Stromelysin-1 expression is activated in vivo by Ets-1 through palindromic head-to-head Ets binding sites present in the promoter.

Regulation of the gene expression of Stromelysin-1 (matrix metalloproteinase-3), a member of the matrix metalloproteinase family, is critical for tissue homeostasis. The Stromelysin-1 promoter is known to be transactivated by Ets proteins through palindromic head-to-head Ets binding sites (EBS), an unusual configuration among metalloproteinase promoters. Patterns of increased co-expression of Stromelysin-1 and Ets-1 genes have been observed in pathological processes such as rheumatoid arthritis, glomerulonephritis and tumor invasion. In this context, we show in a synovial fibroblastic model cell line (HIG-82), which is able to co-express Stromelysin-1 and Ets-1, that the EBS palindrome is essential for the expression of Stromelysin-1. More precisely, using electrophoretic mobility shift assays, DNA affinity purification and chromatin immunoprecipitation, we demonstrate that endogenous Ets-1, but not Ets-2, is present on this palindrome. The use of a dominant-negative form of Ets-1 and the decrease of Ets-1 amount either by fumagillin, an antiangiogenic compound, or by short interfering RNA show that the activation rate of the promoter and the expression of Stromelysin-1 correlate with the level of endogenous Ets-1. Thus, it is the first demonstration, using this cellular model, that endogenously expressed Ets-1 is actually a main activator of the Stromelysin-1 promoter through its effective binding to the EBS palindrome.

Molecular cloning and characterization of the mouse E2F6 gene.

E2F6 is the most recently identified member of the E2F family. In this study, the murine E2F6 gene was cloned and found to consist of eight exons. Analysis of its 5' flanking region revealed two transcription start sites. The proximal promoter region contained no TATA or CAAT box. We also identified a novel E2F6 mRNA containing the alternative exon 2. The E2F6 mRNAs are highly expressed during mouse embryogenesis and are present in all adult tissues examined. Moreover, E2F6 shows a unique expression pattern in synchronized mouse embryonic fibroblasts. E2F6 expression rapidly increases during the G0-G1 transition, reaching its higher level in mid-G1, and remains relatively constant thereafter. These findings suggest that E2F6 may contribute to the regulation of events throughout the cell cycle. Isolation of the murine E2F6 gene is a step toward generation of genetically modified mouse models that will help to understand the functions of E2F6.

Identification of amino acid residues in the ETS transcription factor Erg that mediate Erg-Jun/Fos-DNA ternary complex formation.

Jun, Fos, and Ets proteins belong to distinct families of transcription factors that target specific DNA elements often found jointly in gene promoters. Physical and functional interactions between these families play important roles in modulating gene expression. Previous studies have demonstrated a direct interaction between the DNA-binding domains of the two partners. However, the molecular details of the interactions have not been investigated so far. Here we used the known three-dimensional structures of the ETS DNA-binding domain and Jun/Fos heterodimer to model an ETS-Jun/Fos-DNA ternary complex. Docking procedures suggested that certain ETS domain residues in the DNA recognition helix alpha3 interact with the N-terminal basic domain of Jun. To support the model, different Erg ETS domain mutants were obtained by deletion or by single amino acid substitutions and were tested for their ability to mediate DNA binding, Erg-Jun/Fos complex formation, and transcriptional activation. We identified point mutations that affect both the DNA binding properties of Erg and its physical interaction with Jun (R367K), as well as mutations that essentially prevent transcriptional synergy with the Jun/Fos heterodimer (Y371V). These results provide a framework of the ETS/bZIP interaction linked to the manifestation of functional activity in gene regulation.

The Ets family member Erg gene is expressed in mesodermal tissues and neural crests at fundamental steps during mouse embryogenesis.

The Erg gene belongs to the Ets family encoding a class of transcription factors. To gain new insight on the in vivo functional specificity of the Erg gene within the wide Ets family, we used in situ hybridization to determine its expression pattern during murine embryogenesis. We found that the Erg gene expression predominates in mesodermal tissues, including the endothelial, precartilaginous and urogenital areas. A specific Erg gene expression was also identified in migrating neural crest cells. A comparison with Fli-1, the most closely Erg-related gene, revealed that both gene expressions partially overlap, suggesting that they may contribute to related functions in these tissues. Like other Ets family genes, Erg seems involved in several fundamental developmental steps in murine embryogenesis, including epithelio-mesenchymal transition, cell migration, settlement and differentiation.

Neoplastic AIDS-associated Kaposi's sarcoma cell line KSY-1 cannot transdifferentiate into capillaries.

OBJECTIVE: Kaposi's sarcoma (KS) is an acquired immunodeficiency syndrome (AIDS)-defining neoplasm histologically characterized by proliferation of spindle cells, inflammatory cells, and abundant neovascularization. When the malignant cell line KSY-1 derived from an AIDS-KS tumor is transplanted subcutaneously into nude mice, prominent neovascular features develop. Using this mouse model of neoplastic KS, we set out to determine, using c-ets 1 markers specific for mouse or human tissues, whether vascular growth and inflammatory infiltrate induced by the transplanted KSY-1 cells is of host cell or transplant origin. STUDY DESIGN/METHODS: KS tumors were induced by subcutaneous inoculation of 5 x 10(6) KSY-1 cells/200 microL in immunodeficient mice, and species-specific mouse and human riboprobes of the c-ets 1 protooncogene were used for in situ hybridization to define cell of origin. RESULTS: Five different tumors were examined. Tissue sections from all cases were hybridized with radiolabeled riboprobes for the presence of both mouse and human c-ets 1 mRNA. Tumor cells were labeled with the human c-ets 1 probe, whereas neovascular and inflammatory tissues were of mouse origin. CONCLUSIONS: The finding that vascular but not tumor cells are of host origin supports the model of tumor-induced vascularization via a mechanism of tumor cell-derived cytokine-medicated pathogenesis.

Nuclear localization of a new c-cbl related protein, CARP 90, during in vivo thymic apoptosis in mice.

This study investigates the involvement of the c-cbl protooncogene in thymocyte apoptosis occurring in vivo after hydrocortisone treatment. In the thymus of untreated mice, a few medullary and cortical thymocytes expressed p120cbl, mainly in the cytoplasm. In the cortex, their number and distribution resemble that of apoptotic cells evidenced by TUNEL staining. The expression of Cbl is rapidly increased when apoptosis is triggered by hydrocortisone. This Cbl-specific immunostaining was detected in the nucleus and is due to a Cbl-related 90 kDa protein (CARP 90). These results show that a c-cbl product could localize in the nucleus and suggest that it could be involved as a regulator of thymic apoptosis.

Molecular phylogeny of the ETS gene family.

We have constructed a molecular phylogeny of the ETS gene family. By distance and parsimony analysis of the ETS conserved domains we show that the family containing so far 29 different genes in vertebrates can be divided into 13 groups of genes namely ETS, ER71, GABP, PEA3, ERG, ERF, ELK, DETS4, ELF, ESE, TEL, YAN, SPI. Since the three dimensional structure of the ETS domain has revealed a similarity with the winged-helix-turn-helix proteins, we used two of them (CAP and HSF) to root the tree. This allowed us to show that the family can be divided into five subfamilies: ETS, DETS4, ELF, TEL and SPI. The ETS subfamily comprises the ETS, ER71, GABP, PEA3, ERG, ERF and the ELK groups which appear more related to each other than to any other ETS family members. The fact that some members of these subfamilies were identified in early metazoans such as diploblasts and sponges suggests that the diversification of ETS family genes predates the diversification of metazoans. By the combined analysis of both the ETS and the PNT domains, which are conserved in some members of the family, we showed that the GABP group, and not the ERG group, is the one most closely related to the ETS group. We also observed that the speed of accumulation of mutations in the various genes of the family is highly variable. Noticeably, paralogous members of the ELK group exhibit strikingly different evolutionary speed suggesting that the evolutionary pressure they support is very different.

The carboxy-terminal end of the candidate tumor suppressor gene HIC-1 is phylogenetically conserved.

HIC-1 (hypermethylated in cancer), a new candidate tumor suppressor gene located in 17p13.3, encodes a protein with five Krüppel-like C2H2 zinc finger motifs and a N-terminal protein-protein interaction domain called BTB/POZ. These two domains appeared as the only conserved regions found between human HIC-1 and its avian homologue, gammaFBP-B, isolated as a transcriptional repressor of the gammaF-Crystallin gene. We have recloned the HIC-1 gene and found four nucleotide differences within the 3' part of its published coding sequence. The corrected HIC-1 C-terminal end exhibits now significant homology (70%) to the chicken gammaFBP-B C-terminal end and appears thus as a third phylogenetically conserved domain that may serve an important, yet unknown function in HIC-1.

The avian transcription factor c-Rel is expressed in lymphocyte precursor cells and antigen-presenting cells during thymus development.

Transcription factors of the Rel/NF-kappaB family are widely involved in the immune system. In this study, we investigate the in vivo expression of the avian protein c-Rel in the T-cell lineage during thymus development. The majority of thymocytes do not express the c-Rel protein. However, lymphocyte precursor cells that colonize the thymus express the c-Rel protein shortly after their homing in the organ and before they begin to differentiate. c-Rel is also detected in different subsets of antigen-presenting cells such as epithelial cells, dendritic cells, and macrophages. In vitro studies have shown that Rel/NF-kappaB proteins are sequestered in an inactive form in the cytoplasm by interaction with the IkappaBalpha inhibitory protein. By immunocytochemistry, we show that in vivo c-Rel is localized in the cytoplasm of antigen-presenting cells but in both the cytoplasm and nucleus of lymphocyte precursor cells. The cytoplasmic localization of c-Rel in antigen-presenting cells correlates with a high expression of IkappaBalpha, whereas the nuclear localization of c-Rel in lymphocyte precursor cells correlates with a much lower expression of IkappaBalpha. These results suggest that c-Rel might be constitutively activated in lymphocyte precursor cells.

[Similarities between angiogenesis and neoplasm invasiveness ]. / Similitudes entre les mécanismes de néoangiogenèse et d'invasion tumorales.

We have shown that given cytokines are capable of inducing the expression of transcription factors of the Ets family in two very distinct cell types: 1) endothelial cells of blood vessels, but only during neovascularization, and 2) fibrocytic cells from stroma surrounding tumors, but only if these tumors bear characteristics of invasiveness. In such cases, the fibrocytic cells also express some metalloproteinases (collagenase 1, urkinase plasminogen activator, sometimes stromelysin1). In ex vivo reconstruction experiments, we demonstrate that the corresponding genes are directly up-regulated by the Ets family transcription factors, often associated with the transcription complex Jun/Fos. The proteinases are thought to dismantle the stroma and allow invasive tumors to proceed toward further expansion. We speculate that inactivation of the Ets factors could seriously hamper both neovascularization and tumor expansion.

Targeting of PML/RARalpha is lethal to retinoic acid-resistant promyelocytic leukemia cells.

Acute promyelocytic leukemia (APL) cells, containing the t(15;17) rearrangement, express the fusion protein, PML/RARalpha. Clinically, patients respond to all-trans retinoic acid (ATRA) through complete remissions associated with myeloid maturation of leukemic cells. This clinical ATRA response of APL is linked to PML/RARalpha expression. Unfortunately, these remissions are transient and relapsed APL is often ATRA-resistant. The role PML/RARalpha plays in the growth and maturation of these APL cells with acquired ATRA resistance has not been fully explored. This study uses an ATRA-resistant NB4 cell line (NB4-R1) to investigate the contribution of PML/RARalpha expression to ATRA resistance. Targeting of PML/RARalpha in NB4-R1 cells was undertaken using two approaches: homologous recombination and hammerhead ribozyme-mediated cleavage. Reducing PML/RARalpha protein in NB4-R1 cells rendered these cells more sensitive to ATRA. These cells were growth-inhibited in ATRA, apoptosis was induced, and there was no apparent signaling of differentiation. Sequence analysis identified a mutation in the ligand binding domain (LBD) of the RARalpha portion of PML/RARalpha. Results show that these retinoid-resistant NB4 cells require persistent PML/RARalpha expression for leukemic cell growth. Taken together, these findings can account for why these cells do not respond to ATRA and how reduction of PML/RARalpha abrogates the antiapoptotic effect it confers to these leukemic cells.

Erg proteins, transcription factors of the Ets family, form homo, heterodimers and ternary complexes via two distinct domains.

The ets genes family encodes a group of proteins which function as transcription factors under physiological conditions. We report here that the Erg proteins, members of the Ets family, form homo and heterodimeric complexes in vitro. We demonstrate that the Ergp55 protein isoform forms dimers with itself and with the two other isoforms, Ergp49 and Ergp38. Using a set of Erg protein deletion mutants, we define two distinct domains independently involved in dimerization. The first one is located in the amino-terminal part of the protein containing the pointed domain (PNT), conserved in a subset of Ets proteins. The second one resides within the ETS domain, the DNA-binding domain. We also show that the Erg protein central region behaves as an inhibitory domain of dimerization and its removal enhances the Ergp55 transactivation properties. Furthermore, Ergp55 forms heterodimers with some other Ets proteins. Among the latter, we show that Fli-1, Ets-2, Er81 and Pu-1 physically interact with Erg. Finally, we show that the formation of the previously described ternary complex Ergp55/Fos/jun is mediated by ETS domain and Jun protein, while the ternary complex Ergp49/Fos/Jun is mediated by Fos protein.

The H19 TATA-less promoter is efficiently repressed by wild-type tumor suppressor gene product p53.

The developmentally regulated H19 gene displays several remarkable properties: expression of an apparently non-translated mRNA, genomic imprinting (maternal allele only expressed), relaxation of the imprinting and/or epigenetic lesions demonstrated in some tumors. Despite several observations after relaxation of imprinting status of the gene, data on trans and cis-acting factors required for the human H19 gene expression are still missing. As a first approach to address identification of factors involved in the regulation of the gene, we found that cells from a p53 antisense-transfected HeLa clone displayed increased amounts of H19 transcripts when compared to the non-transfected cells. Moreover, a HeLa clone stably transfected with a temperature sensitive (ts) 143 Ala p53 mutant exhibited temperature-dependent regulation of H19 expression. This preliminary indication of the repressing effect of the p53 protein on H19 expression has been confirmed by transient cotransfection experiments in HeLa cells, using luciferase surrogate constructs under the control of the 823 bp sequence immediately upstream of the transcription start point of the H19 gene, and different constructs containing sense, antisense or a ts 143 Ala mutant p53 cDNA. We observed an increase of H19 promoter-driven activity in transient cotransfections with the antisense p53 cDNA and the temperature sensitive mutant p53 at the non-permissive temperature, but a decrease with sense wild-type p53 cDNA. Furthermore, the cotransfection experiments were repeated in a cell line lacking endogenous p53. (Calu 6 cells) and the results provided additional evidence for a down regulation of the expression of the H19 gene by the p53 protein.

Isolation and characterization of a chicken homologue of the Spi-1/PU.1 transcription factor.

Spi-1/PU.1 is a member of the Ets family of transcription factors important in regulation of hematopoiesis. We have isolated a chicken cDNA homologuous to the mammalian Spi-1/PU.1 gene with an open reading frame of 250 amino acids (aa). The chicken Spi-1/PU.1 protein is 14 aa and 16 aa shorter than its human and mouse counterparts but is extremely well conserved with 78.8% and 75.2% identity respectively. The carboxy terminal DNA binding region, or ETS binding domain, is 100% identical to that of human and mouse. Some differences with the mammalian homologues are seen in the N-terminal part of the protein and in the PEST connecting domain. However, the differences are mainly conservative and all the features underlying functional aspects seem preserved. The major discrepancy lies in a 12 aa deletion in an already poorly conserved part of the PEST sequence. Spi-1/PU.1 transcripts were detected at high levels in spleen and Fabricius bursa of chick embryos by Northern blot and in situ hybridization. Our results show that the chicken Spi-1/PU.1 protein behaves like a bonafide Spi-1/PU.1 transcription factor in its DNA binding and transactivating properties.

The Ets transcription factors interact with each other and with the c-Fos/c-Jun complex via distinct protein domains in a DNA-dependent and -independent manner.

The transcription factors Fos, Jun, and Ets regulate the expression of human stromelysin-1 and collagenase-1 genes. Recently, we found that ERG, an Ets family member, activates collagenase-1 gene but not stromelysin-1 by physically interacting with c-Fos/c-Jun. Interestingly, ERG binds to stromelysin-1 promoter and represses its activation by ETS2. Here, to investigate the molecular mechanism of this regulation, we have used an in vitro protein-protein interaction assay and studied the transcription factor interactions of ETS2. We found that ETS2 could weakly associate with in vitro synthesized ETS1, c-Fos, and c-Jun and strongly with c-Fos/c-Jun complex and ERG via several distinct ETS2 domains including the C-terminal region that contains the DNA-binding domain. Strikingly, these interactions were stabilized in vitro by DNA as they were inhibited by ethidium bromide. Both the N-terminal region, comprising the transactivation domain, and the C-terminal region of ETS2 associated with ERG and, interestingly, the interaction of ERG through the transactivation domain of ETS2 was DNA-independent. The DNA-dependent interaction of ETS2 with c-Fos/c-Jun was enhanced by specific DNA fragments requiring two Ets-binding sites of the stromelysin-1 promoter. Using the two hybrid system, we also demonstrated that ETS2 interacts with c-Jun or ERG in vivo.

The avian transcription factor c-Rel is induced and translocates into the nucleus of thymocytes undergoing apoptosis.

This study investigates the involvement of the avian transcription factor c-Rel in thymocyte apoptosis occurring either in vivo or in organotypic culture. In vivo, only a few cortical thymocytes express the c-Rel protein. Their number, localization and morphology resemble that of apoptotic cells evidenced by TUNEL staining. In organotypic culture, the expression of c-Rel is induced in medullary thymocytes as apoptosis is triggered. This induction would be post-transcriptional since no increase in the c-rel gene expression is detected. Moreover, c-Rel translocates into the nucleus of medullary thymocytes during the time course of apoptosis. This translocation is preceded by a decrease in ikba expression, the gene which encodes the avian homologue of IkappaBalpha. Altogether these results suggest that the proto-oncogene c-rel could take an active part in apoptosis of cortical thymocytes occurring in vivo during T-cell selection as well as in experimentally-induced apoptosis of medullary thymocytes.

Ligand binding was acquired during evolution of nuclear receptors.

The nuclear receptor (NR) superfamily comprises, in addition to ligand-activated transcription factors, members for which no ligand has been identified to date. We demonstrate that orphan receptors are randomly distributed in the evolutionary tree and that there is no relationship between the position of a given liganded receptor in the tree and the chemical nature of its ligand. NRs are specific to metazoans, as revealed by a screen of NR-related sequences in early- and non-metazoan organisms. The analysis of the NR gene duplication pattern during the evolution of metazoans shows that the present NR diversity arose from two waves of gene duplications. Strikingly, our results suggest that the ancestral NR was an orphan receptor that acquired ligand-binding ability during subsequent evolution.

Isolation and characterization of transformed human T-cell lines infected by Epstein-Barr virus.

Epstein-Barr virus (EBV) is a human lymphotropic virus whose main targets have traditionally been described as B lymphocytes and epithelial cells. Here we report the isolation and characterization of largely monoclonal transformed human T-cell lines infected by EBV. The transformed T cells expressed CD2, CD3, and either CD4 or CD8 surface molecules and more generally displayed the phenotype of naive T cells with a complete and clonal rearrangement of the T-cell receptor. None of the cell lines expressed B cells, natural killer, or myeloid antigens or had immunoglobulins genes rearrangement. They grew in the absence of growth factor; however, they all secreted interleukin-2 after mitogenic activation. Polymerase chain reaction (PCR) analysis showed the presence of EBV DNA in all these cell lines. Moreover, Southern blot analysis of one of these cell lines shows the presence of circular episomic EBV DNA, and by Northern blot or reverse transcriptase-PCR analysis, only the expression of Epstein-Barr nuclear antigen-1 (EBNA-1) and latent membrane protein-1 (LMP-1) genes was detected. Finally, the complete transformed phenotype of this T-cell line was shown by its injection into nude or recombination activating gene 2 (RAG2)-deficient mice that led to the formation of solid tumors.

The ETS1 transcription factor is expressed during epithelial-mesenchymal transitions in the chick embryo and is activated in scatter factor-stimulated MDCK epithelial cells.

In embryos and in human tumors, the expression of the ETS1 transcription factor correlates with the occurrence of invasive processes. Although this was demonstrated in cells of mesodermal origin, the expression of ETS1 was not detected in epithelial cells. In the present study, we show that during early organogenesis in the chick embryo, ETS1 mRNA expression was transiently induced in epithelial structures, during emigration of neural crest cells and dispersion of somites into the mesenchymal sclerotome. In contrast, the expression of ETS1 was not detected in situations where epithelial layers stayed cohesive while forming a new structure, such as the dermomyotome forming the myotome. The involvement of ETS1 in epithelial cell dissociation was examined in MDCK epithelial cells stimulated by scatter factor/hepatocyte growth factor (SF/HGF), a potent inducer of cell dissociation and motility. SF/HGF was found to stimulate ETS1 mRNA and protein expressions, and these increases coincided with the dispersion of cells and the expression of protease mRNAs, such as urokinase-type plasminogen activator and collagenase, but not with the protease inhibitor, plasminogen activator inhibitor type 1. Furthermore, we showed that SF/HGF was able to induce a transcriptional response involving ETS1 by using artificial as well as cellular promoters, such as the urokinase-type plasminogen activator and collagenase 1 promoters, containing RAS-responsive elements with essential ETS-binding sites. These data demonstrate expression of ETS1 during epithelial-mesenchymal transitions in the developing embryo and show that ETS1 can act as a downstream effector of SF/HGF in MDCK epithelial cells. Taken together, these data identify ETS1 as a molecular actor of epithelia cell dissociation.

Thyroid hormone receptor genes of neotenic amphibians.

Since thyroid hormones play a pivotal role in amphibian metamorphosis we used PCR to amplify DNA fragments corresponding to a portion of the ligand-binding domain of the thyroid hormone receptor (TR) genes in several neotenic amphibians: the obligatory neotenic members of the family Proteidea the mudpuppy Necturus maculosus and Proteus anguinus as well as two members of the facultative neotenic Ambystoma genus: the axolotl Ambystoma mexicanum and the tiger salamander Ambystoma tigrinum. In addition, we looked for TR genes in the genome of an apode Typhlonectes compressicaudus. TR genes were found in all these species including the obligatory neotenic ones. The PCR fragments obtained encompass both the C and E domains and correspond to alpha and beta genes. Their sequences appear to be normal, suggesting that there is no acceleration of evolutionary rates in the TR genes of neotenic amphibians. This result is not surprising for Ambystomatidae, which are known to respond to T3 (3,3',5-triiodothyronine) but is not in agreement with biochemical and biological data showing that Proteidea cannot respond to thyroid hormones. Interestingly, by RT-PCR analysis we observed a high expression levels of TRalpha in gills, intestine, and muscles of Necturus as well as in the liver of Ambystoma mexicanum, whereas TRbeta expression was only detected in Ambystoma mexicanum but not in Necturus. Such a differential expression pattern of TRalpha and TRbeta may explain the neoteny in Proteidea. The cloning of thyroid-hormone-receptor gene fragments from these species will allow the molecular study of their failure to undergo metamorphosis.