Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 9 de 9
Filter
Add more filters










Database
Language
Publication year range
1.
Biochem J ; 358(Pt 2): 447-55, 2001 Sep 01.
Article in English | MEDLINE | ID: mdl-11513744

ABSTRACT

The HIRA gene encodes a nuclear protein with histone-binding properties that have been conserved from yeast to humans. Hir1p and Hir2p, the two HIRA homologues in Saccharomyces cerevisiae, are transcriptional co-repressors whose action resides at the chromatin level and occurs in a cell-cycle-regulated fashion. In mammals, HIRA is an essential gene early during development, possibly through the control of specific gene-transcription programmes, but its exact function remains to be deciphered. Here we report on the subnuclear distribution and cell-cycle behaviour of the HIRA protein. Using both biochemical and immunofluorescence techniques, a minor fraction of HIRA was found tightly associated with the nuclear matrix, the material that remains after nuclease treatment and high-salt extraction. However, most HIRA molecules proved extractable. In non-synchronized cell populations, extraction from chromatin necessitated 300 mM NaCl whereas 150 mM was sufficient in mitotic cells. Immunofluorescence staining and microscopic examination of mitotic cells revealed HIRA as excluded from condensed chromosomes, confirming a lack of association with chromatin during mitosis. Western-blot analysis indicated that HIRA molecules were hyper-phosphorylated at this point in the cell cycle. Metabolic labelling and pulse-chase experiments characterized HIRA as a stable protein with a half-life of approx. 12 h. The mitotic phosphorylation of HIRA could provide the dividing cell with a way to retarget HIRA-containing multi-protein complexes to different chromatin regions in daughter compared with parental cells.


Subject(s)
Cell Cycle Proteins , Nuclear Matrix/metabolism , Nuclear Proteins/metabolism , Saccharomyces cerevisiae Proteins , Transcription Factors/metabolism , Amino Acid Motifs , Cell Extracts , Chromosomes/metabolism , Evolution, Molecular , Fluorescent Antibody Technique , Half-Life , HeLa Cells , Histone Chaperones , Humans , K562 Cells , Mitosis , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Phosphorylation , Repressor Proteins/genetics , Saccharomyces cerevisiae/genetics , Transcription Factors/chemistry , Transcription Factors/genetics
2.
Biochim Biophys Acta ; 1517(3): 376-83, 2001 Feb 16.
Article in English | MEDLINE | ID: mdl-11342215

ABSTRACT

The human HIRA protein is encoded from a region of chromosome 22q that is critical for the DiGeorge syndrome and the velocardiofacial syndrome. We have previously reported that it directly interacts with core histones, with a novel histone-binding protein, HIRIP3, and during mouse embryogenesis, with the developmentally regulated homeodomain protein Pax3, suggesting a promoter-targeted function at the chromatin level. We here report on HIRA-interacting protein 5 (HIRIP5), a small acidic protein that interacted with HIRA in a double-hybrid screen performed in yeast and in in vitro protein interaction experiments. HIRIP5 has highly conserved homologs in both prokaryotes and eukaryotes, including the NFU1 gene product which has been implicated in iron metabolism in mitochondria of the yeast Saccharomyces cerevisiae. By radioactive in situ hybridization, the HIRIP5 gene was mapped to the 2p13-p15 chromosomal region, separate from any region previously associated with DiGeorge syndrome.


Subject(s)
Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle Proteins , Iron-Sulfur Proteins/biosynthesis , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Conserved Sequence , DNA, Complementary/genetics , HeLa Cells , Histone Chaperones , Humans , In Situ Hybridization , In Vitro Techniques , Mice , Molecular Sequence Data , Nuclear Proteins/genetics , Phylogeny , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Transcription Factors/genetics , Two-Hybrid System Techniques
3.
Mol Cell Biol ; 18(9): 5546-56, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9710638

ABSTRACT

The human HIRA gene has been named after Hir1p and Hir2p, two corepressors which together appear to act on chromatin structure to control gene transcription in Saccharomyces cerevisiae. HIRA homologs are expressed in a regulated fashion during mouse and chicken embryogenesis, and the human gene is a major candidate for the DiGeorge syndrome and related developmental disorders caused by a reduction to single dose of a fragment of chromosome 22q. Western blot analysis and double-immunofluorescence experiments using a specific antiserum revealed a primary nuclear localization of HIRA. Similar to Hir1p, HIRA contains seven amino-terminal WD repeats and probably functions as part of a multiprotein complex. HIRA and core histone H2B were found to physically interact in a yeast double-hybrid protein interaction trap, in GST pull-down assays, and in coimmunoprecipitation experiments performed from cellular extracts. In vitro, HIRA also interacted with core histone H4. H2B- and H4-binding domains were overlapping but distinguishable in the carboxy-terminal region of HIRA, and the region for HIRA interaction was mapped to the amino-terminal tail of H2B and the second alpha helix of H4. HIRIP3 (HIRA-interacting protein 3) is a novel gene product that was identified from its HIRA-binding properties in the yeast protein interaction trap. In vitro, HIRIP3 directly interacted with HIRA but also with core histones H2B and H3, suggesting that a HIRA-HIRIP3-containing complex could function in some aspects of chromatin and histone metabolism. Insufficient production of HIRA, which we report elsewhere interacts with homeodomain-containing DNA-binding factors during mammalian embryogenesis, could perturb the stoichiometric assembly of multimolecular complexes required for normal embryonic development.


Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cell Cycle Proteins , Chromosomal Proteins, Non-Histone , Histones/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Antibodies , Base Sequence , Binding Sites , Carrier Proteins/biosynthesis , Cell Line , Chick Embryo , Chickens , Chromosomes, Human, Pair 22 , Cloning, Molecular , DiGeorge Syndrome/genetics , Glutathione Transferase/biosynthesis , HeLa Cells , Histone Chaperones , Humans , Mice , Molecular Sequence Data , Nuclear Proteins/chemistry , Peptide Fragments/chemistry , Recombinant Fusion Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Transcription Factors/chemistry , Transcription, Genetic , Tumor Cells, Cultured
4.
Biochim Biophys Acta ; 1306(1): 5-8, 1996 Apr 10.
Article in English | MEDLINE | ID: mdl-8611624

ABSTRACT

A full-length cDNA has been isolated for the murine homolog of the human HIRA protein, a member of the HIR family of nuclear proteins that is encoded from the chromosome 22 region critical for the DiGeorge syndrome. This family also contains Hir1p and Hir2p, two proteins identified as regulators of histone gene transcription in yeast. The murine and human amino acid sequences are 95.3% identical, with a striking 99.2% identity in the N-terminal WD repeat domain that is characteristic of the family. The two cDNAs are highly conserved within the coding regions, but also in the entire 5' untranslated region and in a strikingly long stretch of nucleotides in the 3' untranslated region.


Subject(s)
Cell Cycle Proteins , DNA, Complementary/genetics , Mice/genetics , Multigene Family , Nuclear Proteins/genetics , Transcription Factors/genetics , Amino Acid Sequence , Animals , Base Sequence , Chromosomes, Human, Pair 22/genetics , Gene Library , Histone Chaperones , Humans , Molecular Sequence Data , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity
5.
Hum Mol Genet ; 3(2): 335-9, 1994 Feb.
Article in English | MEDLINE | ID: mdl-8004105

ABSTRACT

As part of a general effort to identify new genes mapping to disease-associated regions of human chromosome 22, we have isolated heterogeneous nuclear RNA from somatic cell hybrids selected for their chromosome 22 content. Inter-Alu PCR amplification yielded a series of human DNA fragments which all detected evolutionarily-conserved sequences. The centromere-most gene fragment candidate, XEN61, was shown to lie centromeric to the chromosome 22 breakpoint in the X/22-33-11TG somatic cell hybrid. This region, which is still devoid of characterized genes, overlaps with the critical region for the cat eye syndrome (CES), a developmental disorder associated with chromosomal duplication within 22pter-q11.2. Gene dosage analysis performed on DNA from six CES patients consistently revealed the presence of four copies of XEN61. A fetal brain cDNA clone, 61EW, was identified with XEN61 and entirely sequenced. The deduced protein is the E subunit of vacuolar H(+)-ATPase. This 31 KDa component of a proton pump is essential in eukaryotic cells as it both controls acidification of the vacuolar system and provides it with its main protonmotive force. RT-PCR experiments using oligonucleotides designed from the 61EW cDNA sequence indicated that the corresponding messenger is widely transcribed.


Subject(s)
Chromosomes, Human, Pair 22 , Genes , Proton-Translocating ATPases/genetics , Vacuoles/enzymology , Base Sequence , Centromere , Chromosome Mapping , Genetic Markers , Humans , Hybrid Cells , Molecular Sequence Data , Proton-Translocating ATPases/chemistry , RNA, Heterogeneous Nuclear/genetics
6.
Cancer Res ; 53(16): 3655-7, 1993 Aug 15.
Article in English | MEDLINE | ID: mdl-8339272

ABSTRACT

Peripheral neuroectodermal tumors include the differentiated neuroepithelioma and the undifferentiated Ewing's tumor. Despite clinical and pathological differences, both malignancies are characterized by a t(11;22)(q24;q12) translocation which is observed in > 90% of the cases. Molecularly, the translocation is underlaid by a rearrangement between the EWS and Fli-1 genes on chromosomes 22 and 11, respectively. Because of the difficulties in the differential diagnosis between various small round cell tumors of childhood, including Ewing's tumor, a molecular diagnostic assay would be desirable. A prerequisite for predicting the reliability of such a test resides in the molecular elucidation of the peripheral neuroectodermal tumor cases which do not exhibit the prototypical translocation. We have analyzed one such case of Ewing's tumor-derived cell line with a t(11;22;14)(q24;q12;q11) translocation. An EWS-Fli-1 fusion transcript was evidenced by polymerase chain reaction amplification of a reverse transcription product obtained from total RNA. Direct sequencing was performed to demonstrate that the molecular rearrangement in this particular Ewing sample resulted in a fusion transcript similar to those observed in tumors with the prototypical translocation.


Subject(s)
Chromosomes, Human, Pair 11 , Chromosomes, Human, Pair 14 , Chromosomes, Human, Pair 22 , DNA, Neoplasm/chemistry , DNA-Binding Proteins/chemistry , Proto-Oncogene Proteins , Sarcoma, Ewing/genetics , Trans-Activators/chemistry , Translocation, Genetic/genetics , Base Sequence , DNA-Binding Proteins/genetics , Humans , Karyotyping , Molecular Sequence Data , Polymerase Chain Reaction , Proto-Oncogene Protein c-fli-1 , RNA, Messenger/chemistry , RNA, Neoplasm/chemistry , Recombinant Fusion Proteins/analysis , Sarcoma, Ewing/chemistry , Trans-Activators/genetics , Transcription, Genetic , Tumor Cells, Cultured
7.
Mol Immunol ; 28(11): 1163-70, 1991 Nov.
Article in English | MEDLINE | ID: mdl-1835757

ABSTRACT

Glycosphingolipids added to the cell culture medium can be incorporated into the plasma membrane and interfere with the growth of certain cell types. In the past years, previous reports have shown that gangliosides, a class of glycosphingolipids bearing sialic acid can inhibit antigen or mitogen induced T cell proliferative responses in vitro. We report here that the inhibition of PHA induced proliferation by the trisialoganglioside GT1b was not reversed by addition of exogenous IL-1, IL-2, TPA and calcium ionophore. Furthermore, GT1b did not affect IL-2 production by activated T cells. In addition, GT1b ganglioside could also decrease strongly the expression of the T cell antigens CD3, CD2, CD4, CD8 and the alpha/beta T cell receptor antigenic complex whereas it did not affect HLA-class I antigens. By contrast, GT1b modulated only partially membrane expression of activation antigens such as CD25 (Tac) and transferrin receptor and increased the expression of HLA-class II antigens. Moreover CD25 messenger RNA induction was not affected by GT1b treatment of PHA-stimulated T cells. Our results demonstrate that gangliosides, in spite of their anti-proliferative capacity and their modulation effect on T cell antigen membrane expression, do not prevent the progression of T cells into early stages of the activation process.


Subject(s)
Gangliosides/pharmacology , Lymphocyte Activation/drug effects , T-Lymphocytes/physiology , Antigens, CD/biosynthesis , B-Lymphocytes/drug effects , Blotting, Northern , Calcimycin/pharmacology , Cells, Cultured , Histocompatibility Antigens Class I/biosynthesis , Humans , Immunophenotyping , In Vitro Techniques , Interleukin-1/pharmacology , Interleukin-2/biosynthesis , Interleukin-2/pharmacology , Phytohemagglutinins , RNA, Messenger/biosynthesis , Receptors, Antigen, T-Cell, alpha-beta/biosynthesis , Receptors, Transferrin/biosynthesis , Tetradecanoylphorbol Acetate/pharmacology
8.
Lymphokine Cytokine Res ; 10(3): 165-72, 1991 Jun.
Article in English | MEDLINE | ID: mdl-1715762

ABSTRACT

T cell proliferation, in the presence of monocytes, triggered either by an anti-CD3 monoclonal antibody (mAb) or by a mitogenic pair of anti-CD2 mAbs was inhibited either by the calcium chelator EGTA or the calcium channel blocker nifedipin. Antibodies against interleukin-1 (IL-1) inhibited T cell proliferation in both mitogenic systems. However inhibition achieved by anti-IL-1 beta Ab was greater than by anti-IL-1 alpha Ab while the combination of both anti-rIL-1 alpha Ab + anti-rIL-1 beta could completely inhibit the CD3-triggered T cell proliferation. On the other hand, IL-1 production by LPS-stimulated monocytes was strongly decreased both by EGTA and nifedipin. Northern blot analysis showed that this inhibition paralleled a decrease of IL-1 alpha and beta messenger RNA (mRNA) expression only in the presence of EGTA but not in the presence of nifedipin. These results indicate that EGTA acted at the transcriptional level while nifedipin acted at a yet undefined posttranscriptional level. Thus, it is suggested that the impairment of T cell proliferation by calcium inhibitors could result not only from an effect on Ca2+ influx in T cells but also from interfering with the function of accessory cells, such as the production of IL-1.


Subject(s)
Calcium/physiology , Egtazic Acid/pharmacology , Interleukin-1/physiology , Lymphocyte Activation , Monocytes/immunology , Nifedipine/pharmacology , T-Lymphocytes/immunology , Antibodies, Monoclonal , Blotting, Northern , Cell Communication , Cells, Cultured , DNA Probes , Dose-Response Relationship, Drug , Gene Expression/drug effects , Humans , Interleukin-1/genetics , Interleukin-1/immunology , Kinetics , Monocytes/drug effects , RNA/genetics , RNA/isolation & purification , RNA, Messenger/genetics
9.
Int Immunol ; 2(12): 1159-66, 1990.
Article in English | MEDLINE | ID: mdl-2151025

ABSTRACT

Undifferentiated nasopharyngeal carcinoma (NPC) is tightly associated with the Epstein-Barr virus (EBV) and very heavily infiltrated with T lymphocytes. We demonstrated recently that NPC epithelial cells produce immuno-regulatory molecules, including the Blast-2/CD23 antigen, which is induced in B lymphocytes upon infection by EBV. We demonstrate here that CD23 expression is a non-constant but highly specific feature of epithelial cells from NPC. The C15 and C17 NPC tumor cells express mainly the b form of CD23, which is known to be non-lineage-specific and IL-4-inducible. C17 cells were found also to weakly express the a form of CD23, which has been described as B cell-specific. In addition, several factors potentially released in vivo by tumor infiltrating lymphocytes (TILs) are able to regulate CD23 expression in NPC cells. In particular, we found that IL-4 was a potent inducer of CD23 expression in C15 cells, as shown at both the protein and the mRNA levels. These results, together with the already reported expression of class II MHC antigens and the release of IL-1 by NPC cells, suggest that the interactions between TILs and malignant cells are a key factor in NPC pathogenesis and development.


Subject(s)
Antigens, CD/analysis , Antigens, Differentiation, B-Lymphocyte/analysis , Carcinoma/immunology , Nasopharyngeal Neoplasms/immunology , Receptors, Fc/analysis , Animals , Antibodies, Monoclonal , Antigens, Differentiation, B-Lymphocyte/genetics , Carcinoma/pathology , Epithelium/immunology , Humans , Interferon-gamma/pharmacology , Interleukin-4/pharmacology , Lymphocytes, Tumor-Infiltrating/immunology , Mice , Nasopharyngeal Neoplasms/pathology , RNA, Messenger/analysis , Receptors, Fc/genetics , Receptors, IgE , Recombinant Proteins
SELECTION OF CITATIONS
SEARCH DETAIL
...