|18 December 2000, Volume 19, Number 55, Pages 6482-6489|
|Table of contents Previous Article Next [PDF]
|The role of Fli-1 in normal cell function and malignant transformation|
|Amandine HL Truong1,2 and Yaacov Ben-David1,2|
1Division of Cancer Biology Research, Sunnybrook and Women's College Health Sciences Centre & Toronto-Sunnybrook Regional Cancer Centre (TSRCC), 2075 Bayview Avenue, S-Wing, Room S216, Toronto, Ontario, Canada M4N 3M5
2Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9
Correspondence to: Y Ben-David, Division of Cancer Biology Research, Sunnybrook and Women's College Health Sciences Centre & Toronto-Sunnybrook Regional Cancer Centre (TSRCC), 2075 Bayview Avenue, S-Wing, Room S216, Toronto, Ontario, Canada M4N 3M5
Aberrant expression of the Fli-1 transcription factor following genetic mutation has been recognized as a seminal event in the initiation of certain types of malignant transformation. Indeed, the etiology of a number of virally induced leukemias, including Friend virus-induced erythroleukemia, has been associated with Fli-1 overexpression. The clinical relevance of Fli-1 becomes apparent in human Ewing's sarcoma in which Fli-1 is the target of a characteristic chromosomal translocation. As such, Fli-1 has generated considerable interest over the past several years for its role in malignant transformation and tumor progression. This review will present a synopsis of the current research on Fli-1 with emphasis on its function in malignant transformation. Moreover, the possible role of Fli-1 in cellular proliferation, differentiation and survival, as well as the recent development of transgenic and knock-out mice to investigate the function of Fli-1 will be discussed. Finally, the significance of identifying target genes that are regulated by Fli-1 and their role in cellular function will be reviewed. Oncogene (2000) 19, 6482-6489.
Fli-1; transcription factor; gene regulation; cancer
Fli-1 was first identified as a common site for retroviral integration in Friend virus-induced erythroleukemias (Ben-David et al., 1991a). For two decades, the Friend virus model of leukemogenesis has been used to study the multi-stage nature of cancer. Two separate isolates of Friend virus, termed FV-A and FV-P, have been identified, both existing as complexes of two distinct viral species. The first is a unique replication defective spleen focus-forming virus (SFFV-A and SFFV-P) and the second is a common replication competent Friend murine leukemia virus (F-MuLV) (Ben-David and Bernstein, 1991b). Dissection of the mechanisms underlying Friend-virus induced erythroid transformation has led to the identification of the Spi-1 locus as a preferential site for proviral integration of FV-A and FV-P (Moreau-Gachelin et al., 1988). Spi-1 was subsequently shown to be identical to PU.1, a member of the ets family of transcription factors (Gobel et al., 1990).
The helper virus (F-MuLV) alone has been shown to induce erythroleukemias when injected into susceptible strains of newborn mice (Silver and Kozak, 1986). Analysis of the sites of proviral integration has revealed rearrangements at the Fli-1 locus in most F-MuLV-induced erythroleukemias (Ben-David et al., 1990; Sels et al., 1992). Similarly, proviral insertion near the Fli-1 gene was also detected in early hematopoietic cells by the 10A1 viral isolate of MuLV (Ott et al., 1994), in granulocytic leukemia induced by the Graffi virus (Denicourt et al., 1999) and in non-T and non-B lymphomas induced by the Cas-Br-E virus (Bergeron et al., 1991). Interestingly, the orientation of F-MuLV proviral integration within the Fli-1 locus is in opposite orientation to the direction of transcription, while integration by the Cas-Br-E virus has been shown to be in the same transcriptional orientation, implying that these two viruses activate Fli-1 expression by two distinct mechanisms. Like Spi-1/PU.1, the identity of the transcriptional unit activated at the Fli-1 locus corresponds to a novel transcription factor that belongs to the ets oncogene family (Ben-David et al., 1991a). Interestingly, similar to Fli-1 and Spi-1/PU.1, the v-ets oncogene has also been shown to induce erythroid transformation as a result of a fusion of the gag and myb proteins of the E26 avian retrovirus (Leprince et al., 1983). Taken together, it seems likely that members of the ets family of transcription factors are implicated in malignant transformation (reviewed by Blair in this issue).
Fli-1 characteristics and its position amongst other ets genes
Subsequent to the cloning of mouse Fli-1, the human Fli-1 gene was isolated by sequence homology (Prasad et al., 1992; Watson et al., 1992). Both human and mouse Fli-1 encode two proteins, p51 (452aa) and p48 (419aa). Pulse-field gel analysis has localized the Fli-1 gene within 240 kb of the Ets-1 locus on mouse chromosome 9 and on human chromosome 11q23, suggesting that these two ets genes arose by gene duplication from a common ancestral gene (Ben-David et al., 1991a; Watson et al., 1992). Human Fli-1 contains nine exons which extend over approximately 120 kb (Selleri et al., 1994). A comparison of the amino acid sequences of Fli-1 revealed an 81% homology to the ets-related protein Erg-2 (Prasad et al., 1992). Both Fli-1 and Erg-2 contain two regions designated as the 5' and 3' ets domains which are homologous to those found in the Ets-1 and Ets-2 proteins (Figure 1). The 5' ets domain of Fli-1 (amino acids 121-196) shows 82% sequence identity to Erg-2 and 59-60% to c-Ets-1 and Ets-2.
A Fli-1 specific region (FLS), which is absent in the Erg-2 protein, has been localized within amino acids 205-292. Analysis of the secondary structure of Fli-1 revealed the presence of helix 1-loop-helix 2 (H-L-H) structures in the 5' and 3' ets domains that are also present in Erg-2 (Figure 1) (Rao et al., 1993). The FLS and 5' ets domains contain sequences responsible for transcriptional activation and are herein referred to as the ATA domain. In addition, a region located in the carboxy-terminus of Fli-1, the CTA domain (amino acids 402-452), is also involved in transcriptional activation. The FLS and CTA domains contain sequences which resemble turn-loop-turn (T-L-T) secondary structures.
The presence of H-L-H and T-L-T structures in the ATA and CTA domains suggest that these structures contribute to the transcriptional activity of the Fli-1 protein, likely through interactions with other transcription factors. In the absence of the ATA domain, the CTA domain's ability to activate transcription is significantly impaired. Interestingly, in the absence of the CTA domain, the ATA domain alone results in a significant increase in the transcription of a downstream reporter gene, when compared to the wild-type Fli-1 protein (Rao et al., 1993). As such, it has been suggested that the CTA region may serve simultaneously as a transcriptional activator and repressor (Rao et al., 1993). Recently, mice engineered to lack the CTA domain of Fli-1 by homologous recombination were shown to express negligible to low levels of the mutant of Fli-1 mRNA and protein (Spyropoulos et al., 2000). Furthermore, the recombinant Fli-1 protein lacking the CTA domain displayed only 50-60% of the transcriptional activity of wild-type Fli-1, providing further evidence of the CTA domain's involvement in transcriptional activation. The reduced levels of mutant Fli-1 in these mice seems to suggest that the CTA domain in fact also acts in a self-regulatory fashion to regulate Fli-1 expression. Finally, the 3' ets domain (amino acids 277-360) which is highly conserved between Fli-1 and Erg-2 (98% homology) is found to be responsible for sequence specific DNA-binding activity (Figure 1).
To further understand the DNA binding properties of Fli-1, the structure of human Fli-1 bound to DNA was determined using multidimensional NMR spectroscopy. These analyses have shown that the 3' ets domain of Fli-1 consists of three -helices and a four-stranded -sheet that resembles the structures of the class of helix-turn-helix DNA-binding proteins found in the catabolite activator protein of Escherichia coli, as well as those of several eukaryotic DNA binding proteins including H5, HNF-3/fork head, and the heat shock transcription factor (Liang et al., 1994a,b). A comparison of the Fli-1 3' ets domain to other structures has suggested that this 3' ets domain uses a new variation of the winged helix-turn-helix motif for binding to DNA (Liang et al., 1994b).
Expression of Fli-1 in tumors and tissues
Although Fli-1 is expressed at high levels in F-MuLV-induced erythroleukemias and to a lesser extent in FV-P/FV-A-induced erythroleukemias (Ben-David et al., 1991a), its expression has only been detected in a subset of human erythroleukemia cell lines and cell lines derived from other epithelial cancers (Klemsz et al., 1994; Watson et al., 1992). In adult tissues, however, Fli-1 expression is detected at high levels in hematopoietic tissues and at lower level in some non-hematopoietic tissues such as the lung, the heart, and the ovaries (Ben-David et al., 1991a; Watson et al., 1992). Interestingly, in macrophages, the level of Fli-1 transcription is significantly down-regulated in response to the inflammatory mediators lipopolysaccharide (LPS) and interferon- as well as the differentiation-inducing agents retinoic acid and forskolin (Klemsz et al., 1994). As such, a reduction in Fli-1 expression may be required during immune activation or cellular differentiation. During development, Fli-1 is preferentially expressed in hematopoietic cells, endothelial cells and in the mesenchyme which is mainly derived from neural crest cells (Melet et al., 1996). In zebrafish embryos, Fli-1 expression is detected in sites of vasculogenesis suggesting a role for Fli-1 in blood vessel formation (Brown et al., 2000). The avian Fli-1 gene is specifically expressed during embryogenesis in a subset of neural crest cells giving rise to developing mesenchyme (Mager et al., 1998, see also review by Remy and Balzinger in this issue).
Promoter activity and alternative splicing of Fli-1
The promoters of both mouse and human Fli-1 genes have been isolated and sequence analysis revealed a high level of conservation between the two species (Barbeau et al., 1996; Dhulipala et al., 1998). Several regulatory elements conserved between these two species have been found in the Fli-1 promoter which include binding sites for GATA-1, ets, Sp1, c-Myc, Oct-3, TBP and AP1 (Barbeau et al., 1999; Dhulipala et al., 1998). Starck et al. (1999) demonstrated that in F-MuLV induced-erythroleukemias that have acquired an insertionally activated Fli-1, initiation of transcription started at positions -398 and -204 (Figure 2). However, in erythroleukemias bearing an intact Fli-1 gene, the major site for transcriptional initiation is located at the -204 region (Starck et al., 1999). The promoter activity is localized within position -270 to -41 of the Fli-1 gene, which contains a GATA consensus site as well as two ets binding sites (EBSs). Indeed, introducing mutations within these EBSs abolished the promoter activity. Screening for factors that bind these regions has led to the identification of Spi-1/PU.1, which was shown to activate Fli-1 transcription in erythroleukemic cells by virtue of this interaction.
An alternative spliced form of Fli-1, referred to as Fli-1b, has also been identified and its expression is mainly detected in two human B cell leukemias (Dhulipala et al., 1998). This splice form uses the alternative exon 1b, whose synthesis is initiated 1.8 kb upstream of the other Fli-1 transcripts (Figure 2). It is likely that the upstream sequences of the main Fli-1 initiation site are capable of potentiating promoter activity (Starck et al., 1999). However, the promoter activity of the Fli-1 5' sequences was not detected by others (Barbeau et al., 1996). Although this lack of promoter activity could be explained by the sensitivity of methods used for the analysis, it was shown that the sequences within intron 1 of Fli-1 contain several GATA and EBSs sequences that are capable of increasing the transcription of a minimal promoter (Barbeau et al., 1999). Cumulatively, it seems that regulation of Fli-1 transcription is a complex process involving a number of factors and several regulatory regions.
The complexity of Fli-1 transcription further extends to its translation. Fli-1 mRNA encodes two isoforms, one of 51 and the other 48 kDa. Recently, it has been demonstrated that synthesis of the 51 and 48 kDa isoforms were initiated from the codons AUG +1 and AUG+100 of Fli-1, respectively (Sarrazin et al., 2000). They demonstrated that the expression of these two isoforms is regulated by two short overlapping 5' upstream open reading frames beginning at two highly conserved upstream initiation codons, AUG -41 and GUG -37 as well as by two highly conserved stop codons, UGA +35 and UAA +15.
Regulation of gene expression by Fli-1
Fli-1 binds to DNA in a sequence specific manner
Due to the sequence homology between the 3' ets domains of Fli-1 and Erg-2, it is probable that these proteins recognize a similar DNA binding consensus sequence. Indeed, it has been shown to be the Ets recognition sequence (CCGGAAGT) found within the Drosophila E74 promoter (Rao et al., 1993; Zhang et al., 1993). Other studies have demonstrated that the Ets-1 and Ets-2 consensus sequences are also recognized by Fli-1 (Klemsz et al., 1994; Watson et al., 1992). By utilizing the epitope-tagging strategy, it was later determined that the optimal DNA binding sequence for Fli-1 is ACCGGAAG/aT/c (Mao et al., 1994). This result was confirmed in a recent study which used biochemical selection of randomized sequences both upstream and downstream of the central GGA element (Szymczyna and Arrowsmith, 2000). This study also suggests that the bases flanking the GGA motif synergistically contribute to binding specificity among different ets proteins. The transcriptional activation of promoters containing these sequences has been shown to be up-regulated by Fli-1 in vitro, reflecting the sequence-specific nature of Fli-1 regulation.
As will be discussed below, Fli-1 is a major target for gene translocation in human Ewing's sarcoma (Delattre et al., 1992). The product of this translocation is a fusion protein that consists of the C-terminal region of Fli-1 and the N-terminus of a putative RNA-binding protein of the EWS gene (Figure 3). An examination of the DNA binding properties of Fli-1 and EWS/Fli-1 has revealed that both proteins recognize the same consensus DNA binding sequence which had previously been identified for Fli-1 (Bailly et al., 1994; Mao et al., 1994). However, it appears that the EWS/Fli-1 protein is a more potent transactivator than the wild-type protein (Bailly et al., 1994; May et al., 1993). Thus, in Ewing's sarcomas, the weak transactivation domain of Fli-1 is replaced by a powerful transactivation domain, thereby resulting in the enhanced expression of certain target genes and ultimately uncontrolled cell growth.
Identification of target genes for Fli-1
Analysis of the promoter sequences for EBSs resulted in the identification of several target genes for Fli-1. The chicken GATA-1 promoter contains several EBSs and was the first example of a gene transcriptionally induced by Ets1, Ets2 and Fli-1, as shown by transient transfection assays using COS and HeLa cells (Seth et al., 1993). In contrast, others have shown that ectopic expression of Fli-1 in chicken erythroblasts isolated from bone marrow did not result in a change in GATA-1 expression (Pereira et al., 1999). Interestingly, it has been recently reported that expression of Fli-1 in the human erythroleukemia cell line K562 results in a drastic reduction in the expression of GATA-1 coincidental with an inhibition in differentiation (Athanasiou et al., 2000). A negative correlation between the expression of Fli-1 and GATA-1 was also detected in a murine erythroleukemia cell line undergoing terminal differentiation by erythropoietin (Epo) (Tamir et al., 1999). These results raise the possibility that Fli-1 may activate or suppress the transcription of GATA-1, and that this regulation is cell-type specific.
The expression of Fli-1 has been detected in megakaryocytes and platelets (Bastian et al., 1999) and its ectopic expression in K562, a Fli-1 deficient erythroleukemia cell line resulted in megakaryocytic differentiation (Athanasiou et al., 1996). Interestingly, mice deficient in Fli-1 expression displayed a significant reduction in megakaryocyte numbers (Hart et al., 2000; Spyropoulos et al., 2000), suggesting a role for Fli-1 in megakaryopoiesis. Consistent with these results, a Fli-1 binding site has been identified in the promoters of several megakaryocytic-specific genes including glycoprotein IX (Bastian et al., 1999), the thrombopoietin receptor (MPL) (Deveaux et al., 1996) and glycoprotein IIb (GpIIb) (Bastian et al., 1999). Transcription from these promoters has been shown to be induced by Fli-1 in human erythroleukemic cells.
Members of the ets family of proteins have been previously implicated in angiogenesis (the formation of blood vessels from pre-existing ones). In addition to the contribution of Fli-1 to the hematopoietic cell lineage, Fli-1 expression has also been detected in endothelial cells (EC) (Melet et al., 1996). Mice with a null mutation at the Fli-1 locus die at day 11.5 of embryogenesis, due to a loss of vascular integrity (Hart et al., 2000), raising the possibility that Fli-1 is involved in the regulation of genes critical for neoangiogenesis. For instance, the human heme oxygenase gene, whose expression has been shown to play a role in EC proliferation and angiogenesis, contains several EBS sites that can be activated by Fli-1 as well as Erg-1 and Ets-1 (Deramaudt et al., 1999). Furthermore, mice lacking Fli-1 expressed significantly lower levels of Tek/Tie-2, an endothelial-specific receptor tyrosine kinase that has been shown to be critical for angiogenesis during development (Hart et al., 2000).
Additional promoters containing EBSs were identified and their transcription were shown to be induced by Fli-1 overexpression. These promoters include the human stress response gene GADD153 (Seth et al., 1999), the immunoglobulin (Ig) heavy-chain (Rivera et al., 1993) and the chicken anti-apoptotic gene Bcl-2 (Pereira et al., 1999). In addition, in vitro expressed Fli-1 protein was shown to up-regulate transcription of the Tenascin-C (TN-C) gene in cooperation with Sp1 (Shirasaki et al, 1999). In contrast, Fli-1 appears to suppress transcription of the Retinoblastoma (Rb) gene through direct binding competition with the protein complex RBF-1 on the ets site of the Rb promoter (Tamir et al., 1999).
Protein-protein interactions involving Fli-1
Members of the ets gene family such as ELK1 and SAP1 are capable of forming ternary complexes with SRF on the serum responsive elements (SRE) of the c-Fos promoter (Hipskind et al., 1991; Price et al., 1995). Interestingly, Fli-1 and EWS/Fli-1 have also been found to form ternary complexes with SRF on the Egr1 (Watson et al., 1997) and c-Fos SRE (Watson et al., 1997; Dalgleish and Sharrocks, 2000). Two regions surrounding the DNA binding domain (amino acids 220-229 and 392-411) have been identified (Figure 1) as the critical sites required for this interaction (Dalgleish and Sharrocks, 2000). However, it appears that Fli-1 interacts with SRF in a unique manner that differs from other ets family members.
An interesting protein-protein interaction has recently been demonstrated using the yeast two-hybrid system between Fli-1 and the transcription factor Tel, a new member of the ets oncogene family (Kwiatkowski et al., 1998). The Tel gene was originally identified due to a translocation which places it next to the platelet-derived growth factor receptor chain (PDGFR) in chronic myelomonocytic leukemia (CMML) (Golub et al., 1994). Tel has also been shown to be translocated next to other genes such as AML-1 in childhood pre-B cell acute lymphoblastic leukemia (ALL), as well as several other genes (for review, see Rowley, 1999; Rubnitz et al., 1999). In vivo and in vitro assays have demonstrated that the binding of wild-type Tel to Fli-1 inhibits Fli-1's transactivation ability without affecting its DNA binding properties. However, the Tel-AML-1 fusion protein was unable to inhibit Fli-1 transactivation (Kwiatkowski et al., 1998). Interestingly, in a number of cases involving Tel translocations, the remaining allele is deleted. It has therefore been hypothesized that the inactivation of Tel function as a result of translocation may remove its inhibitory effect on Fli-1 mediated transactivation, thereby contributing to the malignant phenotype in human leukemias. Fli-1 transactivation has also been inhibited by the overexpression of the retinoic acid receptor (RAR) in human erythroleukemic cells (Darby et al., 1997). It appears that Fli-1 and RAR can reciprocally repress the other's transcriptional activation. In addition, Fli-1 has also been reported to interfere with the functions of the thyroid and glucocorticoid receptors. While the mechanism of this repression has not yet been determined, it has been suggested that the indirect interaction of Fli-1 with RAR is through a bridging factor that remains to be identified.
Fli-1 activation is involved in malignant transformation
As Fli-1 has been shown to be a target of proviral integration in F-MuLV-induced erythroleukemia and subject to a translocation in human Ewing's sarcoma, the mechanisms underlying malignant transformation by this transcription factor have been the focus of many studies. During the induction of F-MuLV induced-erythroleukemias, it appears that insertional activation of Fli-1 is the first required genetic alteration, followed by mutations in the p53 tumor suppressor gene (Howard et al., 1993). Erythroleukemias induced due to Fli-1 activation were shown to proliferate in culture primarily in response to the growth factor erythropoietin (Epo). This observation suggests that there exists a synergistic cooperation between Epo signaling and Fli-1 which is necessary for erythroid transformation. As the dependency of erythroleukemic cells on Epo for proliferation seems critical, it was not surprising that these tumor cells eventually acquired Epo independence by autocrine production of this growth factor (Howard et al., 1996).
Secretion of Epo in turn results in the constitutive stimulation of the Epo-R and hence continuous activation of the signaling pathways downstream of this receptor. Studies of a number of systems have provided useful insight on the role of Fli-1 with respect to Epo-R signaling and cellular differentiation. For instance, ectopic expression of Fli-1 in primary avian erythroblasts was shown to prevent terminal differentiation despite their stimulation with Epo (Pereira et al., 1999). In addition, expression of Fli-1 in these cells increased their self-renewal capacity. Consistent with these findings, studies using a unique murine erythroleukemia cell line revealed that a transient downregulation of Fli-1 levels is required as the cells undergo terminal differentiation by Epo (Tamir et al., 1999). Ectopic expression of Fli-1 in these cells inhibits this downregulation, and consequently, the cells are unable to differentiate, but rather remain in a proliferative state in response to Epo stimulation. Fli-1 seemingly alters the responsiveness of erythroid cells to Epo, promoting their self-renewing potential rather than their maturation. In fact, the study of Epo-R signal transduction pathways that are activated by Epo stimulation in these cells has demonstrated that ectopic expression of Fli-1 in HB60-S cells results in the constitutive activation of the Shc/ras pathway, a pathway that has been implicated in self-renewal. Apparently, activation of this pathway overrides the Jak2/Stat5 pathway that is activated when the cells undergo terminal differentiation (Zochodne et al., 2000). Finally, the role of Fli-1 in blocking erythroid differentiation has been supported by two additional works in which Fli-1 was ectopically expressed in erythroleukemia cell lines. Fli-1 overexpression in the human erythroleukemia cell line K562 was shown to block differentiation in response to a chemical inducer by inhibiting expression of erythroid-associated transcription factors, such as GATA-1 (Athanasiou et al., 2000). Moreover, Fli-1 overexpression in a Friend virus-induced erytholeukemia cell line that expresses low levels of endogenous Fli-1 resulted in the inhibition of maturation in response to a chemical inducer of erythroid differentiation (Starck et al., 1999). Hence, these experiments propose a role for Fli-1 in modulating the response of erythroid progenitor cells to Epo stimulation. As such, aberrant Fli-1 activation can enhance malignant transformation by promoting uncontrolled cell division.
Additional evidence implicating Fli-1 in carcinogenesis derives from recent work demonstrating that Fli-1 is able to down-regulate the expression of the Retinoblastoma (Rb) protein at the transcriptional level (Tamir et al., 1999). Rb has been well established as a cell cycle regulator, modulating transition into the S phase. It binds to an array of transcription factors thereby repressing them from transcribing proteins required for cell cycle progression. As such, the Fli-1 down-regulation of Rb leads to the transition of the cell through the S phase.
While it seems that activation of Fli-1 has significant ramifications on erythroid proliferation, it is likely that genetic disruption of this gene would result in more profound cellular defects than previously thought. Indeed, overexpression of Fli-1 in fibroblasts has been shown to inhibit apoptosis (Yi et al., 1997). In addition, the increase in expression of Fli-1 in erythroblasts was concomitant with an improvement in cellular survival due to an increase of their apoptotic threshold, maybe through the up-regulation of Bcl-2 (Pereira et al., 1999). It therefore appears that the activation of the Fli-1 gene in erythroid cells would disrupt cellular homeostasis by promoting cell cycle progression as well as prolonging cell survival. These events are associated with the up-regulation and down-regulation of several target genes (Figure 4). Identification and characterization of additional genes that are targeted by Fli-1 is likely to shed light on the molecular mechanisms underlying this course of events.
The chimeric EWS/Fli-1 product function as a potent transforming oncogene
Although Fli-1 is overexpressed in a number of erythroleukemia cell lines, genetic events which ultimately lead to the accumulation of the Fli-1 gene have yet to be implicated in human carcinogenesis. However, the clinical relevance of Fli-1 becomes evident in Ewing's sarcoma. Indeed, this pediatric disease is characterized by a genetic rearrangement in which Fli-1 becomes juxtaposed 3' to the EWS gene (Delattre et al., 1992). The resulting fusion product is known as the EWS/Fli-1 oncoprotein and is a hallmark of this pathological disorder. Structurally, its amino terminus derives from the EWS protein fused to the carboxyl terminus of the Fli-1 protein. As such, it retains its DNA-binding specificity as a result of the conservation of its 3' Ets-DNA-binding domain. Such a translocation results in the generation of a chimeric protein whose transcriptional activation potential is much greater than wild-type Fli-1 (Bailly et al., 1994; May et al., 1993). Interestingly, a recent study by Jaishankar et al. has demonstrated that certain point mutations within the Fli-1/3' ets domain of the chimeric product abolish its DNA binding activity while retaining its oncogenic potential (Jaishankar et al., 1999). It is therefore suggested that a portion of the oncogenic activity of EWS/Fli-1 is independent of Fli-1 DNA-binding activity.
As EWS/Fli-1 is present in 95% of Ewing's sarcomas (Delattre et al., 1994) and in other tumors of the Ewing's sarcoma family, clinically, detection of this genetic alteration by RT-PCR in patient specimens has provided a powerful tool in confirming the diagnosis of this childhood disease (Peter et al., 1995; Pfleiderer et al., 1995). Recent studies have demonstrated that the characteristic chromosomal translocation of Ewing's sarcoma leads to the production of two common fusion proteins (Figure 3). Type 1 fusion which consists of 60% of all products, results from the juxtaposition of the first seven exons of EWS to exons 6-9 of Fli-1, while type 2 retains the fifth exon of Fli-1. The presence of the former product is associated with a better prognosis than the latter. Intriguingly, recent work which compared the transactivation potential of these two fusion products has shown that type 1 fusion is a less potent transcriptional activator than type 2 despite their comparable DNA-binding potential (Lin et al., 1999).
As the overexpression of EWS/Fli-1 but not Fli-1 resulted in the transformation of NIH3T3 cells, Denny's group used the method of subtractive hybridization to hunt for the target genes of EWS/Fli-1 responsible for this transformation. This analysis resulted in the identification of three genes: EAT-2 (a novel SH2 domain containing protein), mE2-C (a cyclin-selective ubiquitin conjugating enzyme) and MFNG (manic fringe gene involved in somatic development). The expression of these genes was shown to be upregulated by EWS/Fli-1 but not by Fli-1 (Arvand et al., 1998; May et al., 1997; Thompson et al., 1996). However, it still remains to be determined whether these genes are directly regulated by EWS/Fli-1 or whether their induction is a consequence of cellular transformation. In addition to these genes, the indirect upregulation of the c-Myc gene has also been demonstrated by EWS/Fli-1 (Bailly et al., 1994). TGF- type II receptor is an additional target gene whose promoter contains a binding site for both Fli-1 and EWS/Fli-1. In normal cells, this receptor is required to transduce the growth inhibitory signal upon TGF- binding to its cognate receptor. It appears that EWS/Fli-1 suppresses the transcription of TGF- type II receptor subsequently conferring TFG- resistant phenotype to the transformed cells (Hahm et al., 1999). In contrast to EWS/Fli-1, Fli-1 activates the transcription of the TGF- type II receptor gene. Since both EWS/Fli-1 and Fli-1 recognize a similar DNA binding site, the difference in gene regulation is likely mediated by the different transactivation domains of these proteins.
Due to the differences in the oncogenic potential of Fli-1 and EWS/Fli-1, the protein-protein interactions involving EWS and Fli-1 were investigated. hsRPB7, a regulatory subunit of RNA polymerase II, was shown to bind to EWS/Fli-1 but not to Fli-1 (Petermann et al., 1998). Domain swapping experiments demonstrated that the replacement of EWS with hsRPB7 could restore the transactivation ability of the chimera. It is therefore suggested that the interaction of EWS/Fli-1 with RNA polymerase II may selectively enhance promoter activity, which may account for the increase in the transactivation ability of EWS/Fli-1 over wild-type Fli-1.
The role of Fli-1 in development
The majority of the evidence which implicates Fli-1 in malignancy has been gathered by in vitro analysis and work performed on established cell lines. In order to study the function of Fli-1 in a more biologically relevant context, the effects of overexpressing this transcription factor in mice was investigated using the transgenic approach (Zhang et al., 1995). The results of this study revealed that Fli-1 plays a regulatory role in lymphoid cell function and its overexpression led to the development of an immunological renal disease in mice, which included splenomegaly, B-cell hyperplasia and hypergammaglobulinemia. This was also accompanied by the abnormal production of autoreactive lymphocytes and antibodies, a phenotype similar to mice models of autoimmune disorders.
The generation of Fli-1-/- mice was the next critical step in elucidating the role of this transcription factor in development. One of the first targeting strategies used involved replacing exon 2 of the protein with a neomycin cassette, thereby causing a frameshift and abolishing the expression of Fli-1 (Melet et al., 1996). Surprisingly, the phenotype observed was mild and included a defect in thymic development, accompanied by a delay in the onset of Friend-virus induced erythroleukemia. However, these investigators were able to demonstrate that this phenotype was a consequence of the generation of a novel truncated protein, a product of alternative splicing around the neomycin cassette. The truncated protein, which retained the functionality of the full-length protein, was able to attenuate the phenotype of the transgenic mouse, as this novel protein was able to carry out the required functions of Fli-1. Recently, homologous recombination technology has been used to disrupt the Fli-1 gene in embryonic stem cells (Spyropoulos et al., 2000). The product of this mutated gene is a truncated protein which lacks the functional CTA domain required for transcriptional activation. Embryos expressing this mutant form of Fli-1 displayed a defect in hematopoiesis and suffered from hemorrhaging of the CNS and brain at E11.0, ultimately leading to embryonic lethality shortly thereafter. In another study, ES cells lacking the DNA binding domain of the Fli-1 gene were generated (Hart et al., 2000). Mice generated from these ES cells were found to be embryonic lethal on day E11.5. Fli-1-/- homozygotes displayed poor vascularization accompanied by a block in megakaryocyte development. Interestingly, work done in zebrafish provides further support of Fli-1 involvement in vasculogenesis as it demonstrates that Fli-1 expression is required in early vessel formation (Brown et al., 2000). Collectively, these studies demonstrate that Fli-1 expression is critical for embryogenesis and hematopoiesis (for review of ets family knockout and mutant mice, see Bartel and Spyropoulos in this issue).
Summary and perspectives
Since the initial cloning of Fli-1 and the demonstration of its involvement in human Ewing's sarcoma, significant advances have been made in the study of the underlying mechanisms of transformation by Fli-1 and its role in cellular processes. Among these are the ability of Fli-1 to block differentiation and apoptosis, its involvement in cellular proliferation, its requirement during hematopoiesis, and its ability to alter Epo/Epo-receptor signaling. Although several target genes that can account for some of these functions have already been identified (Figure 4), many questions remain unanswered. For instance, the mechanism by which a Fli-1 deficiency in mice leads to embryonic lethality has yet to be determined. Also, the mechanisms by which Fli-1 alters the downstream signaling cascade of the Epo-receptor as well as the way by which Fli-1 overexpression increases cell survival remain to be elucidated. As the knock-out phenotype of Fli-1 has proven the necessity of this transcription factor in vivo, answers to these questions may perhaps provide insight in establishing a physiological role for Fli-1 in ensuring cellular homeostasis. In turn, the understanding of the molecular pathways following the induction of transcriptional activity by Fli-1 may be key to the understanding of the mechanisms underlying malignancy.
This review is dedicated to Dr Takis S Papas who is a pioneer in the field and whose contributions have been instrumental in the discovery and understanding of the Ets family of transcription factors. The authors would like to thank Dr Brian Pak, Dr Rachel Higgins, Jennifer Tran and Zubin Master for their comments on the manuscript. This work is supported in part by grants from National Cancer Institute of Canada and Medical Research Council of Canada to Y Ben-David.
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Figure 1 Diagram of the functional domains located within the Fli-1 protein. Both human and murine Fli-1 consist of 452 amino acids (aa) which contain the following domains: ATA=amino-terminal transcriptional activation domain, FLS=Fli-1 specific domain, CTA=carboxy-terminal transcriptional activation domain, H-L-H=helix-loop-helix structure, and T-L-T=turn-loop-turn structure. The positions of the SRF interactive domains are shown by arrows
Figure 2 Schematic representation of the Fli-1 promoter and its alternative spliced forms. Solid arrows show the initiation site for three alternative spliced forms (Fli-1b, Fli-1 -204, and Fli-1 -398) that are given with respect to the last AUG located on exon 1(+1). The alternative spliced form Fli-1b transcript has only been detected in pre-B cells and not in erythroleukemic cells. The Fli-1 -398 transcript is only expressed in F-MuLV-induced erythroleukemia cell lines while the Fli-1 -204 transcript appears in all Friend virus-induced erythroleukemias. The location of the AUG codons which initiate the 51 kDa (+1) and 48 kDa (+100) Fli-1 proteins are shown by open arrows. The open boxes represent the 5' noncoding sequences and the hatched and solid boxes represent the Fli-1 coding sequences. Vertical arrows show the locations of the GATA-1 site and the two EBSs in exon 1. The EBSs appear to play a critical role in the expression of the Fli-1 -204 transcript
Figure 3 The two main fusion products caused by the t(11;22) chromosomal translocation that is characteristic of Ewing's Sarcoma. The product of this fusion is an N-terminal domain composed of the EWS protein, which functions as a transcriptional activator fused to the C-terminal Fli-1 portion, which retains its DNA-binding activity. The type 1 fusion, which lacks the 5th exon of the Fli-1 gene, has been associated with a better clinical prognosis than type 2 fusion. Type 1 fusion has also been shown to be a weaker transcriptional activator than type 2 fusion despite their similar DNA-binding abilities
Figure 4 Proposed model of the effects of Fli-1 on cellular processes in erythroblasts. Ectopic expression of Fli-1 has been shown to be associated with decreased expression of Rb and increased expression of Bcl-2, possibly leading to a block in apoptosis. In addition, Fli-1 overexpression appears to inhibit differentiation of erythroleukemic cells, and is accompanied by a decrease in Rb and GATA-1 expression. Finally, Fli-1 expression has been shown to alter the Epo-responsiveness of erythroblasts. This results in the activation of the Shc/ras signal transduction pathway, thereby enhancing the proliferative capacity of erythroid cells. Overall, it appears that the inhibition of apoptosis, the block in commitment for terminal differentiation as well as the acceleration of proliferation are required during erythroid transformation by Fli-1
|18 December 2000, Volume 19, Number 55, Pages 6482-6489|
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