RESUMEN

This review focuses on the major protein moiety of chromosomes, i.e., the histone proteins, on the contribution of their posttranslational modification to structural and functional chromatin dynamics, on the acetylation and methylation of lysine residues, and on the phosphorylation of serine or threonine with respect to various steps during the cell cycle.

Asunto(s)

Ensamble y Desensamble de Cromatina/fisiología , Cromatina/metabolismo , Mitosis/fisiología , Fase S/fisiología , Animales , Cromatina/genética , Humanos

RESUMEN

Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informative and readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the use of descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure.

RESUMEN

Although ubiquitously present in chromatin, the function of the linker histone subtypes is partly unknown and contradictory studies on their properties have been published. To explore whether the various H1 subtypes have a differential role in the organization and dynamics of chromatin we have incorporated all of the somatic human H1 subtypes into minichromosomes and compared their influence on nucleosome spacing, chromatin compaction and ATP-dependent remodeling. H1 subtypes exhibit different affinities for chromatin and different abilities to promote chromatin condensation, as studied with the Atomic Force Microscope. According to this criterion, H1 subtypes can be classified as weak condensers (H1.1 and H1.2), intermediate condensers (H1.3) and strong condensers (H1.0, H1.4, H1.5 and H1x). The variable C-terminal domain is required for nucleosome spacing by H1.4 and is likely responsible for the chromatin condensation properties of the various subtypes, as shown using chimeras between H1.4 and H1.2. In contrast to previous reports with isolated nucleosomes or linear nucleosomal arrays, linker histones at a ratio of one per nucleosome do not preclude remodeling of minichromosomes by yeast SWI/SNF or Drosophila NURF. We hypothesize that the linker histone subtypes are differential organizers of chromatin, rather than general repressors.

Asunto(s)

Adenosina Trifosfato/química , Cromatina/química , Proteínas Cromosómicas no Histona/química , Proteínas de Drosophila/química , Regulación de la Expresión Génica , Histonas/genética , Histonas/fisiología , Proteína 4 de Unión a Retinoblastoma/química , Factores de Transcripción/química , Animales , Ensamble y Desensamble de Cromatina , Cromosomas/metabolismo , Drosophila melanogaster/metabolismo , Células HeLa , Histonas/química , Humanos , Microscopía de Fuerza Atómica/métodos , Nucleosomas/química , Nucleosomas/metabolismo , Unión Proteica , Estructura Terciaria de Proteína

RESUMEN

H1 histone subtype genes differ in their expression patterns during the different stages of the cell cycle interphase. While the group of replication-dependent H1 histone subtypes is synthesized during S phase, the replacement histone subtype H1.0 is also expressed replication-independently in non-proliferating cells. The present study is the first report about the analysis of the cell cycle-dependent expression of all five replication-dependent H1 subtypes, the replacement histone H1.0 and the ubiquitously expressed subtype H1x. The expression of these H1 histone subtypes in HeLa cells was analyzed on mRNA level by quantitative real-time RT-PCR as well as on protein level by immunoblotting. We found that after arrest of HeLa cells in G(1) phase by treatment with sodium butyrate, the mRNA levels of all replication-dependently expressed H1 subtypes decreased, but to very different extent. During S phase the individual replication-dependently expressed H1 subtypes show similar kinetics regarding their mRNA levels. However, the variations in their protein amounts partially differ from the respective RNA levels which especially applies to histone H1.3. In contrast, the mRNA as well as the protein level of H1x remained nearly unchanged in G(1) as well as during S phase progression. The results of the present study demonstrate that the cell cycle-dependent mRNA and protein expression of various H1 subtypes is differentially regulated, supporting the hypothesis of a functional heterogeneity.

Asunto(s)

Proliferación Celular , Histonas/metabolismo , Butiratos/farmacología , Línea Celular Tumoral , Fase G1 , Células HeLa , Humanos , ARN Mensajero/metabolismo , Fase S

RESUMEN

The negative cofactor 2 (NC2) is a protein complex composed of two subunits, NC2alpha and NC2beta, and plays a key role in transcription regulation. Here we investigate whether each subunit contains a nuclear localization signal (NLS) that permits individual crossing of the nuclear membrane or whether nuclear import of NC2alpha and NC2beta depends on heterodimerization. Our results from in vitro binding studies and transfection experiments in cultured cells show that each subunit contains a classical NLS (cNLS) that is recognized by the importin alpha/beta heterodimer. Regardless of the individual cNLSs the two NC2 subunits are translocated as a preassembled complex as co-transfection experiments with wild-type and cNLS-deficient NC2 subunits demonstrate. Ran-dependent binding of the nuclear export receptor Crm1/exportin 1 confirmed the presence of a leucine-rich nuclear export signal (NES) in NC2beta. In contrast, NC2alpha does not exhibit a NES. Our results from interspecies heterokaryon assays suggest that heterodimerization with NC2alpha masks the NES in NC2beta, which prevents nuclear export of the NC2 complex. A mutation in either one of the two cNLSs decreases the extent of importin alpha/beta-mediated nuclear import of the NC2 complex. In addition, the NC2 complex can enter the nucleus via a second pathway, facilitated by importin 13. Because importin 13 binds exclusively to the NC2 complex but not to the individual subunits this alternative import pathway depends on sequence elements distributed among the two subunits.

Asunto(s)

Fosfoproteínas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Línea Celular , Ácidos Grasos Insaturados/farmacología , Regulación de la Expresión Génica , Humanos , Carioferinas/metabolismo , Ratones , Señales de Localización Nuclear , Fosfoproteínas/genética , Multimerización de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/genética , Proteína Exportina 1

RESUMEN

The histone fold is a structural element that facilitates heterodimerization, and histone fold heterodimers play crucial roles in gene regulation. Here, we investigated the nuclear import of two human histone fold pairs, which belong to the H2A/H2B family: CHRAC-15/CHRAC-17 and p12/CHRAC-17. Our results from in vitro nuclear import assays with permeabilized cells and in vivo cotransfection experiments reveal that importin 13 facilitates nuclear import of both histone fold heterodimers. Using glutathione S-transferase pulldown experiments, we provide evidence that heterodimers are required for efficient binding of importin 13 because the monomers alone do not significantly interact. Mutational analysis shows that stepwise substitution of basic amino acid residues conserved among the histone fold subunits leads to a progressive loss of importin 13 binding and nuclear accumulation of CHRAC-15/CHRAC-17 and p12/CHRAC-17. The distribution of basic amino acid residues among the histone fold subunits essential for nuclear uptake suggests that heterodimerization of the histone fold motif-containing proteins forms an importin 13-specific binding platform.

Asunto(s)

Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Secuencias de Aminoácidos , Cromatina/química , Citoplasma/metabolismo , Análisis Mutacional de ADN , Dimerización , Glutatión Transferasa/metabolismo , Células HeLa , Histonas/química , Humanos , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Pliegue de Proteína , Proteínas Recombinantes/química

RESUMEN

H1 histones are progressively phosphorylated during the cell cycle. The number of phosphorylated sites is zero to three in late S phase and increases to five or six in late G2 phase and M phase. It is assumed that this phosphorylation modulates chromatin condensation and decondensation, but its specific role remains unclear. Recently, it was shown that the somatic H1 histone subtype H1.5 becomes pentaphosphorylated during mitosis, with phosphorylated threonine 10 being the last site to be phosphorylated. We have generated an antiserum specific for human H1.5 phosphorylated at threonine 10. Immunofluorescence labeling of HeLa cells with this antiserum revealed that the phosphorylation at this site appears in prometaphase and disappears in telophase, and that this hyperphosphorylated form of H1.5 is mainly chromatin-bound in metaphase when chromatin condensation is maximal. In search of the kinase responsible for the phosphorylation at this site, we found that threonine 10 of H1.5 can be phosphorylated by glycogen synthase kinase-3 in vitro, but not by cyclin-dependent kinase 1/cyclin B and cyclin-dependent kinase 5/p35, respectively. Furthermore, addition of specific glycogen synthase kinase-3 inhibitors led to a reduction in phosphorylation at this site both in vivo and in vitro.

Asunto(s)

División Celular , Glucógeno Sintasa Quinasa 3/metabolismo , Histonas/metabolismo , Treonina/metabolismo , Proteína Quinasa CDC2/metabolismo , Quinasa 5 Dependiente de la Ciclina/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Células HeLa , Humanos , Fosforilación

RESUMEN

The lysine-rich H1 histone family in mammals includes eleven different subtypes, and thus it is the most divergent class of histone proteins. The central globular H1 domain asymmetrically interacts with DNA at the exit or entry end of the nucleosomal core DNA, and the C-terminal domain has a major impact on the linker DNA conformation and chromatin condensation. H1 histones are thus involved in the formation of higher order chromatin structures, and they modulate the accessibility of regulatory proteins, chromatin remodeling factors and histone modification enzymes to their target sites. The major posttranslational modification of H1 histones is phosphorylation, which reaches a peak during G2 and mitosis. Phosphorylation is, however, also involved in the control of DNA replication and it contributes to the regulation of gene expression. Disruption of linker histone genes, initially performed in order to delineate subtype-specific functions, revealed that disruption of one or two H1 subtype genes is quantitatively compensated by an increased expression of other subtypes. This suggests a functional redundancy among H1 subtypes. However, the inactivation of three subtypes and the reduction of the H1 moiety in half finally resulted in a phenotypic effect. On the other hand, studies on the role of particular subtypes at specific developmental stages in lower eukaryotes, but also in vertebrates suggest that specific subtypes of H1 participate in particular systems of gene regulation.

Asunto(s)

Cromatina/química , Cromatina/metabolismo , Histonas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Histonas/química , Histonas/genética , Humanos , Mamíferos/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo

RESUMEN

BACKGROUND: Histone H1x is a ubiquitously expressed member of the H1 histone family. H1 histones, also called linker histones, stabilize compact, higher order structures of chromatin. In addition to their role as structural proteins, they actively regulate gene expression and participate in chromatin-based processes like DNA replication and repair. The epigenetic contribution of H1 histones to these mechanisms makes it conceivable that they also take part in malignant transformation. METHODS: Based on results of a Blast data base search which revealed an accumulation of expressed sequence tags (ESTs) of H1x in libraries from neuroendocrine tumours (NETs), we evaluated the expression of H1x in NETs from lung and the gastrointestinal tract using immunohistochemisty. Relative protein and mRNA levels of H1x were analysed by Western blot analysis and quantitative real-time RT-PCR, respectively. Since several reports describe a change of the expression level of the replacement subtype H1.0 during tumourigenesis, the analysis of this subtype was included in this study. RESULTS: We found an increased expression of H1x but not of H1.0 in NET tissues in comparison to corresponding normal tissues. Even though the analysed NETs were heterogenous regarding their grade of malignancy, all except one showed a considerably higher protein amount of H1x compared with corresponding non-neoplastic tissue. Furthermore, double-labelling of H1x and chromogranin A in sections of pancreas and small intestine revealed that H1x is highly expressed in neuroendocrine cells of these tissues. CONCLUSION: We conclude that the high expression of histone H1x in NETs is probably due to the abundance of this protein in the cells from which these tumours originate.

Asunto(s)

Carcinoma Neuroendocrino/metabolismo , Neoplasias Gastrointestinales/metabolismo , Neoplasias Pulmonares/metabolismo , Células Neuroendocrinas/metabolismo , Proteínas Nucleares/metabolismo , Western Blotting , Carcinoma Neuroendocrino/patología , Cromogranina A , Proteínas de Unión al ADN , Neoplasias Gastrointestinales/patología , Biblioteca de Genes , Humanos , Inmunohistoquímica , Intestino Delgado/metabolismo , Intestino Delgado/patología , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/patología , Proteínas Nucleares/genética , Páncreas/metabolismo , Páncreas/patología , Proteínas de Unión al ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Lugares Marcados de Secuencia

RESUMEN

Until a few years ago, the H1 histones were exclusively considered to be the architectural proteins of chromatin involved in chromatin condensation. However there is now increasing data to support the hypothesis that the H1 subtypes are involved in genomic integrity and that they may have unexpected functional roles in various biological processes such as in differentiation and DNA repair, apoptosis and lifespan. Moreover, the H1 histones are phosphorylated to a great extent. Recent work has implicated phosphorylation of H1 in the regulation of chromatin remodeling. In light of the fact that chromatin reorganization and heterochromatin formation has been shown to take place during ageing and senescence, in the present investigation, we have analyzed the changes that take place in the somatic H1 linker histone subtype profile and their phosphorylation states in human peripheral blood lymphocytes as a function of donor age. Results from this work show that there is a significant age-related dephosphorylation of H1.4 and H1.5 and an increase in the heterochromatin protein HP1alpha as a function of donor age. These results indicate that dephosphorylation of H1 histones may be related to an increase in senescence-associated heterochromatin formation during the in vivo ageing of human peripheral blood lymphocytes.

Asunto(s)

Envejecimiento/sangre , Histonas/sangre , Linfocitos/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Western Blotting/métodos , Senescencia Celular/fisiología , Homólogo de la Proteína Chromobox 5 , Heterocromatina/metabolismo , Humanos , Persona de Mediana Edad , Fosforilación

RESUMEN

DNA cleavage is a biochemical hallmark of apoptosis. In humans, apoptotic DNA cleavage is executed by DNA fragmentation factor (DFF) 40. In proliferating cells DFF40 is expressed in the presence of its chaperone and inhibitor DFF45, which results in the formation of the DFF complex. Here, we present a systematic analysis of the nuclear import of the DFF complex. Our in vitro experiments demonstrate that the importin alpha/beta-heterodimer mediates the translocation of the DFF complex from the cytoplasm to the nucleus. Both DFF subunits interact directly with the importin alpha/beta-heterodimer. However, importin alpha/beta binds more tightly to the DFF complex compared with the individual subunits. Additionally, the isolated C-terminal regions of both DFF subunits together bind importin alpha/beta more strongly than the individual C termini. Our results from in vivo studies reveal that the C-terminal regions of both DFF subunits harbor nuclear localization signals. Furthermore, nuclear import of the DFF complex requires the C-terminal regions of both subunits. In more detail, one basic cluster in the C-terminal region of each subunit, DFF40 (RLKRK) and DFF45 (KRAR), is essential for nuclear accumulation of the DFF complex. Based on these findings two alternative models for the interaction of importin alpha/beta with the DFF complex are presented.

Asunto(s)

Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/fisiología , Núcleo Celular/metabolismo , Desoxirribonucleasas/metabolismo , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Citoplasma/metabolismo , Células HeLa , Humanos , Señales de Localización Nuclear/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiología , alfa Carioferinas/metabolismo , beta Carioferinas/metabolismo

RESUMEN

BACKGROUND INFORMATION: H1 histones are a protein family comprising several subtypes. Although specific functions of the individual subtypes could not be determined so far, differential roles are indicated by varied nuclear distributions as well as differential expression patterns of the H1 subtypes. Although the group of replication-dependent H1 subtypes is synthesized during S phase, the replacement H1 subtype, H1 degrees , is also expressed in a replication-independent manner in non-proliferating cells. Recently we showed, by protein biochemical analysis, that the ubiquitously expressed subtype H1x is enriched in the micrococcal nuclease-resistant part of chromatin and that, although it shares common features with H1 degrees , its expression is differentially regulated, since, in contrast to H1 degrees , growth arrest or induction of differentiation did not induce an accumulation of H1x. RESULTS: In the present study, we show that H1x exhibits a cell-cycle-dependent change of its nuclear distribution. This H1 subtype showed a nucleolar accumulation during the G(1) phase, and it was evenly distributed in the nucleus during S phase and G(2). Immunocytochemical analysis of the intranucleolar distribution of H1x indicated that it is located mainly in the condensed nucleolar chromatin. In addition, we demonstrate that the amount of H1x protein remained nearly unchanged during S phase progression, which is in contrast to the replication-dependent subtypes. CONCLUSION: These results suggest that the differential localization of H1x provides a mechanism for a control of H1x activity by means of shuttling between nuclear subcompartments instead of a controlled turnover of the protein.

Asunto(s)

Nucléolo Celular/metabolismo , Fase G1 , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/metabolismo , Ciclo Celular/fisiología , Línea Celular , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN , Histonas/genética , Humanos , Proteínas Nucleares/genética , Fosfoproteínas/metabolismo , Isoformas de Proteínas/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribosomas/metabolismo , Transcripción Genética , Nucleolina

RESUMEN

In gastrointestinal stromal tumors (GISTs), mutually exclusive gain-of-function mutations of KIT and PDGFRA are associated with different mutation-dependent clinical behavior. Taking into account the well-known different clinical behavior of GISTs from the stomach or the intestine, the aim of the current study is to evaluate the mutation- and site-dependent effects on mRNA and protein expression of KIT and PDGFRA in a large series of primary GISTs. Fresh-frozen tissue of 53 primary GISTs from gastric (75%) or intestinal (25%) sites were analyzed for mutation of KIT or PDGFRA using direct sequencing. Furthermore, KIT and PDGFRA mRNA and protein expression were determined using quantitative RT-PCR and quantitative densitometric evaluation of Western blot data. Each tumor either had a mutation of KIT (79%) or PDGFRA (21%). All GISTs with PDGFRA mutation were from gastric sites. Mutation-dependently, GISTs with KIT mutation had a significantly higher expression of KIT and at the same time a significantly lower expression of PDGFRA compared to GISTs with PDGFRA mutation. Site-dependently, gastric GISTs had a significantly higher expression of PDGFRA and a significantly lower expression of KIT compared to intestinal GISTs. Additionally, even if the KIT-mutated GISTs alone were considered, a significantly higher expression of PDGFRA could be observed in gastric than in intestinal tumors. We also found a significant correlation between a higher protein expression of PDGFRA and longer disease-free survival. The correlation of gastric site and PDGFRA mutation with higher PDGFRA expression and longer disease-free survival suggests different regulatory roles of KIT and PDGFRA gene expression on the control of cell proliferation, and, thereby on clinical behavior. The higher PDGFRA expression in gastric GISTs possibly contributes to the well-known site-dependent clinical behavior.

Asunto(s)

Tumores del Estroma Gastrointestinal/genética , Expresión Génica , Neoplasias Intestinales/genética , Proteínas Proto-Oncogénicas c-kit/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Neoplasias Gástricas/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Supervivencia sin Enfermedad , Femenino , Tumores del Estroma Gastrointestinal/metabolismo , Tumores del Estroma Gastrointestinal/mortalidad , Tumores del Estroma Gastrointestinal/patología , Regulación Neoplásica de la Expresión Génica , Alemania/epidemiología , Humanos , Neoplasias Intestinales/metabolismo , Neoplasias Intestinales/mortalidad , Neoplasias Intestinales/patología , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidad , Neoplasias Gástricas/patología , Células del Estroma/metabolismo , Células del Estroma/patología , Tasa de Supervivencia

RESUMEN

Changes in chromatin structure, histone phosphorylation and cleavage of DNA into nucleosome-size fragments are characteristic features of apoptosis. Since H1 histones bind to the site of DNA cleavage between nucleosomal cores, the question arises as to whether the state of H1 phosphorylation influences the rate of internucleosomal cleavage. Here, we tested the relation between DNA fragmentation and H1 phosphorylation both in cultured cells and in vitro. In Jurkat cells, hyperosmotic mannitol concentration resulted in apoptosis, including nucleosomal fragmentation, whereas apoptosis induction by increased NaCl concentration was not accompanied by DNA fragmentation. However, both treatments induced dephosphorylation of H1 histones. In contrast, treatment of Raji cells with alkylphosphocholine led to induction of apoptosis with internucleosomal fragmentation, albeit without notable histone H1 dephosphorylation. These results demonstrate that dephosphorylation of H1 histones is neither a prerequisite for nor a consequence of internucleosomal cleavage. Moreover, we observed with an in vitro assay that the known enhancing effect of H1 histones on the activity of the apoptosis-induced endonuclease DFF40 is independent of the subtype or the phosphorylation state of the linker histone.

Asunto(s)

Apoptosis/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Histonas/metabolismo , Línea Celular Tumoral , Desoxirribonucleasas/efectos de los fármacos , Desoxirribonucleasas/metabolismo , Histonas/química , Histonas/aislamiento & purificación , Humanos , Células Jurkat/efectos de los fármacos , Presión Osmótica , Fosforilación , Proteínas de Unión a Poli-ADP-Ribosa , Estaurosporina/farmacología

RESUMEN

The nuclear import of H1 linker histones is mediated by a heterodimer of transport receptors, known as importinbeta and importin7. Interestingly, both importins separately interact with H1, but only as a dimer they facilitate the translocation through the nuclear pore. We identified the H1 binding site of importin7, comprising two extended acidic loops near the C terminus of importin7. The analysis of the H1 import complex assembly by means of isothermal titration calorimetry revealed that the formation of a receptor heterodimer in vitro is an enthalpy-driven process, whereas subsequent binding of H1 to the heterodimer is entropy-driven. Furthermore, we show that the importinbeta binding domain of importin7 plays a key role in the activation of importin7 by importinbeta. This process is allosterically regulated by importinbeta and accounts for a specific tuning of the activity of the importinbeta.importin7 heterodimer. The results presented here provide new insights into cellular strategies to even energy balances in nuclear import and point toward a general regulation of importinbeta-related nuclear import processes.

Asunto(s)

Núcleo Celular/metabolismo , Histonas/metabolismo , Carioferinas/metabolismo , Modelos Biológicos , Receptores Citoplasmáticos y Nucleares/metabolismo , beta Carioferinas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Sitios de Unión , Pollos , Dimerización , Entropía , Humanos , Unión Proteica , Estructura Secundaria de Proteína , Xenopus laevis

RESUMEN

The replication of eukaryotic genomes necessitates the coordination of histone biosynthesis with DNA replication at the onset of S phase. The multiple histone H4 genes encode identical proteins, but their regulatory sequences differ. The contributions of these individual genes to histone H4 mRNA expression have not been described. We have determined, by real-time quantitative PCR and RNase protection, that the human histone H4 genes are not equally expressed and that a subset contributes disproportionately to the total pool of H4 mRNA. Differences in histone H4 gene expression can be attributed to observed unequal activities of the H4 gene promoters, which exhibit variations in gene regulatory elements. The overall expression pattern of the histone H4 gene complement is similar in normal and cancer cells. However, H4 genes that are moderately expressed in normal cells are sporadically silenced in tumor cells with compensation of expression by other H4 gene copies. Chromatin immunoprecipitation analyses and in vitro DNA binding assays indicated that 11 of the 15 histone H4 genes interact with the cell cycle regulatory histone nuclear factor P, which forms a complex with the cyclin E/CDK2-responsive co-regulator p220(NPAT). These 11 H4 genes account for 95% of the histone H4 mRNA pool. We conclude that the cyclin E/CDK2/p220(NPAT)/histone nuclear factor P signaling pathway is the principal regulator of histone H4 biosynthesis.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , Regulación de la Expresión Génica , Histonas/genética , Fase S/genética , Fase S/fisiología , Animales , Secuencia de Bases , Línea Celular , Secuencia de Consenso , Replicación del ADN , Perfilación de la Expresión Génica , Humanos , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Transducción de Señal

RESUMEN

The members of the H1 histone family can be classified into three groups, which are the main class subtypes expressed in somatic cells, the developmental- and tissue-specific subtypes, and the replacement subtype H1(o). Until now, the subtype H1x was not classified, since it has not yet been thoroughly examined. The results of this study show that H1x shares similarities but also exhibits slight differences in its biochemical behaviour in comparison to the main class H1 histones. In HeLa cells it is located in the nucleus and partially associated with nucleosomes. Nevertheless, it is, like H1(o), mainly located in chromatin regions that are not affected by micrococcal nuclease digestion. Further common features of H1x and the replacement histone H1(o) are that the genes of both subtypes are solitarily located and give rise to polyadenylated mRNA. However, comparison of the inducibility of their expression revealed that their genes are regulated differentially.

Asunto(s)

Regulación de la Expresión Génica/efectos de los fármacos , Histonas/genética , Histonas/metabolismo , Secuencia de Aminoácidos , Animales , Azacitidina/farmacología , Butiratos/farmacología , Línea Celular , ADN/análisis , ADN/metabolismo , Células HL-60 , Células HeLa , Histonas/clasificación , Humanos , Datos de Secuencia Molecular , Nucleosomas/genética , Percloratos , Alineación de Secuencia , Tretinoina/farmacología , Células U937

RESUMEN

Posttranslational modifications of histones have an integral function in the structural and functional organization of chromatin. Several changes in the modification state of histones could be observed after induction of apoptosis with topoisomerase inhibitors and other inducers. Most of these studies include the analysis of the state of phosphorylation of histones, and the results are to some extent controversial, depending on cell lines and agents used. In the present study we compared the kinetics of the dephosphorylation of H1 and H3 histones with apoptosis markers after treatment of leukemic cell lines with topoisomerase inhibitors. In parallel, we determined cell cycle parameters in detail. Dephosphorylation of both histone classes started within 1 h of induction, and no direct correlation with timing and intensity of the investigated apoptotic features could be observed. In contrast, we show that the effect of topoisomerase inhibitors on the state of H1 and H3 phosphorylation is not directly related to apoptosis, but reflects the changes in the cell cycle distribution of cells treated with these inducers.

Asunto(s)

Ciclo Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Histonas/metabolismo , Inhibidores de Topoisomerasa , Línea Celular Tumoral , ADN-Topoisomerasas/metabolismo , Humanos , Fosforilación/efectos de los fármacos

RESUMEN

The transcriptional activator NF-Y is a heterotrimeric complex composed of NF-YA, NF-YB, and NF-YC, which specifically binds the CCAAT consensus present in about 30% of eukaryotic promoters. All three subunits contain evolutionarily conserved core regions, which comprise a histone fold motif (HFM) in the case of NF-YB and NF-YC. Our results of in vitro binding studies and nuclear import assays reveal two different transport mechanisms for NF-Y subunits. While NF-YA is imported by an importin beta-mediated pathway, the NF-YB/NF-YC heterodimer is translocated into the nucleus in an importin 13-dependent manner. We define a nonclassical nuclear localization signal (ncNLS) in NF-YA, and mutational analysis indicates that positively charged amino acid residues in the ncNLS are required for nuclear targeting of NF-YA. Importin beta binding is restricted to the monomeric, uncomplexed NF-YA subunit. In contrast, the nuclear import of NF-YB and NF-YC requires dimer formation. Only the NF-YB/NF-YC dimer, but not the monomeric components, are recognized by importin 13 and are imported into the nucleus. Importin 13 competes with NF-YA for binding to the NF-YB/NF-YC dimer. Our data suggest that a distinct binding platform derived from the HFM of both subunits, NF-YB/NF-YC, mediates those interactions.

Asunto(s)

Factor de Unión a CCAAT/química , Núcleo Celular/metabolismo , Carioferinas/metabolismo , Subunidades de Proteína/metabolismo , beta Carioferinas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Unión Competitiva , Factor de Unión a CCAAT/genética , Factor de Unión a CCAAT/aislamiento & purificación , Factor de Unión a CCAAT/metabolismo , Secuencia Conservada , Dimerización , Femenino , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Microinyecciones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oocitos , Unión Proteica , Subunidades de Proteína/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Xenopus laevis

RESUMEN

MOTIVATION: Histone proteins play important roles in chromosomal functions. They are significantly evolutionarily conserved across species, which suggests similarity in their transcription regulation. The abundance of experimental data on histone promoters provides an excellent background for the evaluation of computational methods. Our study addresses the issue of how well computational analysis can contribute to unveiling the biologically relevant content of promoter regions for a large number of mammalian histone genes taken across several species, and suggests the consensus promoter models of different histone groups. RESULTS: This is the first study to unveil the detailed promoter structures of all five mammalian histone groups and their subgroups. This is also the most comprehensive computational analysis of histone promoters performed to date. The most exciting fact is that the results correlate very well with the biologically known facts and experimental data. Our analysis convincingly demonstrates that computational approach can significantly contribute to elucidation of promoter content (identification of biologically relevant signals) complementing tedious wet-lab experiments. We believe that this type of analysis can be easily applied to other functional gene classes, thus providing a general framework for modelling promoter groups. These results also provide the basis to hunt for genes co-regulated with histone genes across mammalian genomes.

Asunto(s)

Algoritmos , Evolución Molecular , Histonas/genética , Modelos Genéticos , Regiones Promotoras Genéticas/genética , Alineación de Secuencia/métodos , Análisis de Secuencia de ADN/métodos , Animales , Secuencia Conservada , Humanos , Ratones , Filogenia , Ratas , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie