Suppression of the Ewing's sarcoma phenotype by FLI1/ERF repressor hybrids.

Fusion of the 5' half of the Ewing's sarcoma (ES) gene EWS with the DNA-binding domain of several transcription factors has been detected in many human tumors. The t(11;22)(q24;q12) chromosomal translocation is specifically linked to ES and primitive neuroectodermal tumors and results, in the majority of cases, in the fusion of the amino terminus of the EWS gene to the carboxyl-terminal DNA-binding domain of the FLI1 gene. The chimeric protein has been shown to be oncogenic, a potent transcriptional activator, and necessary for the maintenance of the Ewing's phenotype, making it an attractive target for gene therapy. In this study, we demonstrate that the ES transformed phenotype can be suppressed by chimeric transcriptional repressors containing the DNA-binding domain of FLI1 and the regulatory and repressor domain of ERF, a transcription suppressor and member of the ets gene family. The hybrid repressor is expressed at levels comparable with EWS/FLI1, does not affect EWS/FLI1 expression, and exhibits similar DNA-binding specificity but suppresses transcriptional activity. The FLI1/ERF repressor, like the wild-type ERF, is regulated by mitogen-activated protein kinase-dependent subcellular localization. Our data suggest that transformation by EWS/FLI1 may partially be due to activation of specific EWS/FLI1-regulated genes involved in cell proliferation.

FLI-1 is a suppressor of erythroid differentiation in human hematopoietic cells.

The FLI-1 oncogene, a member of the ETS family of transcription factors, is associated with both normal and abnormal hematopoietic cell growth and lineage-specific differentiation. We have previously shown that overexpression of FLI-1 in pluripotent human hematopoietic cells leads to the induction of a megakaryocytic phenotype. In this report we show that FLI-1 also acts as an inhibitor of erythroid differentiation. Following the induction of erythroid differentiation, pluripotent cells express reduced levels of FLI-1. In contrast, when FLI-1 is overexpressed in these cells, the levels of erythroid markers are reduced. The ability of FLI-1 overexpressing cells to respond to erythroid-specific inducers such as hemin and Ara-C is also inhibited, and the uninduced cells show a reduced level of the erythroid-associated GATA-1 transcription factor mRNA. Furthermore, expression of a GATA-1 promoter-driven reporter construct in K562 cells is inhibited by co-transfection with a construct expressing FLI-1. Our results support the hypothesis that FLI-1 can act both positively and negatively in the regulation of hematopoietic cell differentiation, and that inhibition of GATA-1 expression may contribute to FLI-1-mediated inhibition of erythroid differentiation.

Proteins of the ETS family with transcriptional repressor activity.

ETS proteins form one of the largest families of signal-dependent transcriptional regulators, mediating cellular proliferation, differentiation and tumorigenesis. Most of the known ETS proteins have been shown to activate transcription. However, four ETS proteins (YAN, ERF, NET and TEL) can act as transcriptional repressors. In three cases (ERF, NET and TEL) distinct repression domains have been identified and there are indications that NET and TEL may mediate transcription via Histone Deacetylase recruitment. All four proteins appear to be regulated by MAPKs, though for YAN and ERF this regulation seems to be restricted to ERKs. YAN, ERF and TEL have been implicated in cellular proliferation although there are indications suggesting a possible involvement of YAN and TEL in differentiation as well. Other ETS-domain proteins have been shown to repress transcription in a context specific manner, and there are suggestions that the ETS DNA-binding domain may act as a transcriptional repressor. Transcriptional repression by ETS domain proteins adds an other level in the orchestrated regulation by this diverse family of transcription factors that often recognize similar if not identical binding sites on DNA and are believed to regulate critical genes in a variety of biological processes. Definitive assessment of the importance of this novel regulatory level will require the identification of ETS proteins target genes and the further analysis of transcriptional control and biological function of these proteins in defined pathways.

Transcriptional repressor ERF is a Ras/mitogen-activated protein kinase target that regulates cellular proliferation.

A limited number of transcription factors have been suggested to be regulated directly by Erks within the Ras/mitogen-activated protein kinase signaling pathway. In this paper we demonstrate that ERF, a ubiquitously expressed transcriptional repressor that belongs to the Ets family, is physically associated with and phosphorylated in vitro and in vivo by Erks. This phosphorylation determines the ERF subcellular localization. Upon mitogenic stimulation, ERF is immediately phosphorylated and exported to the cytoplasm. The export is blocked by specific Erk inhibitors and is abolished when residues undergoing phosphorylation are mutated to alanine. Upon growth factor deprivation, ERF is rapidly dephosphorylated and transported back into the nucleus. Phosphorylation-defective ERF mutations suppress Ras-induced tumorigenicity and arrest the cells at the G0/G1 phase of the cell cycle. Our findings strongly suggest that ERF may be important in the control of cellular proliferation during the G0/G1 transition and that it may be one of the effectors in the mammalian Ras signaling pathway.

ETS-1 induces increased expression of erythroid markers in the pluripotent erythroleukemic cell lines K562 and HEL.

Members of the ETS gene family are known to be expressed in hematopoietic tissues and cell lines, and there is increasing evidence that ETS proteins may play a role in normal hematopoietic cell development. We demonstrate that ETS-1 can contribute to the development of an erythroid phenotype in vitro. The pluripotent erythroleukemic K562 and HEL cell lines express messages for a number of ETS genes, but only c-ETS-1 levels are elevated in response to treatment with hemin or cytosine arabinofuranoside (Ara-C), agents which induce erythroid differentiation. Furthermore, ETS-1 antisense oligonucleotides inhibit hemoglobinization of cells treated with Ara-C or hemin, and K562 and HEL cells infected with retrovirus expressing the c-ETS-1 gene exhibit a significant increase in erythroid character (as indicated by benzidine staining for hemoglobin (Hb) and surface marker analysis), a dramatic increase in responsiveness to hemin or Ara-C, and a decreased rate of proliferation (20-40% of control rates). In contrast, infection with virus expressing ETS-2 or vector sequences only causes no detectable changes in the proliferation or erythroid character of either the HEL or K562 cell lines. These data indicate a role for ETS-1 in erythroid differentiation.

ERF: genomic organization, chromosomal localization and promoter analysis of the human and mouse genes.

ERF (Ets2 Repressor Factor) is a ubiquitously expressed ets-domain protein that exhibits strong transcriptional repressor activity, has been shown to suppress ets-induced transformation and has been suggested to be regulated by MAPK phosphorylation. We report here the sequence of the mouse gene, the genomic organization of the human and the mouse genes, their chromosomal position and the analysis of the promoter region. Genomic clones encompassing either the human ERF or the mouse Erf gene were isolated and utilized to define their molecular organization. The gene in both species consists of 4 exons over a 10 kb region. Utilizing FISH, somatic cell hybrids and linkage analysis, we identified the chromosomal position of ERF on human chromosome 19q13.1 and on its syntenic region in the mouse, on chromosome 7. Sequence analysis of the mouse gene indicated a 90% identity to the human gene within the coding and promoter regions. The predicted Erf protein is 98% identical to the human protein and all of the identifiable motifs are conserved between the two proteins. However, the mouse protein is three amino acids longer (551 versus 548 aa). The area surrounding the region that is homologous to the 5' end of the human cDNA can serve as a promoter in transfection into eukaryotic cells. This region is highly conserved between the mouse and the human genes. A number of conserved transcription factor binding sites can be identified in the region including an ets binding site (EBS). Interestingly, removal of a small segment that includes the EBS, seriously hampers promoter function, suggesting the ERF transcription may be regulated by ets-domain proteins.

Increased expression of the ETS-related transcription factor FLI-1/ERGB correlates with and can induce the megakaryocytic phenotype.

The human leukemia cell line K562 can be induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) to differentiate along the megakaryocytic pathway, generating morphological changes and increased expression of lineage-specific surface markers. We report that TPA-treated K562 cells also express higher levels of FLI-1/ERGB, a member of the ETS family of transcription factors. Furthermore, introduction of a retroviral construct expressing human FLI-1/ERGB into K562 cells induces changes similar to those seen following TPA treatment, including increased adherence to the surface of the culture vessel and altered size and morphology. Infected cells exhibit higher levels of the megakaryocyte marker CD41a and, to a lesser extent, CD49b. These markers, as well as virally encoded FLI-1/ERGB-specific RNA and protein, are expressed at the highest levels in the attached cell population, while the growth rate of adherent cells is reduced, and the fraction of cells in G0-G1 is increased. FLI-1/ERGB virus-infected cells also exhibit increased expression of hemoglobin, a marker of erythroid differentiation. Our results suggest FLI-1/ERGB plays a role in controlling differentiation and gene expression along the megakaryocyte/platelet pathway, and further implicate ETS-related genes in the control of multiple developmentally regulated hematopoietic genes.

Signal requirements for interleukin 4 promoter activation in human T cells.

We have studied the signal requirements for human IL-4 promoter activation in Jurkat T cells by the use of DNA transfection assays with vectors carrying the IL-4 promoter linked to a reporter gene. Stimulation with calcium (Ca2+) ionophores (ionomycin), but not with phorbol esters (phorbol myristate acetate, PMA) or cyclic AMP elevating agents (prostaglandin E2, PGE2), induced the transcriptional activity of the IL-4 promoter by approximately 3-fold. Costimulation with ionomycin and PGE2 resulted in the same level of IL-4 promoter activity as the stimulation with ionomycin alone. In contrast, costimulation with ionomycin and PMA decreased the activity of the IL-4 promoter by approximately 40% compared to stimulation with ionomycin alone. Induction of Il-4 promoter by ionomycin was partially inhibited (approximately 50% inhibition) in the presence of as high as 2 microgram/ml cyclosporin A (CsA), an inhibitor of the Ca+/calmodulin-dependent phosphatase calcineurin. Under the same conditions, only 0.1 microgram/ml of CsA inhibited by >95% the transactivation of the IL-2 promoter in response to ionomycin and PMA. Transfection of a deletion mutant of the calcineurin catalytic subunit (delta CaM-AI) known to have Ca2+-independent, constitutive phosphatase activity increased IL-4 promoter activity by approximately 14-fold. Stimulation with ionomycin of cells transfected with low doses of delta CaM-AI, further induced IL-4 promoter activity by approximately 2-fold. These results identify the Ca2+-signaling system as a key component of the signal transduction pathway leading to IL-4 promoter activation in Jurkat T cells and suggest a major role of calcineurin in its regulation.

The Gag-Myb-Ets fusion oncogene alters the apoptotic response and growth factor dependence of interleukin-3 dependent murine cells.

Expression of the avian E26-derived Gag-Myb-Ets fusion oncogene in interleukin-3(IL3)-dependent murine hematopoietic cell lines results in a pattern of cell line dependent changes in growth factor-induced proliferation and apoptosis. A drug-selectable retrovirus expressing p135Gag-Myb-Ets induced an erythropoietin(Epo)-responsive phenotype in the cell lines FDC-P2, BaF3 and 32Dc123. Gag-Myb-Ets expression alone did not increase expression of GATA-1 or the Epo receptor(EpoR) in the presence of IL3, and infected cell lines express increased GATA-1 and EpoR only when IL3 was replaced by Epo in the culture media. Indicative of Epo-induced erythroid differentiation, these cells also began to express beta-globin after 3-5 days growth in Epo. Unlike control cells, infected FDC-P2 cells failed to undergo programmed cell death (apoptosis) when transferred from IL3- to Epo-containing media, although a fraction of the cells failed to proliferate following the media shift. Three other IL3-dependent cell lines showed no changes in growth behavior when induced to express the fusion oncogene. Our data shows that Gag-Myb-Ets can have different affects on growth factor pathways depending on the cell background, suggesting a model in which the p135gag-myb-ets fusion oncogene promotes these different responses through its affect on apoptosis.

ERF: an ETS domain protein with strong transcriptional repressor activity, can suppress ets-associated tumorigenesis and is regulated by phosphorylation during cell cycle and mitogenic stimulation.

ERF (ETS2 Repressor Factor) is a novel member of the ets family of genes, which was isolated by virtue of its interaction with the ets binding site (EBS) within the ETS2 promoter. The 2.7 kb ubiquitously expressed ERF mRNA encodes a 548 amino acid phosphoprotein that exhibits strong transcriptional repressor activity on promoters that contain an EBS. The localization of the DNA-binding domain of the protein at the N-terminus and th repression domain at the C-terminus is reminiscent of the organization of ELK1-like members of the ets family; however, there is no significant homology between ERF and ELK1 or any other ets member outside the DNA-binding domain. The repressor activity of ERF can antagonize the activity of other ets genes that are known transcriptional activators. Furthermore, ERF can suppress the ets-dependent transforming activity of the gag-myb-ets fusion oncogene of ME26 virus. Although ERF protein levels remain constant throughout the cell cycle, the phosphorylation level of the protein is altered as a function of the cell cycle and after mitogenic stimulation. The ERF protein is also hyperphosphorylated in cells transformed by the activated Ha-ras and v-src genes and the transcription repressor activity of ERF is decreased after co-transfection with activated Ha-ras or the kinase domain of the c-Raf-1 gene, indicating that ERF activity is probably regulated by the ras/MAPK pathway. Consistent with the in vivo phosphorylation and inactivation by ras, ERF is efficiently phosphorylated in vitro by Erk2 and cdc2/cyclin B kinases, at sites similar to those detected in vivo. Furthermore, a single mutation at position 526 results in the loss of a specific phosphopeptide both in in vivo and in vitro (by Erk2) labeling. Substitution of Thr526 for glutamic acid also decreases the repression ability of ERF. Our data suggest a model in which modulation of ERF activity is involved in the transcriptional regulation of genes activated during entry into G1 phase. Obstruction of the ERF repressor function by the transactivating members of the ets family of genes (i.e.gag-myb-ets) may be essential for the control of genes involved in cell proliferation and may also underlie their tumorigenic effects.

ets-1 in astrocytes: expression and transmitter-evoked phosphorylation.

The ets-1 protein has been primarily studied as a sequence-specific transcriptional regulator that is predominately expressed in lymphoid cells. In this report, we show that ets-1 is also expressed in astrocytes and astrocytoma cells and is regulated during both signal transduction and differentiation. Both isoforms of ets-1, p51 and p42, were found in astrocytes and astrocytoma cells, but whereas expression of p51 was strong, p42, the alternate splice product previously shown to lack the phosphorylation domain, was difficult to detect and was present at a level 10- to 40-fold lower than that of p51. This differed by roughly an order of magnitude from the ratio generally observable in T cells and thymocytes. In two astrocytoma lines of human origin, CCF and 1321N1, ets-1 phosphorylation was stimulated by bradykinin and carbachol, respectively. Glutamate, norepinephrine, and bradykinin elicited phosphorylation of p51 in cultures of primary rat type 1 astrocytes. ets-1 phosphorylation was dramatically blocked by KT5926, an inhibitor of myosin light-chain kinase, suggesting that this kinase may be involved in phosphorylation of ets-1 in vivo. Investigations of retinoic acid-induced differentiation in P19 cells provided further support for a strong correlation of ets-1 with the pathway for astrocyte differentiation.

Structural inferences of the ETS1 DNA-binding domain determined by mutational analysis.

The ets family of transcription factors is characterized by a conserved region that harbors the DNA-binding activity. We performed extensive deletion and mutational analyses, as well as DNA-peptide interaction studies necessary to identify the determinants of the DNA-binding activity of the ETS1 oncoprotein. We found that amino acids beyond the 85 amino acid conserved region are required in order to afford maximum DNA-binding activity in a heterologous system. Mutation throughout the binding domain can have a detrimental effect on binding activity, indicating that proper folding of the entire domain is necessary for DNA binding. A peptide, as small as 37 residues (K37N), derived from the basic region of the ETS1 binding domain, is sufficient to exhibit sequence-specific DNA recognition. Total randomization of Lysine 379, Lysine 381 and Arginine 391 within this region fails to provide functional substitutions, indicating that these specific amino acids within the basic region are required for binding. Transactivation activity of the ETS1 proteins bearing mutations was consistent with their DNA-binding activity, indicating that the primary (if not only) function of this domain is to provide sequence-specific DNA recognition activity. Our mutational analysis, as well as modeling predictions, lead us to propose a helix-turn-helix structure for the basic region of the ETS1 binding domain that is able to interact directly with DNA. We also propose that the hydrophobic alpha-helical region, surrounding tryptophan 338, is fundamental for proper protein folding and functioning of the ets binding domain.

Human ERG-2 protein is a phosphorylated DNA-binding protein--a distinct member of the ets family.

We describe the identification of the ERG-2 gene products using an antibody raised against recombinant human ERG-2 protein. ERG-2 is a nuclear phosphoprotein and binds to purine-rich sequences (C/G)(C/a)GG-AA(G/a)T. ERG-2 protein, with a half-life of 21 h, is considerably more stable than the short-lived ETS-1 or ETS-2 proteins. Its phosphorylation is stimulated by phorbol myristate acetate (PMA), but not by Ca2+ ionophore treatment. ETS-1 protein is phosphorylated by Ca(2+)-dependent events, whereas ERG-2 protein is phosphorylated by activation of protein kinase C, suggesting their involvement in distinct signal transduction mechanisms. The expression of ERG-2 protein is restricted to few cell types and is high in early myeloid cells, indicating that it may function at an early stage of hematopoietic lineage determination. The DNA-binding sequence for ERG-2 protein is identified by using a random oligonucleotide selection procedure. The selected sequence is very similar to the binding sequence determined for human ETS-1 using the same method. Like other ets proteins, ERG-2 is a sequence-specific DNA-binding protein and is expressed at higher levels in early myeloid cells than in mature lymphoid cells. These results suggest that it may act as a regulator of genes required for maintenance and/or differentiation of early hematopoietic cells.

Human ETS1 oncoprotein. Purification, isoforms, -SH modification, and DNA sequence-specific binding.

The human ETS1 proto-oncogene proteins have been isolated from the T-cell leukemia line, CEM, by immunoaffinity chromatography and their identity confirmed by NH2-terminal amino acid sequencing. Incubation of CEM cells with N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) indicates that ETS proteins can be modified in their cellular context and that pretreatment of the cells with N-ethylmaleimide (NEM) protects ETS1 proteins from TLCK modification. These data show that ETS1 proteins can exist in at least two different states, -SH-available and -SH-protected. Renatured human ETS1 has DNA sequence-specific binding to the PEA3 (CAGGAAGT) motif. The ETS1.PEA3 complex can be observed by electrophoretic mobility shift assays (EMSA). Purified ETS1 retards a band which is exactly the same size as a complex that is retarded from nuclear extracts prepared from CEM cells. Reduced ETS1 is required to form the ETS1.PEA3 complex, however; modification of the ETS1 -SH groups by either NEM or by TLCk does not inhibit formation of the complex. The ETS1.PEA3 complex formed with TLCK-modified ETS1 has a slower mobility than the complex formed with unmodified ETS1. Zone sedimentation analysis of purified ETS1 indicates that it is the monomer of ETS1 which binds to the PEA3 oligonucleotide.

High-affinity DNA-protein interactions of the cellular ETS1 protein: the determination of the ETS binding motif.

ETS1 protein purified from CEM cells was used to select its optimum DNA-binding sequence (pu) G/CCaGGA-AGTc (py). The sequence CCGGAAGT (ETS1-3) was preferred 5:1 over CAGGAAGT (PEA3). Quantitative electrophoretic mobility-shift assays (EMSA) indicated that the purified ETS1 protein binds to either ETS1-3 or PEA3 oligonucleotide probes with high affinity (Ka = 0.5-4.0 x 10(10) M-1) and that the purified ETS1 has different binding capacities for ETS1-3 and PEA3 oligonucleotide probes. The ETS1 protein binds 2-5 times more ETS1-3 than PEA3. Competitive binding experiments showed that the ETS1-3 and PEA3 probes effectively compete for the binding of ETS1-3. However, changing the core DNA-binding sequence from GGAA to AGAA eliminates competition. Since the human ETS1 protein selected the same DNA sequence from a mixture of random oligonucleotides as did the Drosophila E74A protein (one of the most divergent members of the ETS family), this strongly suggests that all proteins containing the ETS 85 amino acid domain (sequences which define the ETS family) will bind to the same sequence.

Positive and negative factors regulate the transcription of the ETS2 gene via an oncogene-responsive-like unit within the ETS2 promoter region.

The DNA-protein interactions in the ETS2 promoter have been studied. Three distinct sequence motifs have been identified, each of which interacts with at least two distinctive protein complexes. The GC motif, possessing mirror symmetry, interacts with two ubiquitously identifiable complexes (S and S2); the PEA3 motif interacts with a ubiquitous (H1) and a tissue-specific (H3) complex; the H2 (an AP1-like) motif interacts also with a ubiquitous (H2a) and a tissue-specific (H2b) complex. Mutational analysis and correlation of the presence of defined complexes with the ETS2 mRNA levels indicate that the S, S2, H1, and H2b complexes have positive effects on ETS2 transcription, whereas the H3 and H2a have negative effects. The organization of the PEA3 with the AP1-like motif in the ETS2 promoter resembles the oncogene-responsive unit previously identified in the polyoma virus enhancer region. Our data suggest that cooperation between these two motifs is vital for ETS2 promoter function.

The human ETS1 gene: genomic structure, promoter characterization and alternative splicing.

Genomic clones encompassing the human ETS1 gene were isolated and utilized to define its molecular organization. This gene consists of eight exons spanning over 60 kb. The 5' end of the human ETS1 gene was subcloned and characterized. S1 nuclease, primer extension and RNAase protection analyses of human mRNAs showed multiple transcription initiation sites. DNA sequence analysis indicated a high G + C content in the promoter region and the absence of either a 'TATA' box or a 'CAAT' box. Six consensus recognition sequences for the transcription factor SP1, two AP1 consensus sequences and one consensus AP2 recognition sequence were identified, as well as two GC elements with dyad symmetry. A palindromic region similar to the serum response element of the c-fos gene and two octamer consensus recognition sequences were located upstream of the promoter region. A series of promoter deletion constructs positioned upstream from the bacterial chloramphenicol acetyl transferase gene were transfected into HeLa cells and their functional promoter activity assayed. The deletion constructs identified the 5' boundary for maximum promoter activity at 486 bp upstream of the first initiation site and suggest possible positive and negative regulatory regions. Polymerase chain reaction analysis of ETS1 cDNA identified several amplified products, indicating alternative splicing. In addition to the presence of mRNA products lacking exon VII, products lacking exon IV, as well as ones lacking both exons IV and VII, were found.

Defining target sequences of DNA-binding proteins by random selection and PCR: determination of the GCN4 binding sequence repertoire.

We developed a simple and accurate method to define the sequence recognition properties of DNA-binding proteins. The method employs polymerase chain reaction (PCR) amplification of sequences selected from a mixture of random oligonucleotides by the gel mobility-shift assay. We used this method to define the sequence requirement of the binding domain of the yeast transcriptional activator GCN4. Using a total of 200 ng of purified protein and four cycles of binding and subsequent amplification, we identified the TGA-(C/G)TCA sequence as the binding consensus of GCN4, which is consistent with the previously reported recognition sequence. In addition, our data indicate that GCN4 can bind with lower affinity to sequences that differ from the optimal sequence in one or even two positions. The most common variation was the C to A at position +2. The majority of the substitutions that still allowed binding were 3' to the central C residue indicating that the two sides of the palindromic recognition sequence are not equivalent.

Molecular organization and differential polyadenylation sites of the human ETS2 gene.

The human ETS2 gene is a member of the ets gene family (ETS1, ETS2, ERG, ELK1 and ELK2) with amino acid similarity to the v-ets oncogene of the avian leukemia virus, E26. The ETS2 gene is composed of 10 exons, nine of which contribute to the open reading frame encoding 469 amino acids. The ETS2 gene directs the synthesis of three RNA transcripts, which differ from each other by the length of their 3' ends. This heterogeneity of 3' end is the major reason for the size differences between the transcripts, presumably reflecting utilization of three different polyadenylation signals/sites. The coding regions of all of the ETS2 RNA species are the same length and, thus, should contain the same open reading frame.