RESUMEN
The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD-CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences for different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein-E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle.
Asunto(s)
Factores de Transcripción E2F/química , Proteína de Retinoblastoma/química , Proteína p107 Similar a la del Retinoblastoma/química , Cristalografía por Rayos X , Factores de Transcripción E2F/genética , Factores de Transcripción E2F/metabolismo , Humanos , Dominios Proteicos , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Proteína p107 Similar a la del Retinoblastoma/genética , Proteína p107 Similar a la del Retinoblastoma/metabolismoRESUMEN
In addition to their canonical roles in the cell cycle, RB family proteins regulate numerous developmental pathways, although the mechanisms remain obscure. We found that Drosophila Rbf1 associates with genes encoding components of the highly conserved apical-basal and planar cell polarity pathways, suggesting a possible regulatory role. Here, we show that depletion of Rbf1 in Drosophila tissues is indeed associated with polarity defects in the wing and eye. Key polarity genes aPKC, par6, vang, pk, and fmi are upregulated, and an aPKC mutation suppresses the Rbf1-induced phenotypes. RB control of cell polarity may be an evolutionarily conserved function, with important implications in cancer metastasis.
Asunto(s)
Polaridad Celular/genética , Proteínas de Drosophila/genética , Ojo/metabolismo , Interferencia de ARN , Factores de Transcripción/genética , Alas de Animales/metabolismo , Animales , Animales Modificados Genéticamente , Cadherinas/genética , Cadherinas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Epistasis Genética , Ojo/citología , Ojo/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Discos Imaginales/crecimiento & desarrollo , Discos Imaginales/metabolismo , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo , Larva/citología , Larva/genética , Larva/crecimiento & desarrollo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Unión Proteica , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteína de Retinoblastoma , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/metabolismo , Alas de Animales/citología , Alas de Animales/crecimiento & desarrolloRESUMEN
The retinoblastoma tumor suppressor (RB) and its related family members p107 and p130 regulate cell proliferation through the transcriptional repression of genes involved in cellular G1 to S phase transition. However, RB proteins are functionally versatile, and numerous genetic and biochemical studies point to expansive roles in cellular growth control, pluripotency, and apoptotic response. For the vast majority of genes, RB family members target the E2F family of transcriptional activators as an integral component of its gene regulatory mechanism. These interactions are regulated via reversible phosphorylation by Cyclin/Cyclin-dependent kinase (Cdk) complexes, a major molecular mechanism that regulates transcriptional output of RB/E2F target genes. Recent studies indicate an additional level of regulation involving the ubiquitin-proteasome system that renders pervasive control over each component of the RB pathway. Disruption of the genetic circuitry for proteasome-mediated targeting of the RB pathway has serious consequences on development and cellular transformation, and is associated with several forms of human cancer. In this review, we discuss the role of the ubiquitin-proteasome system in proteolytic control of RB-E2F pathway components, and recent data that points to surprising non-proteolytic roles for the ubiquitin-proteasome system in novel transcriptional regulatory mechanisms.
Asunto(s)
Factores de Transcripción E2F/genética , Complejo de la Endopetidasa Proteasomal/genética , Proteína de Retinoblastoma/genética , Ubiquitina/genética , Proteína Sustrato Asociada a CrK/genética , Proteína Sustrato Asociada a CrK/metabolismo , Quinasas Ciclina-Dependientes/genética , Proteínas de Unión al ADN/genética , Factores de Transcripción E2F/metabolismo , Regulación de la Expresión Génica , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína de Retinoblastoma/metabolismo , Proteína p107 Similar a la del Retinoblastoma/genética , Proteína p107 Similar a la del Retinoblastoma/metabolismo , Transcripción GenéticaRESUMEN
RBf2 is a recently evolved retinoblastoma family member in Drosophila that differs from RBf1, especially in the C-terminus. To investigate whether the unique features of RBf2 contribute to diverse roles in gene regulation, we performed chromatin immunoprecipitation sequencing for both RBf2 and RBf1 in embryos. A previous model for RB-E2F interactions suggested that RBf1 binds dE2F1 or dE2F2, whereas RBf2 is restricted to binding to dE2F2; however, we found that RBf2 targets approximately twice as many genes as RBf1. Highly enriched among the RBf2 targets were ribosomal protein genes. We tested the functional significance of this finding by assessing RBf activity on ribosomal protein promoters and the endogenous genes. RBf1 and RBf2 significantly repressed expression of some ribosomal protein genes, although not all bound genes showed transcriptional effects. Interestingly, many ribosomal protein genes are similarly targeted in human cells, indicating that these interactions may be relevant for control of ribosome biosynthesis and growth. We carried out bioinformatic analysis to investigate the basis for differential targeting by these two proteins and found that RBf2-specific promoters have distinct sequence motifs, suggesting unique targeting mechanisms. Association of RBf2 with these promoters appears to be independent of dE2F2/dDP, although promoters bound by both RBf1 and RBf2 require dE2F2/dDP. The presence of unique RBf2 targets suggest that evolutionary appearance of this corepressor represents the acquisition of potentially novel roles in gene regulation for the RB family.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Genoma , Proteínas Represoras/genética , Ribosomas/metabolismo , Animales , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Drosophila/crecimiento & desarrollo , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/metabolismo , Factor de Transcripción E2F2/antagonistas & inhibidores , Factor de Transcripción E2F2/genética , Factor de Transcripción E2F2/metabolismo , Embrión no Mamífero/metabolismo , Proteínas del Ojo/antagonistas & inhibidores , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Regiones Promotoras Genéticas , Unión Proteica , Interferencia de ARN , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , Proteínas Represoras/metabolismo , Proteína de Retinoblastoma , Análisis de Secuencia de ARN , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The retinoblastoma (RB) tumor suppressor and related family of proteins play critical roles in development through their regulation of genes involved in cell fate. Multiple regulatory pathways impact RB function, including the ubiquitin-proteasome system with deregulated RB destruction frequently associated with pathogenesis. With the current study we explored the mechanisms connecting proteasome-mediated turnover of the RB family to the regulation of repressor activity. We find that steady state levels of all RB family members, RB, p107, and p130, were diminished during embryonic stem cell differentiation concomitant with their target gene acquisition. Proteasome-dependent turnover of the RB family is mediated by distinct and autonomously acting instability elements (IE) located in their C-terminal regulatory domains in a process that is sensitive to cyclin-dependent kinase (CDK4) perturbation. The IE regions include motifs that contribute to E2F-DP transcription factor interaction, and consistently, p107 and p130 repressor potency was reduced by IE deletion. The juxtaposition of degron sequences and E2F interaction motifs appears to be a conserved feature across the RB family, suggesting the potential for repressor ubiquitination and specific target gene regulation. These findings establish a mechanistic link between regulation of RB family repressor potency and the ubiquitin-proteasome system.
Asunto(s)
Proteína de Retinoblastoma/análisis , Proteína de Retinoblastoma/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Secuencia Conservada , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Evolución Molecular , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Transcripción GenéticaRESUMEN
The RB tumor suppressor, a regulator of the cell cycle, apoptosis, senescence, and differentiation, is frequently mutated in human cancers. We recently described an evolutionarily conserved C-terminal "instability element" (IE) of the Drosophila Rbf1 retinoblastoma protein that regulates its turnover. Misexpression of wild-type or non-phosphorylatable forms of the Rbf1 protein leads to repression of cell cycle genes. In contrast, overexpression of a defective form of Rbf1 lacking the IE (ΔIE), a stabilized but transcriptionally less active form of the protein, induced ectopic S phase in cell culture. To determine how mutations in the Rbf1 IE may induce dominant effects in a developmental context, we assessed the impact of in vivo expression of mutant Rbf1 proteins on wing development. ΔIE expression resulted in overgrowth of larval wing imaginal discs and larger adult wings containing larger cells. In contrast, a point mutation in a conserved lysine of the IE (K774A) generated severely disrupted, reduced wings. These contrasting effects appear to correlate with control of apoptosis; expression of the pro-apoptotic reaper gene and DNA fragmentation measured by acridine orange stain increased in flies expressing the K774A isoform and was suppressed by expression of Rbf1ΔIE. Intriguingly, cancer associated mutations affecting RB homologs p130 and p107 may similarly induce dominant phenotypes.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proliferación Celular/fisiología , Drosophila/fisiología , Regulación de la Expresión Génica/genética , Proteína de Retinoblastoma/genética , Alas de Animales/crecimiento & desarrollo , Naranja de Acridina , Animales , Proliferación Celular/genética , Cartilla de ADN/genética , Drosophila/genética , Citometría de Flujo , Mutación Puntual/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The retinoblastoma (RB) family transcriptional corepressors regulate diverse cellular events including cell cycle, senescence, and differentiation. The activity and stability of these proteins are mediated by post-translational modifications; however, we lack a general understanding of how distinct modifications coordinately impact both of these properties. Previously, we showed that protein turnover and activity are tightly linked through an evolutionarily conserved C-terminal instability element (IE) in the Drosophila RB-related protein Rbf1; surprisingly, mutant proteins with enhanced stability were less, not more active. To better understand how activity and turnover are controlled in this model RB protein, we assessed the impact of Cyclin-Cdk kinase regulation on Rbf1. An evolutionarily conserved N-terminal threonine residue is required for Cyclin-Cdk response and showed a dominant impact on turnover and activity; however, specific residues in the C-terminal IE differentially impacted Rbf1 activity and turnover, indicating an additional level of regulation. Strikingly, specific IE mutations that impaired turnover but not activity induced dramatic developmental phenotypes in the Drosophila eye. Mutation of the highly conserved Lys-774 residue induced hypermorphic phenotypes that mimicked the loss of phosphorylation control; mutation of the corresponding codon of the human RBL2 gene has been reported in lung tumors. Our data support a model in which closely intermingled residues within the conserved IE govern protein turnover, presumably through interactions with E3 ligases, and protein activity via contacts with E2F transcription partners. Such functional relationships are likely to similarly impact mammalian RB family proteins, with important implications for development and disease.
Asunto(s)
Proteínas de Drosophila/metabolismo , Lisina/metabolismo , Serina/metabolismo , Treonina/metabolismo , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Sitios de Unión/genética , Western Blotting , Línea Celular , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Proteínas de Drosophila/genética , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Ojo/ultraestructura , Humanos , Lisina/genética , Microscopía Electrónica , Mutación , Fosforilación , Estabilidad Proteica , Proteína de Retinoblastoma , Serina/genética , Treonina/genética , Factores de Transcripción/genética , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismo , Alas de Animales/ultraestructuraRESUMEN
The retinoblastoma (RB) tumor suppressor protein regulates multiple pathways that influence cell growth, and as a key regulatory node, its function is inactivated in most cancer cells. In addition to its canonical roles in cell cycle control, RB functions as a global repressor of RNA polymerase (Pol) III transcription. Indeed, Pol III transcripts accumulate in cancer cells and their heightened levels are implicated in accelerated growth associated with RB dysfunction. Herein we review the mechanisms of RB repression for the different types of Pol III genes. For type 1 and type 2 genes, RB represses transcription through direct contacts with the core transcription machinery, notably Brf1-TFIIIB, and inhibits preinitiation complex formation and Pol III recruitment. A contrasting model for type 3 gene repression indicates that RB regulation involves stable and simultaneous promoter association by RB, the general transcription machinery including SNAPc, and Pol III, suggesting that RB may impede Pol III promoter escape or elongation. Interestingly, analysis of published genomic association data for RB and Pol III revealed added regulatory complexity for Pol III genes both during active growth and during arrested growth associated with quiescence and senescence. This article is part of a Special Issue entitled: Transcription by Odd Pols.
Asunto(s)
Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , ARN Polimerasa III/metabolismo , Proteína de Retinoblastoma/metabolismo , Animales , Humanos , ARN Polimerasa III/genética , Neoplasias de la Retina/metabolismo , Retinoblastoma/metabolismo , Transcripción GenéticaRESUMEN
Multiple myeloma (MM) is a malignant disorder of differentiated B-cells for which standard care involves the inhibition of the proteasome. All clinically used proteasome inhibitors, including the chemotherapeutic drug bortezomib, target the catalytic active sites of the proteasome and inhibit protein proteolysis by competing with substrate binding. However, nearly all (~97%) patients become intolerant or resistant to treatments within a few years, after which the average survival time is less than 1 year. We describe herein the inhibition of the human proteasome via a noncompetitive mechanism by the imidazoline scaffold, TCH-13. Consistent with a mechanism distinct from that of competitive inhibitors, TCH-013 acts additively with and overcomes resistance to bortezomib. Importantly, TCH-013 induces apoptosis in a panel of myeloma and leukemia cell lines, but in contrast, normal lymphocytes, primary bone marrow stromal cells (hBMSC), and macrophages are resistant to its cytotoxic effects. TCH-013 was equally effective in blocking MM cell growth in co-cultures of MM cells with hBMSC isolated from CD138 negative bone marrow (BM) samples of MM patients. The cellular activity translated well in vivo where TCH-013 delayed tumor growth in an MM xenograft model to a similar extent as bortezomib.
Asunto(s)
Antineoplásicos/farmacología , Ácidos Borónicos/farmacología , Resistencia a Antineoplásicos , Imidazoles/farmacología , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Pirazinas/farmacología , Animales , Bortezomib , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Humanos , Proteínas I-kappa B/metabolismo , Imidazoles/química , Ratones , Estructura Molecular , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The retinoblastoma (RB) tumor suppressor protein is a transcriptional cofactor with essential roles in cell cycle and development. Physical and functional targets of RB and its paralogs p107/p130 have been studied largely in cultured cells, but the full biological context of this family of proteins' activities will likely be revealed only in whole organismal studies. To identify direct targets of the major Drosophila RB counterpart in a developmental context, we carried out ChIP-Seq analysis of Rbf1 in the embryo. The association of the protein with promoters is developmentally controlled; early promoter access is globally inhibited, whereas later in development Rbf1 is found to associate with promoter-proximal regions of approximately 2000 genes. In addition to conserved cell-cycle-related genes, a wholly unexpected finding was that Rbf1 targets many components of the insulin, Hippo, JAK/STAT, Notch, and other conserved signaling pathways. Rbf1 may thus directly affect output of these essential growth-control and differentiation pathways by regulation of expression of receptors, kinases and downstream effectors. Rbf1 was also found to target multiple levels of its own regulatory hierarchy. Bioinformatic analysis indicates that different classes of genes exhibit distinct constellations of motifs associated with the Rbf1-bound regions, suggesting that the context of Rbf1 recruitment may vary within the Rbf1 regulon. Many of these targeted genes are bound by Rbf1 homologs in human cells, indicating that a conserved role of RB proteins may be to adjust the set point of interlinked signaling networks essential for growth and development.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Genoma de los Insectos , Proteína de Retinoblastoma/metabolismo , Transducción de Señal/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Genes del Desarrollo , Genes cdc , Homeostasis/genética , Motivos de Nucleótidos , Regiones Promotoras Genéticas , Unión Proteica , Regulón , Proteína de Retinoblastoma/genética , Factores de Transcripción/genéticaRESUMEN
The pathogenesis of rheumatoid arthritis is mainly driven by NF-κB-mediated production of cytokines, such as TNF-α. We report herein that the orally available imidazoline-based NF-κB inhibitor, TCH-013, was found to significantly reduce TNF-α signaling and attenuate collagen antibody induced arthritis in BALB/c mice.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Imidazoles/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Administración Oral , Animales , Artritis Reumatoide/inducido químicamente , Colágeno , Relación Dosis-Respuesta a Droga , Imidazoles/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , FN-kappa B/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The retinoblastoma (RB) tumor suppressor family functions as a regulatory node governing cell cycle progression, differentiation, and apoptosis. Post-translational modifications play a critical role in modulating RB activity, but additional levels of control, including protein turnover, are also essential for proper function. The Drosophila RB homolog Rbf1 is subjected to developmentally cued proteolysis mediated by an instability element (IE) present in the C terminus of this protein. Paradoxically, instability mediated by the IE is also linked to Rbf1 repression potency, suggesting that proteolytic machinery may also be directly involved in transcriptional repression. We show that the Rbf1 IE is an autonomous degron that stimulates both Rbf1 ubiquitination and repression potency. Importantly, Rbf1 IE function is promoter-specific, contributing to repression of cell cycle responsive genes but not to repression of cell signaling genes. The multifunctional IE domain thus provides Rbf1 flexibility for discrimination between target genes embedded in divergent cellular processes.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteolisis , Proteínas Represoras/metabolismo , Proteína de Retinoblastoma/metabolismo , Factores de Transcripción/metabolismo , Ubiquitinación/fisiología , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Regulación de la Expresión Génica/fisiología , Estructura Terciaria de Proteína , Proteínas Represoras/genética , Proteína de Retinoblastoma/genética , Factores de Transcripción/genéticaRESUMEN
The E2F family of transcription factors contributes to oncogenesis through activation of multiple genes involved in cellular proliferation, a process that is opposed by the Retinoblastoma tumor suppressor protein (RB). RB also increases E2F1 stability by inhibiting its proteasome-mediated degradation, but the consequences of this post-translational regulation of E2F1 remain unknown. To better understand the mechanism of E2F stabilization and its physiological relevance, we examined the streamlined Rbf1-dE2F1 network in Drosophila. During embryonic development, Rbf1 is insulated from ubiquitin-mediated turnover by the COP9 signalosome, a multi-protein complex that modulates E3 ubiquitin ligase activity. Here, we report that the COP9 signalosome also protects the Cullin4-E3 ligase that is responsible for dE2F1 proteasome-mediated destruction. This dual role of the COP9 signalosome may serve to buffer E2F levels, enhancing its turnover via Cul4 protection and its stabilization through protection of Rbf1. We further show that Rbf1-mediated stabilization of dE2F1 and repression of dE2F1 cell cycle-target genes are distinct properties. Removal of an evolutionarily conserved Rbf1 C terminal degron disabled Rbf1 repression without affecting dE2F1 stabilization. This mutant form of Rbf1 also enhanced G(1)-to-S phase progression when expressed in Rbf1-containing S2 embryonic cells, suggesting that such mutations may generate gain-of-function properties relevant to cellular transformation. Consistent with this idea, several studies have identified mutations in the homologous C terminal domains of RB and p130 in human cancer.
Asunto(s)
Proteínas Cullin/genética , Replicación del ADN , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Factor de Transcripción E2F1/genética , Complejos Multiproteicos/genética , Péptido Hidrolasas/genética , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Complejo del Señalosoma COP9 , Ciclo Celular/genética , Transformación Celular Neoplásica , Proteínas Cullin/metabolismo , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Factor de Transcripción E2F1/metabolismo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Humanos , Complejos Multiproteicos/metabolismo , Mutación , Péptido Hidrolasas/metabolismo , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteína de Retinoblastoma , Transducción de Señal , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The pathogenesis of rheumatoid arthritis is mainly driven by NF-κB-mediated production of cytokines, such as TNF-α. We report herein that the orally available imidazoline-based NF-κB inhibitor, TCH-013, was found to significantly reduce TNF-α signaling and attenuate collagen antibody induced arthritis in BALB/c mice.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Imidazolinas/uso terapéutico , FN-kappa B/antagonistas & inhibidores , Administración Oral , Animales , Artritis Experimental/inmunología , Imidazolinas/administración & dosificación , Imidazolinas/síntesis química , Ratones , Ratones Endogámicos BALB C , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The human small nuclear RNA (snRNA) and small cytoplasmic RNA (scRNA) gene families encode diverse non-coding RNAs that influence cellular growth and division. Many snRNA and scRNA genes are related via their compact and yet powerful promoters that support RNA polymerase III transcription. We have utilized the human U6 snRNA gene family to examine the mechanism for regulated transcription of these potent transcription units. Analysis of nine U6 family members showed enriched CpG density within the promoters of actively transcribed loci relative to inert genes, implying a relationship between gene potency and DNA methylation. Indeed, both pharmacological inhibition of DNA methyltransferase (DNMT) activity and the forced diminution of DNMT-1, DNMT-3a, and DNMT-3b by siRNA targeting resulted in increased U6 levels in asynchronously growing MCF7 adenocarcinoma cells. In vitro transcription assays further showed that template methylation impedes U6 transcription by RNA polymerase III. Both DNMT-1 and DNMT-3a were detected at the U6-1 locus by chromatin immunoprecipitation directly linking these factors to RNA polymerase III regulation. Despite this association, the endogenous U6-1 locus was not substantially methylated in actively growing cells. However, both DNMT occupancy and low frequency methylation were correlated with increased Retinoblastoma tumor suppressor (RB) expression, suggesting that the RB status can influence specific epigenetic marks.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/metabolismo , Epigénesis Genética , ARN Polimerasa III/metabolismo , ARN Nuclear Pequeño/biosíntesis , Transcripción Genética , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Metiltransferasa 3A , Sitios Genéticos , Células HeLa , Humanos , ARN Polimerasa III/genética , ARN Interferente Pequeño/farmacología , ARN Nuclear Pequeño/genética , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismoRESUMEN
DNA damage induced by ionizing radiation activates the ataxia telangiectasia mutated pathway, resulting in apoptosis or DNA repair. The serine/threonine checkpoint kinase (Chk2) is an important transducer of this DNA damage signaling pathway and mediates the ultimate fate of the cell. Chk2 is an advantageous target for the development of adjuvant drugs for cancer therapy, because inhibition of Chk2 allows normal cells to enter cell cycle arrest and DNA repair, whereas many tumors bypass cell cycle checkpoints. Chk2 inhibitors may thus have a radioprotective effect on normal cells. We report herein a class of natural product derived Chk2 inhibitors, exemplified by indoloazepine 1, that elicit a strong ATM-dependent Chk2-mediated radioprotection effect in normal cells and p53 wt cells, but not p53 mutant cells (>50% of all cancers). This study represents the first example of a radioprotective effect in human cells other than T-cells and implicates a functional ATM pathway as a requirement for IR-induced radioprotection by this class of Chk2 inhibitors. Several of the hymenialdisine-derived analogues inhibit Chk2 at nanomolar concentrations, inhibit autophosphorylation of Chk2 at Ser516 in cells, and increase the survival of normal cells following ionizing radiation.
Asunto(s)
Azepinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirroles/farmacología , Protectores contra Radiación/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Azepinas/síntesis química , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Quinasa de Punto de Control 2 , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Mutación , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Serina-Treonina Quinasas/metabolismo , Pirroles/síntesis química , Radiación Ionizante , Protectores contra Radiación/síntesis química , Serina/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiaciónRESUMEN
The retinoblastoma (RB) transcriptional corepressor and related family of pocket proteins play central roles in cell cycle control and development, and the regulatory networks governed by these factors are frequently inactivated during tumorigenesis. During normal growth, these proteins are subject to tight control through at least two mechanisms. First, during cell cycle progression, repressor potential is down-regulated by Cdk-dependent phosphorylation, resulting in repressor dissociation from E2F family transcription factors. Second, RB proteins are subject to proteasome-mediated destruction during development. To better understand the mechanism for RB family protein instability, we characterized Rbf1 turnover in Drosophila and the protein motifs required for its destabilization. We show that specific point mutations in a conserved C-terminal instability element strongly stabilize Rbf1, but strikingly, these mutations also cripple repression activity. Rbf1 is destabilized specifically in actively proliferating tissues of the larva, indicating that controlled degradation of Rbf1 is linked to developmental signals. The positive linkage between Rbf1 activity and its destruction indicates that repressor function is governed in a manner similar to that described by the degron theory of transcriptional activation. Analogous mutations in the mammalian RB family member p107 similarly induce abnormal accumulation, indicating substantial conservation of this regulatory pathway.
Asunto(s)
Proteínas de Drosophila/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Western Blotting , Línea Celular , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Factores de Transcripción E2F/metabolismo , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Femenino , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Leupeptinas/farmacología , Masculino , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas/genética , Unión Proteica , Estabilidad Proteica , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Proteína p107 Similar a la del Retinoblastoma/genética , Proteína p107 Similar a la del Retinoblastoma/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/genéticaRESUMEN
During blastocyst formation the segregation of the inner cell mass (ICM) and trophectoderm is governed by the mutually antagonistic effects of the transcription factors Oct4 and Cdx2. Evidence indicates that suppression of Oct4 expression in the trophectoderm is mediated by Cdx2. Nonetheless, the underlying epigenetic modifiers required for Cdx2-dependent repression of Oct4 are largely unknown. Here we show that the chromatin remodeling protein Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts. By employing a combination of RNA interference (RNAi) and gene expression analysis we found that both Brg1 Knockdown (KD) and Cdx2 KD blastocysts exhibit widespread expression of Oct4 in the trophectoderm. Interestingly, in Brg1 KD blastocysts and Cdx2 KD blastocysts, the expression of Cdx2 and Brg1 is unchanged, respectively. To address whether Brg1 cooperates with Cdx2 to repress Oct4 transcription in the developing trophectoderm, we utilized preimplantation embryos, trophoblast stem (TS) cells and Cdx2-inducible embryonic stem (ES) cells as model systems. We found that: (1) combined knockdown (KD) of Brg1 and Cdx2 levels in blastocysts resulted in increased levels of Oct4 transcripts compared to KD of Brg1 or Cdx2 alone, (2) endogenous Brg1 co-immunoprecipitated with Cdx2 in TS cell extracts, (3) in blastocysts Brg1 and Cdx2 co-localize in trophectoderm nuclei and (4) in Cdx2-induced ES cells Brg1 and Cdx2 are recruited to the Oct4 promoter. Lastly, to determine how Brg1 may induce epigenetic silencing of the Oct4 gene, we evaluated CpG methylation at the Oct4 promoter in the trophectoderm of Brg1 KD blastocysts. This analysis revealed that Brg1-dependent repression of Oct4 expression is independent of DNA methylation at the blastocyst stage. In toto, these results demonstrate that Brg1 cooperates with Cdx2 to repress Oct4 expression in the developing trophectoderm to ensure normal development.
Asunto(s)
Blastocisto/metabolismo , ADN Helicasas/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Nucleares/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Blastocisto/citología , Factor de Transcripción CDX2 , ADN Helicasas/genética , Metilación de ADN/genética , Ectodermo/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/genética , Ratones , Modelos Biológicos , Proteínas Nucleares/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Unión Proteica , Transporte de Proteínas , Proteínas Represoras/genética , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The mammalian nuclear transcription factor NF-kappaB is responsible for the transcription of multiple cytokines, including the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6). Elevated levels of pro-inflammatory cytokines play an important role in the pathogenesis of inflammatory disorders such as rheumatoid arthritis (RA). Inhibition of the pro-inflammatory transcription factor NF-kappaB has therefore been identified as a possible therapeutic treatment for RA. We describe herein the synthesis and biological activity of a series of imidazoline-based scaffolds as potent inhibitors of NF-kappaB mediated gene transcription in cell culture as well as inhibitors of TNF-alpha and IL-6 production in interleukin 1 beta (IL-1beta) stimulated human blood.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Imidazolinas/síntesis química , Interleucina-6/antagonistas & inhibidores , FN-kappa B/fisiología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Células HeLa , Humanos , Imidazolinas/química , Imidazolinas/farmacología , Técnicas In Vitro , Interleucina-1beta/farmacología , Interleucina-6/biosíntesis , FN-kappa B/genética , Estereoisomerismo , Relación Estructura-Actividad , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
The perinucleolar compartment (PNC) is a nuclear subdomain that is unique to tumor cells, and the percentage of cells in a population containing PNCs (PNC prevalence) indicates the level of malignancy of that population. Here, we utilize anti-cancer drugs and other exogenous stimuli to investigate the structure and function of the PNC. Screening of clinically used anti-cancer drugs revealed two types of drugs disassemble PNCs and do so through their specific molecular actions. Transcription inhibitors reduce PNC prevalence in parallel with RNA polymerase III transcription reduction, and a subset of DNA-damaging drugs and stimuli (UV radiation) disassemble the PNC. Inhibition of cellular DNA damage response demonstrated that the DNA damage itself, not the response or polymerase III inhibition, is responsible for PNC disassembly, suggesting that the maintenance of the PNC is dependent upon DNA integrity. Analyses of the types of DNA damage that cause PNC disassembly show that interstrand DNA base pairing, not strand continuity, is important for PNC integrity, indicating that the PNC components are directly interacting with the DNA. Complementary cell biology experiments demonstrated that the number of PNCs per cell increases with the rounds of endoreplication and that PNCs split into doublets during mid S phase, both of which are phenotypes that are typical of a replicating DNA loci. Together, these studies validate PNC disassembly as a screening marker to identify chemical probes and revealed that the PNC is directly nucleated on a DNA locus, suggesting a potential role for the PNC in gene expression regulation.