1,720,974 research outputs found
Aberrant expression pattern of replication-dependent histone H3 subtype genes in human tumor cell lines
No full textWe have determined the expression pattern of all 11 human replication-dependent histone H3 genes in three fetal human tissues (bladder, liver, and lung) and in eight human cell lines by RNase protection assay. In the fetal human tissues, all 11 genes were expressed to a varied extent. However, the relative contribution of each gene to the total replication-dependent histone H3 mRNA was rather similar in every tissue type. The expression pattern in the fibroblast cell line IMR 90 was similar to the expression pattern in the three fetal tissues. In contrast, the expression patterns varied substantially in seven tumor cell lines: some genes were not expressed at all, and others were expressed much less than in the fetal tissues or the fibroblast cell line. This aberrant expression was different in each of the cell lines tested. In a transient reporter gene assay using the promoters of 6 of the 11 genes, however, the relative activities of the promoters were similar in all cell lines. This indicates that the aberrant expression pattern in the different tumor cell lines is not due to a differential availability of transcription factors. We conclude that the varied expression pattern of the replication-dependent histone H3 genes in the examined human tumor cell lines is most probably due to epigenetic factors, such as the chromosomal context in the different cell lines
Characterisation of nuclear localisation signals of the four human core histones
No full textThe four core histones H2A, H2B, H3 and H4 are transported from the cytoplasm into the nucleus by a receptor-mediated and energy-dependent process. This nuclear transport depends on topogenic signals in the individual histone protein sequences. We have analysed such nuclear localisation signals in the core histones by means of fusion proteins consisting of individual core histones (or fragments thereof) and beta -galactosidase as a reporter protein. The results show that each of the four core histones contains several portions that are capable of mediating nuclear transport. One type of topogenic sequences consists of clustered basic amino acids in the amino terminal segments of each of the core histones. The globular portions of the core histones represent a second type of nuclear localisation signals that could only mediate nuclear transport when the whole protein domains were fused to the beta -galactosidase reporter. Fragments of the globular domains derived from each of the four core histones could not serve as nuclear localisation signals. We conclude that the nuclear targeting of core histones requires information conferred by the globular domain conformation. (C) 2001 Wiley-Liss, Inc
The requirement of H1 histones for a heterodimeric nuclear import receptor
No full textAfter synthesis in the cytoplasm, H1 histones are imported into the nucleus through an energy-dependent process that can be mediated by an importin beta-importin 7 (Impbeta-Imp7) heterodimer. H1 histories contain two structurally different types of nuclear localization signals (NLS). The first type of NLS resides within the unstructured C-terminal domain and is rich in basic amino acids. In contrast, the highly conserved central domain of the H1 histone contains comparatively few basic amino acids but also represents a functional NLS. The competence for the nuclear import of this globular domain seems to be based on its secondary structure. Here, we show that the Impbeta-Imp7 heterodimer is the only receptor for H1 import. Furthermore, we identified the import receptors mediating the in vitro transport of different NLS of the H1 histone. Using the digitonin-permeabilized cell import assay we show that Impbeta is the most efficient import receptor for the globular domain of H1 histones, whereas the heterodimer of Impbeta and Imp7 is the functional receptor for the entire C-terminal domain. However, short fragments of the C-terminal domain are imported in vitro by at least four different importins, which resembles the import pathway of ribosomal proteins and core histones. In addition, we show that heterodimerization of Impbeta with Imp7 is absolutely necessary for their proper function as an import receptor for H1 histories. These findings point to a chaperone-like function of the heterodimeric complex in addition to its function as an import receptor. It appears that the Impbeta-Imp7 heterodimer is specialized for NLS consisting of extended basic domains
Differential expression of human replacement and cell cycle dependent H3 histone genes
No full textHistories are the major protein component of chromatin. Except H4, all histone classes consist of several subtypes. The H3 family includes two replacement histone genes, H3.3A and H3.3B, which both encode the same protein and are expressed independently from the cell cycle. Since the two genes encode an identical protein, we analyzed whether they are differentially expressed. Therefore we cloned, sequenced and characterized the regulatory structures of the H3.3A gene and compared these with the corresponding regions in the H3.3B gene. In contrast to the H3.3B promoter, the promoter region of the H3.3A gene revealed neither a TATA nor any CCAAT boxes but an initiator element and several SP1 binding sequence motifs within an overall GC-rich sequence. Northern blot analysis of RNA from six human cell lines revealed that every cell line expressed each of the H3 isoform genes H3. 1, H3.3A and H3.3B. In contrast, analysis of total RNA from human tissues showed a differential expression of the H3 isoform genes. The H3.3 genes are essentially only expressed in adult tissue, whereas the H3.1 gene is transcribed just in fetal tissue. The functional relevance of the elements identified by sequence analysis was established using a reporter gene assay with deletion constructs of the H3.3A promoter. In this assay a 256 bp fragment was sufficient for the full promoter activity and three promoter segments, each containing SP1 binding motifs, contribute to the H3.3A gene expression. The possible functional relevance of the differences between the two H3.3 genes in structure and expression is discussed. (C) 2003 Elsevier Science B.V. All rights reserved
Core histones and linker histones are imported into the nucleus by different pathways
No full textHistories are the major structural proteins in eukaryotic chromosomes. This group of small very basic proteins consists of the H1 linker histones and the core histones H2A, H2B, H3 and H4. Despite their small size, the nuclear import of histones occurs by an active transport mechanism and not simply by diffusion. Histones contain several nuclear localisation signals (NLS) that can be subdivided into two different types of signal structures. We have previously shown that H1 histones are transported by a heterodimeric import receptor complex consisting of importin beta and importin 7, and we now describe the receptors required for the import of the core histones. Competition experiments using the in vitro transport assay indicate that the import pathway of the core histones differs from that of the linker histones and of nuclear proteins with classical NLS. In vitro binding assays show that each of the import receptors importin beta, importin 5, importin 7 and transportin, has the capacity to bind to any of the four core histones. Reconstitution experiments with recombinant factors indicate that each of these factors can independently serve as an import receptor for each of the core histones
Subunits of the heterotrimeric transcription factor NF-Y are imported into the nucleus by distinct pathways involving importin beta and importin 13
No full textThe 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 (HIM) 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 P-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 P 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 HIM of both subunits, NF-YB/NF-YC, mediates those interactions
Human replication-dependent histone H3 genes are activated by a tandemly arranged pair of two CCAAT boxes
No full textWe have analysed the transcriptional regulation of the human histone H3 genes using promoter deletion series, scanning mutagenesis, specific mutagenesis and electrophoretic mobility-shift assay experiments. The promoters of five of the six examined histone H3 genes showed near-maximal activity at lengths of 133-227 bp: H3/d 198 bp, H3/h 147 bp, H3/k 133 bp, H3/m 227 bp, H3/n 140 bp (exception H3/i). To search for functional cis-elements within these regions, we performed scanning mutagenesis of the two histone H3 promoters H3/k and H3/m. Mutagenesis revealed that the functional framework of the histone H3 promoters consists of a TATA box and two tandemly arranged CCAAT boxes in relatively fixed positions. Alterations of the distance between the CCAAT boxes and of the distance between the CCAAT boxes and the TATA box resulted in significant loss of activity. In electrophoretic mobility-shift assay experiments, the factor CBF (CCAAT-binding factor)/NF-Y (nuclear factor-Y) bound to isolated CCAAT boxes of the H3/k promoter. This suggests that an initiation complex is formed on the histone H3 promoter that has a defined structure and limited flexibility, consisting of two molecules of CBF/NF-Y and further (general or specific) transcription factors
Rapid dephosphorylation of H1 histones after apoptosis induction
No full textH1 histones are involved in the formation of higher order chromatin structures and in the modulation of gene expression. Changes in chromatin structure are a characteristic initial feature of apoptosis. We therefore have investigated the histone H1 pattern of the human leukemic cell line HL60 undergoing programmed cell death, as induced by topoisomerase I inhibition. Histone H1 proteins were isolated and analyzed by high performance liquid chromatography and capillary zone electrophoresis. DNA fragmentation after apoptosis induction was monitored by agarose gel electrophoresis. The patterns of the three H1 histone subtypes extractable from apoptotic HL60 cells significantly differed from those of control cells in showing a decrease of phosphorylated H1 subtypes and an increase of the respective dephosphorylated forms. This dephosphorylation of H1 histones could be observed already 45 min after apoptosis induction and preceded internucleosomal DNA cleavage by approximately 2 h. We conclude that during apoptotic DNA fragmentation, the H1 histones become rapidly dephosphorylated by a yet unknown protein phosphatase
Topoisomerase inhibitor induced dephosphorylation of H1 and H3 histones as a consequence of cell cycle arrest
No full textPosttranslational 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
- …
