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J Ghysdael

Publications and source records attributed to J Ghysdael.

At least 73 records · Page 4Linked to original sources
PubMed1992

Characterization of Spi-B, a transcription factor related to the putative oncoprotein Spi-1/PU.1.

We have cloned a human cDNA from a new gene, spi-B, on the basis of its homology with the DNA-binding domain of the Spi-1/PU.1 putative oncogene product. spi-B codes for a protein of 262 amino acids presenting 43% overall identity with Spi-1. Its highly basic carboxy-terminal region exhibits 34% sequence identity with the DNA-binding domain of the Ets-1 protein. We showed that the Spi-B protein is able to bind the purine-rich sequence (PU box) recognized by Spi-1/PU.1 and to activate transcription of a reporter plasmid containing PU boxes. Chromosome in situ hybridization allowed us to map spi-B to the 19q13.3-19q13.4 region of the human genome. spi-B, like spi-1, was found to be expressed in various murine and human hematopoietic cell lines except T lymphoid cell lines.

Amino Acid Sequence↗
PubMed1992

Half-site spacing and orientation determines whether thyroid hormone and retinoic acid receptors and related factors bind to DNA response elements as monomers, homodimers, or heterodimers.

The receptors for thyroid hormone (T3R) and retinoic acid (RAR) are members of a nuclear receptor subfamily that are capable of recognizing similar DNA sequences. Native response elements for T3R and RAR consist of two or more putative half-site binding motifs organized as imperfect direct or inverted repeats separated by different sized nucleotide gaps. To clarify how T3R, RAR, and related factors recognize DNA response elements, we analyzed the interaction of purified receptors with a series of inverted and direct repeats of an idealized AGGTCA half-site separated by different sized nucleotide gaps. Our results indicate that RAR and T3R can bind to half-sites as monomers and, depending on the orientation and distance between half-sites, also bind as homodimers or T3R-RAR heterodimers. T3R also binds to certain DNA elements as a heterodimer with one or more nuclear factors from eucaryotic cells. Thus, the orientation and spacing of half-sites play a central role in determining which configuration of receptors and nuclear factors will interact with a specific DNA element. This along with the ability of these factors to participate in reversible protein-protein interactions serve to broaden and diversify the responses mediated by T3R, RAR, and related members of this nuclear receptor subfamily.

Base Sequence↗
PubMed1992

The leukaemia oncogene v-erbA: a dominant negative version of ligand dependent transcription factors that regulates red cell differentiation?

The v-erbA oncogene of avian erythroblastosis virus alters the growth properties and arrests differentiation of chick erythroid progenitor cells. The v-erbA protein is a mutated, ligand independent version of the c-erbA/T3R alpha chick receptor for T3, a ligand dependent transcriptional regulator. In reconstituted systems using idealized hormone responsive elements, over-expressed v-erbA acts as a dominant repressor of transcription mediated by liganded c-erbA/T3R alpha. This property seems to account for at least part of the phenotype of AEV transformed erythroid cells and for the transcriptional repression of some erythrocyte specific genes. However, v-erbA is likely to interfere with regulatory circuits other than those directly regulated by T3 receptors. Aspects of this hypothesis are discussed in the context of available evidence for the role of T3 and other hormones in erythroid progenitor cells proliferation/differentiation.

Animals↗
PubMed1992

Analysis of the DNA binding and transcriptional activation properties of the Ets1 oncoprotein.

The c-ets1 gene product (Ets1) is the prototype of a family of sequence-specific transcriptional activators which have been implicated in various developmental processes and in the response of cells to a variety of extracellular stimuli. We report here a structure-function analysis of the DNA binding and transcriptional activation properties of Ets1. The minimal region required for specific DNA binding is located at the carboxy-terminus of Ets1, a domain highly conserved in all known members of the Ets family. Transcriptional activation by Ets1 in mammalian cells requires an additional domain of 110 amino acids characterized by a high content of acidic residues and localized in the amino-terminal half of the protein. This domain also functions as a transcriptional activation domain in yeast cells when linked to the heterologous DNA binding domain of Gal4. In contrast to its conservation in Ets1 proteins across vertebrate species, this activation domain is not conserved in other members of the Ets family. These results indicate that an important level of specificity between different members of the Ets family may reside in the differential interactions of their respective activation domains with distinct general transcription factors or different associated coactivators.

Base Sequence↗
PubMed1991

Functional expression of the human growth factor activatable Na+/H+ antiporter (NHE-1) in baculovirus-infected cells.

We constructed a recombinant baculovirus, based on Autographa californica nuclear polyhedrosis virus, containing the human Na+/H+ antiporter cDNA under control of the polyhedrin promoter. When infected with this recombinant baculovirus, the Sf9 cell line, derived from Spodoptera frugiperda, expresses a fully functional Na+/H+ antiporter as measured by the generation of an amiloride-sensitive Na+ influx in response to an acid load. The Na+/H(+)-exchange activity, not detectable in Sf9 cells, emerges 18 h after infection and continues to increase over the next two days to reach a maximal value about 20-fold higher than in cultured mammalian fibroblasts. Parallel to this activity, infected cells express a single immunoreactive polypeptide of 85 kDa that represents a non-glycosylated form of the 110-kDa mature human antiporter. We estimated that only 10% of the expressed protein is in a functional state. Not only is the antiporter expressed in insect cells phosphorylated, but also, like in mammalian cells, phosphorylation is increased in response to phorbol esters and okadaic acid. Moreover, tumor promoters apparently modify the same antiporter site in both insect and mammalian cells. We conclude that, with this high level of functional expression and apparently conserved signaling machinery, the present system opens the way to the biochemistry of the transporter including identification of the growth factor stimulated phosphorylation sites.

Animals↗
PubMed1991

Ligation of membrane Ig leads to calcium-mediated phosphorylation of the proto-oncogene product, Ets-1.

Recent studies have demonstrated that the nuclear protein, Ets-1, which is preferentially expressed in lymphocytes, binds to the long terminal repeat of Moloney murine sarcoma virus and HTLV-1 and regulates gene expression. The association of Ets-1 with DNA has been shown to be lost when the protein is phosphorylated. Thus, Ets-1 may regulate gene expression in lymphocytes and this activity may be determined by its phosphorylation state. To address the possibility that Ets-1 activity may be altered by membrane (m) Ig-mediated signal transduction, we analyzed the effect of mIgM and mIgD ligation on the phosphorylation state of Ets-1. Monoclonal anti-IgM or anti-IgD antibody stimulation of normal mouse B cells led to increased phosphorylation of Ets-1 within 2 min. This response was absolutely dependent on calcium mobilization and could be induced by elevation of intracellular free calcium using the calcium ionophore, ionomycin. Calcium release from intracellular stores was sufficient to mediate the phosphorylation of Ets-1. Treatment of resting B cells with IL-4, TGF beta-1, IFN-gamma, anti-class I, or anti-class II antibodies did not induce Ets-1 phosphorylation. In summary, calcium mobilization from intracellular stores after mIgM or mIgD ligation provides a necessary and sufficient signal for activation of Ets-1 phosphorylation. This phosphorylation event may act in the alteration of gene expression during B cell activation.

Animals↗
PubMed1991

The bovine leukemia virus tax gene contains an enhancer sequence.

Transactivator proteins of the bovine leukemia (BLV) and human T-lymphotropic (HTLV) viruses increase long terminal repeat (LTR)-directed viral gene expression and act as immortalizing oncogenes in tissue culture. We report here that the BLV tax gene sequence contains an enhancer-like activity. The X long open reading frame was cloned up-stream of the beta-globin promoter linked to the chloramphenicol acetyltransferase (CAT) gene. In the presence of tax sequences, up to sevenfold enhancement of CAT expression was observed. A computer-assisted homology search revealed the presence of a consensus enhancer core sequence (GTGTTGTTGGTTG) into the third exon of the 2.1-kb X mRNA. These studies demonstrate that the tax gene contains a transcriptional enhancer which could be involved in early viral gene expression in vivo.

Animals↗
PubMed1991

Nuclear oncogenes.

Ample evidence has accumulated in recent years to establish that most, if not all, nuclear proto-oncogenes are in fact sequence-specific DNA-binding proteins that modulate gene expression. Their synthesis or activity is modulated by extracellular signals or by cross talk between different classes of transcription factors.

Animals↗
PubMed1991

Sequence-specific interaction of the Ets1 protein with the long terminal repeat of the human T-lymphotropic virus type I.

We recently demonstrated that members of the c-ets proto-oncogene family, Ets1 and Ets2, are sequence-specific transcriptional activators of the human T-lymphotropic virus type I (HTLV-I) long terminal repeat (LTR). We now report that the HTLV-I LTR contains two distinct Ets1-responsive regions, ERR-1 and ERR-2. Expression of Ets1 with reporter plasmids containing ERR-1 or ERR-2 upstream of a basal promoter resulted in an increase in transcriptional activity. By gel mobility shift assay, the interaction of Ets1 with the downstream ERR-1-binding region was found to be more stable than its interaction with the upstream ERR-2 region. By DNase I footprint, gel mobility shift, and methylation interference analyses, ERR-1 was found to contain two Ets1 binding sites, ERE-A and ERE-B. A recombinant Ets1 protein was found to bind with higher affinity to ERE-A than to ERE-B. Binding of Ets1 to these sites appears to result in a specific and sequential protection of a 37-nucleotide sequence of the HTLV-I LTR from -154 to -118. In view of the high-level expression of Ets1 in lymphoid cells, the c-ets proto-oncogenes encode transcription factors which could play an important role in both basal and Tax1-mediated HTLV-I transcription.

Animals↗
PubMed1991

v-erbA oncogene abrogates growth inhibition of chicken embryo fibroblasts induced by retinoic acid.

Retinoic acid inhibits chicken embryo fibroblast (CEF) proliferation by altering the G1 phase of the cell cycle with induction of a strong increase in the generation time. This growth-inhibitory response to retinoic acid is abrogated by expression of the v-erbA oncogene, suggesting an interference between retinoic acid receptors and the v-ErbA oncoprotein. Moreover, CEF expressing either the v-src, v-jun or v-fos oncogenes are also insensitive to retinoic acid treatment. In contrast, CEF expressing either the v-myc, v-myb-ets, v-mil, v-sea or v-erbB oncogenes are still sensitive to retinoic acid. These data strongly suggest functional interferences between the retinoic acid receptors and the AP-1 transcription factor complex in the control of expression of genes involved in CEF proliferation.

Animals↗
PubMed1991

Cloning, sequencing, and expression of two Xenopus laevis c-ets-2 protooncogenes.

In a general approach to identify genes important in the control of genetic expression during development of Xenopus laevis, two complementary DNAs corresponding to two different c-ets-2 genes were cloned and sequenced. One of these complementary DNAs appears to be almost full length. The two variant genes differ in their overlapping sequences by 87 nucleotide substitutions, leading to 17 amino acid modifications in the proteins, 8 of them being conservative. All but one of these changes map outside of the 142 COOH-terminal residues, a region critical for nuclear localization and DNA binding in the ets proteins. Features potentially important for the biological activity of the gene products are conserved. Two transcripts (3.2 and 1.7 kilobases) with maternal characteristics are detected at a constant level from stages II/III of oogenesis to stage 10 of embryogenesis. They later decline to hardly detectable levels at stages 30-40. Variable amounts of the same transcripts are observed in many adult tissues. All of these characteristics support the idea that the ets-2 gene products play an important role during embryogenesis, as well as in adult life. Indeed, they act as ubiquitous transcriptional activators, as recently demonstrated by several investigators (C. V. Gunther, J. A. Nye, R. S. Bryner, and B. J. Graves, Genes & Dev., 4: 667-679, 1990; R. Bosselut, J.F. Duvall, A. Gegonne, M. Bailly, A. Hemar, J. Brady, and J. Ghysdael, EMBO J., 9: 3137-3144, 1990; R. Wasylyk, C. Wasylyk, P. Florès, A. Bègue, D. Leprince, and D. Stéhelin, Nature (Lond.), 346: 191-193, 1990).

Amino Acid Sequence↗
PubMed1991

Ets1, when fused to the GAL4 DNA binding domain, efficiently enhances galactose promotor dependent gene expression in yeast.

Ets1, the translation product of the c-ets1 proto-oncogene and the related Ets2 protein, act as sequence-specific transcriptional factors in transient transfection experiments in animal cells. We report here that in S. cerevisiae, expression of a lacZ test gene placed under the control of the GAL1 promoter is stimulated efficiently by a fusion protein in which the chicken Ets1 sequence starting from amino acid 37, is linked to the DNA binding domain of the yeast GAL4 transcriptional activator. This suggests that Ets1 contains one or more intrinsic transcription activation domain(s). However, the GAL4 integral of Ets1 fusion protein was unable to restore growth of a gal4 deletion mutant on galactose, implying that the fusion product cannot substitute for GAL4 enhancement on all GAL genes.

DNA-Binding Proteins↗
PubMed1990

The product of the c-ets-1 proto-oncogene and the related Ets2 protein act as transcriptional activators of the long terminal repeat of human T cell leukemia virus HTLV-1.

The c-ets-1 proto-oncogene and the related c-ets-2 gene encode related nuclear chromatin-associated proteins which bind DNA in vitro. To investigate the possibility that Ets1 and Ets2 are transcriptional activators, we analyzed the ability of these proteins to trans-activate promoter/enhancer sequences in transient co-transfection experiments. A CAT construct driven by the long terminal repeat of the human T cell leukemia virus, HTLV-1 was found to be trans-activated by both Ets1 and Ets2 in NIH3T3 and HeLa cells. The increased levels of CAT activity were paralleled by increased levels of correctly initiated CAT mRNA. Mutant Ets1 proteins unable to accumulate in the nucleus were found to be inactive. An ets-responsive sequence between positions -117 and -160 of the LTR was identified by analyses of a series of 5' deletion mutants of the HTLV-1 LTR and of dimerized versions of specific motifs of the LTR enhancer region. Using a gel shift binding assay, Ets1 was found to bind specifically to an oligonucleotide corresponding to region -117 to -160. This sequence, which also contributes to Tax1 responsiveness of the HTLV-1 LTR, is characterized by the presence of four repeats of a pentanucleotide sequence of the type CC(T/A)CC. Competition experiments show that integrity of repeats 1 and 4 is important for Ets1 binding. These results show that Ets1 and Ets2 are sequence-specific transcriptional activators. In view of the high level expression of Ets1 in lymphoid cells, Ets1 could be part of the transcription complex which mediates the response to Tax1 and the control of HTLV-1 replication. More generally, Ets1 and Ets2 could regulate transcription of cellular genes.

Animals↗
PubMed1990

Phosphorylation of the v-erbA protein is required for its function as an oncogene.

The v-erbA oncogene of avian erythroblastosis virus (AEV) encodes a ligand-independent mutated version of the chicken c-erbA alpha-encoded thyroid hormone receptor. The v-erbA gene product, a 75-kD gag/v-erbA fusion protein, is phosphorylated on Ser-16/17 of its v-erbA-encoded domain, and phosphorylation at this site is increased in vivo after activation of either the PKA or PKC signal transduction pathways. To test the hypothesis that phosphorylation of Ser-16/17 regulates gag/v-erbA protein function, mutant proteins in which Ser-16/17 had been changed to alanine or threonine residues were analyzed for their ability to inhibit erythroid differentiation of ts v-erbB or ts v-sea-transformed erythroblasts at nonpermissive temperature. Conversion of Ser-16/17 into alanine, although not affecting nuclear localization or DNA binding of the gag/erbA protein, prevented phosphorylation of the v-erbA-encoded domain of the protein both in unstimulated cells or after stimulation by PKA and PKC activators. The nonphosphorylatable AA-gag/v-erbA protein proved unable to inhibit temperature-induced differentiation of ts v-erbB and ts v-sea-transformed erythroblasts and to block expression of the erythrocyte-specific genes band 3 and carbonic anhydrase II. Back mutation of these alanine residues to serine resulted in the recovery of both normal phosphorylation levels and wild-type biological activity. In contrast, substitution of Ser-16/17 for threonine, which preserved phosphorylation in unstimulated cells but not PKA- and PKC-enhanced phosphorylation, resulted in a partially active gag/v-erbA protein. These results, together with the fact that the protein kinase inhibitor H7 resulted in both a dose-dependent inhibition of gag/v-erbA protein phosphorylation and the induction of terminal differentiation of AEV-transformed erythroblasts show that phosphorylation of gag/v-erbA protein is required for full biological activity. These results support the hypothesis that phosphorylation of the gag/v-erbA protein is important for transcriptional repression of at least some of its target genes in erythroid cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Rapid and transient expression of Ets2 in mature macrophages following stimulation with cMGF, LPS, and PKC activators.

We reported previously that Ets2 is expressed in normal and transformed macrophages. We show here that the expression of both c-ets-2 mRNA proteins is induced rapidly and transiently in chicken nondividing bone marrow-derived macrophages but not in E26-transformed myeloblasts in response to chicken myelomonocytic growth factor (cMGF), an avian hematopoietic growth factor required for survival, proliferation, and colony formation of avian myeloid cells. c-ets-2 expression is also rapidly induced in chicken bone marrow-derived macrophages, human monocytes, and mouse peritoneal macrophages in response to LPS and/or PKC activators. The rapid induction of Ets2 after treatment of chicken bone marrow-derived macrophages by cMGF is blunted after down-regulation or inactivation of PKC, suggesting a role of PKC in the cMGF-induced signal transduction pathway. Because Ets2 is localized in the nucleus of macrophages and binds to DNA in vitro, the kinetics of its expression suggest a role for Ets2 in the transduction within the nucleus of specific signals received at the cell membrane and involved in securing the survival and/or the development of functional competence of these cells.

Animals↗