MOLECULAR CARCINOGENESIS 55:1411–1423 (2016)

The TFG-TEC Oncoprotein Induces Transcriptional Activation of the Human b- Via Modification of the Promoter Region

Ah-young Kim, Bobae Lim, JeeHyun Choi, and Jungho Kim* Laboratory of Molecular and Cellular Biology, Department of Life Science, Sogang University, Seoul, Korea

Recurrent translocations are the hallmark of many human cancers. A proportion of human extraskeletal myxoid chondrosarcomas (EMCs) are associated with the characteristic chromosomal translocation t(3;9)(q11–12;q22), which results in the formation of a chimeric in which the N-terminal domain of the TRK-fused gene (TFG) is fused to the translocated in extraskeletal chondrosarcoma (TEC; also called CHN, CSMF, MINOR, NOR1, and NR4A3) gene. The oncogenic effect of this translocation may be due to the higher transactivation ability of the TFG-TEC chimeric protein; however, downstream target of TFG-TEC have not yet been identified. The results presented here, demonstrate that TFG-TEC activates the human b-enolase promoter. EMSAs, ChIP assays, and luciferase reporter assays revealed that TFG-TEC upregulates b-enolase transcription by binding to two NGFI-B response element motifs located upstream of the putative transcription start site. In addition, northern blot, quantitative real-time PCR, and Western blot analyses showed that overexpression of TFG-TEC up-regulated b-enolase mRNA and protein expression in cultured cell lines. Finally, ChIP analyses revealed that TFG-TEC controls the activity of the endogenous b-enolase promoter by promoting H3 . Overall, the results presented here indicate that TFG-TEC triggers a regulatory gene hierarchy implicated in cancer cell metabolism. This finding may aid the development of new therapeutic strategies for the treatment of human EMCs. © 2015 Wiley Periodicals, Inc. Key words: TFG-TEC; human b-enolase gene; transcription activation; chromosomal translocation; fusion protein; human extraskeletal myxoid chondrosarcoma

INTRODUCTION that binds DNA with a sequence specificity indistin- Over the last decade, chromosomal abnormalities, guishable from that of parental TEC and shows in particular recurrent chromosome translocations, enhanced transcriptional activation potential, indi- have been recognized as typical characteristics of cating that the amino terminal portion of TFG-TEC human cancers. Human extraskeletal myxoid chon- provides additional transactivation properties [9]. In drosarcomas (EMCs) are aggressive and rare soft addition, the TFG-TEC protein self-associates and this tissue tumors; although, EMCs arise primarily in the event is dependent upon its coiled–coil domain musculature, most commonly in the thigh and (amino acids 97–124), AF1 domain (amino acids knee, their histogenetic origins are unknown [1–3]. 275–562), and DNA-binding domain (DBD; amino Most human EMCs harbor characteristic transloca- acids 563–655). The mutant TFG-TEC (AAAA) protein, tions, such as t(9;22)(q22;q12) or t(9;17)(q22;q11.2), in which the nuclear localization signal (NLS; 612 615 which involve the gene encoding the translocated in KRRR ) is disrupted, and is able to inhibit TFG- extraskeletal chondrosarcoma (TEC; also called TEC-mediated transcriptional activation in vivo [10]. CHN, CSMF, MINOR, NOR1, and NR4A3) protein Although, TFG-TEC may play a critical role in the (9q22), and either the gene encoding, the Ewing’s formation of human EMCs by modulating the sarcoma protein (22q12) or the gene encoding human TATA-binding protein-associated factor II 68 (17q11.2) [1,3–6]. In addition, the t(9;15)(q22; Grant sponsor: National Research Foundation (NRF) of Korea; q21) translocation, which fuses TEC to the tran- Grant number: 2013R1A1A2009478; Grant sponsor: Bio and Medical scription factor TCF12, has also been reported in Technology Development Program; Grant number: human EMCs, although this translocation is much 2012M3A9B4028763; Grant sponsor: Priority Research Centers Program; Grant number: 2009-0093822; Grant sponsor: Ministry of less common than the t(9;22)(q22;q12) and t(9;17) Education, Science, and Technology (q22;q11.2) translocations [7]. *Correspondence to: Laboratory of Molecular and Cellular Biology, Recently, a proportion of human EMCs were found Department of Life Science, Sogang University, Seoul 121-742, Korea. to harbor a characteristic t(3;9)(q11–12;q22) translo- Received 13 February 2015; Revised 29 July 2015; Accepted 3 August 2015 cation that involves the N-terminal domain (NTD) of DOI 10.1002/mc.22384 the (TFG) at 3q11–12 and the TEC gene at 9q22 [8]. Our Published online 27 August 2015 in Wiley Online Library group demonstrated that TFG-TEC is a nuclear protein (wileyonlinelibrary.com).

ß 2015 WILEY PERIODICALS, INC. 1412 KIM ET AL. transcription of specific target gene(s) required for of regulation of and signal transduc- tumorigenesis, the precise mechanism by which TFG- tion. Enolase (also known as 2-phospho-D-glycerate TEC allows cells to transform that has not yet been hydrolase or phosphopyruvate hydratase) catalyzes the defined. reversible dehydration of 2-phosphoglycerate to phos- The NTRK1 gene (also known as MTC, TRK, TRK1, phoenolpyruvate during glycolysis [25,26]; in this TRKA, Trk-A, or p140-TrkA) encodes a membrane- second glycolytic reaction, enolase generates a com- bound receptor tyrosine kinase that belongs to the pound with high phosphoryl group transfer poten- neurotrophic tyrosine kinase receptor family. Upon tial [27]. Enolase is found in all organisms, and stimulation by growth factors and cytokines, NTRK1 vertebrates exhibit enolase , including non- activates a range of signaling pathways downstream neuronal a-enolase (also called enolase 1 or ENO1), of Ras and phosphatidylinositol 3-kinase [11]. The b-enolase (also called enolase 3 or ENO3), which is TFG gene was originally identified as a fusion partner predominantly found in muscle, and neuron-specific of the NTRK1 gene in human papillary thyroid g-enolase (also called or ENO2) [26,28]. These carcinoma [12] and is also involved in another enolase isozymes homo- or hetero-dimerize to form aa, chromosome translocation (involving the ALK ab, ag, bb,andgg dimers [26,29]. gene) in anaplastic large-cell lymphomas [13]. The Although, most human cancers harbor metabolic TFG gene is located on the long (q) arm of human re-programming characteristics, a link between TFG- , and encodes a ubiquitously expressed TEC and metabolic gene regulation in human EMCs cytoplasmic protein [12]. The complete TFG cDNA has not been described; therefore, the aim of this comprises of 1,677 bp and encodes a protein of 400 study was to characterize the biological function of amino acids that contains putative functional do- TFG-TEC in human EMCs and its contribution to mains, including a Phox and Bem1p (PB1) domain, a cancer cell metabolism. The presence of TFG-TEC coiled–coil domain, and a serine/proline/tyrosine/ binding sites in the human b-enolase promoter raised glycine/glutamine-rich region [14]. TFG interacts the possibility that TFG-TEC might regulate b-enolase with the NF-kB essential modulator and TRAF family expression in human EMCs. Experiments using member-associated NF-kB activator , suggest- human b-enolase promoter constructs showed that ing that it may play a key role in NF-kB regulation [15]. TFG-TEC transactivated the b-enolase gene in vitro, In addition, a primary structure analysis revealed and overexpression of TFG-TEC activated endoge- multiple myristoylation sites in the TFG protein, nous b-enolase gene expression in vivo. The data indicating that it may localize to the cell mem- presented here, suggest that TFG-TEC is responsible brane [14]. However, the biological role of TFG in for the activation of b-enolase expression, and the normal cells is largely unknown. subsequent triggering of a regulatory gene hierarchy TEC, which is the human homolog of the rat gene implicated in cancer cell metabolism. These findings encoding neuron-derived orphan receptor 1 [16], is may aid the development of a therapeutic strategies also known as CHN [1] or MINOR [17]. The TEC cDNA for human EMCs that act by modulating cancer cell was initially isolated from primary cultures of metabolism. apoptotic rat forebrain neurons [16] and encodes a novel orphan nuclear receptor belonging to the MATERIALS AND METHODS steroid/thyroid receptor gene superfamily [1,3]. TEC was also identified as a fusion partner of the gene Materials and General Methods encoding the Ewing’s sarcoma protein in human Restriction endonucleases, calf intestinal alkaline EMCs [3]. The TEC gene is located at 9q22 [18], spans phosphatase, the Klenow fragment of DNA polymer- approximately 40 kb, and contains eight . Exons ase I, and T4 DNA ligase were purchased from New 1 and 2 corresponds to the 50-UTR of the mature TEC England Biolabs, Ipswich, MA. PfuTurbo polymerase mRNA [18]. Although, the biological role of TEC was purchased from Stratagene, La Jolla, CA and remains unknown, previous studies have shown that [g-32P] ATP (3000 Ci/mmol) was obtained from constitutive expression of the gene induces massive PerkinElmer Life Sciences, Waltham, MA. The prepa- cell death in thymocytes [19], suggesting that TEC ration of plasmid DNA, restriction digestions, may play a role in cell proliferation by regulating its agarose gel electrophoresis, DNA ligations, bacterial downstream target genes. transformations, and SDS–PAGE were performed It has been known for decades that cancer cells have using standard methods [30]. Sub-clones generated an altered metabolism and higher rate of glycolysis than from the PCR products were sequenced using the non-malignant cells [20,21]. Inhibition of molecules chain termination method and double-stranded DNA involved in tumor cell glycolytic energy production is templates to ensure the absence of mutations. providing a framework for the discovery of new selective and targeted anti-cancer compounds [22,23]. Alteration Constructs of energy metabolism by oncogenes is also an important The pCMV-Tag2A/TFG-TEC, pCMV-Tag2A/TFG component of the development of human can- (NTD), pCMV-Tag2A/TEC, and pCMV-Tag2A/TFG- cers [20,24]; this process may involve complex patterns TEC (AAAA) expression plasmids have been described

Molecular Carcinogenesis HUMAN b-ENOLASE INDUCTION BY TFG-TEC 1413 previously [9,31]. To generate the pEnob-Luc reporter underlined), and the pEnob-Luc (BS2m) construct, in plasmid, the human b-enolase promoter (nucleotides which the BS2 sequence was mutated from 50- 787 to þ79 relative to the start of transcription; TGACCTTT-30 to 50-TGACCTCT-30 (a mutation site Figure 1) was amplified from human genomic DNA by underlined), the human b-enolase promoter (787 to PCR using the following primers: hEnob-787(s), 50- þ79) was amplified as described above and cloned GATCGGTACCACAAAAGGCCACCCGTCC-30 (KpnI into the T-Blunt vector using the T-BluntTM PCR site underlined) and hEnobþ79(as), 50-GATCCTC- Cloning Kit (SolGent Co., Ltd., Seoul, Korea) to yield GAGCTGGAAGATTCACCCGAC-30 (XhoI site under- T-Blunt/Enob (789/þ79). Site-directed mutagenesis lined). The amplified promoter fragment was cloned was then performed using the method reported by into the KpnI-XhoI sites of the promoterless pGL3- Lim et al. [9], with some modifications, and the Basic vector (Promega, Madison, WI) to generate following mutagenic primers: 50-mBS1, 50-TG- pEnob-Luc. GCCTTTCCTAGAGGTCACTCATTCCTCT-30 (a muta- To generate the pEnob-Luc (BS1m) construct, in tion site underlined) and 30-mBS1, 50-AGAGGAATGA which the BS1 sequence was mutated from 50- GTGACCTCTAGGAAAGGCCA-30 (a mutation site AAAGGTCA-30 to 50-AGAGGTCA-30 (a mutation site underlined) or 50-mBS2, 50-GGGATAGTAGTTGACCT

Figure 1. In silico analysis of the human b-enolase promoter. The 50 TRK-fused gene-translocated in extraskeletal chondrosarcoma (TFG- flanking region of the human b-enolase gene (nucleotides 771 to TEC) recognition elements are shown in bold and labeled as TFG-TEC þ79, relative to the start of transcription) was analyzed using the BS1 (329 to 322) and TFG-TEC BS2 (176 to 169). The positions TRANSFAC program. Putative regulatory elements and transcription of the forward and reverse primers used in the ChIP experiment (ChIP-F factor binding sites are underlined and the names of the corresponding and ChIP-R, respectively) are indicated by dashed lines. The transcrip- transcription factors are shown below the sequence. The two putative tion start site (þ1) is denoted by an arrow.

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CTGGTAAGGGGGC-30 (a mutation site underlined) supplemented with 10% heat-inactivated bovine and 30-mBS2, 50-GCCCCCTTACCAGAGGTCAAC- serum (Gibco, Grand Island, NY), penicillin (Gibco), TACTATCCC-30 (a mutation site underlined). The and streptomycin (Gibco). mutated T-Blunt/Enob (789/þ79) plasmid was digested with KpnI and XhoI, and the mutated b- ChIP Assays enolase promoter fragment was cloned into the same ChIP assays were performed according to the manu- site of the promoterless pGL3-Basic vector (Promega) facturer’s protocol (#17-295, Upstate Biotechnology, to generate pEnob-Luc (BS1m) or pEnob-Luc (BS2m). Lake Placid, NY). Briefly, transfected cells were fixed To generate pEnob-Luc (BS1m BS2m), in which both with 1% formaldehyde and genomic DNA was sheared. the BS1 and BS2 sequences were mutated, as described Polyclonal rabbit, anti-enhanced GFP (EGFP), antibodies above, the T-Blunt/Enob (789/þ79) plasmid under- (A11122, Life Technologies, Carlsband, CA), normal went sequential site-directed mutagenesis reactions rabbit IgG (sc-2027, Santa Cruz Biotechnology, Inc., using the 50-mBS1 and 30-mBS1 primers, followed by Santa Cruz, CA) or polyclonal anti-acetyl H3 antibodies the 50-mBS2 and 30-mBS2 primers. The mutated (06-588, Millipore, Billerica, MA) were added to the plasmid was then digested with KpnI and XhoI, and sheared chromatin. To detect precipitated genomic DNA, the mutated b-enolase promoter fragment was cloned PCR analyses were performed using specific primers into the same sites of the promoterless pGL3-Basic (50Enob-ChIP, 50-GGCCTTTCCTAAAGGTCAC-30;and vector to generate pEnob-Luc (BS1m BS2m). 30Enob-ChIP, 50-CTCAGGCTGCGCATTTATC-30)and the resulting PCR products were subjected to agarose EMSAs gel electrophoresis. The human b-enolase proximal promoter regions used for EMSAs (Probes I–IV) were amplified by PCR Dual-Luciferase Reporter Gene Assays from human genomic DNA isolated from HEK293T Cells were transiently transfected with plasmids using cells using the following primers: Probe I: Probe the VivaMagicTM Transfection Reagent (Vivagen) and 1921 (s), 50-AAGAAGAGTTTGGCCAAA-30 and luciferase assays were performed using the Dual-lucifer- Probe 1422 (as), 50-AGGCTCCGGAAGCCGCCC-30; ase Assay System (Promega). Renilla luciferase activity Probe II: Probe 1421 (s), 50-GGGCTAGGGG- was used to normalize the transfection efficiency. CAAGGGGT-30 and Probe 922 (as), 50-GCCATAC- CAGCCGACCGG-30; Probe III: Probe 921 (s), Northern Blotting 50-TGCAGAGGAGCTCGGGGC-30 and Probe 422 Total RNAs were prepared using TRIzol reagent (as), 50-CTGGGCGGGCCTCTGTCC-30; and Probe (Invitrogen) and aliquots (10 mg/lane) were separated IV: Probe 421 (s), 50-CGATCTGAGCATGTGTGG-30 on 6% formaldehyde-1.5% agarose gels. The RNA was and Probe þ79 (as), 50-CTGGAAGATTCACCCGAC-30. transferred to Hybond nylon membranes (Amer- The promoter fragments were cloned into the T-Blunt sham) and cross-linked to the membrane using a GS vector using the T-BluntTM PCR Cloning Kit to Gene Linker UV Chamber (Bio-Rad, Hercules, CA). generate T-Blunt/Probe I, T-Blunt/Probe II, T-Blunt/ The b-enolase probe (nucleotides þ871 to þ1152, Probe III, and T-Blunt/Probe IV. These plasmids were relative to the start of transcription) was amplified digested with EcoRI and treated with alkaline phos- from human b-enolase cDNA by PCR using the phatase to remove the 50-phosphate groups. The hEnobþ871(s) (50-GAAGAAGGCCTGCAACTGTC-30) synthetic oligonucleotide probes (0.5 ng each) were and hEnobþ1152(as) (50-ACTTGCGTCCAGCAAA- prepared by end labeling with [g-32P] ATP using T4 GATT-30) primers, and then cloned into the polynucleotide kinase. GST alone and the GST-fused pCRTM2.1-TOPO vector (Invitrogen, Carlsband, CA) DBD of TFG-TEC were overexpressed and purified to yield pCRTM2.1-TOPO/Enob (þ871/þ1152). EcoRI from Escherichia coli [10]. DNA-binding reactions digested DNA fragments of pCRTM2.1-TOPO/Enob containing radiolabeled probe and recombinant (þ871/þ1152) were dephosphorylated with alkaline GST or GST-DBD were performed at 48C for 30 min phosphatase, gel purified, and labeled with 32P using in binding buffer containing 10 mM Tris–HCl (pH the Prime-It II Random Primer Labeling Kit (Stra- 8.0), 40 mM KCl, 6% glycerol, 1 mM DTT, 0.05% NP- tagene). Hybridizations were performed overnight at 40, and 10 ng/ml poly(dI dC) (dI dC). The mixtures 688C using radiolabeled b-enolase probe in Expres- were then separated on 4% polyacrylamide gels sHyb Solution (Clontech, Palo Alto, CA). The blots (acrylamide/bisacrylamide ratio, 37:1) in 0.5 TBE were washed twice at 688C with 2 SSC/0.1% SDS (44.5 mM Tris–HCl, 44.5 mM boric acid, and 1 mM and the radiolabeled bands were visualized by EDTA) for 2–3 h at 48C and 150 V. The gels were dried autoradiography. and exposed to Kodak X-Omat film at 708C with an intensifying screen. Reverse Transcription (RT) and Quantitative Real-Time PCR EGFP-positive HEK293T and C28/I2 cells were Cell Culture isolated by FACS. Total RNAs were extracted from HEK293T cells and human C28/I2 juvenile costal the FACS-isolated populations using TRIzol reagent, chondrocyte cells were maintained in DMEM according to the manufacturer’s protocol. The RNAs

Molecular Carcinogenesis HUMAN b-ENOLASE INDUCTION BY TFG-TEC 1415 were reverse-transcribed with an oligo (dT) primer the human b-enolase promoter. Four probes that using the SuperScript First-strand Synthesis System spanned different regions of the human b-enolase (Life Technologies). Quantitative real-time PCR was promoter, namely Probe I (nucleotides 1921 to performed using the Mx3000P QPCR System (Agilent 1422), Probe II (nucleotides 1421 to 922), Probe Technologies, Palo Alto, CA) and SYBR Green Master III (nucleotides 921 to 422), and Probe IV Mix (EnzynomicS, Daejeon, Korea), according to the (nucleotides 421 to þ79; Figure 2A), were generat- manufacturer’s instructions. As an internal control, ed by PCR amplification, TA vector cloning, and the level of the mRNA encoding hypoxanthine- restriction enzyme digestion of the human b-enolase guanine phosphoribosyl transferase (HPRT) was deter- promoter. Probe IV contained the BS1 and BS2 mined by real-time PCR and used to correct for elements. Each probe was incubated with or without experimental variation. The following primers were recombinant GST, or the GST-fused DBD of TFG-TEC used: b-enolase:50-GAAGAAGGCCTGCAACTGT-30 (GST-DBD). To confirm that similar amounts of and 50-ACTTGCGTCCA GCAAAGATT-30;andHPRT: protein were used in each experiment, aliquots of 50-GGACCCCACGAAGTGTTGG-30 and 50- the proteins were fractionated by 15% SDS–PAGE CTGGCGATGTCAATAGGACTCCAG-30. The relative and visualized by Coomassie Blue staining expression levels of the downstream target genes were (Figure 2B). Protein-DNA complexes were observed quantified by normalizing to that of endogenous HPRT in the reaction containing GST-DBD and Probe IV using the DCT method [32]. (Figure 2C, lanes 19 and 20), but not in any of the other reactions. The appearance of two complexes Western Blotting (binary and ternary) in the reaction, containing Cell extracts were resolved by SDS–PAGE (8–15%), Probe IV and GST-DBD, suggested the existence of at transferred to a PVDF membrane, and immunoblot- least two TFG-TEC recognition elements within the ted with an anti-Flag (M2, Sigma–Aldrich, St. Louis, human b-enolase promoter. However, because TFG- MO), anti-EGFP (Invitrogen), anti-b-enolase (79–755, TEC can self-associate [10], we cannot exclude the ProSci, Inc., Poway, CA), or anti-GAPDH (V-18, Santa possibility that two self-associated TFG-TEC proteins Cruz Biotechnology) antibody. Reactive bands were are bound to a recognition sequence within the detected by chemiluminescence using Western Light- human b-enolase promoter. ning reagent (PerkinElmer Life Sciences). To confirm the in vitro EMSA findings, and determine whether TFG-TEC is capable of binding RESULTS to the BS1 and BS2 elements in the human b-enolase promoter in vivo, ChIP assays were performed using b The Human -Enolase Proximal Promoter Contains Two nuclear extracts of HEK293T cell lines, that were Putative TFG-TEC cis-Elements transfected with a recombinant vector expressing Cancer cells are characterized by a re-programming of EGFP alone or EGFP-tagged TFG-TEC (EGFP-TFG- energy metabolism process [33]. To understand, the TEC). The immunoprecipitated DNA samples were mechanism of regulation of cancer cell metabolism by amplified by PCR using a set of primers (ChIP-F and TFG-TEC, and to identify targets of this protein that play ChIP-R), that flanked the BS1 and BS2 elements in a critical role in the pathogenesis of human EMCs, we the human b-enolase promoter (Figure 1). As shown performedaninsilicoanalysisoftheupstreamgenomic in Figure 2D, the proximal promoter region of the sequencesofpotentialtargetgenesusingtheTRANS- human b-enolase gene was precipitated with EGFP- FAC database (http://www.gene-regulation.com). TFG-TEC in vivo; however, this amplified product In our previous study, we demonstrated that TFG- was not detected in extracts of HEK293T cells TEC can bind to the NGFI-B response element (NBRE) expressing EGFP alone. Taken together, these results motif (50-AAAGGTCA-30) [10]. NBREs at positions suggest that TFG-TEC binds to the proximal pro- 329 to 322 (sense orientation, 50-AAAGGTCA-30) moter of the human b-enolase gene both in vivo and and 176 to 169 (antisense orientation, 50- in vitro. TGACCTTT-30), relative to the predicted start of To confirm the specificity of binding of TFG-TEC to transcription, were identified in the immediate 50 the human b-enolase promoter, a series of EMSAs were flanking region of the human b-enolase gene (Figure 1); performed with recombinant GST-DBD and radio- these motifs were named BS1 and BS2, respectively. In labeled synthetic oligonucleotides containing the BS1 addition, several putative transcription factor binding or BS2 motif (Figure 3A). A single protein–DNA sites, including those for Pdx-1, GATA-1, HSF, v-Myb, complex was obtained in reactions containing oligo- and SP1, were identified in the region upstream of the nucleotides harboring the WT BS1 or BS2 sequence human b-enolase gene (Figure 1). (Figures 3B and C, lane 2). Production of this complex was abrogated, when an adenine residue (underlined) b 0 0 TFG-TEC Binds Specifically to the Human -Enolase in BS1 (5 -AAAGGCT-3 ) or a thymine residue (under- Proximal Promoter lined) in BS2 (50-TGACCTTT-30) was mutated to EMSAs were used to determine, whether TFG-TEC guanine or cytosine, respectively (Figures 3B and C, is capable of binding to the BS1 and BS2 elements in lane 4).

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Figure 2. Binding of TRK-fused gene-translocated in extraskeletal radiolabeled probes shown in (A) and the recombinant GST-fusion chondrosarcoma (TFG-TEC) to the human b-enolase promoter. (A) proteins shown in (B). Control reactions (C) contained probe but no A schematic illustration of the human b-enolase promoter probes protein. Lanes 1, 6, 11, and 16: no protein; lanes 2, 7, 12, and 17: used in the EMSA shown in (C). The putative TFG-TEC binding sites 200 mg of GST; lanes 3, 8, 13, and 18: 400 mg of GST; lanes 4, 9, (BS1 and BS2) in the b-enolase promoter are indicated. The 14, and 19: 200 mg of GST-DBD; and lanes 5, 10, 15, and 20: positions are numbered according to the predicted transcription 400 mg of GST-DBD. The positions of the free probe and protein- start site reported previously [48]. (B) Quantification of the GST- DNA complexes are indicated. (D) ChIP analyses of the binding of fusion proteins used in the EMSA shown in (C). One microgram of enhanced GFP (EGFP)-tagged TFG-TEC to the human b-enolase each GST-fusion protein was fractionated by 15% SDS–PAGE and promoter in vivo. The ChIP assays were performed using normal visualized by Coomassie Blue staining. Lane 1, GST alone; lane 2, IgG and an anti-EGFP antibody. The precipitates were examined by GST-fused DNA-binding domain of TFG-TEC (GST-DBD). (C) An PCR using primers that flanked the region of the human promoter EMSA showing the ability of TFG-TEC to bind to the human containing the BS1 and BS2 elements (see Figure 1). The results are b-enolase promoter in vitro. The EMSA was performed with the representative of four independent experiments.

Next, competition EMSAs were performed using the BS1 and BS2 NBREs in the human b-enolase GST-DBD, radiolabeled oligonucleotides containing promoter. BS1 or BS2, and unlabeled oligonucleotides containing an 8 bp consensus NBRE motif (50- TFG-TEC Induces Transcriptional Activation of the Human 0 b GAGTTTTAAAAGGTCATGCTAATTTGG-3 , the NBRE -Enolase Promoter sequence is underlined) or a mutant version of the To determine whether the BS1 and BS2 motifs in motif in which one of the adenine residues was the human b-enolase promoter mediate a transcrip- converted to guanine (50-GAGTTTTAAgAGGTCATGC- tional response to TFG-TEC, HEK293T cells were TAATTTGG-30, the substitution is in small letter). co-transfected with an expression vector harboring Incubation with a 5- or 10-fold excess of the WT Flag-tagged TFG-TEC (pCMV-Tag2A/TFG-TEC) and a unlabeled competitor probe abolished the ability of reporter plasmid (pEnob-Luc) containing the GST-DBD to bind to the BS1 and BS2 motifs human b-enolase proximal promoter region (787 (Figures 3B and C, lanes 5 and 6), but incubation to þ79, relative to the start of transcription) with the unlabeled mutant probe did not upstream of a luciferase gene (Figure 4A). Whereas (Figures 3B and C, lanes 7 and 8). Overall, these co-transfection of cells with pEnob-Luc and empty results suggest a specific interaction of TFG-TEC with expression vector (pCMV-Tag2A) resulted in a low-

Molecular Carcinogenesis HUMAN b-ENOLASE INDUCTION BY TFG-TEC 1417

Figure 3. The specificity of binding of TRK-fused gene-translocated BS2 (C) motifs. EMSAs were performed with the recombinant GST- in extraskeletal chondrosarcoma (TFG-TEC) to the human b-enolase fused DNA-binding domain of TFG-TEC (GST-DBD) and the radio- promoter. (A) The nucleotide sequences of the oligonucleotides used labeled oligonucleotides shown in (A). Competition experiments were for EMSAs. The two putative TFG-TEC binding sites corresponding to performed with a 5- (lane 5) or 10-fold (lane 6) excess of unlabeled WT BS1 (50-AAAGGTCA-30) and BS2 (50-TGACCTTT-30) in the human oligonucleotide, or a 5- (lane 7) or 10-fold (lane 8) excess of unlabeled b-enolase promoter are shown as bold and underlined characters. The mutant oligonucleotide. The positions of the free probe and protein– positions of the mutations in the mutant probes are indicated by DNA complexes are indicated. The results are representative of four lowercase letters. (B, C) Specific binding of TFG-TEC to the BS1 (B) and independent experiments.

level production of luciferase (Figure 4B, upper downstream of the human b-enolase promoter was panel, lane 1), overexpression of Flag-TFG-TEC not activated significantly by deletion of the TEC increased the luciferase activity by up to sevenfold (Flag-TFG [NTD]) or TFG (Flag-TEC) region of TFG- in a dose-dependent manner (Figure 4B, upper TEC, and by mutation of the NLS sequence in the panel, lanes 2–6). Western blot analyses of extracts TEC region (Flag-TFG-TEC [AAAA]: Figure 4C, of the transfected cells confirmed that the TFG-TEC upper panel). These results suggest that both the protein level was higher in the cells transfected with TFG and TEC regions of the TFG-TEC, as well as its pCMV-Tag2A/TFG-TEC than those transfected with NLS, are important for the ability of the protein to pCMV-Tag2A (Figure 4B, lower panel). transactivate the human b-enolase gene. Next, to define the functional regions of TFG-TEC Finally, to confirm that the observed transactiva- required for transcriptional activation of the tion of the human b-enolase promoter by TFG-TEC human b-enolase gene, HEK293T cells were tran- was mediated through the BS1 and BS2 motifs, siently transfected with pEnob-Luc and pCMV- pEnob-Luc constructs containing mutations in one Tag2A vectors harboring Flag-tagged TFG-TEC or both of these sites were generated (Figure 5A, left deletion mutants (Figure 4A). Expression of the panel). HEK293T cells were co-transfected with these exogenous TFG-TEC derivatives was confirmed by reporter constructs and pCMV-Tag2A/TFG-TEC, and Western blotting (Figure 4C, lower panel). Trans- dual-luciferase assays were performed. Mutation of activation of the luciferase gene located BS1 or BS2 reduced the TFG-TEC-induced

Molecular Carcinogenesis 1418 KIM ET AL.

Figure 4. Transactivation of the human b-enolase promoter by the analyses of extracts of the cells used for the luciferase assays (8% gel for TRK-fused gene-translocated in extraskeletal chondrosarcoma (TFG- the detection of Flag-TFG-TEC and 12% gel for the detection of TEC) oncoprotein. (A) Schematic illustrations of the reporter and enhanced GFP [EGFP]). The middle blot shows an overexposed (OE) blot expression plasmids used in this study. The pEnob-Luc reporter plasmid used to detect low levels of TFG-TEC protein. Similar results were contained the luciferase gene downstream of the human b-enolase obtained in four independent experiments. The expression level of promoter (nucleotides 787 to þ79, relative to the start of EGFP was used as an internal control to monitor transfection efficiency. transcription). The expression vectors harboring Flag-tagged TFG- (C) Identification of the regions of TFG-TEC required for transcriptional TEC, TFG (N-terminal domain; NTD), TEC, or TFG-TEC (AAAA) are also activation of the human b-enolase promoter. HEK293T cells were co- shown. The positions of the amino acids are indicated below each transfected with 5 ng of a plasmid expressing Renilla luciferase, 100 ng construct. In the TFG-TEC (AAAA) construct, the nuclear localization of pEnob-Luc, and 200 ng of vector only (pCMV-Tag2A) or the signal in TEC was mutated from 612KRRR615 to 612AAAA615. (B) expression plasmids harboring deletion mutants of Flag-tagged TFG- Transcriptional activation of the human b-enolase promoter by TFG- TEC shown in (A). The data are represented as the mean plus standard TEC. HEK293T cells were co-transfected with 100 ng of pEnob-Luc and error of four experiments. Statistical significance was determined using 0, 25, 50, 100, 200, or 400 ng of the Flag-TFG-TEC expression plasmid. unpaired Student's t tests. P < 0.01 versus vector control and The total amount of DNA transfected into the cells was adjusted by the P < 0.05 versus Flag-TFG-TEC. The lower panel shows Western blot addition of the indicated amounts of empty expression vector (pCMV- (WB) analyses of extracts of the cells used for the luciferase assays (12% Tag2A). Data are represented as the mean plus standard error of gel for the detection of Flag-TFG-TEC and its derivatives, and 15% gel representative duplicate experiments. Statistical significance was for the detection of EGFP). Similar results were obtained in four determined using unpaired Student's t tests. P < 0.01 and P independent experiments. The expression level of EGFP was used as an < 0.001 versus vector only. The lower panel shows Western blot (WB) internal control.

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Figure 5. Mutational analysis of the human b-enolase promoter. (A) plasmid expressing Renilla luciferase. Data are represented as the mean The effects of mutation of the BS1 and BS2 motifs in the human plus standard error of representative duplicate experiments. Similar b-enolase promoter on its transactivation by TRK-fused gene-trans- results were obtained in four independent experiments. Statistical located in extraskeletal chondrosarcoma (TFG-TEC). Site-directed significance was determined using unpaired Student's t tests. #P < 0.01 mutagenesis was used to convert BS1 (50-AAAGGTCA-30) to BS1m compared with vector control; P < 0.05 and P < 0.01 versus WT (50-AgAGGTCA-30) and BS2 (50-TGACCTTT-30) to BS2m (50-TGACCTcT- pEnob-Luc. (B) Western blot analyses of TFG-TEC levels in extracts of 30). The right panel shows the results of luciferase assays of HEK293T the cells used in (A) (8% gel for the detection of Flag-tagged TFG-TEC cells that were co-transfected with 100 ng of the indicated WT or and 12% gel for the detection of EGFP). Similar results were obtained in mutant pEnob-Luc reporter plasmid, 200 ng of vector only (pCMV- four independent experiments. The expression level of enhanced GFP Tag2A) or the pCMV-Tag2A/TFG-TEC expression vector, and 5 ng of a (EGFP) was used as an internal control. production of luciferase by approximately 30–40% Northern blot analyses revealed that the expression (Figure 5A, right panel). In addition, dual mutation level of the human b-enolase mRNA was significantly of BS1 and BS2 reduced the luciferase activity by higher in HEK293T cells, expressing EGFP-TFG-TEC approximately 85% (Figure 5A, right panel). A than those expressing EGFP (Figure 6A). These results Western blot analysis showed that the differences in were confirmed by quantitative real-time PCR analy- the transactivation potentials of the reporter genes ses, which showed that the human b-enolase mRNA fused to the human b-enolase promoters were not level was fourfold higher in HEK293T cells expressing due to differences in the amounts of TFG-TEC EGFP-TFG-TEC than those expressing EGFP protein (Figure 5B). (Figure 6B). Similar results were also obtained, when the experiments were performed in the C28/I2 TFG-TEC Induces Transcriptional Activation and Epigenetic chondrocyte cell line (Figures 6D and E). We also Modification of the Endogenous Human b-Enolase defined the functional regions of TFG-TEC required Promoter for transcriptional activation of the endogenous b- To determine whether TFG-TEC modulates expres- enolase gene. To determine, whether the TFG-TEC sion of the endogenous human b-enolase gene, deletion mutants influence the induction of the HEK293T cells were transfected with expression endogenous human b-enolase gene, expression vec- vectors harboring EGFP or EGFP-TFG-TEC, and tors harboring TFG (NTD), TEC, and TFG-TEC (AAAA) EGFP-positive cells were isolated by FACS 48 h later. derivatives were transfected into HEK293T cells

Molecular Carcinogenesis 1420 KIM ET AL.

Figure 6. Activation of endogenous b-enolase gene expression by transfection was performed at least four times. The data are TRK-fused gene-translocated in extraskeletal chondrosarcoma (TFG- represented as the mean plus standard error, relative to the expression TEC). (A, B, D, E) Northern blot (A, D) and quantitative real-time PCR (B, level of b-enolase in cells expressing EGFP only. Statistical significance E) analyses of human b-enolase mRNA levels in HEK293T cells (A, B) was determined using an unpaired Student's t test. P < 0.05 versus and human C28/I2 chondrocytes (D, E) that were transfected with control cells expressing EGFP. (C, F) Western blot analyses of human expression vectors harboring enhanced GFP (EGFP) or EGFP-TFG-TEC. b-enolase protein levels in extracts of HEK293T (C) and C28/I2 (F) cells EGFP-positive transfected cells were isolated by FACS. (A, D) Ethidium that were transfected with expression vectors harboring EGFP or EGFP- bromide (EtBr) staining of the agarose gels used for northern blotting TFG-TEC (12% gel for the detection of b-enolase and 15% gel for the showed that equal amounts of total RNA were loaded into each lane. detection of GAPDH). The positions of the pre-stained molecular-mass (B, E) The expression level of the mRNA encoding hypoxanthine- markers are indicated. Similar results were obtained in three guanine phosphoribosyl transferase (HPRT) mRNA was used as a independent experiments. The expression level of GAPDH was used control to normalize the quantitative real-time PCR results. Each as a loading control.

(Supplemental Figure S1A), and EGFP-positive cells with the expression plasmid harboring EGFP-TFG- were isolated by FACS 48 h later. Unlike full-length TEC. Together with the results of EMSAs and lucifer- TFG-TEC, quantitative real-time PCR analyses showed ase assays, these results demonstrate that TFG-TEC that neither the mutant with deletion of the TEC activates the human b-enolase promoter and drives region (EGFP-TFG [NTD]) nor the NLS sequence expression of the gene and protein in vivo. mutant (EGFP-TFG-TEC [AAAA]) activated endoge- We analyzed the histone H3 acetylation status of nous human b-enolase gene expression (Supplemental the b-enolase proximal promoter region in HEK293T- Figure S1B). Interestingly, the mutant with deletion of cells and C28/I2 cells expressing EGFP or EGFP-TFG- the TFG region of TFG-TEC (EGFP-TEC) had a reduced TEC. In this experiment, chromatin was cross-linked transactivation ability in comparison to full-length and then immunoprecipitated with an anti-acetyl TFG-TEC in vivo (Supplemental Figure S1B). Similar H3 antibody. Semi-quantitative RT-PCR analyses of results were obtained in human C28/I2 chondrocytes the immunoprecipitates using primers specific for (Supplemental Figure S1C). These results suggest, that the human b-enolase promoter revealed that the both the TFG and TEC regions of TFG-TEC, as well as b-enolase promoter was hyperacetylated in HEK293T its NLS, are important for the ability of this protein to cells and C28/I2 cells expressing EGFP-TFG-TEC, but transactivate the human b-enolase gene in vivo. not those expressing EGFP alone (Figures 7A and B), Western blot analyses revealed a strong induction of indicating that TFG-TEC may be involved in human b-enolase protein expression after transfection chromatin modification of the human b-enolase of HEK293T (Figure 6C) and C28/I2 (Figure 6F) cells promoter region.

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b-Enolase is responsible for catalysis of the conver- sion of 2-phosphoglycerate to phosphoenolpyruvate during glycolysis. Pyruvate kinase then transfers the high-energy phosphoryl group of phosphoenolpyr- uvate to ADP, resulting in the formation of ATP and pyruvate. Since, the discovery that the metabolism of tumor cells is altered, much effort has been devoted to deciphere the biological meaning of metabolic re-programming. The metabolic changes that occur in cancer cells are characterized by several features, including aerobic glycolysis (also known as the Warburg effect), and enhanced activities of glycolytic [33–35]. Notably, recent evidence has suggested that some oncogenes and tumor suppressor genes are involved in this metabolic re-programming process [23,24,36]; hence, it can be assumed that human EMCs process altered metabolic profiles to fulfill their high demands for ATP. Although, the results presented here demonstrates that, TFG-TEC modulates b-enolase expression (Figure 6), whether or not this oncogene links the induction of key programs involved in metabolic re-programming of human EMCs remains to be established. Figure 7. The association between TRK-fused gene-translocated in extraskeletal chondrosarcoma (TFG-TEC) expression and histone H3 Histone acetylation and deacetylation are associat- acetylation at the human b-enolase promoter region. (A, B) Quantita- ed with increase and decrease in transcriptional tive ChIP analyses of the binding of acetyl-histone to the human b-enolase promoter in HEK293T (A) and C28/I2 (B) cells. The cross- activation, respectively; hence, histone modification linked chromatin was immunoprecipitated (IP) with an anti-enhanced is considered a major mechanism in the regulation of GFP (EGFP) or anti-acetyl H3 antibodies (polyclonal, 06-599, Millipore), many genetic functions [37,38]. As TFG-TEC induced and then analyzed by semi-quantitative RT-PCR using primers specific b for the human b-enolase promoter. Each transfection was performed transcriptional activation of the human -enolase at least three times and similar results were obtained. gene, in vitro and in vivo, we hypothesized that TFG- TEC may increase the acetylation of histone H3 in this genetic region. In agreement with this hypothesis, ChIP analyses revealed that binding of TFG-TEC to DISCUSSION the endogenous b-enolase promoter, in vivo resulted Because TFG-TEC is a nuclear protein that binds in a concomitant increase in associated histone H3 DNA with a sequence specificity indistinguishable acetylation. This result suggests that, TFG-TEC acti- from that of parental TEC and shows greatly increased vates b-enolase transcription by altering the structure transcriptional activation potential [9], it is thought of the chromatin at the b-enolase promoter region and to play a critical role in the formation of human EMCs increase the level of acetylated histone H3. Histone by modulating the transcription of specific TEC target acetylation is a gene regulatory process, in which the genes required for tumorigenesis; however, little is lysine residues in the N-terminal tail protruding from currently known about the downstream targets of the histone core of the nucleosome are acetylat- TFG-TEC and their role in the development of EMCs. ed [39,40]; this process can attract specific proteins to Here, we demonstrate a direct regulatory link between elongated chromatin that has been marked by acetyl the chimeric oncogene TFG-TEC and the metabolic groups [41–43]. Further investigation is required to enzyme b-enolase. The results presented here, suggest determine whether TFG-TEC-induced histone acety- that the TFG-TEC oncoprotein binds to the proximal lation at the human b-enolase promoter region promoter region of the human b-enolase gene in vitro provides a platform for the binding of additional and in vivo. Competition assays using WT and proteins. Future studies will focus on understanding mutant probes revealed that this interaction is both the mechanism by which, TFG-TEC alters the direct and specific. In addition, we provide evidence chromatin accessibility of its downstream target that TFG-TEC activates the human b-enolase promoter genes by histone acetylation, and whether this in vivo and in vitro, and upregulates b-enolase mRNA process allows other proteins to associate with the and protein expression. We also demonstrate, that exposed sites to activate downstream target gene TFG-TEC is involved in chromatin modification of the transcription. human b-enolase gene. Overall, the results presented In a previous study, we showed that TFG-TEC can here suggest that TFG-TEC may regulate one or more self-associate in vivo. The responsible domains map to genes that are involved in glucose metabolism, and the coiled–coil domain, AF1, and DBD, which can may enhance the metabolic capacity in human EMCs. self-associate independently with TFG-TEC [10]. We

Molecular Carcinogenesis 1422 KIM ET AL. also reported, that the KRRR sequence within the DBD National Research Foundation (NRF) of Korea (grant targets the TFG-TEC oncoprotein to the nucleus [9]. number 2013R1A1A2009478), the Bio and Medical Interestingly, a TFG-TEC nuclear localization mutant Technology Development Program (grant number forms heterodimeric complexes with wild-type TFG- 2012M3A9B4028763), and the Priority Research TEC and suppresses its transactivation activity [10]. Centers Program (grant number 2009-0093822) Because the DBD overlaps with the self-association through the National Research Foundation of Korea domain in the TFG-TEC fusion protein, it could be funded by the Ministry of Education, Science, and interesting to determine whether DNA binding is Technology. dependent on self-association in human b-enolase gene expression. However, the functional significance REFERENCES of TFG-TEC self-association remains undefined, al- 1. Clark J, Benjamin H, Gill S, et al. 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