RESUMEN
Nuclear structures ND10/PML NBs are linked to multiple processes, including the maintenance of intranuclear homeostasis by sequestering proteins into "nuclear depot." This function presumes release of proteins from PML NBs and their redistribution to the alternative, supposedly "active" locations, in response to the external stress application. To further investigate this nuclear depot function, we focused on the intranuclear distribution of protein Daxx that in normal conditions is mainly accumulated at PML NBs, and has a minor association with centromeres and pericentromeres (CEN/periCEN). Here we report that application of physiological Heat Shock (HS) changes this balance forcing very robust and reversible accumulation of Daxx on CEN/periCEN heterochromatin. Heterochromatin architecture is essential for the proper orchestration of nuclear processes, while transcription from this part of genome is required for its maintenance. To understand functional consequences of Daxx deposition at CEN/periCEN, we tested for Daxx-dependency of heterochromatin transcription. Depletion of Daxx reduces accumulation of CEN RNA in normal conditions and periCEN RNA after HS application. Searching for the mechanism of Daxx-dependent regulation of heterochromatin transcription, we found that depletion of Daxx decreases incorporation of transcription-associated histone H3 variant, H3.3, into both CEN and periCEN. Surprisingly, HS-induced deposition of Daxx does not further elevate incorporation of H3.3 into CEN/periCEN that remained steady during stress and recovery. Instead, depletion of Daxx leads to HS-induced changes in the balance of epigenetic modifications at heterochromatin, most dramatically elevating levels of active H3K4Me2 modification at periCEN. We propose dualistic function of Daxx-containing complexes at CEN/periCEN: (1) regulation of H3.3 loading in normal conditions and (2) protection of epigenetic status upon stress-induced accumulation, thus collectively guarding epigenetic identity of CEN/periCEN heterochromatin.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Centrómero/metabolismo , Heterocromatina/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular , Proteínas Co-Represoras , Histonas/metabolismo , Humanos , Chaperonas Moleculares , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , ARN/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Estrés Fisiológico , TemperaturaRESUMEN
Schroedinger's book 'What is Life?' is widely credited for having played a crucial role in development of molecular and cellular biology. My essay revisits the issues raised by this book from the modern perspective of epigenetics and systems biology. I contrast two classes of potential mechanisms of epigenetic stability: 'epigenetic templating' and 'systems biology' approaches, and consider them from the point of view expressed by Schroedinger. I also discuss how quantum entanglement, a nonclassical feature of quantum mechanics, can help to address the 'problem of small numbers' that led Schroedinger to promote the idea of a molecular code-script for explaining the stability of biological order.
Asunto(s)
Epigénesis Genética/fisiología , Inestabilidad Genómica/fisiología , Física/historia , Biología de Sistemas/historia , Animales , Historia del Siglo XX , Historia del Siglo XXI , HumanosRESUMEN
BACKGROUND: Foamy virus Bel1/Tas trans-activators act as key regulators of gene expression and directly bind to Bel1 response elements (BRE) in both the internal and the 5'LTR promoters leading to strong transcriptional trans-activation. Cellular coactivators interacting with Bel1/Tas are unknown to date. RESULTS: Transient expression assays, co-immunoprecipitation experiments, pull-down assays, and Western blot analysis were used to demonstrate that the coactivator p300 and histone acetyltransferase PCAF specifically interact with the retroviral trans-activator Bel1/Tas in vivo. Here we show that the Bel1/Tas-mediated trans-activation was enhanced by the coactivator p300, histone acetyltransferases PCAF and SRC-1 based on the crucial internal promoter BRE. The Bel1/Tas-interacting region was mapped to the C/H1 domain of p300 by co-immunoprecipitation and pull-down assays. In contrast, coactivator SRC-1 previously reported to bind to the C-terminal domain of p300 did not directly interact with the Bel1 protein but nevertheless enhanced Bel1/Tas-mediated trans-activation. Cotransfection of Bel1/Tas and p300C with an expression plasmid containing the C/H1domain partially inhibited the p300C-driven trans-activation. CONCLUSIONS: Our data identify p300 and PCAF as functional partner molecules that directly interact with Bel1/Tas. Since the acetylation activities of the three coactivators reside in or bind to the C-terminal regions of p300, a C/H1 expression plasmid was used as inhibitor. This is the first report of a C/H1 domain-interacting retroviral trans-activator capable of partially blocking the strong Bel1/Tas-mediated activation of the C-terminal region of coactivator p300. The potential mechanisms and functional roles of the three histone and factor acetyltransferases p300, PCAF, and SRC-1 in Bel1/Tas-mediated trans-activation are discussed.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de los Retroviridae/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Línea Celular , Línea Celular Tumoral , Proteínas de Unión al ADN/fisiología , Células HeLa/química , Células HeLa/metabolismo , Histona Acetiltransferasas , Humanos , Riñón/química , Riñón/citología , Riñón/embriología , Riñón/metabolismo , Mutación/genética , Mutación/fisiología , Péptidos/metabolismo , Mapeo de Interacción de Proteínas/métodos , Estructura Terciaria de Proteína , Elementos de Respuesta/genética , Proteínas de los Retroviridae/fisiología , Spumavirus/genética , Transactivadores/fisiología , Factores de Transcripción p300-CBPRESUMEN
Packing of the eukaryotic genome into chromatin poses an accessibility problem for the DNA repair machinery. Chromatin structure has to be changed for the repair to occur, and we are beginning to discover how different chromatin modifying mechanisms facilitate DNA repair in the chromatin context. On the other hand, the repair-related changes in chromatin should be transient, and a particular chromatin state should be able to survive the repair process. Defects in the proper maintenance of chromatin states after repair could be a factor in the aging process, as well as in other pathologies.
Asunto(s)
Cromatina/genética , Reparación del ADN/genética , Animales , Cromatina/fisiología , Reparación del ADN/fisiología , HumanosRESUMEN
The LIM-domain-binding protein Ldb1 is a key factor in the assembly of transcriptional complexes involving LIM-homeodomain proteins and other transcription factors that regulate animal development. We identified Ssdp proteins (previously described as sequence-specific, single-stranded-DNA-binding proteins) as components of Ldb1-associated nuclear complexes in HeLa cells. Ssdp proteins are associated with Ldb1 in a variety of additional mammalian cell types. This association is specific, does not depend on the presence of nucleic acids, and is functionally significant. Genes encoding Ssdp proteins are well conserved in evolution from Drosophila to humans. Whereas the vertebrate Ssdp gene family has several closely related members, the Drosophila Ssdp gene is unique. In Xenopus, Ssdp encoded by Drosophila Ssdp or mouse Ssdp1 mRNA enhances axis induction by Ldb1 in conjunction with the LIM-homeobox gene Xlim1. Furthermore, we were able to demonstrate an interaction between Ssdp and Chip (the fly homolog of Ldb1) in Drosophila wing development. These findings indicate functional conservation of Ssdp as a cofactor of Ldb1 during invertebrate and vertebrate development.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas , Proteínas de Xenopus , Células 3T3 , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células HeLa , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas con Dominio LIM , Proteínas con Homeodominio LIM , Ratones , Proteínas Mitocondriales , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Células Vero , Xenopus/embriología , ZixinaRESUMEN
High mobility group nucleosome-binding (HMGN) proteins belong to a family of nuclear proteins that bind to nucleosomes and enhance transcription from chromatin templates by altering the structure of the chromatin fiber. The intranuclear organization of these proteins is dynamic and related to the metabolic state of the cell. Here we report that approximately 50% of the HMGN proteins are organized into macromolecular complexes in a fashion that is similar to that of other nuclear activities that modify the structure of the chromatin fiber. We identify several distinct HMGN-containing complexes that are relatively unstable and find that the inclusion of HMGN in the complexes varies according to the metabolic state of the cell. The nucleosome binding ability of HMGN in the complex is stronger than that of the free HMGN. We suggest that the inclusion of HMGN proteins into metastable multiprotein complexes serves to target the HMGN proteins to specific sites in chromatin and enhances their interaction with nucleosomes.