Proteins of the ETS Family with Transcriptional Repressor Activity

George Mavrothalassitis*,1 and Jacques Ghysdael2

1School of Medicine, University of Crete and IMBB-FORTH, Voutes, Heraklion, Crete 714-09, Greece; 2CNRS UMR 146, Institut Curie, Centre Universitaire, Bat 110, 91405 Orsay, France

ETS proteins form one of the largest families of signal- tors by (i) aecting chromatin structure and DNA- dependent transcriptional regulators, mediating cellular accessibility at the region of the gene of interest and (ii) proliferation, dierentiation and tumorigenesis. Most of directly interfering with the basic transcriptional the known ETS proteins have been shown to activate machinery. There is evidence to suggest that ETS- transcription. However, four ETS proteins (YAN, ERF, domain proteins may include paradigms for each of NET and TEL) can act as transcriptional repressors. In these categories. three cases (ERF, NET and TEL) distinct repression ETS domain encoding genes, the prototype of which domains have been identi®ed and there are indications is the v-ets oncogene of avian leukemia virus E26 that NET and TEL may mediate transcription via (Nunn et al., 1983; Leprince et al., 1983) are present in Histone Deacetylase recruitment. All four proteins all the multicellular organisms analysed so far appear to be regulated by MAPKs, though for YAN (Lautenberger et al., 1992; Degnan et al., 1993; Laudet and ERF this regulation seems to be restricted to ERKs. et al., 1999) The identi®cation of v-ets related genes in YAN, ERF and TEL have been implicated in cellular the last 15 years has established the ETS family as one proliferation although there are indications suggesting a of the largest families of transcriptional regulators, possible involvement of YAN and TEL in dierentiation with diverse functions and activities (for recent reviews as well. Other ETS-domain proteins have been shown to see: Ghysdael and Boureux, 1997; Papas et al., 1997; repress transcription in a context speci®c manner, and Dittmer and Nordheim, 1998; Ghosh and Kolodkin, there are suggestions that the ETS DNA-binding domain 1998). ETS proteins are characterized by a conserved may act as a transcriptional repressor. Transcriptional DNA-binding domain (ETS domain) which binds repression by ETS domain proteins adds an other level in DNA sequences centered over a GGAA/T core motif the orchestrated regulation by this diverse family of (EBS: ETS binding site, Karim et al., 1990) and have transcription factors that often recognize similar if not been shown to mediate cellular proliferation, dier- identical binding sites on DNA and are believed to entiation and tumorigenesis (for reviews see Watson et regulate critical genes in a variety of biological al., 1990; Seth et al., 1992; Macleod et al., 1992; processes. De®nitive assessment of the importance of Wasylyk et al., 1993; Ghysdael and Boureux, 1997; this novel regulatory level will require the identi®cation Dittmer and Nordheim, 1998; Papas et al., 1997; of ETS proteins target genes and the further analysis of Ghosh and Kolodkin, 1998). Most of the known ETS transcriptional control and biological function of these proteins have been shown to activate transcription, proteins in de®ned pathways. Oncogene (2000) 19, however in the last few years an increasing number of 6524 ± 6532. transcriptional repressors has been identi®ed (O'Neill et al., 1994; Sgouras et al., 1995; Maira et al., 1996; Keywords: ets; repression; transcription; proliferation Lopez et al., 1999; Chakrabarti and Nucifora, 1999) Furthermore, a number of ETS proteins have been suggested to activate or repress transcription in a Introduction context speci®c manner (Zinck at al., 1993; Borras et al., 1995; Sieweke et al., 1996; Foos et al., 1998; Xing The importance of transcriptional repression in the et al., 2000; Li et al., 2000). regulation of gene expression in bacteria, was recog- Our current knowledge on ETS proteins indicates nized almost 40 years ago by Jacob and Monod and that at any given time multiple members of this family since then has been established as a major mechanism exist in any given cell, all of which can recognize in transcriptional regulation. Transcriptional repressor similar, and many of them identical EBS response proteins aect their target genes through direct or elements. The function and speci®city of ETS proteins indirect DNA-binding at the regulatory regions of the is controlled at several levels. First, the expression of gene of interest and repress transcription by a variety several ETS genes is either highly tissue-speci®c or of distinct mechanisms (for recent reviews see regulated in response to speci®c extracellular signals Knoep¯er and Eisenman 1999; Bird and Wole 1999; (for review see: Ghysdael and Boureux, 1997; Wasylyk Maldonado et al., 1999). A repressor protein can and Nordheim 1997). Second, speci®c intracellular inhibit transcription of selective genes by (i) displacing signaling pathways have been shown to directly an activator from the DNA, and (ii) masking/blocking impinge upon the activity of particular ETS protein the activation domain of a transcriptional activator. In by regulating their subcellular compartmentalization addition a repressor protein can actively inhibit (Le Gallic et al., 1999; Ducret et al., 1999), their DNA- transcription, independently of transcriptional activa- binding activity (Rabault and Ghysdael, 1994; Martin et al., 1996; Cowley and Graves, 2000) or their transactivation potential (Marais et al., 1993; O'Hagen *Correspondence: G Mavrothalassitis et al., 1996; McCarthy et al., 1997). Finally, regulation ETS repressors G Mavrothalassitis and J Ghysdael 6525 of many promoters/enhancers by ETS proteins often expression is also observed in ventral neuroectoderm critically depend upon their interaction with unrelated development and in other stages during development. transcription factors on composite DNA elements Hypomorphic yan mutants exhibit an increased (Dalton and Treisman, 1992; Janknecht et al., 1993; number of R7 photoreceptor cells as a result of Sev/ Treisman, 1994; Giese et al., 1995; Wolberger, 1998; Ras1 signaling, whereas activated yan mutants inhibit Goetze et al., 2000). These and other mechanism are neuronal and mesoderm dierentiation (Rebay and presumed to accommodate the multitude of ETS- Rubin, 1995; Lai et al., 1997). However, other studies domain proteins in a cell without apparent con¯icts. utilizing a null yan mutation indicate that the primary However, it would be dicult to envision today a strict role of yan may be to inhibit cells from entering the cell gene expression regulation without the presence of cycle in the absence of the appropriate signal (Rogge et transcriptional repressors that ensure transcription al., 1995). This would be consistent with the blockade of a given gene. To that extent ETS proteins hyperproliferative phenotype observed in yan/aop with transcriptional repressor activity may be vital to mutants. This is also consistent with the increase in our understanding of the orchestrated presence and the apoptosis induced by activated yan at the morphoge- role of ETS-domain proteins in transcriptional regula- netic furrow of the eye disk when a cell receives a tion. This review is focusing on ETS genes that exhibit proliferation signal while entry into G1 is blocked by transcriptional repressor activity (Figure 1) and their the activated yan. A role of yan in proliferation rather possible role in speci®c biological processes. than dierentiation is further supported by the identi®cation of a master key gene in eye development (Halder et al., 1995a,b) although regulation of this YAN gene by yan cannot be excluded (Rebay et al., 2000). Most members of the RTK signaling pathways are The ®rst ETS domain protein identi®ed, initially as a highly conserved through evolution from the receptors suppressor (Lai and Rubin, 1992) and later as a to the MAPKs (for a comprehensive review see Lewis transcriptional repressor (O'Neill et al., 1994) was the et al., 1998). Yan is highly conserved in structure, Drosophila gene yan/aop. yan was originally identi®ed function, expression pattern and regulation between in an RTK signaling screening as a negative regulator two Drosophila sibling species arguing for its important of R7 photoreceptor neuron dierentiation in the role (Price and Lai, 1999). However, transcription Drosophila developing eye. Later studies have shown factors exhibit considerable diversity throughout evolu- that yan can act as an inhibitor of dierentiation in tion that increases with genome expansion, and to that multiple developmental processes, preventing neuronal extent it may not be surprising that no yan orthologues and nonneuronal cell types from responding inappro- have been identi®ed in vertebrates. The closest YAN priately to external signals (Rebay and Rubin, 1995). homologue in vertebrates is TEL (Golub et al., 1994), yan has been shown to be downstream of, and an ETS-domain protein that also exhibits transcrip- negatively regulate the Sevenless, the EGF-receptor tional repressor activity (see below). and the FGF-receptor pathways. It is believed to be In transient transfection assays, YAN can antag- involved in the regulation of the transition between onize the activity of the Pointed-P2, a transactivator of undierentiated and dierentiated cells as a result of the ETS family (O'Neil et al., 1994; Treier et al., 1995). extracellular signals. In the Drosophila developing eye Genetically, YAN antagonizes Pointed proteins and D- yan is expressed in all undierentiated cells and is Jun activities in several developmental pathways in down regulated as cells dierentiate. This pattern of response to the activation of the ERK or JNK protein kinases (O'Neil et al. 1994; Riesgo-Escovar et al. 1994; Treier et al., 1995). However, no bona ®de YAN target genes have been identi®ed so far, and no information is available on the YAN repression domain and the possible mechanisms by which it represses transcription. In the absence of extracellular signals YAN is nuclear and presumably represses transcription of its target genes. Upon RTK activation, YAN has been proposed to be phosphorylated by MAPK and degraded probably in the cytoplasm (Rebay and Rubin, 1995), thus allowing the activation of YAN- regulated genes by other ETS-domain transcription factors, like the Drosophila Pointed (Scholz et al., 1993; Klambt, 1993). This provides a model where upon receptor stimulation, a binary system of ETS-domain proteins, one of which is inhibited the other is activated by the same signal, switch the transcription on a given gene from the repressed state to the activated state. Figure 1 A schematic representation of ETS-domain proteins with transcriptional repression activity. The hatched box indicate Such a system would insure the strict and concerted the ETS DNA binding domain and the gray box the repression control of YAN/Pointed target genes, consistent with domains identi®ed within dierent proteins. There is no homology the severity of the yan and pointed phenotypes in between the repressor domains from dierent subfamilies and Drosophila. De®nitive assessment of this model requires between the two repressor domains of NET and TEL. The dots over TEL indicate posible involvement of the B/pointed domain the identi®cation of YAN/Pointed-regulated genes. The in repression. The question marks in YAN indicate the lack of completed sequencing of the entire Drosophila genome, experimental evidence for autonomous repression domain(s) in combination with the rapidly developing DNA-array

Oncogene ETS repressors G Mavrothalassitis and J Ghysdael 6526 technology should provide valuable answers both on ERF can suppress oncogenic transformation of the possible interplay of Yan and Pointed on speci®c NIH3T3 cells by several oncogenes. Wild type ERF promoters, as well as some insights on the role of these can suppress E26-induced transformation (Sgouras et genes in cellular proliferation and dierentiation. al., 1995). The transforming potential of the Gag-Myb- Ets oncoprotein of the E26 virus both in this system and in hematopoietic progenitors has been shown to ERF depend on the v-ets DNA-binding domain (Yuan et al., 1989; Rossi et al., 1996), suggesting that common or ERF (ETS2 Repressor Factor) is a ubiquitously cooperating pathways and/or targets may be controlled expressed member of the ETS gene family. Besides by the two proteins. Thus a binary system similar to the conserved ets-DNA-binding domain (Sgouras et al., the yan/pointed may also be in place in vertebrates 1995), ERF presents no other homology with other where ERF would be the yan analogue while the ETS proteins, except PE-1 (Klemsz et al., 1994) with pointed role should be attributed to one of the which they form a new subclass of ETS proteins. ERF members of the ETS1 sub-family of ETS genes. is a strong transcriptional repressor and represses Transforming members of the ETS family regulated transcription via a distinct domain at the carboxyl- by RAS/ERK signaling would also appear as appro- terminus of the protein, that can not be found in PE-1. priate partners for ERF, however, this hypothesis This domain has no recognizable pattern suggestive of needs to be con®rmed. its mode of action and the molecular mechanisms Mutants of ERF that have the MAPK phosphoryla- involved in ERF-mediated repression are unknown. tion sites mutated to Alanine are insensitive to ERK However, there is a report indicating that ERF phosphorylation and constitutively nuclear. These mediated repression may not involve the Sin3/NcoR mutants can block RAS-induced transformation of system (Heinzel et al., 1997). The ERF repression NIH3T3 cells in contrast to wt ERF. This would domain has been transferred to a number of other provide some genetic con®rmation for the role of ERF DNA binding domains and transcription factors within the RAS-signaling pathway. Expression of the (GAL4, ETS1, FLI1, c-MYC, NFkB) resulting an same mutants in normal cells induces a block in the G1 array of transcriptional repressors with diverse speci- phase of the cell cycle, while overexpression of the wt ®city and functions (Sgouras et al., 1995; Athanasiou et ERF has minimal eect on cell cycle progression. This al., 2000; and G Mavrothalassitis, unpublished ob- is consistent with the nuclear localization of ERF in servations). A multitude of native and arti®cial G0/G1 arrested cells and its immediate nuclear export promoters have been shown to be repressed by ERF upon mitogenic stimulation, suggesting that ERF may in a DNA-binding-dependent manner. The Prolactin be controlling RAS-responsive genes required for cell gene promoter in particular, appears to be regulated by cycle re-entry or progression (Le Gallic et al., 1999). ERF in a biological relevant way (Day et al., 1998). The ubiquitous expression of ERF suggests that However, since these experiments made use of transient components of the cell cycle machinery may be under transfection assays, it remains to be seen to what extent the transcriptional repression of ERF. This would these genes are actual ERF targets in vivo. provide a direct link between RAS signaling and the Like yan, ERF is downstream of the RAS/ERK cell cycle machinery. Indeed, cell cycle arrest induced signaling pathway and is directly regulated by ERK via by constitutively active mutants of ERF can be phosphorylation. ERK physically associates with, and alleviated by the overexpression of speci®c Cyclins or phosphorylates ERF at multiple sites both in vitro and loss of the Retinoblastoma protein (G Mavrothalassitis in vivo. Phosphorylation by ERK results in the rapid unpublished observations), lending further support for export of ERF from the nucleus. Unlike YAN, the this model. However, loss of function mutation and protein is stable in the cytoplasm and reaccumulates in identi®cation of candidate target genes are required to the nucleus upon ERK inactivation (Le Gallic et al., evaluate such a possibility. 1999). ERF cellular localization is exclusively dependent on export rates with the import being unaected by the phosphorylation state of the protein. Nuclear NET export is Leptomycin B-sensitive suggesting the involvement of the Exportin/RanGTP-dependent path- NET/SAP-2/ERP (Giovane et al., 1994; Lopez et al., way, though ERF does not possess an identi®able 1994; Price et al., 1995) is a member of the TCF nuclear export signal (NES). Domain mapping in- subfamily of ETS-domain proteins that are capable to dicates that at least two regions of the protein are form ternary complex with DNA and the Serum required for nuclear export, consistent with the Response Factor (SRF). Like the other TCFs, NET extended phosphorylation mediating ERF export (G includes an amino-terminal ETS domain (A domain) Mavrothalassitis, unpublished observations). It is not harboring the DNA-binding activity, a B domain clear if subcellular localization is the only function of mediating the TCF ± SRF interaction, and a carboxy- ERF regulated by ERK phosphorylation. Mutation of terminal C domain that can activate transcription upon an ERK phosphorylation site within the ERF repres- phosphorylation by MAPKs. However, this activation sion domain to Glutamic acid, to mimic the phos- appears to be weaker as compared to the C domains of phorylated state of the protein, decreases the repressor the other TCFs (Price et al., 1995). Unlike other TCFs, activity (Sgouras et al., 1995). This decrease can not be NET exhibits strong transcriptional repressor activity attributed to DNA binding or nuclear accumulation (Maira et al., 1996), though preliminary reports from A suggesting additional regulation at the repression level. Sharrock's lab (Univ. of Manchester, UK, personal However, this hypothesis should be further evaluated communication) indicate that ELK1 may also harbor a in the context of the ERF repression mechanism. repression domain distinct of that found in NET. NET

Oncogene ETS repressors G Mavrothalassitis and J Ghysdael 6527 has two domains that have been shown capable to stimuli. However, it is not known if the same targets suppress transcription when transferred to a hetero- are recognized by NET and the other TCFs. In this logous DNA-binding domain, one of which can model NET may be substituted by other TCFs in a interact with the CtBP co-repressor (Criqui-Filipe et signaling-dependent manner and allow transcriptional al., 1999). This provides a possible mechanism for activation. Alternatively, NET may be controlling a inhibition by Net, which under proper conformation subset of SRF-responsive genes that are quantitatively could interact with CtBP and recruit HDAC on the regulated in a signal-dependent manner. In support of promoters of its target genes. It is not clear by which this hypothesis, there are data suggesting that in the mechanism the other NET inhibitory domain may context of the whole protein, NET DNA-binding may mediate transcriptional repression. Interestingly, upon be dierent from that of the other TCFs (Price et al., RAS/ERK activation NET like the other TCFs can 1995). In addition, dierent TCFs may have dierent also activate transcription, providing a paradigm where eciency in the formation of ternary complexes a single protein can repress or activate the transcription (Fitzsimmons et al., 1996). This would argue that of a gene depending on extracellular signals (Giovane distinct genes are regulated by the dierent TCFs. et al., 1994). GETS-1a, a TCF isolated from gold®sh, is However, given the ability of all TCFs to form similar also capable to repress or activate transcription of a complexes and aect the same reporters, it would be given promoter in a signal-dependent maner (Goldman reasonable to assume that in a given cell the et al., 1998). Although it is not known if NET-like transcription of speci®c genes may be regulated by factors have the same targets in their basal and induced TCFs in a signal dependent manner and in a way states, such a hypothesis is not inconceivable. similar to the one suggested for YAN/Pointed. As with An additional interesting feature of NET is that it other ETS genes, the analysis of loss of function is the only known ETS-domain protein that contains mutants and the identi®cation of target genes will a Leucine-rich nuclear export signal (NES) within the greatly improve our understanding on the role of this ®rst helix of the ETS DNA-binding domain. This gene and the mechanism of their action. NES is functional when transferred to a heterologous protein and is involved in the export of NET from the nucleus in response to stress activated kinases, in TEL particular JNK (Ducret et al., 1999; B Wasylik, IGMCB, France, personal communication). It re- TEL (also known as ETV-6) is one of the few ETS mains to be determined how dierent combinations genes known to be associated with human malignan- of the three subclasses of MAPK may aect NET cies, and the only one of them with transcriptional localization and activity. It is not evident if any of repression activity. It was originally identi®ed by virtue the three subclasses may be dominant over the of its rearangement in speci®c cases of chronic others, and in which in vivo context this regulation myelomonocytic leukemia presenting a may be in eect. Nonetheless, NET may be a t(5,12)(q33;p13) chromosomal translocation (Golub et paradigm of a transcription factor that (depending al., 1994). Since then, TEL has been shown to be on the signaling context) can either repress, or frequently rearranged in a wide variety of leukemia and activate or even be neutral in the regulation of its in solid tumors (see below). TEL, together with the target genes. Thus, NET may regulate expression of a recently identi®ed TEL-B (or TEL-2), a gene encoding given promoter not only in a qualitative but also in a a protein closely related to that encoded by TEL, quantitative way, providing a paradigm for a mode de®nes a new sub-class of the ETS family in mammals of regulation distinct from the binary model (on ± o) (Potter et al., 2000; Poirel et al., 2000). TEL appears to usually suggested for the function of transcription be ubiquitously expressed in mouse and human tissues factors. whereas TEL-B/TEL-2 expression is more restricted A NET splicing variant designated NET-b (Giovane (Golub et al., 1994; Wang et al., 1997, 1998; Potter et et al., 1997) contains only the A and B domains, and al., 2000; Poirel et al., 2000). TEL is essential to mouse only one of the repression domains (NID). This variant development as its inactivation by homologous can also act as a repressor of transcription. However, recombination is lethal at day 10.5 ± 11.5 of embryonic NET-b does not contain the C domain and its activity development. At that age profound defects are seen in is not regulated by RAS signaling. It is unclear if NET- yolk sac angiogenesis, and speci®c areas of apoptotic b responds to other signaling pathways and if its cell death can be identi®ed in the embryo, suggesting a distinct carboxyl-terminal region has a functional role. widespread role of TEL in several developmental It is also not known if it is regulated by subcellular processes (Wang et al., 1997). The analysis of localization as NET. NET-b can be detected in the cell hematopoietic progenitors at various developmental nucleus, where it can presumably act as repressor of stages in chimeras obtained from mouse blastocysts EBS-regulated genes. A third splice variant designated after transfer TEL7/7 embryonic stem (ES) cells NET-c (Giovane et al., 1997) that also contains demonstrates an essential postnatal role of TEL in domains A, B, NID, but not C, can be identi®ed by establishing hematopoiesis of all lineages in adult bone PCR and can also act as an inhibitor of transcription. marrow (Wang et al., 1998). In contrast, TEL7/7 ES However, it is not clear if this gene product can be cells contribute normally to embryonic (yolk-sac identi®ed in cells or is the result of aberrant splicing. derived) and fetal de®nitive (liver-derived) hematopoi- Despite its intriguing regulation and multiple gene esis, indicating a speci®c and non redundant function products, little is known about the biological role of of TEL in either migration or homing of hematopoietic NET. Similar to other TCFs, NET can form ternary progenitors to the adult bone marrow micro-environ- complexes with SRF and convey immediate early ment or in the response of these cells to bone marrow transcriptional responses to a number of extracellular stroma derived signals.

Oncogene ETS repressors G Mavrothalassitis and J Ghysdael 6528 TEL encodes two nuclear polypeptides of 452 (TEL- TEL by unrelated oligomerization domains, including M1) and 409 (TEL-M43) amino-acids residues, respec- that of GAL4 or the leucine zipper of the EBV tively. These two forms arise from alternative transla- ZEBRA, restores both self-association and repression tion initiation at two successive AUG initition codons of EBS-dependent transcription at levels close to the in the TEL mRNA, where TEL-M1 is most abun- wild type protein (Lopez et al., 1999). An additional dantly expressed in the vast majority of cell types repression domain was identi®ed which encompasses analysed (Poirel et al., 1997). The ETS domain of TEL the ETS domain and the 50 amino-acids immediately is most highly related to that of TEL-2/TEL-B (85% upstream of the ETS domain (Lopez et al., 1999). identity). Compared to the majority of the other ETS Although some of the published data are sometimes domains both TEL and TEL-2/TEL-B present primary dicult to reconcile in their details, all converge to amino-acid sequence deviations in the a1 helix, present indicate that TEL-mediated repression is likely to be at the extreme amino-terminus of the ETS domain, and dependent upon the recruitment of co-repressors at the end of the a3 recognition helix. Yet, the isolated ultimately linked to histone (protein) deacetylases. ETS domain of TEL and TEL-B/TEL-2 binds First, mSin3A was found to associate with both conventional EBS in vitro (Poirel et al., 1997, 2000; overexpressed TEL and its B/pointed domain fused to Potter et al., 2000) and regulate EBS-driven transcrip- GALDBD with Sin3A binding correlating with the tion (see below). However, in vitro, the DNA binding repressive properties of this GALDBD fusion protein activity of full-length TEL is considerably lower as (Fenrick et al., 1999). Using in vitro translated proteins compared to that of its isolated ETS domain, or overexpressed TEL, in vivo interactions with SMRT suggesting that the DNA binding properties of TEL or N-CoR were also observed and increased dosage of are subject to an intramolecular repressive mechanism either of these co-repressors was found to increase TEL as frequently observed in other ETS proteins (for repressive properties (Guidez et al., 2000; Chakrabarti review, see Ghysdael and Boureux, 1997; Graves and and Nucifora, 1999). TEL-mediated repression of EBS- Petersen, 1998). Besides its conserved ETS domain, driven transcription is clearly dependent upon the TEL and TEL-B/TEL-2 share with a large subgroup of recruitment of protein deacetylases since it is strongly ETS family members another conserved domain known inhibited by Trichostatin A (TSA), a deacetylase as the B domain (Boulukos et al., 1990), pointed inhibitor (R Lopez and J Ghysdael, unpublished domain (Klambt, 1993) or NCR domain (Poirel et al., observations). Whether the interaction of TEL with 1997). The function of this domain in other ETS speci®c co-repressors is regulated or whether distinct proteins is not clear, although in some instances it has repressor complexes are involved in dierent cell been shown to modulate the transcriptional activation lineages or in the regulation of dierent genes remains properties of speci®c members of the family such as to be established. Furthermore, there is no evidence ETS-1 or FLI-1 (for review, see Ghysdael and that the recruitment of co-repressors/deacetylases Boureux, 1997). The structure of the B/pointed domain complexes is the sole mechanism used by TEL to of ETS-1 has been determined recently by NMR and repress transcription. Finally, given the fact that the shown to fold into a ®ve-helix bundle (Slupsky et al., activity of TEL is clearly under conformational 1998). ETS-1 is monomeric in solution whereas TEL control, and since ETS proteins have been shown to assemble into oligomers, a property which is exclu- play architectural role in multiprotein complexes in sively mediated by its B/pointed domain (Jousset et al., speci®c promoter/enhancers, it cannot be excluded that 1997). TEL-B/TEL-2 also self-associates in vitro and in TEL could also play a role in transcriptional vivo through its B/pointed domain and associates with activation. TEL as well (Potter et al., 2000; Poirel et al., 2000). Only limited information is available concerning the When bound to the EBS of either arti®cial or natural transcriptional regulatory properties of TEL-B/TEL-2, promoters, both TEL-M1 and TEL-M43 isoforms were except that its central domain, although completely found to strongly repress promoter activity (Lopez et unrelated to that of TEL, also functions as transpor- al., 1999). Repression of EBS-driven transcription by table repression domain when fused to the GAL4DBD TEL was found to require an oligomeric protein since (Poirel et al., 2000). It would be important to monomeric forms of TEL generated either by deletion investigate whether this domain is functional in the of the self-association domain or its substitution by the context of the full-length TEL-B/TEL-2 and whether homologous B/pointed domain of monomeric ETS1 the repressive properties of TEL-2/TEL-B also depend failed to repress (Lopez et al., 1999). TEL is an active upon the recruitment of deacetylase or upon a repressor as several transportable domains were completely dierent mechanism. Since TEL and TEL- identi®ed through the study of fusion proteins between B/TEL-2 can form heteromers in vivo this could various portions of TEL and the DNA binding domain introduce additional ¯exibity in repression mediated of the S. cerevisiae GAL4 (GALDBD). Two studies by the members of the TEL subfamily. converge to demonstrate that the large central domain The Drosophila YAN encodes an ETS protein which of TEL (encoded by the large TEL exon 5; Baens et al., is the most closely related to TEL and TEL-B/TEL-2 1996), which extends between the B/pointed domain in both its ETS domain and in its B/pointed domain. and the ETS domain includes a repression domain As detailed above, activation of the ERK and JNK (Lopez et al., 1999; Chakrabarti and Nucifora, 1999). protein kinases induces the relocalisation of YAN from In these transient assays, the isolated B/pointed the nucleus to the cytoplasm, and the mutation of domain also scored as a weak repression domain several ERK/JNK consensus phosphorylation sites into (Chakrabarti and Nucifora, 1999; Fenrick et al., 1999). Alanine result in a constitutively active (repressive) Whether this domain eectively functions as repression YAN. TEL is also a phosphoprotein in vivo with the domain in the context of TEL itself is not clear, TEL-M1 and TEL-M43 isoforms subject to both because the replacement of the B/pointed domain of common and distinct phosphorylation events (Poirel

Oncogene ETS repressors G Mavrothalassitis and J Ghysdael 6529 et al. 1997; R Lopez and J Ghysdael, unpublished ization of TEL-AML1 prevents AML1 to interact with observations). TEL repressive properties can be its normal co-activators (e.g. p300) is unknown. Both relieved in response to the activation of as yet the oligomerization-dependent repression domain of uncharacterized signaling pathaways laying down- TEL (and/or the B/pointed domain), and a Sin3A stream of protein tyrosine kinases (R Lopez and J binding site localized downstream of the runt domain Ghysdael, unpublished observations). Whether this are involved in TEL-AML1-induced repression (Fen- regulation depends upon the modi®cation of TEL rick et al., 1999; Uchida et al., 1999). Sin3A binds phosphorylation is under investigation. considerably more stably to TEL-AML1 than to either AML1 or TEL, suggesting that the strong repressive properties of TEL-AML1 depend upon deregulated TEL-derived oncoproteins Sin3A binding (Fenrick et al., 1999). However, the mechanism involved in TEL-AML1-mediated repres- In man, over a dozen chromosomal translocations sion is likely to be more complex. Unlike AML1, TEL- involving TEL are associated with leukemia and, more AML1 has been shown to also interact with NcoR rarely solid tumors (for review, see Rubnitz et al., through the TEL central repression domain, suggesting 1999). These translocations most often result in the that the repressive activity of TEL-AML1 on AML1 expression of fusion proteins. In one category, chimeric target genes involves altered regulation (Guidez et al., proteins are formed between the amino-terminal 2000). Of note, most of the studies of TEL-AML1 domain of TEL, including always its self-association made use of AML1-regulated promoters/enhancers domain, and the catalytic domain of protein tyrosine which are unlikely to be relevant to TEL-AML1 kinases (PDGFRb, ABL, JAK2, TRK-C). In these induced leukemia. It remains to be seen to what extent chimeric proteins, the B/pointed domain of TEL the oncogenic properties of TEL-AML1 depend upon remains functional, induces self-association and con- these biochemical properties. stitutively activates the tyrosine kinase activity of the fused catalytic domains. These properties are central to the transforming properties of these fusion oncopro- Context speci®c repression teins in several cellular models and to their leukemo- genic properties in animal models (Carroll et al., 1996; The majority of ETS-domain proteins are believed to Golub et al., 1996; Jousset et al., 1997; Lacronique et be transcriptional activators. However interaction with al., 1997; Schwaller et al., 1998; Ritchie et al., 1999; other proteins can minimize or eliminate their Tomasson et al., 1999; Carron et al., 2000). transactivating eect as in the case of ETS1/EAP1 The t(12;22)(p13;q11) translocation found in several (Li et al., 2000) and ETS1/MAF-B (Sieweke et al., cases of myeloid leukemia leads to the expression of a 1996). This could represent a system where a MN1-TEL fusion protein in which the carboxy-terminal transcriptional activator can become a repressor via part of TEL including the ETS domain is fused to MN1 its interaction with cell type speci®c factors. Aberrant sequences. Although the biochemical properties of these expression of ETS genes can suppress tumorigenic fusion proteins have not been reported. MN1-TEL phenotypes as in the case of ETS1 (Huang et al., 1997) would be expected to bind EBS response elements in and ETS2 (Foos et al., 1998). This suppression may be genes regulated by TEL or other members of the ETS due to either activation of alternative pathways or to family to deregulate their expression. In that respect, competition of endogenous factors for binding on MN1-TEL is similar to the EWS-FLI1/EWS-ERG and speci®c target genes. Association of aberrantly ex- TLS-ERG fusion genes expressed as the result of pressed ETS genes in these promoters could disrupt recurrent chromosomal translocations in Ewing sarco- multiprotein complexes and inactivate transcription. mas and leukemias, respectively (for review, see Overexpression of PEA3 can suppress overexpressed Ghysdael and Boureux, 1997). HER-2/neu in breast cancer cells (Xing et al., 2000) The most frequent chromosomal translocation found although it does not aect normal HER-2/neu in childhood leukemia is the t(12;21)(p13;q22) in B-cell expression in other breast cancer cells. Finally, ALL. This translocation results in the fusion of TEL although EWS-FLI1 fusions have been shown to be with AML1 to generate a TEL-AML1 fusion protein strong transcriptional activators, they can repress in which the ®rst 336 amino-acid residues of TEL, transcripsion of the TGFb-RII gene promoter (Hahm including the B/pointed domain and the central et al., 1999; Im et al., 2000). Although most of the repression domain, are fused to all but the ®rst 20 above situations involve systems in which ETS genes amino-acid residues of AML1 (Golub et al., 1995; are over- or miss-expressed, they provide an indication Romana et al., 1995a,b). AML1 (also known as that ETS-domain proteins with transactivation activity, CBFA2) is the human ortholog of the mouse Core could also act as repressors depending on the cell and/ Binding Factor (CBF), a transcriptional activator of or the promoter context (Roy et al., 1998). This could the Runt family (for review, see Ito and Bae, 1997). be accomplished either by competing with other ETS AML1, together with an unrelated CBFb subunit, proteins for speci®c sites or by binding on speci®c binds speci®c DNA sequences through its 128 amino- promoters and have their activity modulated by other acids runt domain and functions either as transcrip- cell type speci®c proteins. Thus the activity of ETS tional activator or repressor, depending on the genes should be carefully determined in a context promoter/enhancer tested. The analysis of the activity speci®c manner. of TEL-AML1 on model AML1 target genes indicates Multiple investigators in the ®eld have shown that that TEL-AML1 is a transcriptional repressor (Hiebert an isolated ETS DNA-binding domain can act as a et al., 1996; Fears et al., 1997; Uchida et al., 1999; transcriptional repressor, and the hypothesis is that it Fenrick et al., 1999). Whether TEL-induced oligomer- competes for binding with the endogenous ETS-

Oncogene ETS repressors G Mavrothalassitis and J Ghysdael 6530 domain proteins. Although this is a plausible hypothesis, it can not be reconciled with the observations that in several cases the observed inhibition by an ETS DNA-binding domain is stronger than the inhibition observed after the mutation or total elimination of the EBS response element on the promoter of the reporter. This would argue for a form of active repression by the isolated ETS DNA-binding domain. Preliminary Figure 2 A simple model suggesting a role for ETS-domain results from A Sharrock's lab (Univ. of Manchester, proteins with transcriptional repression activity. In the absence of stimulation genes required for entry and progression into the G1 UK, personal communication) indicate that the ELK1 phase of the cell cycle are repressed by ETS-domain transcription DNA-binding domain and possibly other ETS DNA- factors after DNA binding and recruitment of co-repressors. binding domains may be capable to interact with the Upon proper signaling the repressors are modi®ed in order to Sin3/Histone Deacetylase system. This could explain promote transcription of these genes by co-activator recruitment, repression by miss-expressed ETS genes, since in the or are released from the DNA to allow gene activation by other ETS-domain transcription factors absence of the correct signals the corresponding proteins could act as repressors. If the ETS domain has an active transcriptional repressor function, would Buskirk and Schupbach, 1999). To that extent multiple also argue strongly that ETS target genes should be ETS genes would be required to integrate these signals expressed only when dictated by speci®c signals. in dierent cell types to either block or promote cell However, the observed inhibition by the ETS domain cycle progression. It is also conceivable that multiple could also be due to disturbance in the normal components of the cell cycle machinery are under the assembly of transcription factor complexes at the transcriptional control of dierent ETS genes, that promoter or in the exposure of protein domains that would be consistent with a distinct and cell type do not normally mediate protein ± protein interactions. speci®c phenotype associated with each of them. The If these ®ndings are con®rmed for wt ETS proteins role of ETS genes in hematopoiesis supports this expressed at endogenous levels, will change our hypothesis. Today it is largely believed that hemato- perception of ETS-domain proteins as transcriptional poietic dierentiation is a stochastic eect (for reviews regulators, in the sense that they not only promote but see Ogawa, 1999; Metcalf, 1999; Busslinger et al., also permit the transcription of their target genes in a 2000). Yet over-expression or elimination ETS genes is signal dependent manner. known to result in phenotypes associated with expansion or elimination of certain cell lineages, arguing for a cell type speci®c survival/proliferative Concluding remarks signal provided by these genes (for a review see Spyropoulos, this issue). It is conceivable that this Thus far, the identi®ed members of the ETS family may be a general function of ETS family genes. The with transcriptional repressor activity are either fact that most if not all know ETS genes are regulated ubiquitous or widely expressed genes, and in all tested by extracellular signals, would further argue that they cases their activity is regulated by mitogenic signals. may not be associated with cellular dierentiation. This Two of these (YAN and ERF) may be involved in cell activity could be expected to be intrinsic to the cycle entry or progression. A third repressor, TEL, presence or absence of a given gene in a given cell. may also has a similar function as suggested by by the However, without the production and careful examina- induced apoptosis observed in TEL7/7 embryos and tion of loss of function mutations, without the its alledged tumor suppressor function (Kim et al., identi®cation of ETS target genes and without better 1996). Finally, a similar role could be envisioned for understanding of the cell cycle machinery and dier- NET given its role as a TCF, one of the components entiation processes, the question of the involvement of responsible for immediate transcriptional response to the ETS domain proteins in proliferation, dierentia- mitogenic stimulation. If such a hypothesis is true it tion, both or none, will remain open. would appear that ETS proteins with transcriptional repression activity may be primarily involved in ensuring the balance between cellular proliferation and dierentiation in dierent cell types and develop- Acknowledgments mental stages, in response to extracellular signals We would like to apologize for the many omissions of the (Figure 2). From systems like the eects of EGF on work of our colleagues in the ®eld. In order to reduce the size of the reference list we have referred to a number of Drosophila development, it appears that the same previous review articles on ETS genes and eliminate some extracellular signals can elicit distinct responses, of the primary publications. This work is dedicated to TS depending on cell type and developmental stage (for Papas, a dear mentor and friend, and a pioneer in the ®eld recent reviews see: Young and Wesley, 1997; Schweit- of ets gene research, who left us prematurely. This work zer and Shilo, 1997; Dominguez et al., 1998; Van was supported by the EU grant HRPN-CT2000-00083.

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Oncogene