Biomedical subjects

J Ghysdael

Publications and source records attributed to J Ghysdael.

At least 55 records · Page 3Linked to original sources

PubMed1994

Repression of AP-1-stimulated transcription by c-Ets-1.

The transcriptional activities of c-Ets-1 and v-Ets and their functional interaction with the AP-1 factor c-Jun were investigated. Several recombinant Ets proteins were produced and purified either from bacteria or from insect cells. Plasmid DNAs that contained the polyoma virus enhancer Ets/AP-1 element were used as templates for in vitro transcription assays in the presence of HeLa nuclear extract and various combinations of the Jun and Ets proteins. Under these conditions full-length c-Ets-1 on its own does not markedly influence transcription but abolishes the strong transcriptional stimulation normally elicited by Jun. This repression depends on the Ets-binding site and on specific features of c-Ets-1 structure, as both v-Ets and a natural splicing variant c-Ets-1 (delta VII) fail to inhibit Jun activity. These findings suggest that c-Ets may act both as a transcriptional repressor or activator depending on promoter context and splicing pattern.

Alternative Splicing↗

PubMed1994

A transcriptional regulatory element is associated with a nuclease-hypersensitive site in the pol gene of human immunodeficiency virus type 1.

Analysis of the chromatin organization of the integrated human immunodeficiency virus type 1 (HIV-1) genome has previously revealed a major constitutive DNase I-hypersensitive site associated with the pol gene (E. Verdin, J. Virol. 65:6790-6799, 1991). In the present report, high-resolution mapping of this site with DNase I and micrococcal nuclease identified a nucleosome-free region centered around nucleotides (nt) 4490 to 4766. A 500-bp fragment encompassing this hypersensitive site (nt 4481 to 4982) exhibited transcription-enhancing activity (two- to threefold) when it was cloned in its natural position with respect to the HIV-1 promoter after transient transfection in U937 and CEM cells. Using in vitro footprinting and gel shift assays, we have identified four distinct binding sites for nuclear proteins within this positive regulatory element. Site B (nt 4519 to 4545) specifically bound four distinct nuclear protein complexes: a ubiquitous factor, a T-cell-specific factor, a B-cell-specific factor, and the monocyte/macrophage- and B-cell-specific transcription factor PU.1/Spi-1. In most HIV-1 isolates in which this PU box was not conserved, it was replaced by a binding site for the related factor Ets1. Factors binding to site C (nt 4681 to 4701) had a DNA-binding specificity similar to that of factors binding to site B, except for PU.1/Spi-1. A GC box containing a binding site for Sp1 was identified (nt 4623 to 4631). Site D (nt 4816 to 4851) specifically bound a ubiquitously expressed factor. These results identify a transcriptional regulatory element associated with a nuclease-hypersensitive site in the pol gene of HIV-1 and suggest that its activity may be controlled by a complex interplay of cis-regulatory elements.

B-Lymphocytes↗

PubMed1994

Cooperative binding of Ets-1 and core binding factor to DNA.

Two phorbol ester-inducible elements (beta E2 and beta E3) within the human T-cell receptor beta gene enhancer each contain consensus binding sites for the Ets and core binding factor (CBF) transcription factor families. Recombinant Ets-1 and purified CBF bound individually to beta E2 and beta E3, in which the Ets and core sites are directly adjacent. In this report, we show that CBF and Ets-1 bind together to beta E2 and beta E3 and that Ets-1-CBF-DNA complexes are favored over the binding of either protein alone to beta E2. Formation of Ets-1-CBF-DNA complexes increased the affinity of Ets-1-DNA interactions and decreased the rate of dissociation of CBF from DNA. Ets-1-CBF-DNA complexes were not observed when either the Ets or core site was mutated. The spatial requirements for the cooperative interaction of Ets-1 and CBF were analyzed by oligonucleotide mutagenesis and binding site selection experiments. Core and Ets sites were coselected, and there appeared to be little constraint on the relative orientation and spacing of the two sites. These results demonstrate that CBF and Ets-1 form a high-affinity DNA-binding complex when both of their cognate sites are present and that the relative spacing and orientation of the two sites are unimportant. Ets and core sites are found in several T-cell-specific enhancers, suggesting that this interaction is of general importance in T-cell-specific transcription.

Animals↗

PubMed1994

DNA-binding and transcriptional activation properties of the EWS-FLI-1 fusion protein resulting from the t(11;22) translocation in Ewing sarcoma.

The 5' half of the EWS gene has recently been described to be fused to the 3' regions of genes encoding the DNA-binding domain of several transcriptional regulators, including ATF1, FLI-1, and ERG, in several human tumors. The most frequent occurrence of this situation results from the t(11;22)(q24;q12) chromosome translocation specific for Ewing sarcoma (ES) and related tumors which joins EWS sequences to the 3' half of FLI-1, which encodes a member of the Ets family of transcriptional regulators. We show here that this chimeric gene encodes an EWS-FLI-1 nuclear protein which binds DNA with the same sequence specificity as the wild-type parental FLI-1 protein. We further show that EWS-FLI-1 is an efficient sequence-specific transcriptional activator of model promoters containing FLI-1 (Ets)-binding sites, a property which is strictly dependent on the presence of its EWS domain. Comparison of the properties of the N-terminal activation domain of FLI-1 to those of the EWS domain of the fusion protein indicates that EWS-FLI-1 has altered transcriptional activation properties compared with FLI-1. These results suggest that EWS-FLI-1 contributes to the transformed phenotype of ES tumor cells by inducing the deregulated and/or unscheduled activation of genes normally responsive to FLI-1 or to other close members of the Ets family. ES and related tumors are characterized by an elevated level of c-myc expression. We show that EWS-FLI-1 is a transactivator of the c-myc promoter, suggesting that upregulation of c-myc expression is under control of EWS-FLI-1.

Amino Acid Sequence↗

PubMed1994

Participation of Ets transcription factors in the glucocorticoid response of the rat tyrosine aminotransferase gene.

We have previously shown that two remote glucocorticoid-responsive units (GRUs) of the rat tyrosine aminotransferase (TAT) gene contain multiple binding sites for several transcription factor families, including the glucocorticoid receptor (GR). We report here the identification of two novel binding sites for members of the Ets family of transcription factors in one of these GRUs. One of these binding sites overlaps the major GR-binding site (GRBS), whereas the other is located in its vicinity. Inactivation of the latter binding site leads to a twofold reduction of the glucocorticoid response, whereas inactivation of the site overlapping the GRBS has no detectable effect. In vivo footprinting analysis reveals that the active site is occupied in a glucocorticoid-independent manner, in a TAT-expressing cell line, even though it is located at a position where there is a glucocorticoid-dependent alteration of the nucleosomal structure. This same site is not occupied in a cell line that does not express TAT but expresses Ets-related DNA-binding activities, suggesting the existence of an inhibitory effect of chromatin structure at a hierarchical level above the nucleosome. The inactive Ets-binding site that overlaps the GRBS is not occupied even in TAT-expressing cells. However, this same overlapping site can confer Ets-dependent stimulation of both basal and glucocorticoid-induced levels when it is isolated from the GRU and duplicated. Ets-1 expression in COS cells mimics the activity of the Ets-related activities present in hepatoma cells. These Ets-binding sites could participate in the integration of the glucocorticoid response of the TAT gene with signal transduction pathways triggered by other nonsteroidal extracellular stimuli.

Animals↗

PubMed1994

c-ErbA, but not v-ErbA, competes with a putative erythroid repressor for binding to the carbonic anhydrase II promoter.

The carbonic anhydrase II (CAII) gene is the only known gene identified as direct target for v-ErbA-mediated repression in avian erythroleukemic cells transformed by Avian Erythroblastosis Virus (AEV). This gene is transcriptionally activated by thyroid hormone (T3) in normal erythrocytic cells. In this work we have analysed the molecular basis of the transcriptional control of the CAII gene by c-ErbA and v-ErbA. We show that several domains in the promoter control hormonal regulation of transcription. One domain proximal to the TATA box mediates T3 response but contains no identified binding site for c-ErbA. An other domain termed PAL2 is approximately 600 bp upstream the transcription initiation site and contains a c-ErbA binding site. We show that when it is associated to a heterologous promoter this site mediates transcriptional repression in erythrocytic cells but not in HeLa cells. Moreover, this site binds a nuclear erythrocyte-specific factor that we called NFX, which is different from c-ErbA. heterodimers between c-ErbA and the 9-cis retinoic acid receptor (RXR) compete with NFX for binding to PAL2. In contrast, v-ErbA alone or in association with RXR is a very poor competitor and is unable to chase NFX out of the PAL2 site. We propose that NFX is a transcription repressor whose activity is inhibited by c-ErbA but not v-ErbA. This mechanism might contribute to the overall regulation of the carbonic anhydrase II promoter. These data illustrate another possible mechanism through which v-ErbA might antagonize the function of c-ErbA in controlling gene expression.

Animals↗

PubMed1994

Myb and Ets related transcription factors are required for activity of the human lck type I promoter.

The lck gene, which encodes a lymphoid-specific Src family tyrosine kinase, is transcribed from two promoters that are differentially utilized during T cell development. We have shown previously that the human lck type I promoter, which is preferentially expressed in immature thymocytes, requires a binding site (-97 to -90) for the Ets family of transcription factors for its activity in Jurkat T leukemia cells. Three putative Myb binding sites (-86 to -82, -77 to -72 and -59 to -54) were analysed for their ability to activate the lck type I promoter. In vitro assays demonstrated specific binding of purified, bacterially expressed c-Myb DNA binding domain to the Myb (-59 to -54) site. Transient transfection assays using the site-directed mutants of the lck type I promoter in Jurkat cells revealed that mutation of the Myb (-59 to -54) site abolished transcriptional activity. In transiently transfected HeLa cells, the lck type I promoter was activated by co-transfection with a vector that expresses c-Myb. This c-Myb dependent activation required the presence of intact Myb and Ets binding sites, indicating that the expressed c-Myb functions with endogenous Ets related transcription factors to activate the lck type I promoter. This effect was further enhanced by co-transfection with vectors that express either Ets1 or Ets2. These results demonstrate that Myb and Ets related transcription factors synergistically activate the human lck type I promoter.

Base Sequence↗

PubMed1994

junB promoter regulation: Ras mediated transactivation by c-Ets-1 and c-Ets-2.

The Jun gene family encode components of the AP-1 transcription factor complex that regulate a variety of TRE-containing target promoters. Expression of family members is induced by a wide variety of extracellular stimuli and thought to be important in mediating cellular proliferation and differentiation. We have localized cis-acting DNA sequences in the murine junB promoter capable of mediating transcriptional activation by the proto-oncogene products c-Ets-1 and c-Ets-2. We show by promoter deletion analysis that multiple elements located between -848 and -574, and between -196 and -91 can mediate transactivation by ETS-family members in different cell types. In vitro DNA binding assays indicate that the elements identified can specifically interact with c-Ets-1 protein. Furthermore, we show that ETS-transactivation of a variety of reporter constructs is dramatically enhanced by introduction of oncogenic Ha-ras. The activation of Ras by extracellular stimuli invokes a phosphorylation cascade that includes the downstream mitogen-activated protein (MAP) kinase p44ERK-1. We further show that addition of activated p44ERK-1 MAP kinase can also enhance ETS-transactivation of junB promoter reporter constructs. Here we propose that ETS-family members play a role in the activation of junB transcription by a Ras-stimulated signal transducing pathway that includes MAP kinase(s).

Animals↗

PubMed1994

Dual control of myc expression through a single DNA binding site targeted by ets family proteins and E2F-1.

NIH3T3 cells expressing a mutant colony-stimulating factor-1 receptor (CSF-1R) containing a phenylalanine for tyrosine substitution in the tyrosine kinase domain at codon 809 exhibit defective myc regulation and do not enter S phase when stimulated by CSF-1. Enforced expression of either ets-1 or ets-2 in these cells restores their mitogenic response, albeit less efficiently than myc itself, suggesting that ets proteins may regulate c-myc expression. Ets-1 transactivates reporter genes driven by the human and mouse c-myc promoters through the binding site for the transcription factor E2F, the latter being required for E1A- and serum-induced c-myc expression. Analysis of E2F-1 sequences identified a minimal DNA binding domain that is related to those of ets proteins. Although E2F and ets proteins interact with similar consensus DNA binding sites, in vitro binding assays revealed that E2F can bind DNA as a homodimer, whereas ets proteins bind these sites as monomers. E2F and ets proteins do not form heterodimers in vitro and do not transactivate c-myc synergistically. Thus, E2F-1 and ets family members may independently regulate c-myc transcription through the same binding site at different times following growth factor stimulation.

Amino Acid Sequence↗

PubMed1993

A single amino-acid substitution in the Ets domain alters core DNA binding specificity of Ets1 to that of the related transcription factors Elf1 and E74.

Ets proteins form a family of sequence specific DNA binding proteins which bind DNA through a 85 aminoacids conserved domain, the Ets domain, whose sequence is unrelated to any other characterized DNA binding domain. Unlike all other known Ets proteins, which bind specific DNA sequences centered over either GGAA or GGAT core motifs, E74 and Elf1 selectively bind to GGAA corecontaining sites. Elf1 and E74 differ from other Ets proteins in three residues located in an otherwise highly conserved region of the Ets domain, referred to as conserved region III (CRIII). We show that a restricted selectivity for GGAA core-containing sites could be conferred to Ets1 upon changing a single lysine residue within CRIII to the threonine found in Elf1 and E74 at this position. Conversely, the reciprocal mutation in Elf1 confers to this protein the ability to bind to GGAT core containing EBS. This, together with the fact that mutation of two invariant arginine residues in CRIII abolishes DNA binding, indicates that CRIII plays a key role in Ets domain recognition of the GGAA/T core motif and lead us to discuss a model of Ets proteins--core motif interaction.

Amino Acid Sequence↗

PubMed1993

Characterization of a hepatoma mRNA transcribed from a third promoter of a 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-encoding gene and controlled by ets oncogene-related products.

6-Phosphofructo-2-kinase (EC 2.7.1.105)/fructose-2,6-bis-phosphatase (EC 3.1.3.46) catalyzes the synthesis and degradation of fructose 2,6-bisphosphate, a ubiquitous stimulator of glycolysis. The liver (L-type) and muscle (M-type) mRNAs for this bifunctional enzyme arise from distinct promoters of the same gene. We have now characterized in rat hepatoma FTO2B cells another mRNA, which is transcribed from a third promoter of that gene. This F-type mRNA is present in fetal rat liver and muscle, in rat placenta, and in several established rat cell lines. The F promoter contains no TATA box but contains several binding sites for Sp1 and for members of the ets oncogene family. Transfection of FTO2B cells with constructs containing the intact or mutagenized F promoter showed that its activity depends mainly on one of these sites. This site bound a heteromeric FTO2B cell protein indistinguishable from the ets-related GA binding protein alpha/ankyrin-repeats GA binding protein beta transcription factor.

Animals↗

PubMed1993

Synergistic activation of the HTLV1 LTR Ets-responsive region by transcription factors Ets1 and Sp1.

Ets1 is the prototype of a family of transcriptional activators whose activity depends on the binding to specific DNA sequences characterized by an invariant GGA core sequence. We have previously demonstrated that transcriptional activation by Ets1 of the long terminal repeat (LTR) of human T cell lymphotropic virus type 1 is strictly dependent on the binding of Ets1 to two sites, ERE-A and ERE-B, localized in a 44 bp long Ets-responsive region (ERR1). We report here that the activity of ERR1 as an efficient Ets1 response element in HeLa cells also depends on the integrity of an Sp1 binding site localized immediately upstream of ERE-A. The response to Ets1 of an element restricted to the SP1/ERE-A binding sites is also strictly dependent on both the Ets1 and Sp1 binding sites. In vitro, Sp1 and Ets1 are shown to cooperate to form a ternary complex with the SP1/ERE-A element. Reconstitution experiments in Drosophila melanogaster Schneider cells show that Ets1 and Sp1 act synergistically to activate transcription from either the ERR1 or the SP1/ERE-A elements and that synergy requires the binding of both Sp1 and Ets1 to their cognate sites. SP1/ERE-A elements are found in the enhancer/promoter region of several cellular genes, suggesting that synergy between Ets1 and Sp1 is not restricted to the ERR1 region of the HTLV1 LTR. These results strengthen the notion that Ets1 as well as other members of the Ets family usually function as components of larger transcription complexes to regulate the activity of a variety of viral and cellular genes.

Animals↗

PubMed1993

Combinatorial generation of variable fusion proteins in the Ewing family of tumours.

Balanced translocations involving band q12 of human chromosome 22 are the most frequent recurrent translocations observed in human solid tumours. It has been shown recently that this region encodes EWS, a protein with an RNA binding homologous domain. In Ewing's sarcoma and malignant melanoma of soft parts, translocations of band 22q12 to chromosome 11 and 12 result in the fusion of EWS with the transcription factors FLI-1 and ATF1, respectively. The present analysis of 89 Ewing's sarcomas and related tumours show that in addition to the expected EWS-FLI-1 fusion, the EWS gene can be fused to ERG, a transcription factor closely related to FLI-1 but located on chromosome 21. The position of the chromosome translocation breakpoints are shown to be restricted to introns 7-10 of the EWS gene and widely dispersed within introns 3-9 of the Ets-related genes. This heterogeneity generates a variety of chimeric proteins that can be detected by immuno-precipitation. On rare occasions, they may be associated with a truncated EWS protein arising from alternate splicing. All 13 different fusion proteins that were evidenced contained the N-terminal domain of EWS and the Ets domain of FLI-1 or ERG suggesting that oncogenic conversion is achieved by the linking of the two domains with no marked constraint on the connecting peptide.

Amino Acid Sequence↗

PubMed1993

GATA and Ets cis-acting sequences mediate megakaryocyte-specific expression.

The human glycoprotein IIB (GPIIB) gene is expressed only in megakaryocytes, and its promoter displays cell type specificity. We show that this specificity involved two cis-acting sequences. The first one, located at -55, contains a GATA binding site. Point mutations that abolish protein binding on this site decrease the activity of the GPIIB promoter but do not affect its tissue specificity. The second one, located at -40, contains an Ets consensus sequence, and we show that Ets-1 or Ets-2 protein can interact with this -40 GPIIB sequence. Point mutations that impair Ets binding decrease the activity of the GPIIB promoter to the same extent as do mutations that abolish GATA binding. A GPIIB 40-bp DNA fragment containing the GATA and Ets binding sites can confer activity to a heterologous promoter in megakaryocytic cells. This activity is independent of the GPIIB DNA fragment orientation, and mutations on each binding site result in decreased activity. Using cotransfection assays, we show that c-Ets-1 and human GATA1 can transactive the GPIIB promoter in HeLa cells and can act additively. Northern (RNA) blot analysis indicates that the ets-1 mRNA level is increased during megakaryocyte-induced differentiation of erythrocytic/megakaryocytic cell lines. Gel retardation assays show that the same GATA-Ets association is found in the human GPIIB enhancer and the rat platelet factor 4 promoter, the other two characterized regulatory regions of megakaryocyte-specific genes. These results indicate that GATA and Ets cis-acting sequences are an important determinant of megakaryocytic specific gene expression.

Base Sequence↗

PubMed1993

Requirement of an ETS-binding element for transcription of the human lck type I promoter.

The requirement for cis-acting DNA sequences for transcriptional activity of the human lck type I promoter was investigated in two human cell lines that express type I transcripts, the leukemic T-cell line, Jurkat, and the colon carcinoma line, SW620. Transient transfection assays in Jurkat and SW620 cells revealed negative and positive cis-acting regulatory elements in the lck type I promoter between -570 and -480 and between -128 and -63 respectively. For the latter, a triple point mutation of a sequence, GCAGGAAGT, from -99 and -91 resulted in complete loss of lck type I promoter activity in both Jurkat and SW620 cells. In vitro binding assays indicated that this sequence, denoted the ETS-binding element or EBE, can interact with the lymphoid-specific transcription factor ETS-1. Thus, a protein(s) in the ETS family appears to be required for transcription of the lck type I promoter in T cells and may be important for the activation of the lck gene in human colon carcinoma.

Adult↗

PubMed1992

A phorbol ester response element within the human T-cell receptor beta-chain enhancer.

The activity of the T-cell receptor beta-chain gene enhancer is increased by activators of the protein kinase C pathway during T-cell activation. Analysis of mutant enhancer constructs identified two elements, beta E2 and beta E3, conferring phorbol ester inducibility. Multimerized beta E2 acted in isolation as a phorbol ester-responsive element. Both beta E2 and beta E3, which contain a consensus Ets-binding site, were shown to bind directly to the product of the c-ets-1 protooncogene. Both regions also bound a second factor, core-binding factor. Mutation of the beta E2 Ets site abolished the inducibility of the beta E2 multimer. beta E2 and beta E3 Ets site mutations also profoundly affected activity and inducibility of the enhancer. In contrast, enhancer activity but not its inducibility was affected by mutation of the beta E2 core-binding factor site. Cotransfection studies showed that Ets-1 specifically repressed activity of the multimerized beta E2 element and the complete T-cell receptor beta-chain enhancer. These data show that the T-cell receptor beta-chain enhancer responds to protein kinase C-mediated activation signals via a functional domain, composed of two elements, which contains binding sites for Ets transcription factors and which is negatively regulated by Ets-1.

Base Sequence↗

PubMed1992

Identification of nucleotide preferences in DNA sequences recognised specifically by c-Ets-1 protein.

The protooncogene Ets-1 is a member of the c-Ets family of genes originally identified through their sequence homology to the v-ets gene of the avian erythroblastosis virus E26. Ets-like factors are characterised by a conserved 85 amino acid domain which appears to be essential for binding to purine rich DNA sequences. Sequences binding to Ets-1 were selected from a random oligonucleotide pool by immunoprecipitation and amplified using the Polymerase Chain Reaction. Oligonucleotides enriched by this procedure were cloned in plasmids and sequenced. Alignment of DNA sequences revealed GGAA and GGAT cores at about a 1.4:1 ratio. Preferred sequences were identified both 5' and 3' of the GGAW core, extending the binding site to ACMGGAWRTT. Analysis of the flanking sequences associated with GGAA and GGAT cores revealed differences which may have compensated for the generally lower affinity of binding sites containing a GGAT core. Lastly mutational analysis of one particular Ets-1 binding site was used to establish the relative importance for binding of some nucleotides within the core and to show that Ets-1 and the closely related Ets-2 proteins bind to similar sequences.

Animals↗

PubMed1992

Myb protein binds to multiple sites in the human T cell lymphotropic virus type 1 long terminal repeat and transactivates LTR-mediated expression.

The members of the c-myb proto-oncogene family encode sequence-specific transcriptional activators. In T cells, expression of c-myb and the related B-myb gene is induced following mitogenic stimulation. Using a purified recombinant protein, we report here that the human T cell lymphotropic virus type 1 (HTLV-1) LTR contains six specific binding sites for Myb. We also show that HTLV-1 LTR chloramphenicol acetyl transferase reporter plasmids are specifically transactivated by c-Myb. These data suggest a role for members of the Myb family as a link between transcriptional activation of the HTLV-1 LTR and T cell activation events.

Base Sequence↗