The Id2 gene is a novel target of transcriptional activation by EWS-ETS fusion proteins in Ewing family tumors

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Abstract

We report here that the Id2 (inhibitor of DNA binding 2) gene is a novel target of transcriptional activation by EWS–FLI1 and EWS–ERG, two fusion proteins that characterize Ewing family tumors (EFTs). To identify downstream targets of these EWS–ETS fusion proteins, we introduced EWS–ETS fusion constructs into a human fibrosarcoma cell line by retroviral transduction. cDNA microarray analysis revealed that Id2 expression was up-regulated by introducing the EWSETS fusion gene but not by the normal full-length ETS gene. An Id2 promoter-luciferase reporter assay showed that transactivation by EWS–ETS involves the minimal Id2 promoter and may function in cooperation with c-Myc within the full-length regulatory region. A chromatin immunoprecipitation assay revealed direct interaction between the Id2 promoter and EWS-FLI1 fusion protein in vivo. Significantly higher expression of Id2 and c-Myc was observed in all of the six EFT cell lines examined compared to six other sarcoma cell lines. Moreover, high levels of Id2 expression were also observed in five of the six primary tumors examined. Id2 is generally thought to affect the balance between cell differentiation and proliferation in development and is highly expressed in several cancer types. Considering these previous studies, our data suggest that the oncogenic effect of EWS–ETS may be mediated in part by up-regulating Id2 expression.

Introduction

Ewing family tumors (EFTs), including Ewing's sarcoma and peripheral primitive neuroectodermal tumors, are highly malignant tumors arising in adolescents and young adults. EFT is sometimes difficult to distinguish pathologically from other small round cell tumors, such as neuroblastoma, malignant lymphoma, and some types of rhabdomyosarcoma and osteosarcoma. However, EFTs are characterized by specific balanced chromosomal translocations that result in fusion of the N-terminus of the EWS gene product to the C-terminus of an ETS transcription factor including FLI1, ERG, ETV1, E1AF or FEV (Delattre et al., 1992, 1994; Sorensen et al., 1994; Giovannini et al., 1994; Jeon et al., 1995; Urano et al., 1996; Kaneko et al., 1996; Peter et al., 1997). EWS-FLI1 appears to be essential for maintaining the oncogenic property of tumor cells (Ouchida et al., 1995; Kovar et al., 1996; Tanaka et al., 1997) and has properties that are distinct from normal full-length FLI1 (May et al., 1993a,b). Based on the protein structure of EWS-FLI1, its transforming activity is mainly thought to depend on its DNA binding ability.

TGF-beta type II receptor, mE2-C, EAT-2, PDGF-C, p57KIP2 and c-Myc genes were previously identified as transcriptional targets of EWS–ETSs (May et al., 1997; Hahm et al., 1999; Arvand et al., 1998; Thompson et al., 1996; Zwerner and May, 2001; Dauphinot et al., 2001), although the exact mechanism of tumorigenesis mediated by EWS–ETS remains unclear. To systematically identify other genes that would participate in development of EFTs, we focused on genes that are specifically regulated by EWS–FLI1 and EWS–ERG, but not by FLI1 and ERG, using cDNA microarray analysis. We report here that the Id2 gene encoding a helix–loop–helix protein is a target of transcriptional activation by EWS–FLI1 and EWS–ERG, mediated by their direct interaction with the Id2 promoter. Previous studies showed that Id2 was overexpressed in several types of tumors and that up-regulation of Id2 was mediated by N-Myc, c-Myc or TCF (Lasorella et al., 2000; Rockman et al., 2001). Therefore, our data reveal a novel transcriptional regulation of the Id2 gene and suggest its involvement in the oncogenesis of EFTs.

Results

Establishment of HT-1080 cell populations expressing EWS–ETSs fusion genes

To identify the downstream targets of EWS–ETS fusion genes, we first established HT-1080 cell populations expressing either EWS–ETSs or their normal ETS counterparts. HT-1080 cells were transfected separately with the retroviral expression vector pLNCX containing either ERG (pL-E), FLI1 (pL-F), EWS–ERG (pL-EE), EWS–FLI1 (pL-EF) cDNAs, or with an empty vector pLNCX. Stable drug-resistant transfectants were pooled and named HT-pL-E, HT-pL-F, HT-pL-EE, HT-pL-EF and HT-pL, respectively. Northern and immunoblot analysis revealed expression of both mRNA and protein corresponding to the transfected genes in a pool of stable clones (Figure 1).

Figure 1
figure1

Expression of the transfected FLI1 and ERG genes in HT-1080 cells. (a) Northern blot analysis revealed expression of the expected mRNAs in a pool of HT-1080 cells that were separately transfected with a control retroviral vector pLNCX (lane 1) and its derivatives encoding ERG (pL-E, lane 2), EWS–ERG (pL-EE, lane 3), FLI1 (pL-F, lane 4) and EWS–FLI1 (pL-EF, lane 5), respectively. 10 μg of total RNA were loaded in each lane. Hybridization was performed with a human ERG or FLI1 cDNA fragment as a probe. (b) Immunoblot analysis showed expression of the expected proteins in a pool of HT-1080 cells transfected with a control vector pLNCX (lanes 1 and 4), pL-E (lane 2), pL-EE (lane 3), pL-F (lane 5) and pL-EF (lane 6), respectively. A total of 20 μg of whole cell lysate were loaded in each lane. The proteins were detected with antibodies against the C-terminal region of ERG or FLI1

Specific induction of the Id2 gene by EWS–ETSs fusion genes but not by normal ETSs

In an effort to identify genes that are specifically regulated by EWS–ETSs but not by the normal ETSs transcription factor, we used cDNA microarray analysis to compare the expression profile of cells expressing EWS–FLI1 or EWS–ERG with those expressing FLI1 or ERG. Using this approach, we detected several genes that were reproducibly activated by EWS–FLI1 or EWS–ERG but not by FLI1 or ERG. One of the genes that was specifically induced by EWS–ETS was Id2. Northern blot analysis using an Id2 cDNA probe confirmed that endogenous Id2 expression was increased in cells expressing EWS–FLI1 or EWS–ERG but not FLI1 or ERG (Figure 2a). In addition, accumulation of the endogenous Id2 protein was detected by immunoblot analysis in HT-pL-EF cells (Figure 2b).

Figure 2
figure2

Effect of EWS–ETSs on the expression of Id2 in HT-1080 cells. HT-1080 cells were transfected with each of the pLNCX derivatives (a and b) or pBabe-Puro derivatives (c and d). Total RNA (10 μg) or whole cell lysates (20 μg) from each of the HT-1080 transfectants were analysed by Northern blot (a and c) and immunoblot analysis (b and d), respectively. (a) Northern blot analysis showed prominent expression of Id2 mRNA in HT-1080 cells expressing EWS–ERG (HT-pL-EE) or EWS–FLI1 (HT-pL-EF). (b) Immunoblot analysis showed comparable Id2 protein expression in HT-pL-EF cells. Upper bands are non-specific. (c,d) Northern blot and immunoblot analysis showed that Id2 expression was also up-regulated prominently in HT-1080 cells expressing EWS–ERG (HT-pB-EE) or EWS–FLI1 (HT-pB-EF) from a different retroviral vector, pBabe-Puro

To further confirm that the Id2 gene is specifically induced by EWS–ETSs but not normal ETSs, we used a second retroviral expression vector pBabe-Puro. HT1080 cells were transfected separately with pBabe-Puro either containing ERG (pB-E), FLI1 (pB-F), EWS (pB-EWS), fused EWS–ERG (pB-EE), EWS–FLI1 (pB-EF) cDNA or an empty vector. Stable, puromycin-resistant transformants were pooled and named HT-pB-E, HT-pB-F, HT-pB-EWS, HT-pB-EE, HT-pB-EF and HT-pB, respectively. Immunoblot analysis of the stable clones verified protein expression corresponding to the transfected genes (for example Figure 3a, lane 2). Figure 2c,d show the significantly higher expression of endogenous Id2 mRNA and protein in HT-pB-EE and HT-pB-EF cells relative to the low level of expression in HT-pB-E, HT-pB-F and HT-pB-EWS cells. Therefore, these results demonstrate that the high level expression of Id2 was not due to clonal heterogeneity but was up-regulated as a consequence of EWS–ETSs expression.

Figure 3
figure3

Id2 expression is attenuated by down-regulation of EWS–ETSs. (a) HT-pB-EF cells, which are EWS–FLI1-expressing HT-1080 cells, were separately transfected with a control plasmid (pIRESneo2), antisense ERG-(pIRES-asE) and an antisense EWS–FLI1-expression vector (pIRES-asEF), respectively. Stable transfectants were pooled separately and named HT-pB-EF/neo, HT-pB-EF/asE and HT-pB-EF/asEF (#1 to #10), respectively. Immunoblot analysis was performed to examine the effect of antisense EWS–FLI1 on EWS–FLI1 expression. EWS–FLI1 fusion protein was detected with an anti-FLI1 antibody (C-19). One of the ten HT-pB-EF/asEF clone pools showed attenuated expression of EWS–FLI1 (clone #7) whereas others did not (for example, clones #3 and #9). (b) Attenuated expression of Id2 and c-Myc mRNAs was observed in HT-pB-EF/asEF#7 cells (lane 6) compared to HT-pB-EF/asE (lane 5), HT-pB-EF/neo (lane 4), HT-pB-EF cells (lane 3). A total of 10 μg of total RNA or 20 μg of whole cell lysate were loaded in each lane

Down-regulation of EWS–ETSs by antisense cDNA attenuates Id2 expression

To investigate whether inhibition of EWS–FLI1 expression attenuates Id2 expression, we established antisense-EWS–FLI1 expressing HT-pB-EF cells. HT-pB-EF cells were transfected with an antisense-EWS–FLI1 cDNA expressing vector pIRES-asEF. Stable transfectants were selected and more than one hundred independent clones were pooled separately. Ten different pools of clones were named HT-pB-EF/asEF#1 to HT-pB-EF/asEF#10. Immunoblot analysis revealed that HT-pB-EF/asEF#7 cells were a cell population exhibiting the most significant reduction of EWS–FLI1 expression (Figure 3a). We then examined the expression level of Id2 in HT-pB-EF/asEF cells. In accordance with the low level of expression of EWS–FLI1, significantly attenuated expression of Id2 was observed in HT-pB-EF/asEF#7 cells (Figure 3b). c-Myc mRNA expression was also attenuated coordinately with diminished expression of EWS–FLI1 (Figure 3b). In HT-pB-EF cells transfected with pIRES-asE or an empty pIRESneo2, the expression level of both EWS–FLI1 protein and Id2 mRNA was not affected (Figure 3a,b). These results suggest that EWS–FLI1 is essential for the up-regulation of Id2 expression in this model system.

Identification of specific sites in the Id2 promoter that mediate transcriptional induction by EWS–ETS

The data described above demonstrated that EWS–ETS is able to active Id2 transcription directly or indirectly. To test the regulatory elements of Id2 for transactivation by EWS–ETS, we performed an Id2 promoter-reporter assay. We first searched for a consensus ETS-binding sequence, especially FLI1 (ACCGGAAG/aT/c, Mao et al., 1994), in the Id2 promoter and found three candidate sites at positions −2633, −120 and −60, where +1 represents the transcription start site (Figure 4a). To determine the relation between these sites and Id2 induction mediated by EWS–ETS, we cloned a 2.7-kb Id2 promoter region containing these three ETS-binding sequences and two E-box sequences that are high-affinity c-Myc-binding sites (Figure 4a, CACATG at −1880 and CACGTG at −1570; Lasorella et al., 2000). The 2.7 kb promoter fragment and a series of deletion constructs were inserted upstream of a luciferase reporter gene (Figure 4a) and used in a reporter assay. HCI-H1299 cells were transiently co-transfected with a reporter plasmid together with the expression vector pcDNA-EF, pcDNA-EE, pcDNA-F, pcDNA-E or pcDNA3.1(+) (Figure 4b).

Figure 4
figure4

Transactivation of the Id2 promoter by EWS–ETS and normal ETS. (a) A schematic representation of the Id2 promoter-reporter constructs. Six fragments containing portions of 5′-flanking regions of the Id2 gene were subcloned upstream of a luciferase reporter gene in pGL3-Basic vector. The transcriptional start site was considered +1. Open boxes represent putative ETS-binding sites. The three ETS-binding sites at −2633, −120 and −60 match the consensus sequence at eight of nine, seven of nine and seven of nine nucleotides, respectively. Open circles represent E-box consensus sequences that respond to N-Myc and c-Myc oncoproteins. (b) Luciferase assay for Id2 promoter activity. H1299 cells were co-transfected with each of the reporter plasmids together with either pcDNA-E, pcDNA-EE, pcDNA-F or pcDNA-EF, and a control reporter pRL-TK. Luciferase activity was defined as the ratio of Photinus pyralis luciferase activity from pGL3-Basic derivatives relative to Renilla reniformis luciferase activity that reflected the efficiency of transfection. These experiments were performed in triplicate and repeated at least three independent times. The bars and brackets indicate mean and standard deviation, respectively. (c) Chromatin immunoprecipitation (ChIP) assay of the Id2 promoter. ChIP assay of a genomic fragment (nucleotide position −172 to +16) containing the two potential ETS binding sites in HT-pL (lanes 5 and 6), HT-pL-F (lanes 7 and 8) and HT-pL-EF (lanes 9 and 10) is shown. Immunoprecipitation was performed using anti-FLI1 polyclonal antibody (C-19) against the carboxy terminus of FLI1, followed by PCR amplification (anti-FLI1, lanes 5, 7 and 9). Input chromatin represents a portion of the sonicated chromatin prior to immunoprecipitation (lanes 2–4). ChIP assays in the absence of antibody (no Ab, lanes 6, 8 and 10) were used for controls. DW indicates a no template control (lane 1)

We deduced from the reporter assay that: (1) the pGL3-60 reporter construct encompassing −60 to +35 of the Id2 gene conferred little luciferase activity with any expression vector; (2) Luciferase activity for each reporter co-transfected with an EWS–ETS expressing vector is higher than reporter co-transfected with the normal ETS expressing vector; (3) With regard to the region between −160 to −60, deletion of the ETS-binding site at −120 alone reduced EWS–ETS responsiveness. In addition, EWS–ETS-mediated induction was largely eliminated when the ETS-binding sites at −120 and −60 were excluded from the reporter construct; (4) The region between −1329 to −166 acts as a repressive element for Id2 transcription; (5) The EWS–ETS constructs activate the reporter pGL-Id2-2755 slightly higher than constructs without the Myc-binding sites (pGL-Id2-del980 and pGL-Id2-1329). Altogether, these results strongly suggest that the two EST-binding sites at −120 and −60 are important for EWS–ETS-mediated transcriptional induction. A direct transcriptional activating function of EWS–ETS on a minimal Id2 promoter likely occurs in cooperation with the action of c-Myc on the full-length regulatory region of the Id2 gene.

To determine whether the EWS–FLI1 fusion protein selectively binds to the Id2 promoter in vivo, we performed ChIP assays. The ChIP assay relies on the ability of specific antibodies to immunoprecipitate DNA-binding proteins along with the associated genomic DNA. Immunoprecipitation of a DNA-protein complex using an antibody against FLI1 was performed on cross-linked extract from HT-pL-EF, HT-pL-F and HT-pL cells. We then measured the abundance of genomic DNA containing the Id2 promoter within the immune complexes by PCR amplification. The FLI1 immunoprecipitated complex from HT-pL-EF cells contained a greater amount of amplifiable Id2 promoter than complexes from HT-pL-F or HT-pL cells (Figure 4c). This result suggests that the EWS–FLI1 fusion protein interaction with the Id2 promoter is more substantial than that of the normal FLI1 protein in vivo.

Id2 is highly expressed in EFT cell lines and primary tumors

Although Id2 is likely to be a mediator of EWS–ETSs function in these model cells, this is not necessarily the case in primary EFTs. Therefore, we performed Northern blot analysis with twelve sarcoma cell lines, including six EFTs (A673, NCR-EW2, RD-ES, SCCH196, SK-ES-1 and W-ES), three rhabdomyosarcomas (A204, Hs729T and RD), a fibrosarcoma (HT-1080), an osteosarcoma (SaOS2), and a liposarcoma (SW872). Id2 was prominently expressed in all of the six EFT cell lines examined while only weakly expressed in the other sarcoma cell lines tested (Figure 5a). Interestingly, all 6 EFT cell lines also display high expression of c-Myc relative to the other sarcoma cell lines (Figure 5a). Moreover, in general Id2 and c-Myc expression in primary EFT specimens were higher than those in HT-1080 cells, whereas the relative level of Id2 mRNA was not completely concordant with that of c-Myc in these primary EFT samples (Figure 5b). We also confirmed Id2 expression in primary EFT samples by immunostaining. EFT cell lines cultured on chamber slides were used as a positive control. For example, Id2 expression was detected in SK-ES1 cells (Figure 6a), and incubation of the primary antibody with a specific blocking peptide significantly reduced these signals (Figure 6b). Immunohistochemical staining was also performed on six primary EFT samples. Representative results are shown in Figure 6c,d; the Id2 protein was detected in tumor cells of five of the six EFT samples tested, and the Id2-specific blocking peptide significantly reduced the signals as it did in EFT cell lines. These results show that a high level of Id2 expression is a general feature of EFTs.

Figure 5
figure5

Id2 and c-Myc mRNA levels in sarcomas. (a) Northern blot analysis of Id2 and c-Myc in sarcoma cell lines including six EFTs and six other sarcomas; A-673, NCR-EW2, RD-ES, SK-ES1, SCCH-196 and W-ES (EFTs), A204, Hs729T and RD (rhabdomyosarcomas), HT-1080 (fibrosarcoma), Saos-2 (osteosarcoma), SW872 (liposarcoma). A total of 10 μg of total RNA were loaded in each lane. The probe for c-Myc was hybridized to the same membrane after stripping the membrane of Id2 probe. Id2 and c-Myc mRNA levels were high in EFTs relative to the other sarcoma cell lines. (b) Quantitation of Id2 and c-Myc mRNA in primary EFT specimens. The amount of mRNA is represented as the ratio of the particular mRNA to 18S rRNA. The Id2 mRNA level (left panel) does not completely correlate with c-Myc levels (right panel) in primary EFT specimens

Figure 6
figure6

Detection of the Id2 protein in an EFT cell line and clinical samples. Representative immunohistochemical detection of the Id2 protein in primary EFTs specimens and SK-ES1 cells cultured on a chamber slide with an anti-Id2 antibody C-20. Specificity of Id2-immunoreactivity was demonstrated by competition with an Id2-specific blocking peptide sc-489P (b,d). SK-ES1 cells: (a,b); a primary Ewing sarcoma specimen: (c,d). Original magnification: ×100

Discussion

EWS–ETS fusion genes are clearly associated with tumorigenesis. Therefore, isolation of downstream targets of EWS–ETSs could lead to the identification of genes involved in the initiation and progression of EFTs. Here we identified the Id2 gene as a novel target of transcriptional activation by EWS–ETS proteins. Id2, a helix–loop–helix (HLH) protein is generally thought to affect the balance between cell proliferation and differentiation by functioning as a dominant-negative antagonist of basic helix–loop–helix (bHLH) transcription factors (reviewed in Yokota, 2001; Lasorella et al., 2001; Zebedee and Hara, 2001). Analysis of Id2-deficient mice revealed that Id2 is indispensable for normal development, especially in the generation of peripheral lymphoid organs and natural killer cells (Yokota et al., 1999). In addition, Id2 overexpression has been reported in several human malignancies, such as neuroblastoma with N-Myc gene amplification, pancreatic adenocarcinoma, and colon cancers exhibiting alteration of the β-catenin/TCF pathway (Kleeff et al., 1998; Lasorella et al., 2000; Rockman et al., 2001). In the present study, we showed that Id2 is induced to a significantly higher level in cells expressing EWS–ETS fusions than in cells expressing their corresponding normal ETSs and that induction of Id2 is dependent on the expression of EWS–ETSs in our model cells. Moreover, we observed strong expression of Id2 in Ewing sarcoma cell lines and EFT primary tumors.

Dysfunction of pathways that regulate the cell cycle are critical steps in the development and progression of human malignancy. Molecules involved in one of the major pathways of G1-S regulation, p16, cdk4/cdk6, cyclinD and Rb, are genetically altered in many kinds of tumors. Several studies have demonstrated that p16 is altered in approximately 20% of primary EFTs (Lopez-Guerrero et al., 2001; de Alava et al., 2000; Wei et al., 2000). Lasorella et al. (2000) examined a genetic interaction between Id2 and Rb during development using Id2-Rb double knockout embryos, demonstrating that loss of Id2 rescued the embryonic lethality of homozygous Rb mutation. Therefore, cell-cycle progression induced by EWS–ETS oncoproteins may be mediated in part by inactivation of Rb through overexpression of Id2.

We also found high expression of c-Myc in all of Ewing sarcoma cell lines (Figure 5b), which is consistent with a previous report (Dauphinot et al., 2001). Although EWS–ETS dependent induction of c-Myc is likely indirect (Bailly et al., 1994), overexpression of Id2 can be a result of transcriptional activation by the Myc family (Lasorella et al., 2000). Therefore, Id2 induction may be regulated not only by EWS–ETSs through its ETS-binding sites but also regulated through c-Myc overexpression. In conclusion, our results suggest that EWS–ETS fusion proteins transactivate the Id2 gene in EFT cells in a direct manner and may play an important role in the oncogenesis of EFTs.

Materials and methods

Cell lines and tissue samples

Three Ewing sarcoma cell lines (A-673, RD-ES and SK-ES1), a fibrosarcoma cell line (HT-1080), three rhabdomyosarcoma cell lines (A204, Hs729T and RD), an osteosarcoma cell line (Saos-2), a liposarcoma cell line (SW872) and a non-small cell lung carcinoma cell line (NCI-H1299) were purchased from American Type Culture Collection (USA). One Ewing sarcoma cell line (SCCH-196) was purchased from the Japanese Collection of Research Bioresources (Osaka, Japan). Two Ewing sarcoma cell lines (NCR–EW2 and W–ES) were reported previously (Urano et al., 1998). All cell lines were cultured under conditions recommended by their respective depositors. Tumor specimens were surgically resected from patients in Sapporo Medical University Hospital and Kyoto University Hospital. Biopsy or tumor resection specimens were obtained prior to chemotherapy and radiotherapy and under informed consent from the patients.

Retroviral vector construction and infection

The entire open reading frame of EWS–ERG, EWS–FLI1, ERG and FLI1 cDNA was inserted downstream of a cytomegalovirus promoter in the retroviral expression vector pLNCX (Clontech, Palo Alto, CA, USA) and named pL-EE, pL-EL, pL-E and pL-F, respectively. Another set of retroviral expression vectors using pBabe-Puro (Morgenstern and Land, 1990) instead of pLNCX were constructed and named pB-EE, pB-EF, pB-E, and pB-F, respectively. These constructs were then transfected into a virus packaging cell line (293 10A1) by a calcium phosphate precipitation method. After 48 h, high titer viral stocks were collected as conditioned media. HT-1080 cells were infected with each of the replication-deficient viral stocks and stable transfectants were selected with either geneticin (400 μg/ml) for pLNCX derivatives or puromycin (0.5 μg/ml) for pBabe-Puro derivatives. More than 100 independent transfectants were pooled and used in this study. Expression of the exogenously introduced genes was assessed by Northern blot and immunoblot analysis to confirm their appropriate expression.

cDNA microarray

For cDNA expression arrays, poly(A)+ RNA was isolated with the FastTrack 2.0 mRNA isolation system (Invitrogen, Carlsbad, CA, USA) from each retrovirus-infected HT-1080 human fibrosarcoma cells described above, and used as a template for synthesis of Cy3- or Cy5-labelled cDNA probes. The probes were hybridized to cDNA microarrays containing 23 040 human genes. Microarray construction, hybridization procedures and data analysis were described previously (Ono et al., 2000; Kihara et al., 2001).

Northern blot analysis

For Northern blot analysis, total RNA was isolated from cell lines in the exponential growth phase using TRIZOL (Invitrogen, Carlsbad, CA, USA). Ten micrograms were electrophoresed through a 1% agarose gel containing 2.2 M formaldehyde and blotted onto a nitrocellulose membrane (Schleicher & Schuell Inc., Keene, NH, USA). RNA was visualized with Vistra Green (Amersham Pharmacia Biotech, Buckinghamshire, UK) to ensure that the RNA was intact and loaded in similar amounts, and to confirm equal transfer. Hybridization was performed as described (Ishida et al., 2000). cDNA probes for Northern blot analysis of Id2 and c-Myc mRNA were amplified by RT–PCR with appropriate cDNA pools as templates and the PCR products were sequenced to verify their identity.

Immunoblot analysis

Cellular extracts were prepared in RIPA buffer without SDS. A total of 20 μg of proteins were loaded on SDS-polyacrylamide gels, size-separated by electrophoresis and then electro-transferred onto PVDF membranes (Millipore, Bedford, MA, USA). After staining with Ponceau S (Sigma, Steinheim, Germany) to ensure equal loading, the membrane was blocked with PBS (−) containing 5% BSA for 3 h at room temperature. The membrane was incubated overnight at 4°C with primary antibody then washed and incubated for 1 h with horseradish peroxydase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing the membrane, the detection procedure was performed with an enhanced ECL Plus kit (Amersham Pharmacia Biotech, Buckinghamshire, UK) according to the manufacturer's instructions. The primary antibodies used in this study are as follows: rabbit anti-human ERG-1/ERG-2 polyclonal antibody (C-20, Santa Cruz Biotechnology); rabbit anti FLI-1 polyclonal antibody (C-19, Santa Cruz Biotechnology, Santa Cruz); rabbit anti-human Id2 polyclonal antibody (C-20, Santa Cruz Biotechnology).

Antisense cDNA expression vector

Each entire open reading frame of EWS–FLI1 and ERG cDNA was inserted in the reverse orientation downstream of the cytomegalovirus promoter in pIRESneo2 (Clontech, Palo Alto, CA, USA) and named pIRES-asEF and pIRES-asE, respectively. These plasmids and empty pIRESneo2 were transfected separately into HT-pB-EF cells (HT-1080 cells expressing exogenous EWS–FLI1 gene) using TransIT-LT1 (panVera, Madison, WI, USA). Stable transfectants were selected with both geneticin (400 μg/ml) and puromycin (0.5 μg/ml). More than 100 independent clones were pooled separately, and the expression level of EWS–FLI1 protein in each of the resulting cell populations was assessed by immunoblot analysis using an antibody that recognized the C-terminus of FLI1 (C-19).

Luciferase assay

A 2790 base pair genomic fragment (−2755 to +35) containing the 5′ region of the Id2 gene was amplified by PCR with KOD plus DNA polymerase (Toyobo, Osaka, Japan). An amplified DNA fragment was inserted into the MluI–XhoI site upstream of a luciferase reporter gene in pGL3-Basic vector (Promega, Madison, WI, USA) and sequenced to verify the identity of the amplified DNA. The resulting construct (named pGL-Id2-2755) was enzymatically digested with EcoRI alone or MluI–EcoRI, and re-ligated to make two reporter constructs (named pGL-Id2-del980 and pGL-Id2-1329, respectively). Three different constructs were generated by inserting PCR-amplified fragments of the Id2 promoter into the MluI–XhoI site of pGL3-vector and named pGL-Id2-60, pGL-Id2-100 and pGL-Id2-166 (shown in Figure 3a). ERG, FLI1, EWS-ERG and EWS-FLI1 cDNAs containing the entire open reading frame were inserted separately downstream of the cytomegalovirus promoter in the expression vector pcDNA3.1(+) (Invitrogen, Carlsbad, CA, USA), and were named pcDNA-E, pcDNA-F, pcDNA-EE and pcDNA-EF, respectively.

A total of 1×106 of H1299 cells in 6 cm dishes were co-transfected separately with 1 μg each of the pGL3-Basic derivatives, together with 1 μg of pcDNA-E, pcDNA-F, pcDNA-EE, pcDNA-EF or a control pcDNA3.1(+), and 0.5 μg of a Renilla luciferase internal control reporter plasmid pRL-TK (Promega, Madison, WI, USA) using Lipofectin (Invitrogen, Carlsbad, CA, USA). Cells were harvested 48 h after transfection followed by measurement of luciferase activity using a Dual–Luciferase Reporter Assay System (Promega, Madison, WI, USA). The luciferase activity was defined as the ratio of Photinus pyralis luciferase activity from pGL3-Basic derivatives relative to Renilla reniformis luciferase activity from pRL-TK, which reflected the efficiency of transfection.

Chromatin immunoprecipitation assay

The chromatin immunoprecipitation (ChIP) assay was performed using a ChIP assay kit (Upstate Biotechnology, Lake Placid, NY, USA), as recommended by the manufacturer (Sasaki et al., 2002; Morimoto et al., 2002). Genomic DNA and protein were cross-linked by addition of formaldehyde to 2×106 cells. Cells were lysed in SDS lysis buffer with a protease inhibitor cocktail and then sonicated to generate DNA fragments of approximately 300–1000 bp. After centrifugation, the cleared supernatant was incubated with an anti-FLI1 antibody (C-19). Immune complexes were precipitated, washed, and eluted as recommended. DNA-protein cross-links were reversed by heating and DNA was purified and resuspended in 50 μl of TE buffer. Five microliters of each sample was used as a template for PCR amplification. PCR amplification of the Id2 promoter region that contains the two potential EST binding sites was performed on immunoprecipitated chromatin using the specific primers 5′-ATTGGCTGCGAACGCGGAAG-3′ (forward) and 5′-GGGCTCGGCTCAGAATGAAG-3′ (reverse).

Quantification of Id2 and c-Myc expression by TaqMan real-time RT–PCR

RT–PCR experiments were carried out using a Superscript II RT–PCR system kit according to instructions provided by the manufacturer (Invitrogen, Carlsbad, CA, USA). Total RNA (1 μg) extracted from tumor tissues was reverse transcribed to generate cDNA as recommended. To quantify expression of Id2 and c-Myc, we used TaqMan real-time PCR and a 7700 Sequence Detector (Perkin Elmer Applied Biosystems, Foster City, CA, USA). Reactions contained 2×TaqMan Universal PCR Master Mix, 300 nM of forward and reverse primers and 200 nM of the Taq-Man-probes. 18S ribosomal RNA was quantified as an internal control with Pre-Developed TaqMan Assay Reagent (Perkin Elmer Applied Biosystems). Thermal cycling proceeded with 40 cycles of 95°C for 15 s and 60°C for 1 min. Amount of input RNA were calculated with relative standard curves for both the mRNAs of interest and 18S rRNA.

Immunohistochemical- and immunocytochemical staining

We used a rabbit anti-human Id2 polyclonal antibody C-20 (Santa Cruz Biotechnology) as the primary antibody at a 1 : 200 dilution with or without a competitive C-20-blocking peptide sc-489P (Santa Cruz Biotechnology). For fixation of cells cultured on chamber slides, we incubated cells with 4% paraformaldehyde at room temperature for 15 min. Before incubation with the primary antibody, the sections were heated in Target Retrieval Solution (Dako, Glostrup, Denmark) at 105°C for 10 min. For detection of the bound antibody, we used Histofine SAB-PO kit (Nichirei, Tokyo, Japan). 3,3-diaminobenzidine was then used to visualize the Id2 immunoreactivity. Specific immunoreactivity was demonstrated by competition with Id2 antibody-specific blocking peptide sc-489P (Santa Cruz Biotechnology). Sections were counterstained with Mayer's hematoxylin.

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Acknowledgements

This work was partially supported by a grant from the Ministry of Education, Culture, Sport, Science, and Technology of Japan. Authors are grateful to Drs Joseph F Costello, Goichi Watanabe and Setsuko Ishida for valuable discussion.

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Correspondence to Takashi Tokino.

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Keywords

  • Id2
  • EWS-ETS
  • Ewing family tumor

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