrid to a variety of disease targets and the potential benefits of this approach are discussed.


Asunto(s)
Adenoviridae/genética , Terapia Genética/métodos , Hepatocitos/virología , Herpesvirus Humano 4/genética , Plásmidos , Transducción Genética , Animales , Femenino , Genes Reporteros , Vectores Genéticos/administración & dosificación , Inestabilidad Genómica , Inyecciones Intravenosas , Luciferasas/genética , Luciferasas/metabolismo , Luminiscencia , Ratones , Ratones Desnudos , Imagen de Cuerpo Entero
13.
iScience ; 15: 95-108, 2019 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-31055217

RESUMEN

Thalidomide is a teratogen that causes multiple malformations in the developing baby through its interaction with cereblon (CRBN), a substrate receptor subunit of the CRL4 E3 ubiquitin ligase complex. CRBN was originally reported as a gene associated with autosomal recessive non-syndromic mild mental retardation. However, the function of CRBN during brain development remains largely unknown. Here we demonstrate that CRBN promotes brain development by facilitating the proliferation of neural stem cells (NSCs). Knockdown of CRBN in zebrafish embryos impaired brain development and led to small brains, as did treatment with thalidomide. By contrast, overexpression of CRBN resulted in enlarged brains, leading to the expansion of NSC regions and increased cell proliferation in the early brain field and an expanded expression of brain region-specific genes and neural and glial marker genes. These results demonstrate that CRBN functions in the determination of brain size by regulating the proliferation of NSCs during development.

14.
FASEB J ; 20(7): 935-46, 2006 May.
Artículo en Inglés | MEDLINE | ID: mdl-16675851

RESUMEN

We describe an inducible genetic model for degeneration of midbrain dopaminergic neurons in adults. In previous studies, knock-in mice expressing hypersensitive M2 domain Leu9'Ser (L9'S) alpha4 nicotinic receptors (nAChR) at near-normal levels displayed dominant neonatal lethality and dopaminergic deficits in embryonic midbrain, because the hypersensitive nAChR is excitotoxic. However, heterozygous L9'S mice that retain the neomycin resistance cassette (neo) in a neighboring intron express low levels of the mutant allele (approximately 25% of normal levels), and these neo-intact mice are therefore viable and fertile. The neo cassette is flanked by loxP sites. In adult animals, we locally injected helper-dependent adenovirus (HDA) expressing cre recombinase. Local excision of the neo cassette, via cre-mediated recombination, was verified by genomic analysis. In L9'S HDA-cre injected animals, locomotion was reduced both under baseline conditions and after amphetamine application. There was no effect in L9'S HDA-control treated animals or in wild-type (WT) littermates injected with either virus. Immunocytochemical analyses revealed marked losses (> 70%) of dopaminergic neurons in L9'S HDA-cre injected mice compared to controls. At 20-33 days postinjection in control animals, the coexpressed marker gene, yellow fluorescent protein (YFP), was expressed in many neurons and few glial cells near the injection, emphasizing the neurotropic utility of the HDA. Thus, HDA-mediated gene transfer into adult midbrain induced sufficient functional expression of cre in dopaminergic neurons to allow for postnatal deletion of neo. This produced increased L9'S mutant nAChR expression, which in turn led to nicotinic cholinergic excitotoxicity in dopaminergic neurons.


Asunto(s)
Dopamina/metabolismo , Neuronas/metabolismo , Neuronas/patología , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Sustancia Negra/patología , Adenoviridae , Animales , Animales Modificados Genéticamente , Muerte Celular , Regulación de la Expresión Génica , Locomoción/fisiología , Ratones
15.
Mol Cell Biol ; 22(8): 2788-98, 2002 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-11909971

RESUMEN

Surfaces of human TATA box-binding protein (hsTBP) required for activated transcription in vivo were defined by constructing a library of surface residue substitution mutations and assaying them for their ability to support activated transcription in transient-transfection assays. In earlier work, three regions were identified where mutations inhibited activated transcription without interfering with TATA box DNA binding. One region is on the upstream surface of the N-terminal TBP repeat with respect to the direction of transcription and corresponds to the TBP surface that interacts with TFIIA. A second region on the stirrup of the C-terminal TBP repeat corresponds to the TFIIB-binding surface. Here we report that the third region where mutations inhibit activated transcription in mammalian cells, the convex surface of the N-terminal repeat, corresponds to a surface on TBP that interacts with hsTAF1, the major scaffold subunit of TFIID. Since mutations at the center of the hsTAF1-interacting region inhibit the ability of the protein to support activated transcription in vivo, these results are consistent with the conclusion that an interaction between hsTBP and TAF(II)s is required for activated transcription in mammalian cells.


Asunto(s)
Proteínas Cromosómicas no Histona , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Unión Competitiva , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Células HeLa , Chaperonas de Histonas , Humanos , Técnicas In Vitro , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína de Unión a TATA-Box , Factor de Transcripción TFIIA , Factor de Transcripción TFIIB , Factor de Transcripción TFIID , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción TFII/metabolismo , Activación Transcripcional , Transfección
16.
Cell Host Microbe ; 22(6): 789-800.e5, 2017 Dec 13.
Artículo en Inglés | MEDLINE | ID: mdl-29241042

RESUMEN

The N-terminal half of adenovirus e1a assembles multimeric complexes with host proteins that repress innate immune responses and force host cells into S-phase. In contrast, the functions of e1a's C-terminal interactions with FOXK, DCAF7, and CtBP are unknown. We found that these interactions modulate RAS signaling, and that a single e1a molecule must bind all three of these host proteins to suppress activation of a subset of IFN-stimulated genes (ISGs). These ISGs were otherwise induced in primary respiratory epithelial cells at 12 hr p.i. This delayed activation of ISGs required IRF3 and coincided with an ∼10-fold increase in IRF3 from protein stabilization. The induced IRF3 bound to chromatin and localized to the promoters of activated ISGs. While IRF3, STAT1/2, and IRF9 all greatly increased in concentration, there were no corresponding mRNA increases, suggesting that e1a regulates the stabilities of these key activators of innate immune responses, as shown directly for IRF3.


Asunto(s)
Adenoviridae/inmunología , Proteínas E1A de Adenovirus/metabolismo , Interacciones Huésped-Patógeno , Evasión Inmune , Inmunidad Innata , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Oxidorreductasas de Alcohol/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/inmunología , Células Epiteliales/virología , Factores de Transcripción Forkhead/metabolismo , Humanos , Factor 3 Regulador del Interferón/metabolismo , Unión Proteica
17.
Oncogene ; 24(52): 7673-85, 2005 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-16299528

RESUMEN

Adenovirus continues to be an important model system for investigating basic aspects of cell biology. Interactions of several cellular proteins with E1A conserved regions (CR) 1 and 2, and inhibition of apoptosis by E1B proteins are required for oncogenic transformation. CR2 binds RB family members, de-repressing E2F transcription factors, thus activating genes required for cell cycling. E1B-19K is a BCL2 homolog that binds and inactivates proapoptotic BAK and BAX. E1B-55K binds p53, inhibiting its transcriptional activation function. In productively infected cells, E1B-55K and E4orf6 assemble a ubiquitin ligase with cellular proteins Elongins B and C, Cullin 5 and RBX1 that polyubiquitinates p53 and one or more subunits of the MRN complex involved in DNA double-strand break repair, directing them to proteosomal degradation. E1A CR3 activates viral transcription by interacting with the MED23 Mediator subunit, stimulating preinitiation complex assembly on early viral promoters and probably also the rate at which they initiate transcription. The viral E1B-55K/E4orf6 ubiquitin ligase is also required for efficient viral late protein synthesis in many cell types, but the mechanism is not understood. E1A CR1 binds several chromatin-modifying complexes, but how this contributes to stimulation of cellular DNA synthesis and transformation is not clear. E1A CR4 binds the CtBP corepressor, but the mechanism by which this modulates the frequency of transformation remains to be determined. Clearly, adenovirus has much left to teach us about fundamental cellular processes.


Asunto(s)
Adenoviridae/genética , Adenoviridae/fisiología , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/fisiología , Ciclo Celular/fisiología , Regulación Viral de la Expresión Génica , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/fisiología , Apoptosis , Transformación Celular Neoplásica , Genes p53 , Transcripción Genética
18.
J Bone Joint Surg Am ; 85(5): 905-11, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12728043

RESUMEN

BACKGROUND: Bone morphogenetic proteins (BMPs) are now being used as bone-graft substitutes to enhance spinal fusion. However, the large doses of BMP required to induce a spinal fusion in humans suggests that the delivery of these proteins should be improved. We used ex vivo adenoviral gene transfer to create BMP-2-producing bone marrow cells, and these autologous cells were found to induce a posterolateral fusion of the spine in syngeneic rats. METHODS: Intertransverse spinal arthrodesis (L4 and L5) was attempted in ten groups of Lewis rats with 5 x 10 (6) BMP-2-producing rat bone marrow cells (Ad-BMP-2 cells), created through adenoviral gene transfer with guanidine hydrochloride-extracted demineralized bone matrix as a carrier (Group I); 5 x 10 (6) Ad-BMP-2 cells on a collagen sponge carrier (Group II); 10 micro g of recombinant BMP-2 (rhBMP-2) in a guanidine hydrochloride-extracted demineralized bone matrix carrier (Group III); 10 micro g of rhBMP-2 in a collagen sponge carrier (Group IV); autogenous iliac crest bone-grafting (Group V); 5 x 10 (6) beta-galactosidase-producing rat bone marrow cells, created through adenoviral gene transfer with guanidine hydrochloride-extracted demineralized bone matrix as a carrier (Group VI); decortication of the transverse processes alone (Group VII); 5 x 10 (6) uninfected rat bone marrow cells with a guanidine hydrochloride-extracted demineralized bone matrix carrier (Group VIII); guanidine hydrochloride-extracted demineralized bone matrix only (Group IX); or a collagen sponge alone (Group X). Each specimen underwent plain radiography, manual palpation, and histological analysis. RESULTS: All spines in Groups I and II (BMP-2-producing bone marrow cells) and all spines in Groups III and IV were fused at four weeks postoperatively. In contrast, none of the spines in the other groups had fused at a minimum of eight weeks after implantation. Histological analysis of the specimens revealed that the spines that had received BMP-2-producing bone marrow cells (Groups I and II) were filled with coarse trabecular bone postoperatively, whereas those that had received rhBMP-2 (Groups III and IV) were filled with thin, lace-like trabecular bone. All of the other spines, including those that had been treated with autogenous iliac crest bone-grafting (Group V), produced little or no new bone. CONCLUSION: BMP-2-producing bone marrow cells, created by adenoviral gene transfer, produce sufficient BMP to induce an intertransverse fusion in the rat spine model.


Asunto(s)
Proteínas Morfogenéticas Óseas/genética , Terapia Genética/métodos , Fusión Vertebral/métodos , Trasplante de Células Madre , Transducción Genética/métodos , Factor de Crecimiento Transformador beta , Animales , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/metabolismo , Femenino , Radiografía , Ratas , Ratas Endogámicas Lew , Columna Vertebral/diagnóstico por imagen
19.
Cell Host Microbe ; 16(5): 663-76, 2014 Nov 12.
Artículo en Inglés | MEDLINE | ID: mdl-25525796

RESUMEN

Oncogenic transformation by adenovirus small e1a depends on simultaneous interactions with the host lysine acetylases p300/CBP and the tumor suppressor RB. How these interactions influence cellular gene expression remains unclear. We find that e1a displaces RBs from E2F transcription factors and promotes p300 acetylation of RB1 K873/K874 to lock it into a repressing conformation that interacts with repressive chromatin-modifying enzymes. These repressing p300-e1a-RB1 complexes specifically interact with host genes that have unusually high p300 association within the gene body. The TGF-β, TNF-, and interleukin-signaling pathway components are enriched among such p300-targeted genes. The p300-e1a-RB1 complex condenses chromatin in a manner dependent on HDAC activity, p300 lysine acetylase activity, the p300 bromodomain, and RB K873/K874 and e1a K239 acetylation to repress host genes that would otherwise inhibit productive virus infection. Thus, adenovirus employs e1a to repress host genes that interfere with viral replication.


Asunto(s)
Adenoviridae/genética , Proteínas E1A de Adenovirus/metabolismo , Proteína de Retinoblastoma/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Adenoviridae/fisiología , Proteínas E1A de Adenovirus/genética , Transformación Celular Viral , Células Cultivadas , Quimiocina CXCL1/metabolismo , Cromatina/metabolismo , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN , Transducción de Señal , Replicación Viral
20.
J Virol Methods ; 192(1-2): 28-38, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23624118

RESUMEN

Adenoviruses are employed in the study of cellular processes and as expression vectors used in gene therapy. The success and reproducibility of these studies is dependent in part on having accurate and meaningful titers of replication competent and helper-dependent adenovirus stocks, which is problematic due to the use of varied and divergent titration protocols. Physical titration methods, which quantify the total number of viral particles, are used by many, but are poor at estimating activity. Biological titration methods, such as plaque assays, are more biologically relevant, but are time consuming and not applicable to helper-dependent gene therapy vectors. To address this, a protocol was developed called "infectious genome titration" in which viral DNA is isolated from the nuclei of cells ~3 h post-infection, and then quantified by Q-PCR. This approach ensures that only biologically active virions are counted as part of the titer determination. This approach is rapid, robust, sensitive, reproducible, and applicable to all forms of adenovirus. Unlike other Q-PCR-based methods, titers determined by this protocol are well correlated with biological activity.


Asunto(s)
Adenoviridae/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Carga Viral/métodos , Animales , ADN Viral/aislamiento & purificación , Femenino , Vectores Genéticos/aislamiento & purificación , Humanos , Ratones Desnudos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Manejo de Especímenes/métodos
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA