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Biomedical subjects

K Ozato

Publications and source records attributed to K Ozato.

At least 37 records · Page 2Linked to original sources
PubMed2000

IFN consensus sequence binding protein potentiates STAT1-dependent activation of IFNgamma-responsive promoters in macrophages.

IFNgamma, once called the macrophage-activating factor, stimulates many genes in macrophages, ultimately leading to the elicitation of innate immunity. IFNgamma's functions depend on the activation of STAT1, which stimulates transcription of IFNgamma-inducible genes through the GAS element. The IFN consensus sequence binding protein (icsbgamma or IFN regulatory factor 8), encoding a transcription factor of the IFN regulatory factor family, is one of such IFNgamma-inducible genes in macrophages. We found that macrophages from ICSBP-/- mice were defective in inducing some IFNgamma-responsive genes, even though they were capable of activating STAT1 in response to IFNgamma. Accordingly, IFNgamma activation of luciferase reporters fused to the GAS element was severely impaired in ICSBP-/- macrophages, but transfection of ICSBP resulted in marked stimulation of these reporters. Consistent with its role in activating IFNgamma-responsive promoters, ICSBP stimulated reporter activity in a GAS-specific manner, even in the absence of IFNgamma treatment, and in STAT1 negative cells. Indicative of a mechanism for this stimulation, DNA affinity binding assays revealed that endogenous ICSBP was recruited to a multiprotein complex that bound to GAS. These results suggest that ICSBP, when induced by IFNgamma through STAT1, in turn generates a second wave of transcription from GAS-containing promoters, thereby contributing to the elicitation of IFNgamma's unique activities in immune cells.

Animals↗
PubMed2000

Histone acetylation is a checkpoint in FGF-stimulated mesoderm induction.

We have previously demonstrated that the transcription factor, AP-1 (c-jun/c-fos heterodimer), mediates fibroblast growth factor (FGF) signaling during mesoderm induction in Xenopus embryo. In the present studies, we show that histone acetylation is involved in FGF-mediated signaling leading to mesoderm induction. Histone acetylation is a dynamic process regulated by the activities of two histone-modifying enzymes, the histone acetyltransferase(s) and histone deacetylase(s) (HDACs). We found that basal and FGF-regulated activator protein 1 (AP-1) activity in Xenopus embryo is markedly reduced by treatment of trichostatin A (TSA), a specific inhibitor of HDAC. However, activity of another transcription factor, NFkappaB, is enhanced by TSA treatment. AP-1-mediated mesoderm induction in the animal caps is dramatically suppressed by TSA at a dose-dependent manner. This suppression can be rescued by ectopic expression of HDAC3 at early stage. Finally, we found that histone acetylation in animal caps is inhibited by FGF whereas enhanced by TSA (as a control). Therefore, we propose that histone acetylation is a checkpoint for transduction of the FGF/AP-1 signals to induce mesoderm. Published 2000 Wiley-Liss, Inc.

Acetylation↗
PubMed2000

ICSBP directs bipotential myeloid progenitor cells to differentiate into mature macrophages.

During hematopoiesis, myeloid progenitor cells give rise to granulocytes and macrophages. To study the role for ICSBP, a hematopoietic cell-specific transcription factor in myeloid cell development, the gene was introduced into myeloid progenitor cells established from ICSBP-/- mice. ICSBP retrovirus-transduced cells differentiated into mature macrophages with phagocytic activity, which coincided with the induction of specific target DNA binding activity. Similar to macrophages in vivo, ICSBP-transduced cells were growth arrested, expressed many macrophage-specific genes, and responded to macrophage activation signals. Contrary to this, ICSBP transducion led to repression of granulocyte-specific genes and inhibited G-CSF-mediated granulocytic differentiation in these and other myeloid progenitor cells. Together, ICSBP has a key role in the myeloid cell lineage selection and macrophage maturation.

Animals↗
PubMed2000

Modulation of retinoid signalling through NGF-induced nuclear export of NGFI-B.

The retinoid-X receptor (RXR) regulates multiple hormonal pathways through heterodimerization with nuclear receptors such as the all-trans retinoic acid receptor (RAR). The orphan nuclear receptor NGFI-B (also called Nur77) can heterodimerize with RXR. Here we show that nerve growth factor (NGF) induces the phosphorylation of Ser 105 of NGFI-B in PC12 phaeochromocytoma cells, resulting in translocation of the NGFI-B-RXR heterodimer complex out of the nucleus using nuclear export signals within NGFI-B. As a consequence of the redistribution of RXR, the transcriptional activity of RXR-RAR is reduced. NGFI-B-mediated nuclear export of receptors may serve as a mechanism for crosstalk between NGF and retinoid pathways.

Amino Acid Sequence↗
PubMed2000

Activity of the medaka translation elongation factor 1alpha-A promoter examined using the GFP gene as a reporter.

The translation elongation factor 1alpha (EF-1alpha) is known to have several isoforms, which are expressed in a tissue- and stage-specific manner. Two genes encoding EF-1alpha exist per haploid genome in the medaka. In the present study, the promoter activity of the 5'-flanking region of the medaka EF-1alpha-A gene, an isoform of EF-1alpha, was characterized using transgenic techniques. First, using CAT gene as a reporter, it was revealed that about 1.8 kbp 5'-flanking sequence from the transcription initiation site of EF-1alpha-A was sufficient for high-level promoter activity. Second, the green fluorescent protein (GFP) gene fused to this region was introduced into medaka eggs using the microinjection method. Three germline transgenic individuals (one male and two female) were mated with non-transgenic medaka to obtain F1 offspring. In the case of embryonic and adult F1 transgenic individuals, GFP fluorescence was observed in almost all the tissues examined (e.g. kidney, liver, heart, gill, ovary, and testis), except for the skeletal muscle. In the case of F2 transgenic embryos derived from F1 transgenic males and non-transgenic females, the fluorescence was observed from the early gastrula stage. On the other hand, in the case of F2 transgenic embryos derived from F1 transgenic females and non-transgenic males, the fluorescence was observed even at the 1-cell stage, suggesting that this region is transcriptionally active during oogenesis. The usefulness of the EF-1alpha-A promoter as a tool for introducing foreign proteins into oocytes is discussed.

Animals↗
PubMed2000

Expression of GFP in nuclear transplants generated by transplantation of embryonic cell nuclei from GFP-transgenic fish into nonenucleated eggs of medaka, Oryzias latipes.

In order to investigate whether foreign genes can be used as genetic markers of donor nuclei in fish nuclear transplantation, expression of the GFP gene derived from donor nuclei was examined in nuclear transplants in medaka (Oryzias latipes). Embryonic nuclei were obtained from blastula embryos produced by crossing of transgenic fish of the wild-type strain heterozygous for the GFP gene with nontransgenic ones or by mutual crossing between transgenic fish. The GFP gene was driven by the promoter of the medaka elongation factor gene, EF-1alpha-A, which is known to induce GFP expression in many tissues except for the muscle in the transgenic fish. The nuclei were transplanted into nonenucleated unfertilized eggs of the orange-red strain. Adult nuclear transplants were successfully obtained at the rate of about 2% of the operated eggs. They were triploid and had no reproductive potential. The GFP gene was expressed in embryos, fry, and adults of nuclear transplants in a pattern similar to that in the transgenic fish. These results indicate that GFP is useful as a foreign genetic marker of donor nuclei in fish nuclear transplantation.

Animals↗
PubMed2000

A bromodomain protein, MCAP, associates with mitotic chromosomes and affects G(2)-to-M transition.

We describe a novel nuclear factor called mitotic chromosome-associated protein (MCAP), which belongs to the poorly understood BET subgroup of the bromodomain superfamily. Expression of the 200-kDa MCAP was linked to cell division, as it was induced by growth stimulation and repressed by growth inhibition. The most notable feature of MCAP was its association with chromosomes during mitosis, observed at a time when the majority of nuclear regulatory factors were released into the cytoplasm, coinciding with global cessation of transcription. Indicative of its predominant interaction with euchromatin, MCAP localized on mitotic chromosomes with exquisite specificity: (i) MCAP-chromosome association became evident subsequent to the initiation of histone H3 phosphorylation and early chromosomal condensation; and (ii) MCAP was absent from centromeres, the sites of heterochromatin. Supporting a role for MCAP in G(2)/M transition, microinjection of anti-MCAP antibody into HeLa cell nuclei completely inhibited the entry into mitosis, without abrogating the ongoing DNA replication. These results suggest that MCAP plays a role in a process governing chromosomal dynamics during mitosis.

Amino Acid Sequence↗
PubMed2000

Inhibition of p300 histone acetyltransferase by viral interferon regulatory factor.

Kaposi's sarcoma-associated herpesvirus (KSHV) has been consistently identified in Kaposi's sarcomas, body cavity-based lymphomas, and some forms of Castleman's disease. The K9 open reading frame of KSHV encodes a viral interferon regulatory factor (vIRF) which functions as a repressor for cellular interferon-mediated signal transduction and as an oncogene to induce cell growth transformation. We demonstrate that KSHV vIRF directly interacts with cellular transcriptional coactivator p300 and displaces p300/CBP-associated factor from p300 complexes. This interaction inhibits the histone acetyltransferase activity of p300, resulting in drastic reduction of nucleosomal histone acetylation and alteration of chromatin structure. As a consequence, vIRF expression markedly alters cellular cytokine expression, which is regulated by acetylation of nucleosomal histones. These results demonstrate that KSHV vIRF interacts with and inhibits the p300 transcriptional coactivator to circumvent the host antiviral immune response and to induce a global alteration of cellular gene expression. These studies also illustrate how a cellular gene captured by a herpesvirus has evolved several functions that suit the needs of the virus.

3T3 Cells↗
PubMed1999

Ligand-induced recruitment of a histone deacetylase in the negative-feedback regulation of the thyrotropin beta gene.

We have investigated ligand-dependent negative regulation of the thyroid-stimulating hormone beta (TSHbeta) gene. Thyroid hormone (T3) markedly repressed activity of the TSHbeta promoter that had been stably integrated into GH(3 )pituitary cells, through the conserved negative regulatory element (NRE) in the promoter. By DNA affinity binding assay, we show that the NRE constitutively binds to the histone deacetylase 1 (HDAC1) present in GH(3 )cells. Significantly, upon addition of T3, the NRE further recruited the thyroid hormone receptor (TRbeta) and another deacetylase, HDAC2. This recruitment coincided with an alteration of in vivo chromatin structure, as revealed by changes in restriction site accessibility. Supporting the direct interaction between TR and HDAC, in vitro assays showed that TR, through its DNA binding domain, strongly bound to HDAC2. Consistent with the role for HDACs in negative regulation, an inhibitor of the enzymes, trichostatin A, attenuated T3-dependent promoter repression. We suggest that ligand-dependent histone deacetylase recruitment is a mechanism of the negative-feedback regulation, a critical function of the pituitary-thyroid axis.

Base Sequence↗
PubMed1999

Regulation of apoptosis in myeloid cells by interferon consensus sequence-binding protein.

Mice with a null mutation of the gene encoding interferon consensus sequence-binding protein (ICSBP) develop a disease with marked expansion of granulocytes and macrophages that frequently progresses to a fatal blast crisis, thus resembling human chronic myelogenous leukemia (CML). One important feature of CML is decreased responsiveness of myeloid cells to apoptotic stimuli. Here we show that myeloid cells from mice deficient in ICSBP exhibit reduced spontaneous apoptosis and a significant decrease in sensitivity to apoptosis induced by DNA damage. In contrast, apoptosis in thymocytes from ICSBP-deficient mice is unaffected. We also show that overexpression of ICSBP in the human U937 monocytic cell line enhances the rate of spontaneous apoptosis and the sensitivity to apoptosis induced by etoposide, lipopolysaccharide plus ATP, or rapamycin. Programmed cell death induced by etoposide was specifically blocked by peptides inhibitory for the caspase-1 or caspase-3 subfamilies of caspases. Studies of proapoptotic genes showed that cells overexpressing ICSBP have enhanced expression of caspase-3 precursor protein. In addition, analyses of antiapoptotic genes showed that overexpression of ICSBP results in decreased expression of Bcl-X(L). These data suggest that ICSBP modulates survival of myeloid cells by regulating expression of apoptosis-related genes.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Regulation of IFN consensus sequence binding protein expression in murine macrophages.

Recent work has demonstrated that the transcription factor, IFN consensus sequence binding protein (ICSBP), plays a critical role in the capacity of mice to control infection with Toxoplasma gondii and Leishmania major, agents that require highly activated macrophages for their elimination. In this report the regulation of ICSBP mRNA and protein were analyzed in murine macrophages stimulated with LPS and/or IFN-gamma. Like induction of leishmaniacidal activity, LPS and IFN-gamma synergize to induce ICSBP mRNA and protein. Deletion analysis of the ICSBP promoter identified regions that were IFN-gamma responsive, regions that mediate the ability of LPS and IFN-gamma to activate this promoter synergistically, as well as regions that normally repress ICSBP transcription. Finally, exogenous expression of ICSBP, found in previous studies to down-regulate MHC I gene expression, failed to repress basal or IFN-gamma-induced ICSBP transcription. This demonstrates that ICSBP can selectively suppress the expression of IFN-responsive genes. These findings extend in a significant way our understanding of the regulation of ICSBP by LPS and IFN-gamma and provide important clues as to its role in macrophage activation.

Animals↗
PubMed1999

Differential requirement of IFN consensus sequence binding protein for the production of IL-12 and induction of Th1-type cells in response to IFN-gamma.

IFN-gamma exerts multiple biological activities in the modulation of immune responses by the induction of transcription factors. One transcriptional factor of the IFN regulatory factor family found to be critical in regulating IL-12-dependent IFN-gamma production in vivo following infectious challenge has been designated IFN consensus sequence-binding protein (ICSBP). In this study, the role of ICSBP in regulating type 1 responses to T cell-specific stimulation in vitro was assessed. Total splenocytes from ICSBP-/- mice stimulated with soluble anti-CD3 were markedly impaired in the production of IFN-gamma compared with similarly stimulated cells from ICSBP+/+ mice. Consistent with the decrease in IFN-gamma production, splenocytes from ICSBP-/- mice stimulated with anti-CD3 in the presence or absence of IFN-gamma or a soluble CD40 ligand agonist failed to produce IL-12 p40 and IL-12 p70 protein; however, the deficient production of IFN-gamma from ICSBP-/- mice could be restored by the addition of anti-CD28 Ab in an IL-12-independent manner. In contrast to the previous data, production of IFN-gamma from naive CD4+/LECAM-1high cells of ICSBP-/- mice that had been primed in vitro with anti-CD3 was similar to or greater than that of ICSBP+/+ controls. In addition, the presence of IFN-gamma in priming cultures enhanced both priming for IFN-gamma and IL-12 responsiveness from ICSBP-/- CD4+ T cells. Overall, these results provide evidence that ICSBP is differentially required for the ability of IFN-gamma to regulate type 1 cytokine responses from APCs and CD4+ T cells.

Adjuvants, Immunologic↗
PubMed1999

Transplantation of blastula nuclei to non-enucleated eggs in the medaka, Oryzias latipes.

Studies of nuclear transplantation were conducted to establish methods for the production of clones of fish, using a small laboratory fish, medaka, Oryzias latipes. As the first step of the study, single-blastula nuclei of an inbred strain with the wild-type body color were transplanted into non-enucleated unfertilized eggs of an outbred orange red strain. Of 845 operated eggs, 45 hatched into fry exhibiting the wild-type body color, one of the donor markers. Twenty-seven of these nuclear transplants grew to the adult stage and clearly exhibited external secondary sexual characteristics. Fourteen were females and 13 were males. The allozyme analysis of phosphoglucomutase, measurements of relative DNA content by microfluorometry and chromosome counts consistently indicated that the nuclear transplants were triploids that originated from both the diploid donor nuclei and the haploid recipient pronuclei. In the crossing experiments between the nuclear transplants and the orange-red strain, most of the male nuclear transplants were sterile, whereas one male produced a viable offspring with wild-type body color. All of the female nuclear transplants were sterile. Macroscopic observations of their gonads showed that the testes appeared normal and the ovaries appeared degenerated. These features of the reproductive potential and the morphology of gonads also indicated that the nuclear transplants were triploids. These results demonstrated that a basic technique for nuclear transplantation in medaka was established.

Animals↗
PubMed1999

Expression of murine early embryonic antigens, SSEA-1 and antigenic determinant of EMA-1, in embryos and ovarian follicles of a teleost medaka (Oryzias latipes).

Stage-specific embryonic antigen-1 (SSEA-1) and the antigenic determinant of monoclonal antibody EMA-1 are expressed in a stage-specific manner in mouse early embryos. To study whether these antigens generally exist in fish, expression of the antigens was examined in embryos, ovarian follicles, and adult tissues of a teleost medaka (Oryzias latipes), using immunohistochemical techniques. In 1-cell-stage embryos, these carbohydrate antigens were found in numerous cytoplasmic granules in the blastodisc and the cortical cytoplasm. These granules gradually decreased in number as the embryos developed. In 4-cell-stage embryos, the antigens appeared on the cleavage planes and were located on the cleavage planes within the blastoderm in the following cleavage stages. In blastula-stage embryos, the expression was ubiquitously found on the cell surface of blastomeres. At the mid-gastrula stage, the antigens were restricted to the enveloping layer, yolk syncytial layer, and cortical cytoplasm, but were rarely found in deep cells that contribute to formation of the embryonic body. In later-stage embryos and adult fish, the antigens were located in various tissues. In ovarian follicles, the antigens were found in granules of oocytes and granulosa cells. These observations were basically consistent with those in mice; however, expression in 1-cell-stage embryos and ovarian follicles has not been observed in mice. This unexpected finding suggests that the antigens are produced in granulosa cells and transferred to 1-cell-stage embryos via oocytes, and that the antigens involved in the early developmental process are maternally prepared in teleosts.

Animals↗
PubMed1999

Construction of a linkage map of the medaka (Oryzias latipes) and mapping of the Da mutant locus defective in dorsoventral patterning.

Double anal fin (Da) is a medaka with an autosomal semidominant mutation that causes mirror image duplication of the ventral region concentrating on the caudal region. The chromosomal location of the Da gene and its sequence have remained unknown. We constructed a medaka linkage map as a first step to approach positional cloning of the gene. The segregation analysis was performed on the basis of genetic recombination during female meiosis using 134 random amplified polymorphic DNA (RAPD) markers, 13 sequence-tagged sites (STSs), 15 polymorphic sequences from known genes, and the Da gene. One hundred forty-six markers from the above markers segregated into 26 linkage groups. The size of the genome was estimated to be 1776 cM in length. We identified four syntenic regions between medaka and zebrafish (and human) by mapping the known genes and found one of them to be located in close proximity to the Da gene. By mapping the region surrounding the Da gene in high resolution, two markers were detected flanking the Da gene at 0.32 and 0.80 cM. The detected markers providing a vital clue to initiate chromosome walking will lead us to the definite location of the Da gene.

Animals↗
PubMed1999

The histone acetylase PCAF is a phorbol-ester-inducible coactivator of the IRF family that confers enhanced interferon responsiveness.

Transcription factors of the interferon regulatory factor (IRF) family bind to the type I interferon (IFN)-responsive element (ISRE) and activate transcription from IFN-inducible genes. To identify cofactors that associate with IRF proteins, DNA affinity binding assays were performed with nuclear extracts prepared from tissue culture cells. The results demonstrated that the endogenous IRFs bound to the ISRE are complexed with the histone acetylases, PCAF, GCN5, and p300/CREB binding protein and that histone acetylase activities are accumulated on the IRF-ISRE complexes. By testing recombinant proteins, we show that PCAF directly binds to some but not all members of the IRF family through distinct domains of the two proteins. This interaction was functionally significant, since transfection of PCAF strongly enhanced IRF-1- and IRF-2-dependent promoter activities. Further studies showed that expression of PCAF and other histone acetylases was markedly induced in U937 cells upon phorbol ester treatment, which led to increased recruitment of PCAF to the IRF-ISRE complexes. Coinciding with the induction of histone acetylases, phorbol ester markedly enhanced IFN-alpha-stimulated gene expression in U937 cells. Supporting the role for PCAF in conferring IFN responsiveness, transfection of PCAF into U937 cells led to a large increase in IFN-alpha-inducible promoter activity. These results demonstrate that PCAF is a phorbol ester-inducible coactivator of the IRF proteins which contributes to the establishment of type I IFN responsiveness.

Acetyltransferases↗
PubMed1998

The histone acetylase PCAF is a nuclear receptor coactivator.

Whereas the histone acetylase PCAF has been suggested to be part of a coactivator complex mediating transcriptional activation by the nuclear hormone receptors, the physical and functional interactions between nuclear receptors and PCAF have remained unclear. Our efforts to clarify these relationships have revealed two novel properties of nuclear receptors. First, we demonstrate that the RXR/RAR heterodimer directly recruits PCAF from mammalian cell extracts in a ligand-dependent manner and that increased expression of PCAF leads to enhanced retinoid-responsive transcription. Second, we demonstrate that, in vitro, PCAF directly associates with the DNA-binding domain of nuclear receptors, independently of p300/CBP binding, therefore defining a novel cofactor interaction surface. Furthermore, our results show that dissociation of corepressors enables ligand-dependent PCAF binding to the receptors. This observation illuminates how a ligand-dependent receptor function can be propagated to regions outside the ligand-binding domain itself. On the basis of these observations, we suggest that PCAF may play a more central role in nuclear receptor function than previously anticipated.

3T3 Cells↗