RESUMEN
The organization of chromatin within the nucleus influences gene expression during cell differentiation and development. Recent work took advantage of the genome-wide localization of molecular marks on chromosomes to analyze their linear distributions at different length scales. Moreover, chromosome conformation capture techniques detect spatial proximity inside the cell nucleus and allow us to characterize local and long-range chromatin loops as well as interchromosomal contacts. These techniques have improved our understanding of chromatin composition in eukaryotic chromosomes, but the principles governing nuclear organization are still little understood. On the one hand, proteins might localize in stable nuclear structures, such as transcription factories, on which chromatin would have to be targeted to be processed. On the other hand, proteins binding to chromatin might induce the formation of specialized nuclear compartments de novo. Current work is aimed at distinguishing between these possibilities and at elaborating predictive models of chromatin folding.
Asunto(s)
Cromatina/química , Conformación de Ácido Nucleico , Animales , Humanos , Modelos BiológicosRESUMEN
Insulators play important roles in controlling gene activity and maintaining regulatory independence between neighbouring genes. In this article, we show that the enhancer-blocking activity of the insulator present within the LTR retrotransposon Idefix can be abolished if two copies of the region containing the insulator--specifically, the long terminal repeat (LTR)--are fused to the retrotransposon's 5' untranslated region (5' UTR). The presence of this combination of two [LTR-5' UTR] modules is a prerequisite for the loss of enhancer-blocking activity. We further show that the 5' UTR causes flanking genomic sequences to be displaced to the nuclear periphery, which is not observed when two insulators are present by themselves. This study thus provides a functional link between insulators and independent genomic modules, which may cooperate to allow the specific regulation of defined genomic loci via nuclear repositioning. It further illustrates the complexity of genomic regulation within a chromatic environment with multiple functional elements.
Asunto(s)
Regiones no Traducidas 5'/química , Regulación de la Expresión Génica , Elementos Aisladores , Elementos Reguladores de la Transcripción , Retroelementos , Animales , Núcleo Celular/genética , Drosophila/genética , Genoma , Secuencias Repetidas Terminales , TransgenesRESUMEN
CREB-binding protein (CBP) is a coactivator for multiple transcription factors that transduce a variety of signaling pathways. Current models propose that CBP enhances gene expression by bridging the signal-responsive transcription factors with components of the basal transcriptional machinery and by augmenting the access of transcription factors to DNA through the acetylation of histones. To define the pathways and proteins that require CBP function in a living organism, we have begun a genetic analysis of CBP in flies. We have overproduced Drosophila melanogaster CBP (dCBP) in a variety of cell types and obtained distinct adult phenotypes. We used an uninflated-wing phenotype, caused by the overexpression of dCBP in specific central nervous system cells, to screen for suppressors of dCBP overactivity. Two genes with mutant versions that act as dominant suppressors of the wing phenotype were identified: the PKA-C1/DCO gene, encoding the catalytic subunit of cyclic AMP protein kinase, and ash1, a member of the trithorax group (trxG) of chromatin modifiers. Using immunocolocalization, we showed that the ASH1 protein is specifically expressed in the majority of the dCBP-overexpressing cells, suggesting that these proteins have the potential to interact biochemically. This model was confirmed by the findings that the proteins interact strongly in vitro and colocalize at specific sites on polytene chromosomes. The trxG proteins are thought to maintain gene expression during development by creating domains of open chromatin structure. Our results thus implicate a second class of chromatin-associated proteins in mediating dCBP function and imply that dCBP might be involved in the regulation of higher-order chromatin structure.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insecto/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/metabolismo , Proteína de Unión a CREB , Cromatina/genética , Cromosomas/genética , Cromosomas/inmunología , Cromosomas/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Femenino , Eliminación de Gen , Regulación de la Expresión Génica , Genes Reporteros , Masculino , Microscopía Confocal , Microscopía Fluorescente , Neuronas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética , Alas de Animales/anatomía & histología , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismo , Dedos de Zinc/genéticaRESUMEN
cdc2 gene expression is under the control of multiple factors. Although E2F/DP proteins have been reported to play a central role, they cannot account for all aspects of the fine modulation of cdc2 gene expression during cell cycle and embryonic development. To characterize the transcription factors that control cdc2 gene expression during nerve cell differentiation in avians, we have previously cloned the quail cdc2 gene promoter region. We had identified an octamer (CAGGTGGC) containing an E-box, which has important activity in regulating cdc2 transcription. Using in vivo genomic footprinting experiments, we show here that this motif, currently named IG, is the target of binding proteins at different stages of neuroretina development, confirming its importance as a regulatory response element for cdc2 gene expression. A subset of Helix-Loop-Helix family of transcription factors, known as Upstream Stimulatory Factors (USFs) specifically bind to this sequence as dimers. Moreover, our results indicate that USFs transactivate the promoter of cdc2 via the IG motif. These data may help to better understand the mechanisms that control cell division in differentiating nerve cells.
Asunto(s)
Proteína Quinasa CDC2/genética , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Factores de Transcripción/metabolismo , Animales , Células COS , Huella de ADN , Humanos , Codorniz , Retina/metabolismo , Activación Transcripcional , Células Tumorales Cultivadas , Factores Estimuladores hacia 5'RESUMEN
Infection by HTLV-1 has been correlated with the appearance of various proliferative or degenerative diseases. Some of these disorders have been observed in transgenic mice expressing the Tax protein, which is known to transactivate various viral and cellular promoters through interactions with several transcription factors. In this study we show that the C-terminus of this viral oncoprotein represents a motif permitting binding of Tax to the PDZ domains of several cellular proteins. A two-hybrid screen with Tax as bait indeed yielded complementary DNAs coding for six proteins including PDZ domains. Two of them correspond to truncated forms of the PSD-95 and beta1-syntrophin proteins, another clone codes for a protein homologous to the product of the C. elegans gene lin-7. The other three clones code for new human members of the PDZ family of cellular proteins. The interaction of Tax with the products of these clones was confirmed by immunoprecipitation assays in mammalian cells, and analysis of various mutants of Tax established the importance of the C-terminal amino acids for several of these interactions. These data suggest that Tax could perturb the normal function of targeted cellular proteins by strongly interacting with their PDZ domains.
Asunto(s)
Proteínas de Drosophila , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Proteínas de Insectos/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Clonación Molecular , Secuencia de Consenso , ADN Complementario/genética , ADN Complementario/metabolismo , Productos del Gen tax/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Proteínas de Insectos/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mapeo Peptídico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The Tax transactivator of the human T cell leukemia virus type I (HTLV-I) exhibits oncogenic properties. A screen for proteins interacting with Tax yielded a complementary DNA (cDNA) encoding the human Int-6 protein. In mice, the Int-6 gene can be converted into a putative dominant negative oncogene after retroviral insertion. Here, Int-6 was localized in the cell nucleus to give a speckled staining pattern superposed to that of the promyelocytic leukemia (PML) protein. The binding of Tax to Int-6 caused its redistribution from the nuclear domains to the cytoplasm. Thus, Int-6 is a component of the PML nuclear bodies and Tax disrupts its normal cellular localization by binding to it.
Asunto(s)
Núcleo Celular/química , Productos del Gen tax/metabolismo , Proteínas de Neoplasias , Proteínas Nucleares , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/análisis , Animales , Línea Celular , Citoplasma/química , Factor 3 de Iniciación Eucariótica , Productos del Gen tax/análisis , Células HeLa , Humanos , Ratones , Proteína de la Leucemia Promielocítica , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Proteínas Supresoras de TumorRESUMEN
To achieve a better understanding of the mechanism of transactivation by Tax of human T-cell leukemia virus type 1 Tax-responsive element 1 (TRE-1), we developed a genetic approach with Saccharomyces cerevisiae. We constructed a yeast reporter strain containing the lacZ gene under the control of the CYC1 promoter associated with three copies of TRE-1. Expression of either the cyclic AMP response element-binding protein (CREB) or CREB fused to the GAL4 activation domain (GAD) in this strain did not modify the expression of the reporter gene. Tax alone was also inactive. However, expression of the reporter gene was induced by coexpression of Tax and CREB. This effect was stronger with the GAD-CREB fusion protein. Analysis of different CREB mutants with this genetic system indicated that the C-terminal 92 amino acid residues, which include the basic domain and the leucine zipper, are necessary and sufficient to mediate transactivation by Tax. To identify cellular proteins binding to TRE-1 in a Tax-dependent manner, this strain was also used to screen a library of human cDNAs fused to GAD. Of five positive clones isolated from 0.75 x 10(6) yeast colonies, four were members of the CREB/activating transcription factor (ATF) family: CREB, two isoforms of the cyclic AMP-responsive element modulator (CREM), and ATF-1. Interestingly, these three proteins can be phosphorylated by protein kinase A and thus form a particular subgroup within the CREB/ATF family. Expression of ATF-2 in S. cerevisiae did not activate TRE-1 in the presence of Tax. This shows that in a eukaryotic nucleus, Tax specifically interacts with the basic domain-leucine zipper region of ATF-1, CREB, and CREM. The fifth clone identified in this screening corresponded to the Ku autoantigen p70 subunit. When fused to GAD, the C-terminal region of Ku was able to activate transcription via TRE-1 but this activation was not dependent on Tax.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Citocromos c , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/genética , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/metabolismo , Activación Transcripcional , Factores de Transcripción Activadores , Secuencia de Aminoácidos , Animales , Proteínas Sanguíneas/química , Clonación Molecular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/química , Grupo Citocromo c/genética , ADN Complementario , Proteínas de Unión al ADN , Proteínas Fúngicas/metabolismo , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Linfocitos/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , beta-Galactosidasa/biosíntesisRESUMEN
The viral Tax protein, which is encoded by human T-cell leukaemia virus HTLV-I, activates nuclear translocation of the NF-kappa B/Rel transcription factors and relieves cytoplasmic sequestration of RelA and Rel by heterodimerization with NF-kappa B1/p1O5 (refs 1,2). Proteolytic maturation of this precursor protein is performed by the proteasome complex. Here we show that Tax binds specifically to two subunits of the 20S proteasome, HsN3 and HC9. This interaction is weakened with HsN3 and lost for HC9 when a mutant of Tax is substituted that is selectively defective for NF-kappa B activation. Immunoprecipitation shows that p1O5 binds weakly to HC9 and that this interaction is reinforced by Tax. No bridging function of Tax between p1O5 and HsN3 was observed. From these results, we propose that Tax accelerates the proteolytic maturation of P105 by favouring its anchorage to the proteasome.