Molecular biology of the Ets family of transcription factors

Gene 303 (2003) 11–34 www.elsevier.com/locate/gene

Review

Molecular biology of the Ets family of transcription factorsq Tsuneyuki Oikawa*, Toshiyuki Yamada Department of Cell Genetics, Sasaki Institute, 2-2 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan Received 19 August 2002; received in revised form 25 October 2002; accepted 12 November 2002 Received by A.J. van Wijnen

Abstract The Ets family of transcription factors characterized by an evolutionarily-conserved DNA-binding domain regulates expression of a variety of viral and cellular genes by binding to a purine-rich GGAA/T core sequence in cooperation with other transcriptional factors and cofactors. Most Ets family proteins are nuclear targets for activation of Ras-MAP kinase signaling pathway and some of them affect proliferation of cells by regulating the immediate early response genes and other growth-related genes. Some of them also regulate apoptosisrelated genes. Several Ets family proteins are preferentially expressed in specific cell lineages and are involved in their development and differentiation by increasing the enhancer or promoter activities of the genes encoding growth factor receptors and integrin families specific for the cell lineages. Many Ets family proteins also modulate gene expression through protein-protein interactions with other cellular partners. Deregulated expression or formation of chimeric fusion proteins of Ets family due to proviral insertion or chromosome translocation is associated with leukemias and specific types of solid tumors. Several Ets family proteins also participate in malignancy of tumor cells including invasion and metastasis by activating the transcription of several protease genes and angiogenesis-related genes. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Ets transcription factor; Growth; Differentiation; Apoptosis; Leukemia; Tumor cell; Malignancy

1. Introduction The first founding member of the ets (E26 transformation-specific) family gene, v-ets, was originally identified as a gag-myb-ets fusion oncogene of the avian transforming

Abbreviations: AML, acute myelogenous leukemia; APC, adenoma polyposis of colon; DMSO, dimethylsulfoxide; EWS, Ewing sarcoma; GMCSF, granulocyte macrophage colony stimulation factor; HDAC, histone deacetylase; HLH, helix–loop– helix; HTH, helix–turn –helix; ICSBP, interferon consensus sequence binding protein; Ig, immunoglobulin; JNK, c-Jun N-terminal kinase; MAP kinase, mitogen activated protein kinase; MEL, murine erythroleukemia; METS, mitogen Ets transcriptional repressor; MMP, matrix metalloproteinase; MuLV, murine leukemia virus; NBs, nuclear bodies; PDGFR, platelet-derived growth factor receptor; Pip, PU.1 interacting partner; PML, promyelocytic leukemia; PNT, pointed; SRE, serum response element; SRF, serum response factor; SUMO1, small ubiquitin-like modifier 1; TCF, ternary complex factor; TCR, T cell receptor; TEL, translocation ets leukemia; TGF, tumor growth factor; Ubc, ubiquitin-conjugating enzyme; VEGF, vascular endothelial growth factor. q Dedicated to our friend Dr. Takis S. Papas who was a pioneer in Ets research. * Corresponding author. Tel.: þ 81-3-3294-3286; fax: þ81-3-3294-3290. E-mail address: [email protected] (T. Oikawa).

retrovirus E26 which induces both erythroblastic and myeloblastic leukemias in chickens (Leprince et al., 1983). Many cellular homologs were isolated thereafter from Caenorhabditis elegans or Drosophila melanogaster to humans (Hart et al., 2000b; Hsu and Schulz, 2000). So far, approximately 30 members of the family have been identified in mammals which are shown to encode nuclear transcription factors to regulate gene expression. A characteristic feature of this family is that they shear an evolutionarily-conserved Ets domain of about 85 amino acid residues that mediate binding to purine-rich DNA sequences with a central GGAA/T core consensus and additional flanking nucleotides (Graves and Petersen, 1998). Many Ets family proteins are down-stream nuclear targets of the signal transduction cascades. This posttranslational modification of the proteins often changes their DNA binding, transcriptional activities, association with cellular partners, subcellular localization and/or protein stabilities (Sharrocks, 2001). They activate or repress transcription on DNA in cooperation with other members of transcription factors and co-factors to play crucial roles in regulation of a variety of cellular function including growth, apoptosis, development, differentiation and oncogenic

0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. doi: 1 0 . 1 0 1 6 / S 0 3 7 8 - 1 1 1 9 ( 0 2 ) 0 1 1 5 6 - 3

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transformation (Bassuk and Leiden, 1997; Dittmer and Nordheim, 1998; Graves and Petersen, 1998; Sharrocks et al., 1997; Wasylyk et al., 1998). Several lines of evidence have established that protein-protein interactions between Ets family proteins and other cellular partners also modulate gene expression (Li et al., 2000a). We summarize here recent advances in the molecular biology of the Ets family of transcription factors.

2. General features of Ets family proteins 2.1. Structure and expression patterns of Ets family proteins Ets family proteins can be divided into several subfamilies on the basis of their structural composition and their similarities in the DNA-binding Ets domains (Fig. 1). Most of them have the Ets domains in their C-terminal regions. However, several Ets family proteins like the ternary complex factor (TCF) subfamily have the Ets domains in their N-terminal regions. In addition, beside the conserved Ets domain, a subset of Ets family proteins have another evolutionarily-conserved domain called the Pointed (PNT) domain at their N-terminal regions, which forms a helix – loop – helix (HLH) structure for protein-protein

interactions (Kim et al., 2001). Certain Ets family protein such as Fli-1 has two possible transcriptional activation domains at their N- and C-terminal regions, though the Cterminal activation is not as strong as the N-terminal domain (Rao et al., 1993). NMR-analysis of the structure of the Ets domains revealed that it contains three a-helixes (a1 –a3) and four-stranded b-sheets (b1 –b4) arranged in the order a1b1-b2-a2-a3-b3-b4 forming a winged helix – turn– helix (wHTH) topology (Kodandapani, 1996; Donaldson et al., 1996). The third a-helix is responsive to contact to the major groove of the DNA. Different members of the Ets family proteins display distinct DNA binding specificities. The Ets domains and the flanking amino acid sequences of the proteins influence the binding affinity (Graves and Petersen, 1998), and the alteration of a single amino acid in the Ets domain can change its DNA binding specificities (Mo et al., 2000). Furthermore, DNA sequences flanking the GGAA/T central core also affect the DNA binding affinities of Ets family proteins. For an example, changing sequence of 50 -GCGGAAGCG-30 in the MSV (Moloney sarcoma virus) enhancer to GCGGATGCG significantly reduces Elf1 binding but no effect on Ets-1 binding, while altering this sequence to GCGGAAGAA completely abolishes Ets-1 binding but little effect on Elf-1 binding (Wang et al., 1992).

Fig. 1. Schematics of the structure of the members of Ets family proteins. Ets, DNA-binding (Ets) domain; HLH, helix–loop–helix domain (Pointed domain); AD, activation domain; ID, auto-inhibitory domain; RD, repression domain.

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Thus, flanking DNA sequences as well as purine-rich core DNA sequences appear to be important to determine the preferential binding of individual Ets family proteins. Some Ets family proteins are expressed ubiquitously, some in a tissue-specific manner. Expression patterns of representative Ets family proteins are shown in Table 1. For examples, Ets-1 first appears in blood islands of yolk sac and then in several organs including blood vessels, developing brain and bone, and mesodermal cells in organs undergoing morphogenetic processes during mammalian development (Kola et al., 1993). In adult tissues, however, high level Ets-1 expression is restricted in lymphoid tissues. Ets-2 having a high similarity with Ets-1 is expressed in the extra-embryonic trophectoderm during embryonic development (Yamamoto et al., 1998) and is expressed ubiquitously in adult tissues. Erg is initially expressed in embryonic endothelial tissues and later in the kidney, urogenital tracts and hematopoietic cells. Fli-1 is the gene originally isolated from the proviral integration site of Friend murine leukemia virus-induced mouse erythroleukemia cells and it is preferentially expressed in the hematopoietic cell lineages and vascular endothelial cells. GAPBa (GA-binding protein a), a subunit of GABP heterodimerized with the second subunit of non-Ets family protein GABPb, is expressed in a variety of tissues. The TEL (translocation ets leukemia)/ ETV6 (Ets translocation variant 6) gene, originally isolated from the breakpoint of chromosome translocation in human chronic myelomonocytic leukemia cells (Golub et al., 1994), is expressed ubiquitously and is suggested to be essential for maintenance of the vascular network in the yolk sac and adult hematopoiesis in bone marrow (Wang et al., 1997). PEA3 (polyomavirus enhancer-binding activator 3)/E1AF (adenovirus E1A enhancer-binding factor) is mainly expressed in epithelial cells including mammary gland, epidermis and brain but not in hematopoietic cells (Xin et al., 1992). Elf-1 (E74-like factor) is expressed in hematopoietic cells and developing epithelial tissues, whereas ESE-1 (epithelial specific Ets)/ESX (epithelial-restricted with serine box) belonging to the same ELF subfamily is expressed exclusively in epithelial cells and could be a critical regulator of epithelial cell Table 1 Tissue distribution of major Ets family proteins Member

Expressing organs and tissues

Ets-1 Ets-2 Erg Fli-l GABPa TEL PEA3/E1AF Elf-1 ESE-1/ESX PU.1 TCFs

Lymphoid organs, brain, vascular endothelial cells Ubiquitous Vascular endothelial cells, hematopoietic cells, kidney, etc. Hematopoietic cells, vascular endothelial cells Ubiquitous Ubiquitous Epidermis, mammary gland, brain, etc. Hematopoietic cells, liver, kidney, intestine, etc. Epithelial cells B cells, macrophages, neutrophils Ubiquitous

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differentiation (Oettgen et al., 1997). PU.1 is exclusively expressed in hematopoietic cells (Klemsz et al., 1990) and is the most distantly related member of the Ets family protein having only about 40% identity with Ets-1 in its DNAbinding domain. The PU.1 gene is identical to the Spi-1 gene that had been identified in a common site for proviral integration of spleen focus forming virus (SFFV) which is responsible for development of murine erythroleukemias induced by Friend virus (Moreau-Gachelin et al., 1988). Members of TCF subfamily including Elk-1 (Ets-like protein 1), Sap-1 (serum response factor [SRF] accessory protein 1), Net (new Ets) and its splicing variant Net-b are expressed in many tissues (Treisman, 1994). They play important roles in cell growth as nuclear targets of signal transduction from extra-cellular stimulation as stated in Section 3.2. 2.2. Interaction between Ets family and other cellular proteins Ets family proteins regulate gene expression by functional interaction with other transcription factors and co-factors on composite DNA-binding sites. The most well-known example is interaction of Ets family with Jun family proteins on the DNA sequences called the Ras-responsive element (RRE) consisting of an Ets binding site and an AP-1 (Fos/Jun) site. The RRE was originally identified on the polyomavirus enhancer and is located on the sequence in various cellular genes which are responsive to Ras/mitogen activated protein (MAP) kinase signaling (Imler et al., 1988). This element is found in the promoters of matrix metalloproteinase (MMP) genes, which are responsible for organ remodeling and tumor invasion, and the promoters are synergistically activated by Ets-1 and AP-1 with the coactivator CBP/p300 (Fig. 2a). Ets-1 and a pituitary-specific POU homeodomain transcription factor Pit-1/GHF-1 cooperatively enhance the activity of the prolactin and growth hormone genes (Bradford et al., 2000). Phosphorylation of Pit-1 regulates the interaction with Ets-1 (Augustijn et al., 2002). Phosphorylated Elk-1 and Sap-1a cooperatively interact with SRF on serum response element (SRE) to enhance the c-fos promoter activity (Fig. 2b) (see Section 3.2) (Hassler and Richmond, 2001). It is also known that a number of Ets family proteins interact and crosstalk with several transcription factors including AP-1, AML1, LEF-1, Sp1, c-Myb, Pax-5, NF-kB, Stat-5 and Maf-B to co-regulate the expression of cell-type specific genes (Li et al., 2000a). Conversely, there are several cases showing that proteinprotein interactions lead to suppression of the function of Ets family proteins and/or partner proteins. The transcriptional activity of Ets-2 is inhibited by protein-protein interaction with Erg (Basuyaux et al., 1997), and PU.1 and Elf-1 have been reported to bind directly to the tumor suppressor protein Rb (Hagemeire et al., 1993; Wang et al., 1993). Ets-1 and TEL have been reported to interact with Ubc9, a ubiquitin-conjugating enzyme usually involved in

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Fig. 2. Functional cooperation of Ets family proteins with other transcription factors and co-activators on various cellular promoters and enhancers. (a) Ets-1 or PEA-3 activates the MMP (matrix metalloproteinase) promoters in cooperation with AP1 (c-Fos/c-Jun) on the RRE (Ras-response element). (b) TCF (Elk-1, Sap-1) activates the c-fos promoter in cooperation with SRF (serum response factor). (c) PU.1 activates myeloid-specific promoters in cooperation with AML1 and C/EBP. (d) Fli-1 activates megakaryocyte-specific promoters in cooperation with GATA-1. (e) PU.1 activates the immunoglobulin (Ig) heavy (H) chain enhancer in cooperation with E12, Ets-1, TFE3 and Oct-2. (f) Ets-1 activates the T cell receptor (TCR) a enhancer in cooperation with CREB/ATF, TCF-1, AML1 and GATA-3.

proteosome-mediated degradation, through the Pointed domain (the HLH domain). The interaction, however, does not lead to degradation of the proteins but rather leads to modulation of the transcriptional activity of these Ets family proteins (Hahn et al., 1997; Chakrabarti et al., 1999). Some Ets family proteins can change their subcellular localization by interacting with other nuclear proteins. The interaction of TEL with Ubc9 further leads to conjugation to small ubiquitin-like modifier-1 (SUMO-1) and modifies the localization of TEL to cell cycle-specific nuclear speckles called TEL bodies (Chakrabarti et al., 2000). Although SUMO-1-modified proteins are believed to be redirected to a specific subcellular compartment in the nucleus, it is not yet known if other members of Ets family proteins also interact with SUMO-1. Nuclear bodies (NBs) are also unique structures in the nucleus and consist of the promyelocytic leukemia (PML) protein and Sp100 nuclear matrix-associated protein, another component of NBs (Borden, 2002). Besides these proteins, NB contains transcription factors, tumor suppressor gene products such as hypophosphorylated Rb and p53, co-activators, corepressors and chromosomal proteins (Seeler and Dejean, 1999). It has been reported that Ets-1 physically and functionally interacts with Sp100 to alter the morphology of NBs by decreasing the number of NBs and increasing the transcriptional activity of Ets-1 (Wasylyk et al., 2002). Ets-1 also interacts with Daxx, the death-associated protein and a

component of NBs, and is co-localized in the nucleus to repress the transcriptional activation of the matrix metalloproteinase 1 (MMP-1) and bcl-2 genes (Li et al., 2000b). Many Ets family proteins including Elk-1, Ets-1, Ets-2 and ER81 interact with co-activator CBP/p300 to activate transcription (Jayaraman et al., 1999). We have reported that PU.1 interacts with CBP and p300 (Yamamoto et al., 1999, 2002), and also with histone deacetylase-1 (HDAC1) through mSin3A (Kihara-Negishi et al., 2001), suggesting that PU.1 acts as an activator and a repressor for transcription in association with co-activators and corepressors, respectively. TEL has been shown to repress transcription by recruiting co-repressor mSin3A or N-CoR and HDAC-3 but not other HDACs (Wang and Hiebert, 2001). Furthermore, we have recently found that PU.1 directly interacts with MeCP2, a member of methyl-CpGbinding domain proteins, in an mSin3A – HDACs repressor complex (Suzuki et al., preparation). Induction of Fli-1 results in the transcriptional activation of DNA methyltransferase-1 gene expression in K562 erythroleukemia cells treated with interleukin (IL)-6 (Hodge et al., 2001). Moreover, a recent report shows that Erg interacts with ESET (Erg-associated protein with SET domain), a histone H3-specific methyltransferase (Yang et al., 2002). Thus, some of the Ets family proteins may participate in operating chromatin modification and/or DNA methylation to activate or repress transcription.

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3. Ets family proteins involved in cell growth 3.1. Growth factors and growth factor receptors regulated by Ets family proteins Expression of many cell type-specific growth factors, growth factor receptors and integrin families involved in cellular proliferation are controlled by Ets family proteins. Signaling by the growth factors and their receptors finally modulates the functional activities of several Ets family proteins via MAP kinase, Jak/Stat signaling and others. Representative examples of the cytokine or growth factor receptors controlled by Ets family proteins include the GMCSF receptor, M-CSF receptor (c-fms) (Fig. 2c), G-CSF receptor, Toll-like receptor-4, c-fes, IL-1b and FcgRI in myeloid cells (Friedman, 2002), the thrombopoietin receptor c-Mpl (Fig. 2d) in megakaryocytic cells (Deveaux et al., 1996), the immunoglobulin heavy (IgH) (Fig. 2e) and light chains (Igk, Igl) in B cells, and the T cell receptors (TCRa/ b) (Fig. 2f), IL-2 receptor, and CD31 in T cells (Bassuk and Leiden, 1997). Lipopolysaccharide (LPS) stimulates the binding of Ets-1 and Elk-1 to tumor necrosis factor-a (TNFa) promoter in macrophages (Tsai et al., 2000). Expression of hepatocyte growth factor (HGF) receptor, encoded by c-met, is up-regulated by Ets-1 and c-met activation by HGF induces transcription of c-ets-1 via a Ras-MAP kinase signaling pathway, indicating that Ets-1 acts both upstream and downstream of c-met (Gambarotta et al., 1996; Paumelle et al., 2002). The activity of the erb B2/HER-2/neu promoter is enhanced by PEA3, ESE-1/ ESX and Elf-1 in breast cancer cells (Scott et al., 2000). Recent reports suggest that ESE-1/ESX modulates TGFb signaling by regulating transcription of the TGFb type II receptor gene (Chang et al., 2000). 3.2. Signal transduction and Ets family proteins Most Ets family proteins are phosphorylated in response to growth factors and cellular stress. Phosphorylation of the proteins sometimes affects their DNA-binding activities, transcriptional activities and association with other transcription factors. Several members of Ets family proteins usually show negative regulation of DNA binding by intra-molecular interaction. For example, deletion mutants of Ets-1 in the central region and the C-terminal region display enhanced DNA-binding activity 10– 20 times than the full length protein, suggesting that the regions flanking the Ets domain are inhibitory domains for DNA binding (Lin et al., 1992). This autoinhibition is reinforced by phosphorylation of Ets1 by calcium signaling pathway but not by MAP kinase signaling. It is speculated that intramolecular interaction between the inhibitory regions destabilizes protein-DNA complexes by an allosteric mechanism (Jonsen et al., 1996), and that negatively charged phosphate groups promote electrostatic interaction between the phosphorylated region

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and the inhibitory module of Ets-1 (Cowley and Graves, 2000). The autoinhibition of Ets-1 can also be relieved by interactions with partner transcription factors. The mutual activation of DNA binding of Ets-1 and AML1/RUNX1, a runt domain transcription factor, by direct interaction of their autoinhibitory domains has been reported on the T cell receptor (TCR) b-chain enhancer (Kim et al., 1999) and on the Moloney murine leukemia virus enhancer (Goetz et al., 2000). Autoinhibition of DNA binding is also observed in Ets-2 (Wasylyk et al., 1992), PEA3 (Greenall et al., 2001) and ERM (Ets-related molecule PEA-like) (Laget et al., 1996). However, autoinhibitory mechanisms appear to be different among them, since there are no homologous amino acid sequences in their inhibitory domains. TCF subfamily is implicated in regulation of cell proliferation in response to MAP kinase signaling (Gutman and Wasylyk, 1990; Sharrocks, 2001). Phosphorylation of TCFs (Ser383 in Elk-1) by the Erk MAP kinase following serum or TPA stimulation promotes association with serum response factor (SRF) (Gille et al., 1996) and subsequent allosteric changes in TCFs enhance the binding of the complex to the promoters of many immediate early genes including c-fos (Fig. 2b), egr-1, pip-92 and nur77 (Wasylyk et al., 1998). Mutation of Ser383 to alanine in Elk-1 abolishes transcriptional activation by Elk-1 (Cruzalegui et al., 1999). Although Elk-1 and Sap-1 can also act as targets of the stress-activated JNK and p38 MAP kinases, Sap-1 is preferentially phosphorylated by p38 (Janknecht and Hunter, 1997) and Elk-1 is preferentially phosphorylated by JNK (Strahl et al., 1996). Furthermore, it is thought that the response of TCFs to different MAP kinase pathways differs depending on cell types and stimuli (Galanis et al., 2001). Fli-1 also associates with SRF to activate the egr-1 promoter (Mora-Garcia and Sakamoto, 2000). Net is also a member of TCF subfamily but it negatively regulates the cfos and egr-1 promoter activities when it is not phosphorylated by Erk2 MAP kinase. This negative regulation is mediated by recruitment of histone deacetylase (HDAC) via the co-repressor CtBP (E1A C-terminal-binding protein) (Criqui-Filipe et al., 1999). A recent report indicated that recruitment of an mSin3A-HDAC complex in an Elk-1 complex also negatively regulates the c-fos promoter activity (Yang et al., 2001), suggesting that the same Ets family proteins behave as activators or repressors depending on cellular conditions. TEL has a homology with Yan, a repressor Ets family protein for Ras-signal transduction in Drosophila. When Ras-MAP kinase pathway is activated through EGF receptors, Yan is phosphorylated and inactivated concomitant with phosphorylation of another Ets family activator PntP2 to activate target genes essential in eye development (Hsu and Schulz, 2000). In mammalian cells, TEL has been reported to act as a tumor suppressor to induce G1 arrest and to suppress Ras-induced transformation (Rompaey et al., 2000). The function of Ets-1 and Ets-2 is also activated by RasMAP kinase signaling. Mutation of a threonine residue to

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alanine located within the N-terminal Pointed (PNT) domains of Ets-1 and Ets-2 (Thr38 and Thr72, respectively) has been reported to abolish Ras-responsive enhancement of their transcriptional activities (Yang et al., 1996). The PNT domains of Ets-1 and Ets-2 contain Erk2 MAP kinase docking sites, and mutation of these sites prevents Rasmediated enhancement of the transactivation function of these proteins (Seidel and Graves, 2002). The transcriptional activities of other members of Ets family proteins are also modified by phosphorylation. It has been reported that PI3K (phosphoinoside 3 kinase) – Akt signaling is able to enhance the immunoglobulin (Ig) k30 enhancer activity in pre-B cells probably due to an increase in the transcriptional activity of PU.1 but not due to alteration of PU.1 expression, DNA binding, or protein-protein-interaction with Pip (PU.1-interacting partner)/IRF-4 (interferon regulatory factor-4) to recognize the PU.1 and Pip binding sequences in the enhancer. Substitution analysis indicates that Ser41 in the transactivation domain of PU.1 is a phosphorylation site to respond to Akt (Rieske and Pongubala, 2001). For the recruitment of Pip to the Ig k30 enhancer, Ser148 in the PEST domain of PU.1 has to be phosphorylated by casein kinase II (CKII) (Pongubala et al., 1993). Furthermore, JNK (c-Jun N-terminal kinase)/SAPK (stress-activated protein kinase) phosphorylates PU.1 and Spi-B, but Erk-1 phosphorylates Spi-B but not PU.1, suggesting differential regulation of their activities by MAP kinases (Mao et al., 1996). Ets family proteins are nuclear proteins and some of them have nuclear export signals (NES) as well as nuclear localization signals (NLS). Phosphorylation of Ets family proteins changes their subcellular localization in several cases. Stress of UV irradiation or heat shock to cells induces active nuclear exclusion of Net, a member of the TCF subfamily, through phosphorylation of NES that involves JNK/SAPK (Ducret et al., 1999). Similar regulation by changing subcellular localization of proteins has also been reported in Ets-2 repressor factor (ERF), which was originally isolated from the Ets binding site of the c-ets-2 promoter and is believed to be important in the control of cell proliferation during G0/G1 phase of the cell cycle, although Erk2 MAP kinase instead of JNK is involved in this case (Gallic et al., 1999). Thus, post-translational modification of Ets family proteins modulates DNA-binding activities, association with co-regulatory partners, transcriptional activation capacities, and subcellular localization. 3.3. Regulation of cell cycle-related genes by Ets family proteins Ets-1 and Ets-2 do not bind to the serum response element (SRE) of the c-fos promoter even after phosphorylation by MAP kinase but appear to contribute to activate cmyc expression by binding to the Ets consensus site overlapping the E2F site of the promoter (Roussel et al.,

1994). Mitogenic signaling by colony-stimulation factor-1 (CSF-1) and Ras, which activates c-myc expression, is suppressed by a dominant negative mutant of Ets-2 and is restored by c-myc overexpression (Langer et al., 1992), suggesting that Ets-2 also acts as an effector of a signal transduction pathway. Activation of both Ets-1 and Ets-2 via Ras-MAP kinase signaling has been reported to enhance the junB promoter (Coffer et al., 1994). Ets-2 also transactivates the cdc2 (Wen et al., 1995) and cyclin D1 promoters (Albanese et al., 1995). Cdk10, a Cdc2-related kinase regulating G2/M phase of the cell cycle, interacts with the Pointed domain of the Ets-2 but not Ets-1 to inhibit Ets-2-mediated transactivation (Kasten and Giordano, 2001), suggesting involvement of Ets-2 in cell cycle regulation. In this regard, MEF (myeloid Elf-1-like factor), a member of the Elf subfamily proteins, interacts with cyclin A-Cdk2 complex to inhibit the DNA-binding activity of MEF by phosphorylation (Miyazaki et al., 2001). Expression of c-ets-2 is negatively regulated by ERF (Ets2 repressor factor). The repressor activity of ERF is modulated by change in its cellular localization through cell cycle-dependent phosphorylation of the protein (Gallic et al., 1999). Ets family proteins activate tumor suppressor genes in some circumstances, which may reflect the feedback mechanism. It has been recently reported that Ets-1 or Ets-2 activates the promoter of the p16 INK4a gene, whose product inhibits Cdk4 and Cdk6 cyclin-dependent kinases, through an Ets-binding site and contributes to regulation of cellular senescence. In young human diploid fibroblasts, p16 INK4a is expressed at low levels probably due to inhibition of signaling of Ets-2 by relatively high levels of Id1, whereas up-regulation of p16 INK4a is followed by an increase in Ets-1 and the reciprocal reduction of Id1 in senescent cells (Ohtani et al., 2001). E1AF, a human homolog of mouse PEA3, has been found to up-regulate transcription from the p21 WAF1/Cip1 promoter in vitro (Funaoka et al., 1997). This is also true in Ets-1 and Fli-1. Human p53 gene is transcriptionally regulated by Ets-1 and Ets-2 but not Fli-1 by binding to the Ets binding sites on its promoter (Venanzoni et al., 1996). GABPa and Fli-1 can bind to the promoter of the Rb gene to inhibit expression of Rb (Savoysky et al., 1994; Tamir et al., 1999). Function of Ets family proteins are also regulated by protein-protein interactions. The function of TCF is negatively regulated by interaction with Id proteins, a subfamily of HLH transcription factors (Yates et al., 1999). It has also been reported that BRCA1 splice variants BRCA1a/1b also interact with Elk-1, Sap-1 and Fli-1 via their Ets domains, suggesting that one of the mechanisms by which BRCA1a/1b proteins function as tumor suppressors is through inhibition of the expression of Elk-1 targets like the c-fos gene in human breast cancer (Chai et al., 2001). Collectively, not only TCF subfamily but also other Ets subfamilies modulate the expression of many cell cycle-

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related genes including oncogenes and tumor suppressor genes.

4. Ets family proteins involved in apoptosis 4.1. Prevention of apoptosis by Ets family proteins Several Ets family proteins have been shown to be involved in the apoptotic process. Most members of Ets family proteins behave anti-apoptotically. Ets-1 appears to be required for survival and activation of T cells, since c-ets1 2/2 Rag-2 2/2 chimeric mice display a marked decrease in the number of mature T cells and a severe deficiency in proliferation in response to activational signals with increased rates of spontaneous apoptosis in T cells (Muthusamy et al., 1995). B cells from Spi-B-knockout mice poorly proliferate with severe defects in B cell antigen receptor-mediated signaling and exhibit increased rates of apoptosis (Su et al., 1997), suggesting that Spi-B is essential for B cell survival. Expression of c-rel, a member of the Rel/ NF-kB family, is markedly reduced in PU.1 þ/ 2 Spi-B 2/2 mice, and both Spi-B and PU.1 are shown to transactivate the c-rel promoter in B cells (Hu et al., 2001). This is consistent with the previous report that Rel is anti-apoptotic to regulate the expression of anti-apoptotic Bcl-XL and Bfl1/A1 in B cells (Chen et al., 2000; Lee et al., 1999). Thus, anti-apoptotic activity of Spi-B may be partly due to activation of c-rel expression in B cells. Ets-2 and PU.1 rescue apoptosis in macrophages upon deprivation of macrophage colony-stimulating factor (M-CSF), through up-regulation of anti-apoptotic Bcl-XL but not of apoptotic Bcl-Xs (Sevilla et al., 2001). Fli-1 and Erg inhibit apoptosis in NIH/3T3 cells induced by serum depletion or treated with a calcium ionophore (Yi et al., 1997). In this connection, the most recent report indicates that Fli-1 transcriptionally activates the bcl-2 gene expression by binding directly to the upstream regulatory region of the gene (Lesault et al., 2002). It has also been reported that absence of TEL leads to a defect in yolk sac angiogenesis and intra-embryonic apoptosis of mesenchymal and neural cells (Wang et al., 1997), suggesting an important role of TEL in survival of specific types of cells. Thus, many Ets family proteins appear to be anti-apoptotic.

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revealed that Ets-1 is required for the formation of a stable DNA-p53-CBP complex to induce pro-apoptotic genes in the process of UV-induced apoptosis in embryonic stem (ES) cells (Xu et al., 2002). Overexpression of Ets-2 in prostate tumor cells increases apoptosis accompanied by increased levels of p21WAF1/Cip1 (Foos and Hauser, 2000). Furthermore, constitutive expression of Elk-1 triggers apoptosis in Rat-1 fibroblasts and MCF-7 human breast cancer cells when treated with calcium ionophore (Shao et al., 1998). We also showed that overexpression of PU.1 in conjugation with the dimethylsulfoxide (DMSO) in murine erythroleukemia cells induces apoptosis accompanied with downregulation of cmyc and bcl-2 expression (Kihara-Negishi et al., 1998), and with loss of DNA-binding activity of GATA-1 (Yamada et al., 1998) which is an important transcription factor for differentiation and survival of erythroid cells (Cantor and Orkin, 2002). The TEL – PDGFRb (platelet derived growth factor receptor b) fusion protein isolated from patients bearing a t(5;12) chromosome translocation (see Section 6.2.2 and Fig. 4a) induces apoptosis in mouse bone marrowderived Ba/F3 cells through activation of JNK/SAPK signaling pathway (Atfi et al., 1999). There are several reports showing that expression of apoptosis-related genes is directly induced by Ets family proteins. Expression of the Fas ligand (FasL) gene in vascular smooth muscle cells is controlled by cooperative activation between Ets-1 and Sp1 (Kavurma et al., 2002). The Ets-binding sites of the 50 -flanking region of the human caspase-3 genes are necessary to achieve sustained transcriptional activity of caspase-3 (Liu et al., 2002). High expression levels of poly(ADP-ribose) polymerase (PARP) in Ewing’s sarcoma cells is suggested to be due to a strong enhancement of PARP promoter activity by Ets-1 (Soldatenkov et al., 1999). Furthermore, it has also been reported that Ets-1 and Fli-1 regulate expression of GADD 153, which induces growth arrest and apoptosis in response to stress signals or DNA damage (Seth et al., 1999). Whether the Ets family proteins induce or prevent apoptotic cell death may depend on several factors such as expression levels, cellular contexts and the existence of agonistic or antagonistic signals in cells.

5. Ets family proteins involved in lineage development and cell differentiation

4.2. Induction of apoptosis by Ets family proteins 5.1. Hematopoietic differentiation On the other hand, some of the Ets family proteins behave apoptotically under a certain condition of cells. Ets-1 and Ets-2 have been reported to be pro-apoptotic as well as antiapoptotic in some cases. Expression of the p42 spliced variant of Ets-1 promotes Fas-mediated apoptosis by upregulating caspase-1 in human colon cancer cells (Li et al., 1999) and overexpression of Ets-1 in human umbilical vein endothelial cells induces apoptosis under serum-deprived conditions (Teruyama et al., 2001). A recent report has

5.1.1. Differentiation of hematopoietic progenitors Determination of hematopoietic cell differentiation seems to be controlled by coordinate action of extracellular signals and a cascade of appearance of several critical master regulators of transcription factors for specific cell lineages (Graf, 2002; Orkin, 1996; Tenen et al., 1997). SCL/ Tal-1 and Lmo2/Rbtn2 are involved in primitive hematopoiesis in yolk sac, whereas GATA-2, AML1/RUNX1,

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CBFb and c-Myb are involved in definitive hematopoiesis in fetal liver (Shivdasani and Orkin, 1996). Several Ets family proteins are preferentially expressed in certain lineages of hematopoietic cells and also play important roles in their development, commitment and differentiation (Fig. 3). Ets-1, Fli-1 and Erg are initially expressed in the blood island of the yolk sac where hemangioblasts, the common precursors of vascular and hematopoietic lineages, are present (Maroulakou and Bowe, 2000). This may account for the expression of these Ets family proteins in both endothelial cells and a certain lineage of hematopoietic cells. Vav is a guanine nucleotide exchange factor for the Rho/Rac GTPase up-regulated during the transition from primitive to definitive hematopoiesis in the aorta-gonad-mesonephros (AGM) region of the embryo (Okada et al., 1998), and the expression of the vav gene is shown to be regulated by AML1 and PU.1 (Denkinger et al., 2002). TEL is essential for embryonic angiogenesis in the yolk sac and in adult hematopoiesis by controlling the bone marrow microenvironment (Wang et al., 1997). Ets binding sites are also found in the enhancers and/or promoters of the SCL/Tal-1 (Bockamp et al., 1995), c-kit (Ratajczak et al., 1998), IL-3 (Gottschalk et al., 1993) and Tie-2/Tek genes (Dube et al., 1999), all of

which are critical for early hematopoiesis and angiogenesis. Indeed, expression of the Tie-2/Tek gene, which encodes the receptor of angiopoitin-1, is shown to be completely abolished in Fli-1 knockout mice (Hart et al., 2000a). Furthermore, it has been recently shown that the SCL enhancer is directly regulated by Fli-1, Elf-1 and GATA-2 (Go¨ttgens et al., 2002). Expression of PU.1 is observed in CD34þhematopoietic progenitors and differentiation commitment toward myeloid and lymphoid lineages appears to be determined by the expression levels of PU.1, since high PU.1 levels promote macrophage differentiation and relatively low PU.1 levels induce B-cell differentiation when PU.1 2/2 hematopoietic progenitors are restored by retrovirus transduction of PU.1 (DeKoter and Singh, 2000). PU.1 2/2 hematopoietic progenitors fail to express interleukin-7 receptor a (IL-7Ra) transcripts which are essential for B cell differentiation. Analyses of the promoter and chromatin immunoprecipitation experiments revealed that PU.1 directly regulates IL-7Ra promoter in common lymphoid progenitors (DeKoter et al., 2002). 5.1.2. Myeloid cell differentiation Early myeloid differentiation is controlled by PU.1 and other families of transcription factors, such as C/EBPs,

Fig. 3. Schematics of transcriptional regulation of hematopoietic cell differentiation. HAB, hemangioblast; AB, angioblast; EC, Endothelial cell; HSC, hematopoietic stem cell; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; MEP, myeloid-erythroid progenitor; GMP, granulocyticmonocytic progenitor; ProB, pro-B cell; ProT, pro-T cell; Ery, erythrocyte; Meg, megakaryocyte; Neu, neutrophil; MF, macrophage; B, B cell; NK, natural killer cell; T, T cell. Note the levels of PU.1, GATA-1 and other lineage-specific transcription factors are critical to determine the subsequent cell fates of hematopoietic progenitors.

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AML1, retinoic acid receptora (RARa), c-Myb and others (Friedman, 2002). PU.1-knockout mice are deficient in developing mature macrophages and the function of neutrophils, and contain defects in lymphoid cells, especially in B cells (McKercher et al., 1996; Scott et al., 1994). C/EBPa and C/EBP1 knockout mice also have a defect in the development of mature macrophages and neutrophils (Zhang et al., 1997; Yamanaka et al., 1997). Thus, PU.1, as well as the C/EBP family, is a very critical master regulator for determination of the developmental pathway toward the myeloid lineage in hematopoietic cells. With regard to this, we have found that overexpression of PU.1 in erythroid cells results in a lineage shift to macrophages (Yamada et al., 2001). A similar result has also been reported in PU.1-overexpressing hematopoietic precursors, where lymphoid development is blocked but macrophage development is promoted in this system (Anderson et al., 2002). Many myeloid specific promoters have PU.1 and C/EBP binding sites upstream of the transcription start sites (Fig. 2c). These include the genes encoding macrophage colony stimulating factor (M-CSF) receptor (c-fms), granulocyte colony stimulation factor (GCSF) receptor, granulocyte-macrophage colony stimulating factor (GM-CSF) receptor, scavenger receptor, CD11b/ CD18 (Mac-1), myeloperoxidase (MPO), lysozyme, IL-1b, c-fes, neutrophil esterase, gp91phox and gp47phox (phagocyte NADPH oxidase components), and others (Friedman, 2002; Oikawa et al., 1999). Thus, failure in the development of myeloid lineages during fetal liver hematopoiesis in PU.1knockout mice could be partly due to absence of expression of GM-CSF receptor. However, early myeloid genes such as the GM-CSF receptor, G-CSF receptor and MPO genes are expressed in PU.1 2/2 embryos (Olson et al., 1995). In spite of expression of the GM- and G-CSF receptor, PU.1 2/2 myeloid progenitors fail to respond to GM- and G-CSF, although the unresponsiveness to G-CSF is bypassed by signaling with IL-3 in vitro (DeKoter et al., 1998). Furthermore, the expression of genes associated with terminal myeloid differentiation such as M-CSF receptor and CD11 is eliminated in differentiated PU.1 2/2 embryonic stem (ES) cells (Simon et al., 1996). PU.1 2/2 mice develop neutrophils but PU.1-deficient neutrophils fail to respond to chemokines and to generate superoxide ions, and they are ineffective at bacterial uptake and killing (Anderson et al., 1998). Therefore, the earliest events in myelopoiesis occur but terminal myeloid differentiation is blocked in the absence of PU.1, suggesting that PU.1 is essential for macrophage and granulocyte differentiation but not for commitment to myeloid cell lineages. PU.1 plays a role in not only differentiation but also proliferation and prevention of apoptosis in macrophages (Sevilla et al., 2001). PU.1 negatively regulates c-myb promoter activity by binding to an Ets binding site in the promoter during terminal myeloid differentiation (Bellon et al., 1997), which may correspond to the stop of cell proliferation in differentiated macrophages. PU.1 exhibits partial functional

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redundancy with Spi-B but not with Ets-1 or Elf-1 (Garrett-Sinha et al., 2001). This is supported by a report showing that Spi-B rescues macrophage and granulocyte development in the absence of PU.1 (Dahl et al., 2002). C/ EBPa interacts physically with PU.1 to block the function of PU.1 (Reddy et al., 2002) and PU.1 interacts with GATA1 to block the function of GATA-1 (Nerlov et al., 2000) which is an important master regulator for erythroidmegakaryocytic development (Cantor and Orkin, 2002) and eosinophilic differentiation (McNagny and Graf, 2002), suggesting that protein-protein interactions between these master regulators could specify cell fates in hematopoietic cells. ICSBP (interferon consensus sequence binding protein) also plays a critical role in myeloid cell differentiation (Tamura et al., 2000). It has been reported that PU.1 functionally interacts with ICSBP to activate the IL-1b, Toll-like receptor 4, p67phox, gp91phox and other genes in myeloid cells (Marecki et al., 2001; Rehli et al., 2000). Ets-1 and C/EBPa cooperatively transactivate the eos47 promoter whose expression is eosinophil-specific (McNagny et al., 1998). METS (mitogenic Ets transcriptional suppressor)/PE1 is highly related to ERF (Ets-2 repressor factor) and its expression is enhanced during macrophage differentiation. Enhanced expression of METS down-regulates several Rasdependent cell cycle control genes including the c-myc, cdc2 and p54 subunit of DNA primase genes having Ets monomeric binding sites in the promoters, while it does not inhibit Ras-dependent expression of several cell typespecific genes necessary for differentiation, such as the scavenger receptor A (SR-A), gelatinase B/MMP-9 and macrosialin genes having the binding sites recognized by AP-1/Ets ternary complexes in the promoters (Klappacher et al., 2002). Thus, induction of METS expression results in selective displacement of Ets activators from the monomeric binding sites on cell cycle-regulatory genes, thereby inducing cell cycle arrest without affecting expression of the cell type-specific genes. Overexpression of Ets-2 in mouse myeloid leukemia M1 cells induces macrophage differentiation (Aperlo et al., 1996), although the results of c-ets-2-deficient mice suggest that Ets-2 is not essential for hematopoietic cell development (Yamamoto et al., 1998). 5.1.3. Megakaryocytic and erythroid differentiation A member of the Ets family proteins especially essential for development of megakaryocyte appears to be Fli-1, since Fli-1-knockout mice exhibit severe hemorrhage in the brain based on lack of platelets and are lethal at embryonic day 11.5 (Hart et al., 2000a; Spyropoulos et al., 2000). Importance of Fli-1 in megakaryocytic differentiation is also supported by experimental results that show exogenous overexpression of Fli-1 in K562 human erythroleukemia cells can promote megakaryocytic differentiation (Athanasiou et al., 1996). IL-6 treatment of K562 cells also induces

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the expression of endogenous Fli-1 via Stat3 transcription factor and drives the cells toward megakaryocytic differentiation (Hodge et al., 2002). Fli-1 increases the promoter activity of the p21 WAF1 gene, of which overexpression is known to promote the late stage of megakaryocytic differentiation in K562 cells. It has also been shown that TEL, which interacts with Fli-1 to block its function, inhibits megakaryocytic differentiation in K562 cells (Kwiatkowski et al., 2000). Besides Fli-1, other transcription factors including NF-E2 (Shivdasani et al., 1995), MafG (Onodera et al., 2002), GATA-1 (Shivdasani et al., 1997; Vyas et al., 1999) and co-factor FOG-1 (Friend of GATA-1) (Cantor and Orkin, 2002) are also required to induce megakaryocyte-specific genes for megakaryocytic differentiation. Indeed, in concert with GATA-1, Fli-1 transcriptionally activates a number of megakaryocytespecific genes encoding glycoprotein (Gp) Iba (Hashimoto and Ware, 1995), GpIIb (Zhang et al., 1993), GpIII, GpIX (Bastian et al., 1999), platelet factor 4 (PF4) (Minami et al., 1998), von Willebrand factor (Schwachtgen et al., 1997), thromobopoietin receptor c-Mpl (Fig. 2d) (Deveaux et al., 1996), though not only Fli-1 but also Ets-1 or PU.1 has been reported to be able to enhance these promoter activities in vitro. On the other hand, the representative Ets family proteins involved in erythropoiesis are PU.1, Fli-1 and Ets-1. Expression of PU.1 is down-regulated during terminal differentiation of murine erythroleukemia (MEL) cells by treatment with dimethylsulfoxide (DMSO) (Hensold et al., 1996), and enforced expression of PU.1 in MEL cells inhibits erythroid differentiation as we reported (Oikawa et al., 1999; Yamada et al., 1997). In contrast to the inhibitory effect of PU.1 on erythroid differentiation, GATA-1 is an essential positive activator for differentiation and survival of erythroid cells (Weiss and Orkin, 1995) and overexpression of GATA-1 in myeloid cells induces lineage switch toward erythroid cells (Seshasayee et al., 1998). Since there is negative crosstalk between PU.1 and GATA1, loss of GATA-1 function by overexpression of PU.1 in erythroid progenitors appears to be responsible for block of erythroid differentiation (Nerlov et al., 2000). GATA-1 inhibits binding of c-Jun, a critical co-activator for PU.1 transactivation of myeloid promoters, to PU.1 in myeloid cells (Zhang et al., 1999). Thus, PU.1, a master regulator toward the myeloid lineage, and GATA-1, a master regulator toward the erythroid-megakaryocytic lineage, antagonize each other, suggesting that commitment to one cell fate inhibits other cell fates. Fli-1 has also been reported to inhibit erythroid differentiation (Starck et al., 1999). It is speculated that Fli-1 might promote the megakaryocytic differentiation pathway rather than erythroid differentiation and inhibit expression of the Rb gene essential for erythroid differentiation. Furthermore, it has also been proposed that PU.1 and Fli-1 may inhibit erythroid differentiation through functional interference between these Ets family proteins

and nuclear hormone receptors (Darby et al., 1997; Gauthier et al., 1993). In contrast to PU.1 and Fli-1, overexpression of Ets-1 promotes erythroid differentiation of K562 cells (Clausen et al., 1997). Furthermore, it is known that Ets-1 transactivates the transferrin receptor gene essential for erythroid differentiation. Overexpression of MafB, an AP-1like myelomonocyte-specific transcription factor, in the erythroblast cell line down-regulates expression of the endogenous transferrin receptor gene by a direct interaction with Ets-1 and inhibits erythroid differentiation (Sieweke et al., 1996). 5.1.4. Lymphoid cell differentiation Multiple Ets family proteins are expressed in lymphoid cells (Bassuk and Leiden, 1997). These include Ets-1, Ets-2, Erg, Fli-1, Elf-1, GABPa, PU.1 and Spi-B (Anderson et al., 1999). B cell development is controlled by several cell-type specific transcription factors including PU.1, E2A, EBF, Ikaros, Pax-5, Sox-4, Bcl-6, Blimp-1 and XBP-1 (Glimcher and Singh, 1999; Schebesta et al., 2002). PU.1 appears to be one of the most important transcription factors for B cell development, because PU.1-deficient mice show a complete lack of B cells as well as macrophages (Scott et al., 1994; McKercher et al., 1996). As already mentioned, it has been shown that transcription of the IL-7a gene, which is primarily expressed in common lymphoid progenitors and is crucial for B cell differentiation, is directly regulated by PU.1. Furthermore, expression of various B cell-specific genes is recovered in IL-7a-transduced PU.1 2/2 cells (DeKoter et al., 2002). Thus, it is very likely that PU.1 regulates early B cell development partly by controlling the expression of the IL-7Ra gene. Oct-2, a lymphoid-specific octamer binding protein in B cells, and PU.1 itself autoregulate the PU.1 promoter in B cells (Chen et al., 1996), although relief from negative regulation might also be important as suggested by our results that expression of tissue-specific transcription factor genes is extinguished in somatic cell hybrids between different cell lineages (Hitomi et al., 1993; Oikawa et al., 2001b). In pre-B and B cells, PU.1 is implicated in regulating the activities of enhancers and promoters in many B cellspecific genes including the immunoglobulin genes. A minimal domain of the intronic enhancer of immunoglobulin m heavy-chain (IgH) gene enhancer is composed of mA, mE3 and mB that bind Ets-1, TEF3 and PU.1, respectively (Fig. 2e) (Rao et al., 1997). In the Igk and Igl enhancers, PU.1 forms a ternary complex with Pip (PU.1-interacting partner) which binds to the DNA sequences immediately adjacent to the PU.1 binding site (Eisenbeis et al., 1995; Pongubala et al., 1992). Phosphorylation of Ser148 of PU.1 by casein kinase II (CKII) is required for the interaction of PU.1 with Pip on DNA (Pongubala et al., 1993). It is suggested that an interaction of the autoregulatory segment of Pip with PU.1 alleviates intra-molecular inhibition of

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DNA-binding (Brass et al., 1999). PU.1 and Pip is also necessary for pre-B cell-specific expression of the CD20 promoter in cooperation with the basic helix – loop – helix (bHLH) family of transcription factors (Himmelmann et al., 1997). PU.1 is also essential for B cell-specific activity of the promoter of the CD72 gene encoding a transmembrane glycoprotein which plays important roles in B cell activation, proliferation and plasma cell differentiation (Ying et al., 1998). Spi-B likely binds to PU.1 binding sites, since it is closely related to PU.1 with 43% overall similarity and 67% identity at the amino acid level in the C-terminal region including DNA-binding Ets domain (Ray et al., 1992). SpiB is exclusively expressed in lymphoid cells and is detected in the early thymus and spleen. Spi-B expression increases during B cell maturation and decreases during T cell maturation (Su et al., 1996). Unlike PU.1 2/2 mice, Spi-B 2/ 2 mice are viable and possess mature B and T cells. However, these mice exhibit defects in antigen-dependent expansion of B cells, T-dependent immune responses and maturation of germinal centers in the spleen (Su et al., 1997). Thus, it is likely that PU.1 is required for B cell development and Spi-B is required for normal B cell receptor-mediated signal transduction. The Spi-B-related genes, Spi-C and Spi-D, have been isolated. The former is expressed in mature B cells (Bemark et al., 1999) and the latter is expressed abundantly in Leydig and epigonal organs, with low expression in the spleen (Anderson et al., 2001). Precise characterization of the function of these Ets family proteins is unknown. PU.1 is expressed not only in B cells and myeloid progenitors but also in T lineage precursors before commitment events, and it is down-regulated during the pro-T cell stage. Indeed, loss of PU.1 function delays T cell development (Spain et al., 1999). Furthermore, it has been shown that constitutive expression of PU.1 in fetal hematopoietic progenitors down-regulates pre-Ta, Rag-1 and Rag-2 and results in growth inhibition and arrest at the pro-T cell stage (Anderson et al., 2002). The cells expressing the highest levels of PU.1 are prevented from entering the lymphoid developmental pathway, and either differentiate into myeloid cells or die. Thus, T cell lineage commitment appears to be associated with repression of PU.1. As already stated, Ets-1 appears to participate in inhibition of T cell apoptosis although the unimpairment of development of T cells in c-ets-1 2/2 Rag-2 2/2 chimeric mice may be due to compensation by other Ets family proteins. Natural killer (NK) cells express high levels of Ets1, and c-ets-1 2/2 Rag-2 2/2 chimeric mice contain reduced numbers of NK cells, suggesting that Ets-1 is implicated in development of NK cells (Barton et al., 1998). Functional interaction of Ets-1 or Ets-2 with Stat5 is believed to contribute to the response to interleukin-2 (IL-2) in T cells (Ramei et al., 2000). Ets-1 controls several T cell-specific genes including the T cell receptor (TCR) a (Fig. 2f) and

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TCRb genes in cooperation with ATF/CREB, TCF-1, AML1 and GATA-3 (Ho et al., 1990; Halle et al., 1997). Similar cooperation is also observed in the CD8a enhancer (Hambor et al., 1993). Elf-1 likely has a role in T cell proliferation and differentiation. It is markedly induced in T cells by treatment with phorbol ester TPA (Leiden et al., 1992). It has been shown that the regulatory regions of the IL-2 receptor (John et al., 1995; Serdobova et al., 1997) and CD4 (Wurster et al., 1994) genes have Elf-1 target sequences. Elf-1 has also been reported to increase the promoter activity of the terminal deoxynucleotidyl-transferase (TdT) gene (Ernst et al., 1996). 5.2. Vascular endothelial cell differentiation Vascular endothelial cells arise from a common progenitor for hematopoietic and vascular lineages, called hemangioblasts (Choi et al., 1998). Several members of the Ets family proteins including Ets-1, Fli-1, Erg, TEL1 and NERF-2 (new Ets-related factor-2), a member of the Elf subfamily, are reported to be expressed in endothelial cells and their progenitors. They are thought to play crucial roles in vascular development and angiogenesis (Sato, 2001). Angiopoietin-1, which is important for endothelial cell survival and migration in angiogenesis, directly induces expression of NERF-2 that is specifically induced by endothelial cells (Christensen et al., 2002). It has been reported that NERF-2 and Ets-2 show strong transactivation of the Tie promoter, whereas Ets-1 causes low levels of stimulation, and other Ets family proteins give little or no transactivation (Iljin et al., 1999). Vascular endothelial growth factor (VEGF) receptor-1 ( flt-1) is regulated by Ets1 and VEGF can up-regulate the expression of Ets-1 in human umbilical vein endothelial cells. VEGF stimulates the invasiveness of endothelial cells and this VEGF-induced invasiveness is inhibited by Ets-1 antisense oligomers (Chen et al., 1997). Flk-1 is essential for embryonic blood vessel development and tumor angiogenesis. The Ets binding site, as well as the SCL/Tal-1 and GATA binding sites, in the flk1 enhancer are essential for vascular development (Kappel et al., 2000). Ets-1 lowers endothelial cell density at confluence and induces expression of VE-cadherin (Lelievre et al., 2000). The heme oxygenase gene, crucial for proliferation and angiogenesis in endothelial cells can be activated by Ets-1, Fli-1 and Erg (Deramaudt et al., 1999). A recent report shows that Ets-1 activates an autoregulatory loop of expression of the Fli-1 gene in endothelial cells but not in fibroblasts (Lelievre et al., 2002), suggesting Fli-1 might play certain roles in endothelial cells during angiogenesis. TEL is involved in angiogenesis in the yolk sac (Wang et al., 1997). Not only angiogenesis but also development of lymphatic vessels is controlled by Ets family proteins. Nettargeted mutant mice die of respiratory failure soon after birth developing chylothorax due to highly dilated lym-

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phatic vessels accompanied by high expression of egr-1, suggesting Net is involved in vascular development (Ayadi et al., 2001). In addition, it has been reported that there are two Ets binding sites in the mouse and human promoters of the flt-4 (VEGFR-3) gene which is responsible for lymphangiogenesis (Iljin et al., 2001). 5.3. Neuronal differentiation Several Ets family transcription factors are expressed in the central nervous system to perform with neural cell typespecific function. In Drosophila, two Ets family proteins, a positive regulator PntP2 and a negative regulator Yan, have been reported to act antagonistically in R7 photoreceptor neuron induction and differentiation in the eye (O’Neill et al., 1994). Repression activity of Yan is released by phosphorylation of the protein mediated through a RasMAP kinase signaling (Hsu and Schulz, 2000). In mammals, Ets-1 and Ets-2 are expressed in specific regions of the brain (Maroulakou and Bowe, 2000; Valter et al., 1999). Ets-1 is also expressed in astrocytes and its expression is known to be up-regulated during retinoic acid-induced neural differentiation of multipotent mouse embryonic carcinoma P19 cells (Fleischman et al., 1995). Furthermore, Ets-1 and GHF-1/Pit-1, a pituitary-specific POU homeo-domain transcription factor, act synergistically with Ras-MAP kinase signaling to stimulate prolactin promoter activity in pituitary cells, while Ets-2 has no synergistic effect on Ras activation of the promoter (Bradford et al., 1995). This difference is supposed to be based on the lack of functional interactions between Ets-2 and GHF-1/Pit-1. It has been reported that nerve growth factor (NGF) induces neuronal differentiation of rat pheochromocytoma PC12 cells through induction of the brain-specific isoform of Elk-1. This novel short isoform of Elk (sElk-1) plays an opposite role to the wild-type of Elk-1 in neuronal cell differentiation and proliferation (Vanhoutte et al., 2001). Ets family proteins including Ets-1, Ets-2, PEA3, ER81 (ets-related 81), Pet-1 (PC12 ets factor-1), Elk-1 and GABPa are shown to bind to the control regions of the neural genes such as the synapsin II (Petersohn et al., 1995), peripherin (Chang and Thompson, 1996) and d-opioid receptor genes (Sun and Loh, 2001). Expression of the human presenilin gene, whose mutation is related to early onset of Alzheimer’s disease, is also believed to be regulated by Ets-1 and Ets-2 (Pastorcic and Das, 1999). Members of the PEA3 subfamily appear to be involved in development of the central and peripheral nervous system. PEA3 is expressed in specific bundles of motor neurons that innervate limb muscles and appear in afferent sensory neurons of these same muscles (Lin et al., 1998). PEA3-deficient male mice have been reported to be infertile probably due to mechanisms proximal to cavernosal smooth muscle or an ejaculatory dysfunction based on functional disorder of neuron connections (Laing et al., 2000). ER81, a member of the PEA3 subfamily, is also

expressed in distinct neuronal subsets of the developing spinal cord to control a late step in the establishment of functional sensory-motor neuron connections (Arber et al., 2000). ERM (Ets related molecule), also a member of the PEA3 subfamily, is expressed in satellite glia (Hagedorn et al., 2000). Expression of the nicotinic acetylcholine receptor genes in the synapse are regulated by the Ets family proteins, GABPa (Fromm and Burden, 1998) and Pet-1 (McDonough et al., 2000). Pet-1 was isolated from rat adrenal chromaffin-derived PC12 cells (Fyodorov et al., 1998). It has been shown that it binds to the PEA3 site of the serotonin receptor, serotonin transporter and tryptophan hydroxylase genes to regulate their expression (Hendricks et al., 1999). 5.4. Myogenic and osteogenic differentiation It has been reported that the myogenic transcription factor MEF2 potentiates the transactivating ability of PEA3 and overexpression of PEA3 accelerates myogenic differentiation upon serum withdrawal, whereas blocking of PEA3 function delays myoblast fusion (Taylor et al., 1997). Therefore, PEA3 appears to be involved in regulation of myogenesis in cooperation with the MEF2 and MyoD families (Arnold and Winter, 1998). Ets family proteins are also involved in osteogenic differentiation. Ets-1 and Ets-2, as well as c-Fos and AML3, have been reported to play an important role in osteoblast development and bone formation (Raouf and Seth, 2000). Retinoic acid (RA) regulates the expression of the c-ets-1 by binding an RA response element in its promoter in osteogenic progenitor cells (Raouf et al., 2000), and the expression of bone related genes, PTHrP (parathyroid hormone-related peptide) and tenascin-C, is regulated by Ets-1 (Shirasaki et al., 1999). Moreover, expression of osteopontin (OPN), an extracellular matrix (ECM) secreted by osteoblasts, is regulated by Ets-1 and Ets-2 (Sato et al., 1998). Involvement of the Ets subfamily in osteogenesis is supported by a report showing that Ets-2 transgenic mice incur bone malformation (Sumarsono et al., 1996). A novel variant of Egr is shown to be expressed in developing articular chondrocytes by immunohistochemistry and in situ hybridization (Iwamoto et al., 2000). PU.1-knockout mice exhibit osteopetrosis as well as defects in the development of B cells and macrophages (Tondravi et al., 1997), suggesting that PU.1 is involved in the development of osteoclasts derived from the myeloid lineage.

6. Ets family proteins in malignancy 6.1. Activation of the ets family genes by proviral integration in animal tumors Some of the ets family genes can be activated and become oncogenic by retroviral insertion in animal tumors.

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The Friend virus, which consists of the replication competent Friend murine leukemia virus (F-MuLV) and replication defective spleen focus forming virus (SFFV), is known to induce erythroleukemia in mice. In Friend virusinduced erythroleukemia, PU.1 is activated as a result of integration of SFFV provirus near its locus (Moreau-Gachelin, 1994). In F-MuLV-induced erythroleukemia, however, Fli-1 instead of PU.1 is activated as a result of integration of F-MuLV (Ben-David et al., 1991). Infection of PU.1-transducing retrovirus to long-term bone marrow cultures resulted in proliferation of proerythroblast-like cells that were immortalized but were Epo-dependent (Schuetze et al., 1993). Furthermore, PU.1-transgenic mice developed erythroleukemia within the early stage of birth (Moreau-Gachelin et al., 1996). On the other hand, Fli1 transgenic mice did not develop erythroleukemia but rather developed B cell hyperplasia with a progressive immunological renal disease similar to systemic lupus erythematosus (SLE) in humans (Zhang et al., 1995). As we reported, overexpression of PU.1 in murine erythroleukemia (MEL) cells blocks erythroid differentiation (Yamada et al., 1997). Since PU.1 functionally antagonizes GATA-1, a critical zinc finger transcription factor for erythroid differentiation, this interaction may be one molecular mechanism block of erythroid differentiation. Overexpression of Fli-1 in MEL cells also inhibits erythroid differentiation (Starck et al., 1999). Fli-1, but not PU.1, negatively regulates Rb expression by binding an Ets binding site in the promoter (Tamir et al., 1999). It has been demonstrated in Rb-deficient mice that Rb is essential for differentiation of erythroid cells (Hu et al., 1997). Therefore, the block of terminal differentiation and enhanced proliferation of erythroid cells by overexpression of Fli-1 may be partly mediated through transcriptional repression of the Rb gene in F-MuLV-induced murine erythroleukemia. Recently, it has also been reported that Fli-1 enhances the bcl-2 promoter activity (Lesault et al., 2002), suggesting that Fli-1 may rescue the leukemic cells from apoptosis by accumulating anti-apoptotic protein Bcl2 in the cells. 6.2. Ets family genes involved in chromosome translocation in human tumors 6.2.1. Ewing’s tumors Chromosome translocation is frequently associated with human leukemia and solid tumors (Look, 1995). The ets family genes are also involved in chromosome translocation and lead to the production of fusion proteins. One wellknown example is the t(11;22)(q24;q12) translocation found in 95% of human Ewing’s sarcomas. In this translocation, the N-terminal region of EWS, an RNA-binding protein, and the C-terminal region of Fli-1 including Ets domain are fused (Delattre et al., 1992). So far, two types of common fusion proteins have been reported. The first type is formed by juxtaposition of exons I– VII of the EWS gene to exons

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VI – IX of the Fli-1 gene, while the second one is formed by juxtaposition of the same regions of the EWS gene to exons V – IX of the Fli-1 gene. The fusion proteins possess increased transactivation potential in comparison with the wild-type Fli-1 and this activity is thought to contribute to malignant transformation of the cells (Bailly et al., 1994). Indeed, the latter type of fusion protein, which shows stronger transactivation potential than the former, is often associated with poor prognosis (Lin et al., 1999). Overexpression of EWS-Fli-1, but not wild-type Fli-1, transforms NIH/3T3 cells. Several genes encoding for EAT-2, an SH2 domain containing protein, and mE2-C, a cyclinselective ubiquitin conjugating enzyme involved cyclin B destruction, have been shown to be up-regulated by EWSFli-1 but not Fli-1 (Arvand et al., 1998). In addition to these genes, the expression of EWS-Fli-1 leads to a strong upregulation of the c-myc oncogene and a considerable downregulation of the p57 KIP2 tumor suppressor gene (Dauphinot et al., 2001). Expression of the TGFb type II receptor gene is up-regulated by Fli-1 but is down-regulated by EWS-Fli1, implicating that the TGFb type II receptor is a direct target of EWS-Fli-1 (Hahm et al., 1999). It has been recently demonstrated that introduction of EWS-Fli-1 lacking DNAbinding ability, due to point mutation or a deletion in the Ets domain, in NIH/3T3 cells can still accelerate tumor formation (Jaishankar et al., 1999), suggesting that there is also a DNA-binding-independent pathway to malignant transformation. In addition to the Fli-1 gene, the EWS gene is juxtaposed to other ets family genes including Erg, ER81/ETV1, FEV and E1AF in some cases of Ewing’s sarcoma (Mastrangelo et al., 2000). 6.2.2. Human leukemias Another representative example of ets family genes involved in chromosome translocation is the TEL/ETV6 gene. The TEL gene is juxtaposed to several tyrosine kinase genes, including the platelet-derived growth factor receptor b (PDGFRb) gene by t(5;12)(q33;p13) translocation in human chronic myelomonocytic leukemias (Golub et al., 1994), the c-abl gene by t(9;12)(q34;p13) in chronic myelogenous leukemias (CML) and acute lymphoblastic leukemias (ALL) (Golub et al., 1996a), the Jak2 gene by t(9;12)(p24;p13) in T cell and B cell ALL (Lacronique et al., 1997), the FGFR3 (fibroblast growth factor 3) gene by t(4;12)(p16;p13) in a patient with peripheral T cell lymphoma (Yamasaki et al., 2001), the TrkC/NTRK3 (neurotrophin-3 receptor) gene by t(12;15)(p13;q24) in congenital fibrosarcomas (Knezevich et al., 1998), and ARG (c-abl related gene)/ABL2 by t(1;12)(q25;p13) in an acute myelogenous leukemia (AML) line (Iijima et al., 2000). All of the above-mentioned chimeric proteins possess the Nterminal region including the Pointed (PNT) domain for homo- and hetero-dimerization from TEL and the intact tyrosine kinase domains from the partner proteins (Fig. 4a). Self-association through the PNT domain of TEL moiety

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T. Oikawa, T. Yamada / Gene 303 (2003) 11–34

Fig. 4. Molecular mechanisms of cell transformation by chimeric fusion proteins of Ets family. (a) TEL–PDGFRb. Tyrosine kinase is activated by homodimerization of TEL–PDGFRb through the Pointed (PNT) domains of TEL. Ets, DNA-binding (Ets) domain of TEL; TM, transmembrane domain of PDGFRb; Kinase, tyrosine kinase domain of PDGFRb. Small arrows indicate breakpoints. (b) TEL–AML1. AML1 activates gene expression by recruiting the co-activator p300, while TEL–AML1 represses gene expression by recruiting the co-repressor mSin3A, N-CoR and histone deacetylase 3 (HDAC3) through the PNT domain and the central region of TEL. TEL–AML1 acts as a dominant negative mutant against AML1 which is important for hematopoietic cell differentiation. Ets, DNA-binging (Ets) domain of TEL; Runt, DNA-binding (runt) domain of AML1. Small arrows indicate breakpoints.

and subsequent activation of kinase activity of the fusion protein likely contributes to transformation of the cells (Golub et al., 1996b). Actually, TEL – Jak2 transgenic mice show activation of Stat1 and Stat5 and develop T cell lymphoma, confirming that the TEL – Jak2 fusion gene is indeed an oncogene in vivo (Carron et al., 2000). It has also been reported that the chimeric protein tyrosine kinase TEL – TrkC requires both Ras-MAP kinase and PI3 kinaseAkt signaling for fibroblast transformation (Tognon et al., 2001). The second type of chimeric protein is formed by t(12;21)(p13;q22) translocation in childhood B cell-lineage ALL. The chimeric protein consists of the N-terminal region containing the PNT domain and the central repressor domain of TEL and all but the first 20 amino acids of AML1. The TEL – AML1 fusion protein acts competitively with AML1 target genes and shows transcriptional repressive activity (Fig. 4b). It is known that co-repressor mSin3A binds to the PNT domain of TEL (Chakrabarti and Nucifora, 1999) and another co-repressor N-CoR and histone deacetylase HDAC3 bind to the central repression domain of TEL (Wang and Hiebert, 2001). It is thought, therefore, the transcriptional repressive activity of the fusion protein is the result of interaction with these co-repressors. Altered

regulation of the AML1 target genes by the fusion protein is suggested to be one of the molecular mechanisms of B-cell leukemogenesis (Hiebert et al., 1996; Uchida et al., 1999). A recent report shows that TEL – AML1 indeed contributes to leukemogenesis in cooperation with loss of p16 INK4A/ p19ARF tumor suppressors in bone marrow cells transduced with a retroviral vector expressing TEL – AML1 (Bernardin et al., 2002). In contrast to the above examples, t(12;22)(p13;q11) translocation found in myeloid leukemias juxtaposes the Nterminal region of a putative transcription factor MN1 to the C-terminal region including the DNA-binding (Ets) domain of TEL. This MN1– TEL fusion protein has been shown to act as a chimeric transcription factor on a synthetic promoter containing TEL binding sites and transform NIH/3T3 cells (Buijs et al., 2000). In summary, the fusion proteins formed by chromosome translocation involving the TEL gene are classified into three categories depending on their mechanism contributing to malignant transformation of the cells. Additionally, it is worth noting that in leukemic cells with chromosome translocation involving the TEL gene, the remaining TEL allele is frequently lost (Stegmaire et al., 1995). This, along with the fact that TEL has transcriptional repressive activity, leads us to speculate that TEL could be a

T. Oikawa, T. Yamada / Gene 303 (2003) 11–34

tumor-suppressor protein. Indeed, overexpression of TEL in Ras-transformed NIH/3T3 cells results in growth inhibition and reduced malignancy accompanied by transcriptional repression of stromelysin-1 (MMP-3) expression (Fenrick et al., 2000). We have also found the inhibitory effect of TEL on cell growth in K562 cells (Sakurai et al., unpublished observation). Since TEL forms a heterodimer with Fli-1 via their PNT domains (Kwiatkowski et al., 2000), loss of TEL function might result in functional activation of Fli-1. The Erg gene is known to form a chimeric gene with TLS (translocated in liposarcoma)/FUS gene, a docking molecule in the recruitment of RNA polymerase II and serinearginine (SR) splicing factors, by t(16;21)(p11;q22) translocation in several types of myeloid leukemia (Ichikawa et al., 1999). In this case, the weakened transcriptional activity of the fusion protein in comparison with wild-type Erg, is believed to be implicated in transformation of the cells. A recent report proposes that TLS – Erg fusion protein may also lead to various cell abnormalities by interfering with the splicing of important cellular regulators (Yang et al., 2000). Mutations in the PU.1 gene, which is very important for myeloid differentiation as already stated in Section 5.1.2, have been recently reported to be associated with approximately 7% of human acute myeloid leukemia (AML), suggesting that disruption of PU.1 function contributes to the differentiation block found in AML patients (Mueller et al., 2002). 6.3. Ets family proteins involved in malignancy of tumors Up-regulation of expression of the ets-1 gene has been documented in many types of human tumors. Generally, expression levels of Ets-1 correlate well with the grade of invasiveness and metastasis (Behrens et al., 2001; Nakayama et al., 2001) and therefore can be useful for predicting poor prognosis of the cancer patients. Expression of genes encoding for enzymes involved in degradation of the extracellular matrix (ECM), such as MMP-1 (collagenase-1), MMP-3 (stromelysin-1), MMP-7 (matrilysin), and MMP-9 (type IV collagenase/gelatinase B) is regulated by Ets family proteins including Ets-1 and PEA3/E1AF (Fig. 2a). Hence, it is strongly suggested that Ets-1 contributes to tumor invasion and progression through activation of these enzymes. Indeed, expression of these ECM remodeling enzymes is detected along with expressing c-ets-1 mRNA in tumor cells and/or stroma cells (Davidson et al., 2001). Expression of c-ets-1 in both tumor and stroma correlates with poor survival in human ovarian carcinoma (Davidson et al., 2001). Moreover, c-ets-1 is up-regulated and is also involved in enhanced expression of MMP-7 in human hepatocellular carcinoma (HCC), which may contribute to the progression of HCC (Ozaki et al., 2000). Ets-1 is also involved in angiogenesis which is essential for tumor progression. It has been shown that VEGF and bFGF induce

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Ets-1 in endothelial cells as already mentioned, and Ets-1 then confers an angiogenic phenotype to endothelial cells through induction of the urokinase-type plasminogen activator (u-PA) and MMPs. Activation of u-PA is also induced in human fibrosarcoma HT1080 cells by introduction of PU.1 (Kondoh et al., 1998). N-Acetylglucosaminyltransferase V (GnT-V) associated with metastasis of tumors is controlled by Ets-1 (Ko et al., 1999). The microenvironment of a growing tumor mass is significantly oxygendeprived and hypoxia induces expression of c-ets-1 via the activity of hypoxia-inducible factor-1 (HIF-1) (Oikawa et al., 2001a). Thus, chronic hypoxia in rapidly growing tumors results in induction of Ets-1 in tumor cells and/or stroma cells to subsequently induce angiogenesis-related genes. The majority of breast cancer metastasizing to bone secretes parathyroid hormone-related protein (PTHrP) which induces local osteolysis that leads to activation of TGFb. Ets-1 but not ESE-1/ESX nor Elf-1 cooperatively enhances the TGFb/Smad-mediated activation of the PTHrP-P3 promoter in invasive breast cancer cells (Lindemann et al., 2001). PEA3 is often overexpressed in breast cancer and is a downstream target of the HER-2/neu receptor tyrosine kinase (O’Hagan and Hassel, 1998). In contrast to this observation, a recent report shows that PEA3 expression results in inhibition of cell growth and tumor development of HER-2/neu-overexpressing cancer cells (Xing et al., 2000). This suppression is specific for the HER-2/neu gene and expression of the DNA-binding domain of PEA3 is enough to repress HER-2/neu promoter activity, suggesting that PEA3 competes with the transacting factor(s) for the same DNA motif and represses HER-2/neu transcription. Some members of the Elf subfamily are expressed exclusively in epithelial tissues. ESE-1/ESX/ERT (etsrelated transcription factor), a member of this group, is associated with mammary gland development and terminal differentiation of mammary epithelial cells. ESE-1/ESX has been reported to modulate TGFb signaling by regulating transcription of TGFb type II receptor in breast cancer cells (Chang et al., 2000). It is also known that ESE1/ESX is up-regulated during early stages of human ovarian tumorigenesis and its promoter is a downstream target of the HER-2/neu/c-ErbB2 receptor tyrosine kinase (Neve et al., 2002). The inducible prostaglandin synthase cyclooxygenase-2 (cox-2) gene is aberrantly expressed in many human cancers, including colon and breast cancers. A recent analysis of the cox promoter indicates that the NF-IL6 binding site in the promoter is crucial for mediating PEA3 responsiveness in colorectal and mammary cancers (Howe et al., 2001). Furthermore, the PEA3 subfamily renders the MMP-7 promoter responsive to LEF-1 (lymphoid enhancer factor-1) and b-catenin, which is a target of APC mutation and Wnt signaling (Crawford et al., 2001), suggesting that expression of the PEA3 subfamily, in conjugation with the

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Table 2 Phenotypes in mice deficient for the ets family genes Member

Phenotypes

Ets-1

Increase in T cell apoptosis, defects in B cell function Embryonic lethal, defects in extraembryonic tissue (placenta), deficient expression of MMP-9 Embryonic lethal, defects in megakaryopoiesis, decreased numbers of CFU-E and CFU-GM Embryonic lethal, defects in yolk sac angiogenesis, defects in bone marrow hematopoiesis Male infertile (defects in neuron connection?) Defects in synaptic connection to motor neuron Death soon after birth, defects in B cells and macrophages Defects in B cell receptor signaling Death soon after birth, respiratory failure due to dilated lymphatic vessels

Ets-2 Fli-1 TEL/ETV6 PEA3/E1AF ER81/ETV1 PU.1 Spi-B Net

accumulation of b-catenin, contributes to gastrointestinal tumorigenesis.

7. Conclusion and perspective We have summarized recent advances in the molecular biology of the Ets family of transcription factors. Many of the Ets family proteins are involved in the Ras-MAP kinase signaling pathway and they control transcription by binding the Ets-binding sites of the enhancers or promoters of the specific genes in cooperation with other transcription factors and co-factors. They also interact with various types of specific cellular partners to crosstalk with each other, which influence the other signaling pathways such as the Jak/Stat, Smad and Wnt signaling pathways. Table 2 summarizes normal function of some major Ets family proteins

suggested by the results obtained from gene targeting studies. Since some Ets family proteins affect the expression of several oncogenes and tumor suppressor genes by direct regulation of their promoters or by protein– protein interactions and it is evident that they play important roles in cell proliferation, apoptosis and differentiation in normal cells, deregulated expression of Ets family proteins could lead to disruption of these processes and contribute to development and progression of malignant tumors (Table 3). Although significant progress has been achieved for molecular understanding of Ets family proteins, there are still several points to be clarified. First, function of Ets family proteins has to be considered in combination of other cellular proteins, since the function of the same Ets protein sometimes differs in different types of tissues. For example, Fli-1 is induced by Ets-1 in endothelial cells but not in fibroblasts (Lelievre et al., 2002). A large amount of PU.1 is not toxic for macrophages but is toxic for lymphoid cells (Anderson et al., 2002) and erythroid cells (Yamada et al., 1997). Furthermore, Ets-1 is involved in angiogenesis but overexpression of Ets-1 in human umbilical vein endothelial cells induces apoptosis under serum deprived conditions (Teruyama et al., 2001). These differences appear to be based on differences in cellular context, which may include combination of positive and negative crosstalk between the Ets family proteins and partner proteins specific for different cell types. Therefore, the identification of further partners of Ets family proteins and elucidation of functional roles of novel crosstalk between these proteins seem to be one of the important areas in future studies. Secondly, although many potential target genes for Ets family proteins have been identified, most studies have been carried out by use of in vitro assays. The results obtained from the in vitro assays may not be consistent with the in

Table 3 Ets family genes involved in malignant tumors Member

Tumors

Molecular events

Animal tumors Ets-1 PU.1 Fli-1

Erythroblastosis in chicken (E26 virus) Erythroleukemia in mice (SFFV) Erythroleukemia in mice (F-MuLV)

Fusion protein (gag-myb-ets) Overexpression (viral integration) Overexpression (viral integration)

Many invasive tumors Ewing tumor t(11;22)(q24;q12) Ewing tumor t(21;22)(q22;q12) Acute myeloid leukemia t(16;21)(p11;q22) Ewing tumor t(17;22)(q12;q12) Breast cancer Ewing tumor t(7;22)(p22;q12) Breast cancer Acute myeloid leukemia Myelomonocytic leukemia t(5;12)(q33;p13) Acute lymphatic leukemia t(12;21)(p13;q22) Congenital fibrosarcoma t(12;15)(p13;q24)

Overexpression Fusion protein (EWS-Fli-1) Fusion protein (EWS-Erg) Fusion protein (FUS/TLS-Erg) Fusion protein (EWS-E1AF) Overexpression Fusion protein (EWS-ETV1) Overexpression Mutation Fusion protein (TEL–PDGFRb) Fusion protein (TEL–AML1) Fusion protein (TEL–TrkC)

Human tumors Ets-1 Fli-1 Erg E1AF/PEA3 ER81/ETV1 ESE-1/ESX PU.1 TEL

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vivo results. Furthermore, since several Ets family proteins bind to the same Ets binding site in vitro, there may be a functional redundancy among the Ets family proteins in vivo. It is, therefore, important to clarify the in vivo target genes for individual Ets proteins. Chromatin immunoprecipitation method may be useful to identify Ets family proteins bound to the Ets binding sites in vivo in certain tissue types of cells or under certain conditions of cells. Microarray will also serve as a powerful technology to identify such in vivo targets including known and novel genes for individual Ets proteins in specific cell lineages. Since Ets family proteins play very critical roles in various cellular functions including cell growth, development, differentiation and apoptosis, further molecular analyses of the proteins will pave the way not only to understand their precise normal in vivo functions but also to control many human diseases including immunological disorders, vascular diseases and malignant tumors.

Acknowledgements We acknowledge Drs. G. Bernardi and A. van Wijnen for giving us an opportunity to write this review article. We are grateful our colleagues Drs. F. Kihara-Negishi, M. Suzuki and T. Sakurai. We also thank Drs. F. Moreau-Gachelin, R. Maki, D. Kabat, A. Bernstein, T. Golub, R.H., Goodman, Y. Nakatani, I. Kitabayashi for valuable reagents, and Drs. H. Kobayashi, Y. Hashimoto, M. Mochizuki and S. Kohno for their continuous encouragement. Our studies reported here were supported by Grants-in-Aid for Scientific Research on Priority Areas (to T.O.) and for Scientific Research (B) (to T.O.) and (C) (to T.Y.) from the Ministry of Education, Science and Culture of Japan, and by grants from the Uehara Memorial Foundation, Tokyo (to T.O.), and the Naito Foundation, Tokyo, Japan (to T.Y.). Financial support to T.O. from Dr. K. Tanzawa, Sankyo Ltd., Tokyo, Dr. J. Akiyama, OB-GYN Akiyama Memorial Hospital, Hakodate, and Dr. K. Watanabe, Watanabe Clinic, Shizuoka, Japan, is also acknowledged.

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