EWS-Fli1 Up-Regulates Expression of the Aurora A and Aurora B Kinases

Introduction

Ewing family tumor is a rare type of bone and soft tissue tumor, accounting for ∼2% of cases of childhood cancer (1-4). Cytogenetic and molecular analyses of Ewing family tumor indicate that the 5′-region of the EWS gene (from band 22q12) is fused to the Fli1 gene (from band 11q24) or the Erg gene (from band 21q22), both of which are members of the Ets family of transcription factors. Less frequently, chromosomal translocations are observed between the same region of the EWS gene and various other Ets genes, such as ETV-1, E1AF, or FEV (5, 6). Previously, we showed that EWS protein is an RNA-binding protein and that EWS binds preferentially to polyguanylic acid and polyuridylic acid (1). The RNA-binding activity of the EWS protein is located in the RGG box, which lies in the COOH-terminal region, whereas the NH₂-terminal region of the EWS protein regulates RNA-binding activity (1). Ets family transcription factors, characterized by an evolutionarily conserved Ets domain, play important roles in cell development, cell differentiation, cell proliferation, apoptosis, and tissue remodeling (7, 8). The Ets domain of about 85 amino acid residues mediates binding to purine-rich DNA sequences with a central GGAA core consensus sequence (7, 8). Functional characterization of EWS-Fli1 and EWS-Erg chimeric proteins suggests that they act as sequence-specific transcriptional activators and are able to transform cells (1, 9-11). Antagonism of EWS fusion gene expression in tumors results in reduced tumorigenicity and clonogenicity, suggesting that their chimeric products must be expressed above a threshold level to maintain oncogenicity (1, 11). EWS-Fli1 binds to DNA by using the same consensus sequence as Fli1, and the NH₂-terminal region of EWS is capable of activating the promoters of target genes (6, 9). Several target genes of EWS-Fli1 have been identified, such as c-fos, manic fringe, NKX2.2, PTPL1, NR0B1, platelet-derived growth factor C, Id2, thrombospondins, telomerase, and transforming growth factor-β receptor (12-23).

Aurora A and B, a highly conserved family of serine/threonine protein kinases, are tightly regulated during the cell cycle and are overexpressed in many carcinomas, such as hepatocellular, ovarian, esophageal, bladder, breast, prostate, pancreas, and lung cancers (24-34). Human Aurora A, B, and C have been implicated in different aspects of mitosis. The Aurora A gene maps to chromosomal region 20q13.2, which is frequently amplified in a diverse array of human cancers. Aurora A protein localizes to the centrosome and is required for centrosome maturation and spindle formation (35-37). The Aurora B gene maps to human chromosomal region 17q13.1 (38). The Aurora B gene product shows an intracellular localization pattern typical of chromosomal passenger proteins, which relocate from the centromere to the equatorial region along the midzone after onset of anaphase and are required for chromosome segregation and cytokinesis (39-42). The Aurora C gene product is abundant in the testis and has a function in male meiosis (43). Levels of mammalian Aurora A and B proteins are regulated by transcription and protein degradation during the cell cycle (40, 44, 45). Both Aurora A and B are overexpressed at the mRNA and protein levels in a variety of human cancers. Overexpression of both Aurora A and B is linked to centrosome amplification, leading to chromosomal instability and aneuploidy, and consequently malignant transformation (35, 40, 43, 45). Relationships between expression of the Aurora A and B gene products and prognosis or malignant stage have been reported for many types of malignant tumor (13, 24, 25, 46-48). In addition, overexpression of Aurora A can transform rodent fibroblasts, as studied by growth in soft agar and in nude mice (49). Moreover, genetic variants of Aurora A are significantly associated with increased risk of cancer occurrence (49). These results suggest that Aurora kinases have an important role in various cancers.

In the present study, we first examined the relationship between the Aurora A or Aurora B genes and the EWS-Fli1 fusion gene. Knockdown of EWS-Fli1 by small interfering RNA (siRNA) led to reductions in mRNA and protein levels of EWS-Fli1, Aurora A, and Aurora B. In addition, the EWS-Fli1 fusion gene up-regulated the transcription of Aurora A and Aurora B. Both the Aurora A and Aurora B promoters contain positive regulatory elements required for EWS-Fli1 binding. The Aurora A and Aurora B promoters both interact with EWS-Fli1 gene products, as detected by chromatin immunoprecipitation assay. These results strongly suggest that EWS-Fli1 up-regulates Aurora A and Aurora B transcription in Ewing sarcoma.

Results

Knockdown of EWS-Fli1 in TC135 Cells Leads to Reduction of Aurora A and B mRNA or Protein Levels

To understand the relationships between EWS-Fli1 and the mitotic kinases Aurora A and B, their expression levels were investigated in EWS-Fli1 knockdown cells. We confirmed the findings of our previous studies indicating that the expression of EWS-Fli1 protein or mRNA is reduced by EWS-Fli1 siRNA (50, 51). In this study, we also examined by reverse transcription-PCR the levels of Aurora A and B mRNA in TC135 cells treated with EWS-Fli1 siRNA. There were reductions in expression of all of these mRNAs in cells transfected with EWS-Fli1 siRNA compared with those transfected with irrelevant control RNA (Fig. 1A ). Protein levels of Aurora A, Aurora B, and EWS-Fli1 were reduced by transfection with EWS-Fli1 siRNA (Fig. 1B). Knockdown of EWS-Fli1 caused marked decreases in both protein and mRNA of Aurora A and Aurora B, indicating its regulatory role with respect to these kinase genes.

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FIGURE 1.

Knockdown of EWS-Fli1 leads to decreases in mRNA and protein of Aurora A and Aurora B. The Ewing sarcoma cell line TC135, containing the type I EWS-Fli1 fusion gene, was transfected with siRNA for EWS-Fli1 or irrelevant (IR) RNA by using Lipofectamine 2000. After 48 h, total RNA was isolated and subjected to reverse transcription-PCR (RT-PCR) to amplify the cDNA fragments of EWS-Fli1, Aurora A, and Aurora B (A) or protein levels of EWS-Fli1, Aurora A, and Aurora B that were measured by Western blotting (B).

EWS-Fli1 Gene Product Is a Positive Regulator of Aurora A and/or Aurora B Transcription

We examined whether EWS-Fli1 activates the transcription of Aurora A and Aurora B by using the plasmids pGL3A-1486 and pGL3B-1879, respectively. Both promoter regions contain Ets-binding sites (Fig. 2A ). Endogenous EWS-Fli1 protein was detected in TC135 and A673, but not in HT1080, cells (Fig. 2B). These constructs were cotransfected with the phRL-SV40 reference vector into TC135 and A673 cells, which carry EWS-Fli1, or into HT1080 cells, which do not carry EWS-Fli1. For Aurora A, the luciferase activities of pGL3A-1486 were significantly higher than those of empty vector (pGL3) in all three cell lines, as shown in Fig. 2C (P < 0.05). However, there was no significant difference in the luciferase activities of pGL3A-1486 in TC135 and A673 cells. On the other hand, the activity of pGL3A-1486 in HT1080 cells was ∼40% of that in TC135 cells (P < 0.05). For Aurora B, the luciferase activities of pGL3B-1879 were significantly higher than those of pGL3 in all three cell types. Additionally, the luciferase activity in HT1080 cells was only 20% of that in TC135 and A673 cells, as shown in Fig. 2D (P < 0.05). These results indicate that the promoter activities of both Aurora A and Aurora B are significantly up-regulated in TC135 cells, which carry EWS-Fli1, compared with HT1080 cells, which do not, suggesting that Aurora A and Aurora B are genes lying downstream of the transcriptional regulator EWS-Fli1.

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FIGURE 2.

Aurora A and Aurora B promoter activities in TC135, A673, and HT1080 cells. A. Schematic representation of human Aurora A (pGL3A-1486) and Aurora B (pGL3B-1879) promoters containing putative Ets-binding elements (GGAA). Numerals indicate nucleotide number from the transcription initiation site. B. The protein levels of EWS-Fli1 and actin in TC135, A673, and HT1080 cells were measured by Western blotting. Luciferase activities of TC135, A673, or HT1080 cells were measured 48 h after transfection of the promoter constructs pGL3A-1486 (C) or pGL3B-1879 (D) or the control vector pGL3. Results are expressed as the ratio of luciferase activities in each cell extract relative to that in TC135 cells transfected with pGL3A-1486 or pGL3B-1879. All assays were repeated individually at least thrice.

Identification of a Positive Regulatory Region for EWS-Fli1 in the Aurora A and Aurora B Promoters

To confirm the expression levels of EWS-Fli1 in HT1080 cells transfected with EWS-Fli1 expression vector, Western blotting was done (Fig. 3A ). EWS-Fli1 protein was overexpressed in TC135 cells after mock transfection and in HT1080 cells transfected with pEWS-Fli1, but not in HT1080 transfected with empty vector or after mock transfection. On the other hand, Fli1 protein was detected in all HT1080 cell lines examined, but not in TC135 cells. These results show that TC135 has endogenous EWS-Fli1, but not Fli1, whereas HT1080 has endogenous Fli1, but not EWS-Fli1. They also show that transfection of pEWS-Fli1 effectively causes overexpression of EWS-Fli1 protein in HT1080 cells. A series of deletion mutants of the Aurora A and Aurora B promoter regions was generated from pGL3A-1486 and pGL3B-1879 to identify the regions regulated by EWS-Fli1 (Fig. 3B and C). These reporter plasmids were cotransfected with 2 μg of pEWS-Fli1 or empty vector and phRL-SV40 as a reference vector into HT1080 cells. The luciferase activity of pGL3A-1486 in cells cotransfected with pEWS-Fli1 was significantly higher than that in cells cotransfected with empty vector (P < 0.05). Deletion of the sequence from −1486 to −124 in Aurora A did not significantly affect luciferase activity compared with full-length pGL3A-1486. Approximately 50% reduction in activity resulted from deleting the sequence −124 to −75. The reduction induced by deletion of −124 to −75 was greater than that induced by deletion of −75 to +17 (Fig. 3B). These results suggest the presence of an important regulatory sequence between −124 and +17 of the Aurora A promoter. Deletion of the sequence from −1879 to −74 in Aurora B did not affect luciferase activity. Approximately 50% reduction in relative activity was caused by deletion between −74 and +34 (P < 0.05), and a further decrease was caused by deletion between +34 and +171 (P < 0.05). A marginal effect was observed after deletion between +171 and +280 (P = 0.878; Fig. 3C). These results indicate that the regulatory regions for EWS-Fli1 are located in the sequences from −124 to +17 of the Aurora A promoter and from −74 to +171 of the Aurora B promoter.

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FIGURE 3.

Promoter activities of deletion mutants of Aurora A and Aurora B with or without EWS-Fli1. A. Western blotting analysis of EWS-Fli1 and Fli1 expression was done using lysates of TC135 and HT1080 cells with mock transfection and HT1080 cells transfected with pEWS-Fli1 or empty vector. B and C. A series of promoter constructs for Aurora A (B) or Aurora B (C) was cotransfected with phRL-SV40 and pEWS-Fli1 (2 μg) or empty vector (2 μg) into HT1080 cells. Left, schematic representation of the series of deleted promoter regions, containing putative Ets-binding elements, in Aurora A (B) and Aurora B (C). Right, luciferase activities measured with the various deleted promoter constructs indicated on the left. Results are expressed as the ratio of luciferase activities in each cell extract relative to that in cells transfected with pGL3A-1486 (B) or pGL3B-1879 (C). All assays were repeated individually at least thrice.

Effect of Ets Consensus Sequence Mutations in the Aurora A and Aurora B Promoters

Because several groups have reported that the binding site for EWS-Fli1 is the Ets-binding consensus element (GGAA), we examined the effects of point mutations in the Ets-binding element to determine the precise locations of the positive regulatory regions. The experiments above indicated that positive regulatory regions are located from −124 to +17 and from −74 to +280 in pGL3A-1486 and pGL3B-1879, respectively. A computer search revealed one and three Ets-binding sites in the Aurora A and Aurora B promoter regions, respectively. Thus, we generated one promoter mutant (pGL3A-1486-Mut) in pGL3A-1486 for Aurora A, and three mutants (pGL3B-1879-Mut 1, pGL3B-1879-Mut 2, and pGL3B-1879-Mut 3) in pGL3B-1879 for Aurora B, which were then subjected to luciferase assay (Fig. 4 ). The luciferase activity of pGL3A-1486-Mut was markedly decreased to 28% of that of the wild-type promoter (P < 0.05; Fig. 4A). In the Aurora B promoter, we found three Ets-binding sites at positions −71 to −68, +165 to +168, and +265 to +268. The luciferase activity of pGL3B-1879-Mut 1 was significantly reduced to 52% of that of the wild-type promoter (P < 0.05; Fig. 4B). However, the other promoter mutants (pGL3B-1879-Mut 2 and pGL3B-1879-Mut 3) did not show any decrease in luciferase activity compared with the wild-type (P = 0.302 and 0.599, respectively; Fig. 4B). These results confirm that the Aurora A and Aurora B promoters have specific positive regulatory regions at −84 to −81 and −71 to −68, respectively, which are responsible for activation by EWS-Fli1. The results suggest that these Ets-binding sites have an important role in recruiting complexes containing EWS-Fli1 to the Aurora A and Aurora B promoters.

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FIGURE 4.

Promoter activities of mutated Aurora A and Aurora B promoters. Wild-type or mutated promoters of Aurora A (A) or Aurora B (B) were cotransfected with phRL-SV40 and 2 μg of pEWS-Fli1 into HT1080 cells. Left, schematic representation of the various mutants of the Aurora promoters. Wild-type and mutated Aurora A promoter constructs were designated pGL3A-1486 and pGL3A-1486-Mut, respectively. Wild-type and mutated Aurora B promoter constructs were designated pGL3B-1879 and pGL3B-1879-Mut 1, pGL3B-1879-Mut 2, and pGL3B-1879-Mut 3, respectively. The empty vector was pGL3. The mutated Ets sequences and the nucleotide numbers are shown. Right, luciferase activity of the wild-type and several mutated promoters assessed 48 h after transfection. Results are expressed as the ratio of luciferase activities in each cell extract relative to that in cells transfected with the wild-type pGL3A-1486 (A) or pGL3B-1879 (B). All assays were repeated individually at least thrice. Asterisks indicate statistically significant differences by unpaired two-tailed Student's t test.

EWS-Fli1 Gene Products Bind Directly to the Aurora A and Aurora B Promoter Regions

We did chromatin immunoprecipitation assays to investigate whether EWS-Fli1 gene products interact with the Aurora A and Aurora B promoter regions. The TC135 cell line, which expresses the EWS-Fli1 fusion protein, was immunoprecipitated with anti-Fli1 antibody or nonspecific IgG, and the promoter regions of Aurora A and Aurora B were detected by PCR. Bands of the corresponding size were detected from DNA immunoprecipitated with anti-Fli1, but not from DNA immunoprecipitated with control IgG, by using the A-1 or B-1 primer sets (Fig. 5C and G ). On the other hand, DNA fragments were never amplified from DNA immunoprecipitated with anti-Fli1 or control IgG when the A-2 or B-2 primer sets were used (Fig. 5A and E). These results suggest that the 5′-flanking region of Aurora A and Aurora B interacts with EWS-Fli1 in the TC135 cell line. Next, HT1080 cells were cotransfected with wild-type or mutant Aurora promoter constructs in combination with a Flag-tagged EWS-Fli1 expression vector (pFlag/EWS-Fli1). The promoters pGL3A-1486-Mut or pGL3B-1879-Mut 1 were used as mutated promoter constructs. At 48 hours after transfection, each potential component of the EWS-Fli1 complex was precipitated with anti-Flag antibody or nonspecific IgG, and the promoter regions of Aurora A and Aurora B were detected by PCR. When the A-1 primer set was used for PCR, DNA fragments of the Aurora A 5′-flanking region with the expected size were detected in all input samples (Fig. 5D, top). However, no band was detected with nonspecific IgG (Fig. 5D, middle). The complex precipitated with anti-Flag antibody exhibited a band corresponding to the Aurora A promoter only when cells were cotransfected with wild-type pGL3A-1486 and pFlag/EWS-Fli1, but not with the mutant promoter or the empty vector pFlag (Fig. 5D, bottom). On the other hand, when the A-2 primer set was used for PCR, the band was never detected except for the input sample (Fig. 5B). For Aurora B, the DNA fragment was amplified with the B-1 primer set only in the complex from the combination of wild-type pGL3B-1879 and pFlag/EWS-Fli1 (Fig. 5H), a result similar to that obtained with wild-type pGL3A-1486 (Aurora A). These results confirm that complexes containing EWS-Fli1 bind to the Aurora A and Aurora B promoters in vivo and that the binding sites of the EWS-Fli1 gene product in the Aurora A and Aurora B promoters are located at −84 to −81 and −71 to −68, respectively.

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FIGURE 5.

Detection of EWS-Fli1 binding to the Aurora A and Aurora B promoters by chromatin immunoprecipitation. A, C, E, and G. Each potential component of the EWS-Fli1 complex was precipitated with anti-Fli1 antibody from TC135 cells bearing the EWS-Fli1 fusion protein. As a negative control, the same amount of anti-mouse IgG was used for each sample. Immunoprecipitated DNA was analyzed by PCR with primers specific for the 5′ flanking regions of Aurora A [A-2 (−1643; −1489), A-1 (−177; −17)] and Aurora B [B-2 (−1882; −1735), B-1 (−87; +26)]. As an input, the lysate before immunoprecipitation was subjected to PCR with the same primers. HT1080 cells were cotransfected with combinations of reporter plasmids having Aurora A promoters (wild-type or mutated pGL3A-1486; B and D) or Aurora B promoters (wild-type or mutated pGL3B-1879; F and H) and regulator-encoding plasmids, pFlag/EWS-Fli1 (+) or empty vector (−). Bottom, after 48 h, each potential component of the EWS-Fli1 complex was precipitated with anti-Flag antibody. Middle, as a negative control, the same amount of anti-mouse IgG was used for each sample. Immunoprecipitated DNA was analyzed by PCR with primers specific for the 5′ flanking regions of Aurora A or Aurora B, which are A-1 (D) and A-2 (B) or B-1 (H) and B-2 (F), respectively. Top, as an input for each assay, the lysate before immunoprecipitation was subjected to PCR with the same primers.

Discussion

Our present results indicate that knockdown of EWS-Fli1 by siRNA results in reductions in mRNA and protein levels of EWS-Fli1, Aurora A, and Aurora B. In addition, the EWS-Fli1 fusion gene product interacts with the Aurora A and Aurora B promoters and up-regulates the transcription of these genes.

Several groups have studied Aurora promoter activity by using deleted or point-mutated promoters (26, 36, 39, 40, 45). Transcription of Aurora A is positively regulated by E4TF1, a ubiquitously expressed member of the Ets protein family, and the binding site of E4TF1 is located between −85 and −79 upstream, a region that contains the consensus Ets-binding element GGAA (45). TRAP/MED1 has been reported to bind to E4TF1/GABP and to regulate Aurora A promoter activity via an Ets-binding element (44). The promoter activity of Aurora A is associated with Ets2, which belongs to the Ets family of transcription factors, and the regulatory element of Aurora A is the same site as the E4TF1 site (45). However, the Ets family transcriptional factor Ets1 does not bind to the DNA sequence to which E4TF1 binds. In addition, the amount of Aurora A protein is not decreased by siRNA transfection of the Ets family transcription factor Elk1 (28). These results suggest that only some members of the Ets family of transcription factors bind to the promoter region of Aurora A and up-regulate its transcription. In this study, we showed that EWS-Fli1 binds to the same promoter element and up-regulates its transcription. Conversely, the functional relationships between Ets family transcription factors and the Aurora B promoter have not been studied. Here, we identified the EWS-Fli1–binding site in the Aurora B promoter region and showed that the element can up-regulate Aurora B transcription. This is the first report of a relationship between the Ets family transcription factor EWS-Fli1 and Aurora B expression.

In Ewing family tumors, the EWS-Ets fusion product acts as an oncogene and promotes abnormal cellular proliferation (52, 53). EWS-Fli1 up-regulates c-myc, Id2, hTERT, PTPL1, and NR0B1 and down-regulates p27, p57, p21, transforming growth factor-β receptor, and NKX2.2, suggesting that the amounts of many cell cycle regulators are controlled by the EWS-Fli1 fusion protein. Complexes containing EWS-Fli1 have been shown to interact directly with the Ets-binding site (GGAA) of many promoters and to regulate their activity (12-16, 19, 20, 23 53-56), suggesting that the interaction of EWS-Fli1 with the Aurora A and Aurora B promoters may be direct. However, some investigators have reported a new aspect of the tumorigenic mechanism of EWS-Ets in that the DNA-binding domain is not essential for its transforming activity and some gene promoters are activated by EWS-Ets regardless of the lack of direct binding to DNA (18, 55). Nevertheless, our results strongly suggest that the Aurora A and Aurora B genes are the direct targets of the oncogenic EWS-Fli1 fusion protein via Ets-binding elements in their promoters.

Aurora A and Aurora B are important mitotic regulators, and elevated expression of Aurora A and Aurora B has been reported in various human cancers (24-34). In Ewing sarcoma, Aurora A and Aurora B are up-regulated directly by the EWS-Fli1 fusion gene product. The present investigations indicate that Aurora kinases are potential targets for the treatment of Ewing sarcoma.

Materials and Methods

Cell Culture

TC135 and A673 are Ewing sarcoma cell lines carrying EWS-Fli1. TC135 was provided by Dr. T.J. Triche (University of Southern California, Los Angeles, CA) and A673 was purchased from the American Type Culture Collection. HT1080, a fibroblastoma cell line lacking EWS-Fli1, was purchased from the American Type Culture Collection. TC135 and HT1080 cells were maintained in RPMI 1640 (Invitrogen) containing 10% fetal bovine serum, 100 units/mL penicillin G, and 100 μg/mL streptomycin at 37°C under 5% CO₂ (49, 50). A673 cells were maintained in DMEM supplemented with 2 mmol/L l-glutamine and 10% fetal bovine serum at 37°C under 10% CO₂ (57).

siRNA and Reverse Transcription-PCR and Western Blotting

The sequences of the EWS-Fli1 siRNA (5′-AGCAGAACCCTTCCTTATGAC-3′) and irrelevant siRNA (5′-AGTCGACGTCAGCTGAAGGC-3′) were as previously described (50, 51). The former has been reported to decrease both the protein and mRNA levels of EWS-Fli1 (50, 51). The siRNAs were transfected into TC135 cells with Lipofectamine 2000, and total RNA was isolated from the cells 48 h after transfection by using ISOGEN (Nippon Gene). Reverse transcription-PCR was done in 20 μL of first-strand buffer containing 0.2 μg of total RNA and 100 ng of oligo(dT) primer with 0.2 μL of PowerScript (Clontech) for 90 min at 42°C followed by heat inactivation for 5 min at 99°C. The primer sets used were as follows: a 153-bp Aurora A cDNA fragment (5′-TGATGATATCGCCGCGCTCGTCGT-3′ and 5′-CACAGCCTGGATAGCAACGTACAT-3′), a 412-bp β-actin cDNA fragment (5′-TGATGATATCGCCGCCGCGCTCGTCGT-3′ and 5′-CACAGCCTGGATAGCAACGTACAT-3′), and a 347-bp Aurora B cDNA fragment (5′-AGAACTCCTACCCCTGGCCCTAC-3′ and 5′-ATGCTCCACGCCCTCCTTCTCTA-3′). PCR was carried out using KOD Plus (TOYOBO) for 32 cycles at 98°C (10 s), 56°C (30 s), and 68°C (30 s) for Aurora A and Aurora B and at 98°C (10 s), 59°C (30 s), and 68°C (30 s) for β-actin (40). For EWS-Fli1, primers (5′-GTGATACAGCTGGCGGCGTTGGCG-3′ and 5′-GCTGCCCGTAGCTGCTGCTCTGTT-3′) were used to generate a 332 bp cDNA product with 35 cycles at 98°C (10 s), 65°C (30 s), and 68°C (30 s; ref. 51).

Cell harvesting and Western blotting were done as described (50). The EWS/Fli1 fusion protein (68 kDa) and Fli1 (51 kDa) protein were detected by Western blotting analysis using an anti-Fli1 antibody (Santa Cruz Biotechnology). Aurora A and Aurora B proteins were detected with anti-Aurora A (46 kDa; ref. 37) and anti-Aurora B (41 kDa) antibodies (Santa Cruz Biotechnology), respectively.

Plasmid Construction for Aurora A, Aurora B, and EWS-Fli1

The EWS-Fli1 expression vector was constructed as described previously (3). We subcloned the coding region of EWS-Fli1 into pCMV-FLAG (Sigma) to produce pFlag/EWS-Fli1.

To obtain the Aurora A reporter plasmid (pGL3A-1486), the genomic DNA fragment containing −1486 to +354 of the 5′-flanking sequence was amplified by PCR with PAC clone RP5-1167H4 (Children's Hospital Oakland Research Institute) with the primer set 5′-AAAGATCTCACATGAGAGATTAGAGGCTG-3′ and 5′-GGGGGATCCCTCTAGCTGTAAGTAAC-3′ and subcloned into the BamHI site of the pGL3 vector (Promega). The plasmids pGL3A-415 and pGL3A+17, which are deletion mutants of pGL3A-1486, were constructed by using appropriate restriction enzymes. To obtain pGL3A-124 and pGL3A-75, the DNA fragments corresponding to −124 to +354 and to −75 to +354 were amplified by PCR with the primer sets 5′-GGGGGATCCTGGGACTGCCACAGGTCTGG-3′ and 5′-GGGGGATCCCTCTAGCTGTAAGTAAC-3′, and 5′-GGGGGATCCCACCACTTCCGGGTTCTTAG-3′ and 5′-GGGGGATCCCTCTAGCTGTAAGTAAC-3′, respectively. Digested PCR products were subcloned into the BamHI site of pGL3 (45).

For Aurora B, the promoter region containing −1879 to +392 of the human Aurora B gene was subcloned into pGL3 (pGL3B-1879) and its deleted promoters were constructed as previously reported (39). Reporter plasmids with mutant Ets-binding sites were generated by PCR-targeted mutagenesis with a QuikChange kit (Stratagene) in accordance with the manufacturer's instructions (40). In pGL3A-1486 (Aurora A), the primer corresponding to the site from −84 to −81 was 5′-GGCCGTTGGCTCCACCACGCGTGGGTTCTTAGGGAGCAAG-3′. In pGL3B-1879 (Aurora B), the primers used for the sites located from −71 to −68 (pGL3B-1879-Mut 1), +165 to +168 (pGL3B-1879-Mut 2), and +265 to +268 (pGL3B-1879-Mut 3) were 5′-CAGGACATCGAGCCAATGCATGCTAGGCTGGGCGACGAG-3′, 5′-CGCGCACGCCGCAGGGCTACGTGGAGGTAGGGACGATAGC-3′, and 5′-ACCAGCTCGGGCTAGCGCGCGTGGCTCGATCGGTCCAACC-3′, respectively. The original Ets sequences (GGAA or TTCC) are mutated as underlined.

Transfection and Luciferase Assay

Reporter genes were transfected into TC135 or HT1080 cells together with phRL-SV40 (Promega) by using FuGene 6 (Roche; refs. 18, 40) and luciferase activity was measured with a luminometer (VERITAS, Promega). Firefly luciferase activities, derived from pGL3 or pGL3A-1486 or pGL3B-1879, were normalized by comparison with Renilla luciferase activities from phRL-SV40 (40, 45).

Chromatin Immunoprecipitation

Chromatin immunoprecipitation was carried out using a chromatin immunoprecipitation assay kit (Upstate Biotechnology) in accordance with the manufacturer's protocol (18-20, 28, 44). TC135 cells or transfected HT1080 cells were fixed with 1% formaldehyde solution for 10 min at 37°C. Complexes containing Flag/EWS-Fli1 were immunoprecipitated with anti-Flag (Sigma), anti-Fli1 (Santa Cruz Biotechnology), or nonspecific normal rabbit IgG (Santa Cruz Biotechnology).

Primer sequences used to generate Aurora A genomic DNA fragments were as follows: region A-1 (−177; −17), 5′-AGGGGCTGTTGCTTCACCGATAA-3′ and 5′-TGACAACAAACCCGACGCCTTGA-3′; region A-2 (−1643; −1489), 5′-AGGGGCTGTTGCTTCACCGATAA-3′ and 5′-TGACAACAAACCCGACGCCTTGA-3′. Primer sequences used to generate Aurora B genomic DNA fragments were as follows: region B-1 (−87; +26), 5′-GGACATCGAGCCAATGGGAACTA-3′ and 5′-AAACAACTGAATCTGCCACGCCG-3′; region B-2 (−1882; −1735), 5′-TATGCATGTAGAGGCTCAACAAT-3′ and 5′-TCTGGAAGTGAGGGAAGCAT-3′. PCR was carried out using KOD-Fx (TOYOBO) for 20 cycles of 98°C (10 s), 57°C (30 s), and 68°C (30 s) for region A-1 and 98°C (10 s), 59°C (30 s), and 68°C (30 s) for region B-1. As negative controls, Aurora A and Aurora B genomic DNA fragment PCR was carried out using KOD-Fx for 25 cycles of 98°C (10 s), 60°C (30 s), and 68°C (30 s) for regions A-2 and B-2.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.