FLI1 and EWS-FLI1 Function As Ternary Complex Factors and ELK1 And

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FLI1 and EWS-FLI1 function as ternary complex factors and ELK1 and SAP1a function as ternary and quaternary complex factors on the Egr1 promoter serum response elements

Dennis K Watson1, Lois Robinson2, David R Hodge3, Ismail Kola4, Takis S Papas1 and Arun Seth5

1Center for Molecular and Structural Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA; 2ABL, NCI-FCRDC, Frederick Maryland 21702, USA; 3Laboratory of Molecular Oncology, NCI-FCRDC, Frederick Maryland 21702, USA; 4Molecular Genetics and Development Group, Monash University, Melbourne 3168, Australia; 5MRC Group in Periodontal Physiology, and Department of Pathology, University of Toronto, and Laboratory of Molecular Pathology, Women's College Hospital, Toronto, Ontario, Canada M5S 1B2

The ETS gene products are a family of transcriptional for these ETS transcription factors in the regulation of regulatory proteins that contain a highly conserved and the Egr1 gene. structurally unique DNA binding domain, termed the ETS domain. Several ETS proteins bind to DNA as Keywords: ETS; FLI1; EWS-FLI1; Egr1; ternary monomers, however it has been shown that the DNA complex factor (TCF); quaternary complex factor binding activity is enhanced or modulated in the presence (QCF); CArG box; SRE of other factors. By dierential display and whole genome PCR techniques, we have recently shown that the Erg1 gene is a target for ETS proteins. The Egr1 promoter contains multiple ETS binding sites, three of Introduction which exist as parts of two serum response elements (SREI and SREII). The SRE is a cis-element that The ETS family of proteins are important transcrip- regulates the expression of many growth factor respon- tional regulators and contain a highly-conserved DNA- sive genes. ELK1 and SAP1a have been shown to form binding domain (Seth et al., 1992; Nye et al., 1992; ternary complexes with SRF on the SRE located in the c- Watson et al., 1988). Members of the ETS gene family fos promoter. Similarly, we examined whether the ELK1, have been characterized and identi®ed in a broad range SAP1a, FLI1, EWS-FLI1, ETS1, ETS2, PEA3 and of animal species, including Drosophila, sea urchin, PU.1 proteins can form ternary complexes with SRF on Xenopus, avian, murine and humans (Janknecht and the Egr1 SREI and II. Our results demonstrate that Nordheim, 1993; Seth et al., 1992; Wasylyk et al., 1993; indeed ELK1, SAP1a, FLI1 and EWS-FLI1 are able to Watson et al., 1990). The ETS family genes include: v- form ternary complexes with SRF on Egr1 SREs. In ets, ETS1, ETS2, ERG, ELK1, PEA3, SAP1a, Spi-1/ addition, ELK1 and SAP1a can also form quarternary PU.1, GABPa (E4TF1-60), ER71, ER81, FLI1 (which complexes on the Egr1 SREI. However, the proteins we designated as ERGB, Watson et al., 1992), ELF, ETS1, ETS2, PEA3 and PU.1 were unable to form TEL, ERP, PE1, ERF, sea urchin ETS and Erg and ternary complexes with SRF on either the Egr1 or c-fos Drosophila E74, D-Ets2/pointed, D-ets3, D-ets4, D- SREs. Our data demonstrate that FLI1 and EWS-FLI1 Ets6, D-elg and D-yan/pok (Bhat et al., 1996). constitute new members of a subgroup of ETS proteins The ETS DNA-binding domain consists of 85 amino that can function as ternary complex factors and further acids and recent NMR analysis of this domain reveals implicate a novel function for these ETS transcription a novel topography containing three a-helices and a factors in the regulation of the Egr1 gene. By amino acid four-stranded b-sheet, forming a helix ± turn ± helix sequence comparison we found that, in fact, 50% of the structure (Donaldson et al., 1994; Liang et al., 1994; amino acids present in the B-box of SAP1a and ELK1, Werner et al., 1995). The ETS proteins bind to a which are required for interaction with SRF, are identical purine-rich core sequence located in many gene to those present in both FLI1 (amino acids 231 ± 248) and promoter and enhancer sequences (Seth et al., 1993a, EWS-FLI1 proteins. This B-box is not present in ETS1, 1994; Watson et al., 1992). The consensus ETS binding ETS2, PEA3 or PU.1 and these proteins were unable to site (EBS) contains a -GGAA/T- core sequence ¯anked form ternary complexes with SRF and Egr1-SREs or c- by dierent nucleotides that determine the anity and fos SRE. Furthermore, deletion of 194 amino terminal speci®city of binding for a particular ETS protein amino acids of FLI1 did not interfere with its ability to (Ascione et al., 1992; Seth et al., 1993a, 1994). The interact with SRF, in fact, this truncation increased the ETS proteins are able to bind to DNA either directly stability of the ternary complex. The FLI1 protein has a or, in certain cases, DNA-binding activity of an ETS unique R-domain located next to the DNA binding protein can be augmented or stabilized in the presence region. This R-domain may modulate the interaction with of speci®c antibody (Seth et al., 1993b) or other protein SRF, providing a mechanism that would be unique to factors (Dalton and Treisman, 1992; La Marco et al., FLI1 and EWS-FLI1, thus implicating a novel function 1991). The ETS family of transcription factors play important roles during murine embryogenesis. During Correspondence: A Seth Received 8 May 1996; revised 16 September 1996; accepted 16 mouse development ETS1 mRNA is associated with September 1996 bone formation in vertebrae, tails and limbs (Kola et ETS proteins function as ternary and quaternary complex factors DK Watson et al 214 al., 1993; Maroulakou et al., 1994). ETS2 is expressed Results in newly forming cartilage, including in the skull precursor cells and vertebral primordia. Overexpres- In order to isolate novel ETS target genes, we sion of the ETS2 gene in transgenic mice resulted in performed ETS1 binding with MboI cleaved genomic Down's syndrome-like skeletal abnormalities (Sumar- DNA followed by whole genome PCR and isolated sono et al., 1996). various promoter/enhancer fragments that bind to the The ETS gene products induce cellular transforma- ETS1 protein. The bound DNA fragments were tion and are involved in neoplasia. Overexpression of ampli®ed and subcloned into plasmid vectors and ETS1 and ETS2 genes has been shown to transform subjected to DNA sequence and computer analysis. mouse and rat ®broblasts in culture and induce tumors One of the clones we isolated was found to contain in nude mice (Seth and Papas, 1990; Seth et al., 1989; DNA sequences derived from the regulatory regions of Topol et al., 1992). The FLI1 gene is found to be fused the human EGR1 gene. Interestingly, the mouse Egr1 in-frame to the EWS gene at the breakpoint of gene was also identi®ed as an ETS target by dierential translocation t(11 : 22) which is frequently found in display between control cell-lines and transfectants that Ewing sarcomas and peripheral neuroectodermal express high levels of ETS1 (Robinson et al., 1995). tumors (Delattre et al., 1992). The chimeric (EWS- FLI1) protein product contains the amino terminal Binding of FLI1 protein to the Egr1 EBS region of EWS (a potential RNA-binding protein) fused with the carboxy terminal DNA binding domain The mouse Egr1 promoter contains several ETS of FLI1 protein. This chimeric protein is able to binding sites (Figure 1). Two of them are located at transform cells and induces tumors in nude mice position 7414 and 7393 which ¯ank a CArG box (Lessnick et al., 1995; May et al., 1993). Although (SREI). A third site is located at position 792; this the chimeric protein binds to the same EBS, it exhibits EBS is ¯anked by two CArG boxes (SREII). We much higher transactivation activity than the full- synthesized oligonucleotides corresponding to these length FLI1 protein (Bailly et al., 1994; Mao et al., two sites and examined their binding with recombi- 1994; May et al., 1993; Ohno et al., 1993). nant ETS proteins by electrophoretic mobility shift In our continuing eort to investigate the mechan- assays (EMSAs). The ETS1 protein binds to both the isms that control transcriptional regulation and target Egr-SREI and Egr-SREII oligonucleotides (Robinson selection by ETS proteins, we have utilized whole et al., 1995). To examine whether other members of the genome PCR and dierential display techniques to ETS family will also bind to these sites, we used identify and clone promoters/enhancers containing recombinant FLI1 protein expressed in insect cells. functional ETS binding sites (EBS) that are required This protein binds speci®cally to both the Egr-SREI for the proper induction (or repression) of the target and Egr-SREII oligonucleotides (Figure 2, lanes 1 and gene. We found that both of these methods identi®ed 4) because the complex is eectively competed by the Egr1 gene as an ETS target (Robinson et al., 1995). Egr1 is an early growth response gene (also known as NGF1-A, zif268 and Krox-24) that was originally identi®ed by its rapid induction in response a to various growth factors (Christy et al., 1988). Interestingly, when overexpressed in v-sis transformed NIH3T3 cells, Egr1 was able to suppress tumor growth in nude mice (Huang et al., 1994). The Egr1 promoter contains multiple ETS binding sites; in fact, many of the ETS binding sites are located adjacent to b the CArG boxes and constitute the serum response elements (SRE). The expression of the Egr1 gene is controlled by SREs clustered in its promoter region. Two of the SRE motifs in the Egr1 promoter are required for elevated expression during monocyte dierentiation (Kharbanda et al., 1994), and these sites are also necessary for the induction of Egr1 expression during B-cell activation (McMahon and Monroe, 1995). The transcriptional induction of several genes in response to serum growth factors is elicited, in part, by the formation of a ternary complex with SRF on the SRE situated within their promoters. The ternary complex contains the SRE, serum response factor (SRF) and one of several ternary complex factors (TCF) (Treisman, 1994). ELK1 and SAP have been shown to form a ternary complex on the c-fos promoter (Hipskind et al., 1991; Price et al., 1995). Herein we present evidence that in Figure 1 The Egr1 promoter. (a) Diagrammatic representation addition to ELK1 and SAP1a, FLI1 and EWS-FLI1 of the Mouse Egr1 Promoter between 7483 to +22. Location of proteins can also form ternary complexes on the Egr1 CArG and ETS boxes within SREI (distal) and SREII (proximal) are indicated. (b) Wild type and mutant Erg1-SREI (EGR1-I, SREs. We also show that ELK1 and SAP1a can form 7424 to 7375) and Egr1-SREII (EGR1-II, 7114 to 765) quarternary complexes on the Egr1 SREI. oligonucleotide sequences (Tsai-Morris et al., 1988) ETS proteins function as ternary and quaternary complex factors DK Watson et al 215 ERGB/FLI1 ELK1 and SAP1a form ternary complexes with SRF EGR–1–I EGR–1–II on the Egr1-SREII and c-fos SRE. On the Egr1-SREI, ELK1 and SAP1a form ternary and quaternary Monoclonal Antibody ––+––+ complexes, while FLI1 or EWS-FLI1 form only a Excess Oligonucleotide –+––+– ternary complex. Formation of a quaternary complex is due to the presence of two EBS sequences located in III¨ Egr1-SREI which is in contrast to the single EBS present in c-fos and Egr1-SREII. These data further suggest that the mechanism of ternary and quaternary II¨ complex formation with ELK1 and SAP1 on the Egr1- SREI is fundamentally dierent than the c-fos and Egr1-SREII complex. In addition, ELK1 and SAP1a bind dierently than did the EWS-FLI1 and FLI1 on the Egr1-SREI site.

Speci®c interaction of FLI1 and EWS-FLI1 with SRF ¨ I on Egr1-SREI To determine the speci®city of protein-DNA complexes, we performed EMSAs in the presence of various competing oligonucleotides (Figure 4a). The wild type Free Probe— SREI oligonucleotide inhibits the formation of both binary and ternary complex formation with SRF as well as with FLI1 and EWS-FLI1 (lanes 2, 6, 10, 14 and 18). 123456 Competition with excess EBS (lacking a CArG box) Figure 2 Electrophoretic mobility shift assay (EMSA) with FLI1 inhibits the formation of a binary complex with FLI1 protein. EMSA were done using either Egr-SREI or Egr-SREII and EWS-FLI1 (lanes 3 and 19), as well as inhibiting labeled oligonucleotides in the presence (lanes 2 and 5) and the ternary complex (lanes 7 and 15). Incubation in the absence (1, 3, 4 and 6) of excess unlabeled oligonucleotide. Supershift analysis was done with 2 mg of a speci®c monoclonal presence of an SREI oligonucleotide containing a antibody (lanes 3 and 6) (Zhang et al., 1995) mutation in the CArG box (SREM) is able to block the formation of binary complexes with FLI1 or EWS- FLI1 (lanes 4 and 20) and inhibits the ternary complex addition of the unlabeled oligonucleotides (lanes 2 and formation (lanes 8 and 16). Our data provide evidence 5). Similarly, the addition of monoclonal antibody that ternary complex formation by FLI1 and EWS- speci®c for FLI1 produces supershifted complexes FLI1 requires both wild type SRE and SRF protein. (lanes 3 and 6), which are abrogated with the addition Subsequently we performed binding assays using of excess unlabelled probes (data not shown). Although constant amounts of SRF and increasing amounts of the ETS1 protein binds with equal eciency to both either FLI1 or EWS-FLI and vice versa. The results, the single EBS Egr-SREII and dual EBS containing shown in Figure 4b, demonstrate that increasing Egr-SREI sites (data not shown), the FLI1 shows amounts of either SRF or ETS proteins, result in an much higher anity for the dual EBS containing site increase in ternary complex formation. Moreover, present in Egr-SREI. increasing amounts of SRF eects a further decrease in the FLI1 : Egr-SREI and EWS-FLI : SREI binary complexes, suggesting that these interactions are FLI1, EWS-FLI1, ELK1 and SAP1a form ternary speci®c, and that the ternary complex is being complexes with SRF on the Egr1 SREI and SREII generated by FLI : SRF : Egr1-SREI and EWS- ELK1 and SAP1a have been shown to form ternary FLI1 : SRF : Egr1-SREI interactions. Similarly, increas- complexes with SRF on the c-fos SRE. To determine ing amounts of SRF protein in the presence of constant whether FLI1 and EWS-FLI1 can funtion as TCFs, we ELK1 protein results in a signi®cant increase in ternary examined their ability to form ternary complexes with and quaternary complex formation and a reduction in SRF on the c-fos SRE. We found that although EWS- the ELK1 : DNA complex (data not shown). FLI1 is able to form a weak ternary complex, the FLI protein was unable to form a complex (Figure 3a). Dierential contribution of EBS sequences to the We also examined whether these ETS proteins can formation of the ternary and quaternary complexes form ternary complexes on the Egr1 SREI and SREII. Our results show that FLI1, EWS-FLI1, ELK1 and To determine the relative contribution of the two EBS SAP1a proteins are able to interact with SRF on both sequences present in the Egr1-SREI to the binding by the Egr1-SREI and Egr1-SREII sites (Figure 3b and c). ETS proteins and to the formation of ternary and In addition, each of these proteins is able to form a quaternary complexes in the presence of SRF protein, strong binary complex with the Egr1-SREI site (Figure we synthesized several oligonucleotides having muta- 3c). No such strong binary complex is observed with c- tions in each of the EBS sequences (Figure 1) and fos-SRE and Egr1-SREII (Figure 3a and b). Although tested their binding eciency by EMSA (Figure 5). ETS1, ETS2, PEA3 and PU.1 proteins are able to form ELK1 and SAP1a can form binary complexes with a binary complex on Egr1-SREI, they are unable to LMR and LRM oligonucleotides (Figure 5a). Mutation interact with SRF to form a ternary complex (data not of the 5' EBS in the LMR oligonucleotide does not shown). allow for the formation of the quaternary complex ETS proteins function as ternary and quaternary complex factors DK Watson et al 216 a EWS– b EWS– ETS ELK1 SAP1a FLI1 FLI1 ETS ELK1SAP1a FLI1 FLI1

SRF +–+–+–+–+ SRF +–+–+–+–+ TC¨ ¨ TC TC¨ SRF¨

SRF¨ ¨ EWS–FLI1

ELK/SAP1a ¨

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c EWS– ETS ELK1SAP1a FLI1 FLI1

SAF –+–+–+–++ ¨ ¨

TC¨ TC

¨ SRF ¨ EWS–FLI1

ELK1/SAP1a ¨ ¨ FLI1

123456789 Figure 3 FLI1, EWS-FLI1, ELK1 and SAP1a form ternary and quarternary complexes with SRF and Egr1 SREs. Ternary complex analyses were carried out using in vitro expressed ELK1, SAP1a, FLI1 and EWS-FLI1 with SRF protein and labeled SREs from either c-fos (a) or the Egr1 promoter (SREII (b) and SREI (c)). The speci®c ETS protein incubated with labeled oligonucleotides in the presence or absence of SRF is indicated above each lane

which is seen with the wild type Egr1-SREI (LR) the SREI is critical for the formation of ternary oligonucleotide (compare lanes 2 and 4 with lanes 7 complexes for ELK1, SAP1a, EWS-FLI, and FLI1, and 9). In contrast ELK1 and to a lesser extent, SAP1a and quaternary complex formation by ELK1 and are able to form ternary and quaternary complexes SAP1a. (lanes 12 and 14) when the mutation is in the 3' EBS (LRM). Signi®cantly, the interaction of FLI1 and EWS- Localization of the FLI1 domain required for interaction FLI1 (Figure 5b) with Egr1 SREI is unaected by with SRF mutation of the 3' EBS ((LRM) compare lanes 5 and 8; lanes 14 and 17). In contrast, the ability to bind ELK1 and SAP1a contain three regions of sequence directly to the SRE or to form a ternary complex in the similarity: (1) the amino-terminal DNA binding domain: presence of SRF protein is completely abolished by (2) a region (the B-box) that is necessary for protein- mutation in the 5' EBS (LMR). Collectively, from these protein interaction with SRF and (3) a region near the data, it can be concluded that the 5' EBS site present in carboxy-terminus (C box) which contains regulatory ETS proteins function as ternary and quaternary complex factors DK Watson et al 217 a ETS FLI1– EWS–FLI1

Competitor M M M M M SREI EBS SRE SREI EBS SRE SREI EBS SRE SREI EBS SRE SREI EBS SRE SRF ––––++++++++++++––––

TC¨ ¨ TC

SRF ¨ EWS¨ – FLI1

FLI1¨

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b SRF – 33333693369–33 3 FLI1 3 36 9 – 3 3 3 – – ––– –––

EWS–FLI1 – ––––––––3333369 TC¨ ¨ TC

SRF¨

¨ SRF ¨ EWS–FLI1

FLI1¨

12345678910111213141516 Figure 4 Speci®c interaction between FLI1 and EWS-FLI1 and Egr1-SREI. (a) Ternary complex formation was analysed by EMSA using FLI1 and EWS-FLI1 protein. Competition was done with excess unlabeled oligonucleotides: SREI (lanes 2, 6, 10, 14 and 18); EBS (lanes 3, 7, 11, 15 and 19); SREM (mutation in the CArG box, lanes 4, 8, 12, 16 and 20). Positions of protein-DNA complexes are indicated by arrows. (b) Eect of titration of SRF and ETS proteins on ternary complex formation. EMSAs were performed with either increasing amounts of SRF (lanes 6 ± 8, 10 ± 12) or FLI1 (lanes 2 ± 4) or EWS-FLI1 (lanes 14 ± 16). Positions of protein-DNA complexes are indicated by arrows sites for phosphorylation by MAP kinase. Amino acid stronger complex than the full length FLI1 protein, sequence alignment of FLI1 (amino acids 231 ± 248) with suggesting that the amino-terminal end in the FLI1 the B-boxes of SAP1a and ELK1 reveals signi®cant protein contains sequences than can inhibit complex homology in that region (Figure 6a). This region of FLI1 formation. is also retained in the majority of EWS-FLI1 chimeric proteins. A deletion variant of the FLI1 protein (FLI1 (BC)), lacking the 194 amino terminal amino acids, but Discussion retaining the B box homology region, was examined for its ability to form a ternary complex on the Egr1 SREI By dierential display and whole genome PCR and SREII. Although FLI1(BC) is not able to form a technologies we have identi®ed the Egr1 gene binary complex, it is able to form ternary complexes on promoter as a target for the ETS1 and FLI1 the Egr1-SREI and SREII (Figure 6b, lanes 6 and 13). It transcription factors (Robinson et al., 1995). Egr1 is is likely that the FLI1 and EWS-FLI1 proteins interact an early growth response gene containing several serum with SRF through B-box sequences in a manner similar response elements (SRE) in its promoter region. to ELK1 and SAP1a. The FLI1(BC) proteins forms a Transcriptional induction of several genes including c- ETS proteins function as ternary and quaternary complex factors DK Watson et al 218

a LMR LR LRM SRF –2 2 2 – – 2 2 2 – – 2 2 2 – ELK1 22–– –22– ––22––– SAP1a –––2 2––– 22–––22

¨ TC¨

SRF¨

ELK1/SAP1a ¨

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b Oligo LMRLLRLRM MR LR LRM

EWS– EWS– EWS– ETS FLI1 FLI1 FLI1 FLI1 FLI1 FLI1

SRF –++–++–++–++ –++ –++

TC¨ ¨ TC

SRF¨ ¨ SRF ¨ EWS–FLI1

FLI1¨ 123456789101112 13 14 15 16 17 18 Figure 5 Interaction of ETS proteins with the SRF on wild type or mutant Egr1-SREI sites. Gel shift assays were done with ELK1 and SAP1a (a) or FLI1 and EWS-FLI1 (b) using labeled wild type (LR) or mutant (LMR, LRM) oligonucleotides, as indicated above the lanes (see Figure 1 for sequences)

fos in response to serum growth factors is elicited by function as TCF on c-fos and Egr1 SREs. Our results the formation of a ternary complex on the SRE located demonstrated that ELK1, SAP1a, FLI1 and EWS- within its promoter region. The ternary complex is FLI1 can indeed interact eciently with the SRF on formed by the SRE sequence, the SRF protein, and Egr1 SREI and ELK1, SAP1a and EWS-FLI1 can also one of the ETS proteins such as ELK1 or SAP1a that form ternary complexes with SRF on the Egr1 SREII are able to function as ternary complex factors (TCF). and c-fos SRE sequences. Although the FLI1 protein In this report we examined whether in addition to forms a ternary complex with SRF on the Egr1 SREI, ELK1 and SAP1a, six other ETS factors, FLI1, EWS- it is unable to form a ternary complex with the SRF on FLI1, ETS1, ETS2, PEA3 and PU.1, are able to the c-fos SRE. On the other hand the ETS family ETS proteins function as ternary and quaternary complex factors DK Watson et al 219 a

b SREI SREII

EWS–FLI1 ++ ––– – –++– –– –– FLI1 ––++–––––++– –– FLI1 (BC) ––––++–––––++– SRF –+ –+– ++–+– +– ++

¨ TC ¨ SRF ¨

EWS–FLI1¨

¨ FLI1 IV

Figure 7 Schematic representation of the ternary and quarternary complexes formed with the Egr1 SREs. I, II, III. Ternary complexes consist of a SRF dimer (open oval) and the indicated ETS proteins (®lled octagons). IV. A quaternary complex was Figure 6 Localization of SRF-FLI1 interactive domain. (a) formed on the symmetrical Egr1 SREI with two ETS binding Amino acid alignment of FLI1 with the B boxes of SAP1a and sites. Sequence speci®city conferred by the EBS restricts ELK1. Identical amino acids between FLI1 and SAP1a or ELK1 quarternary complex formation to ELK1 and SAP1a are indicated. (b) FLI1(BC), lacking N-terminal 194 amino acids, forms ternary complexes with SRF and Egr1-SREI and SREII. EMSA analyses were performed using labeled SREI or SREII contains two C residues 5' to the core, which creates and EWS-FLI1 (lanes 1, 2, 8 and 9), FLI1 (lanes 3, 4, 10 and 11) or FLI(BC) (lanes 5, 6, 12 and 13) plus (lanes 2, 4, 6, 7, 9, 11, 13 an optimal ETS1, FLI1, ELK1 and SAP1a binding and 14) or minus (lanes 1, 3, 5, 8, 10 and 12) SRF site. On the other hand the right ETS box (CAGGAA) in SREI and the single ETS box (CAGGAA) in SREII are not ¯anked by the optimal nucleotides which are members like ETS1, ETS2, PEA3 and PU.1 do not known to be recognized by ETS1, FLI1, ELK1 and form ternary complexes with SRF on both the Egr1 SAP1a therefore the binding at these two sites is and c-fos SREs. Thus, our study demonstrates that observed to be weaker. Further, in support of these FLI1 and EWS-FLI1 are additional members of the observations, mutation analysis shows that FLI1 and ETS gene family proteins that can function by EWS-FLI1 bind only to the 5' ETS box of the distal formation of ternary complex factors on SREs of Egr1-SREI site, whereas oligonucleotides with wild various promoters. type sequences or mutation in the 3' ETS box have We have shown that the two SREs located in the equivalent binding activities. Egr1 promoter are targets for transcriptional regula- We have shown that ELK1, SAP1a, FLI1 and EWS- tion via binding by ETS family proteins. However, FLI1 proteins interact with SRF and, dependent upon there are dierential preferences in the ETS family the nucleotide sequences of the SREs, may form ternary targeted-binding to these SREs. Speci®cally the distal or quaternary complexes (Figure 7). EWS-FLI1 formed SRE element appears to bind more eciently than does a ternary complex with all three SREs, Egr1-SREI, Egr1- the proximal SRE site. The distal SRE, in the Egr1 SREII and c-fos SRE. FLI1 formed a strong ternary promoter region, contains one CArG box ¯anked by complex with Egr1 SREI and a weak ternary complex two ETS boxes (Egr1-SREI), whereas the proximal with Egr1 SREII. ELK1 and SAP1a generated ternary SRE contains an ETS box ¯anked by two CArG boxes complexes with Egr1-SREII and c-fos SRE, and formed (Erg1-SREII). In addition, these ETS boxes contain both ternary and quaternary complexes with Egr1-SREI dierent nucleotides ¯anking the GGAA core se- (Figure 7). The unique ability of ELK1 and SAP1a quence. In SREI, the left ETS box (CCGGAA) proteins to form quaternary complexes on SRE elements ETS proteins function as ternary and quaternary complex factors DK Watson et al 220 having more than one EBS, supports the idea that the From the data we have presented, it appears that DNA binding speci®city of ETS proteins plays a role in the Egr1 gene is responsive to ETS transcription de®ning target selection to a subset of serum response factors in an SRF independent or dependent manner elements. It is therefore quite likely to generalize that by a speci®c TC formation that depends upon the SREs having dierent ¯anking sequences will be type of ETS factors bound. The ability of TCFs to transcriptionally activated by distinct TCF proteins, bind to SRE elements in the absence of SRF protein thereby further conferring tissue speci®city by this is dependent upon the ¯anking sequences present in molecular variation. the ETS boxes. For example, ELK1 is unable to bind By amino acid sequence comparison we found that, in to the SRE present in the c-fos promoter in the fact,many oftheamino acid sequences present intheBbox absence of SRF (see Figure 3a and (Hipskind et al., of SAP1a and ELK1, which is the region that has been 1991)). Similar to the results we describe for the SRF- shown to interact with SRF, are identical to those present independent binding of ELK1 to the Egr1 SREI in both FLI1 (aa 231 ± 248) and EWS-FLI1. However, this (Figure 3c), ELK1 has been shown by others to be B-box is not present in ETS1, ETS2, PEA3 and PU.1 and able to bind (independently of SRF) to the SRE of these proteins are therefore unable to form ternary the pip92 gene (Latinkic and Lau, 1994). The SRE complexes with SRF and Egr1-SREI or SREII. Thus, it elements in the Egr1 promoter are critical for speci®c is very likely that FLI1 and EWS-FLI1 interact with SRF cellular responses (McMahon and Monroe, 1995). The in this region. Furthermore, deletion of 194 amino- distal SRE element (Egr1-SREI) is required for terminal amino acids of FLI1 does not interfere with its induction of Egr1 during monocytic dierentiation abilitytointeractwithSRFbecauseitstillretainstheB-box (Kharbanda et al., 1994), as well as in B-cell region (aa 231 ± 248). In fact, this truncation signi®cantly maturation (McMahon and Monroe, 1995). Similarly increases the stability of the ternary complex suggesting the proximal SRE (Egr1-SREII) is important for that the N-terminal domain (aa 1-194) may exert NGF induction in PC12 (DeFranco et al., 1993) and inhibitory action on the DNA binding domain of FLI1. cytokine induction (Sakamoto et al., 1994). Such The FLI1 protein has a unique region (R domain) located selective utilization of dierent Egr1 SRE elements next to the DNA binding domain, that is also conserved in may, in part, be due to the speci®c ETS family sea urchin, mouse and human erg-related genes (ELK1, SAP1, FLI1 or EWS-FLI1) transcription (Lautenberger et al., 1992; Qi et al., 1992). This domain factor present and thus contributes to the formation may modulate the interaction with SRF, providing a of the ternary and/or quaternary complexes in mechanism that would be unique to FLI1 and EWS-FLI1. regulating Egr1 gene expression. Ewing's sarcoma is associated with the translocation t(11 : 22) which results in production of a chimeric EWS-FLI1 protein. In the hybrid protein, the N- terminal transactivation domain of EWS, an RNA binding protein, is fused in-frame with the FLI1 DNA Materials and methods binding domain. Recombinant proteins The chimeric EWS-FLI1 protein has biological activities dierent from both EWS or FLI1. The The recombinant ETS1 and FLI1 proteins were obtained FLI1 does not transform cells; whereas, the chimeric from SF-9 cells using ETS1 or FLI1 gene-containing EWS-FLI1 protein exhibits high transforming activity. baculoviral vectors. In addition, ETS1 and FLI1 as well In addition, EWS-FLI is more ecient than FLI1 in as SAP1a, ELK1 and SRF, were produced in vitro using the Promega rabbit reticulocyte system. The SAP1a, transcriptional activation of dierent gene promoters ELK1 and SRF plasmids were generously provided by (May et al., 1993; Lessnick et al., 1995). Since the Dr R Treisman. FLI(BC) was expressed in Ecoliusing normal FLI1 transactivation domain has been sub- the HisTag expression vector, courtesy of Dr Constance stituted with the EWS N-terminal domain, the fusion Fisher. The ETS1 and FLI monoclonal antibodies were protein may be unable to activate a number of wild- produced in mice using bacterially-expressed products type FLI1 target genes and activate others more (Koizumi et al., 1990; Zhang et al., 1995). eciently. In some instances EWS-FLI1 may even inappropriately activate a new set of genes resulting in Electrophoretic mobility shift assay marked growth alteration and cellular transformation. The Egr1 gene appears to have a critical function in the The proteins utilized in gel retardation assays were either obtained from SF-9 cells or prepared in vitro using the regulation of multiple developmental pathways. This rabbit reticulocyte system. The protein : DNA interactions role was ®rst hypothesized based upon the elevated were performed using a 32P-labeled, puri®ed double- Egr1 expression observed during mitogenic stimulation stranded probe (50 000 c.p.m.) containing the Egr1SREI of ®broblasts (Christy et al., 1988), B-cell maturation and Egr1SREII sequences (Figure 1). 1 mgofSF-9nuclear and in the dierentiation of nerve (Milbrandt, 1987), extract or 5 mlofin vitro translated proteins were incubated bone (Suva et al., 1991), and myeloid cells (Coleman et at 48C. After 20 min the protein : DNA complexes were al., 1992; Nguyen et al., 1993). Similar to a regulatory analysed on a non-denaturing 4% polyacrylamide gel role demonstrated for various ETS genes, Egr1 is also containing 0.256TBE; the gel was dried and subjected to involved in B- and T-cell maturation (Perez-Castillo et autoradiography (Seth et al., 1993b). al., 1993). Further support for such an inter-relation- ship between ETS proteins and Egr1 is provided by the Cloning of the EWS-FLI chimeric cDNA from the HTB166 cell observation that both Ets1 and Egr1 gene products are line upregulated upon dierentiation of P19 cells into TheEWS-FLI1chimericgenewasampli®edwithsense neural-type derivatives with retinoic acid (Kola et al., (AGAGGGAGACGGACGTTG) and antisense (AGGC- 1993; Sukhatme et al., 1988). CACATATGTCCTGT) primers using cDNA prepared ETS proteins function as ternary and quaternary complex factors DK Watson et al 221 from the HTB166 cell line mRNA. The ampli®ed EWS-FLI Acknowledgements fragment was subcloned in pBS SK+ and pSG5 vectors We thank Dr Thompson for techincal assistance and Dr (Stratagene). The DNA sequence of the EWS/FLI junction Goukei Pei for the synthesis of oligonucleotides. We also was determined by the dideoxy chain termination method thank Toni Morrison and Judy Yost for assistance in the and found to disrupt the FLI1 protein between amino acids preparation of the manuscript. Part of this work was 197 and 198. funded by a MRC group grant to AS.

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