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The TFG-TEC Oncoprotein Induces Transcriptional Activation of the Human B-Enolase Gene Via Chromatin Modification of the Promoter Region

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The TFG-TEC Oncoprotein Induces Transcriptional Activation of the Human B-Enolase Gene Via Chromatin Modification of the Promoter Region MOLECULAR CARCINOGENESIS 55:1411–1423 (2016) The TFG-TEC Oncoprotein Induces Transcriptional Activation of the Human b-Enolase Gene Via Chromatin 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 chromosome 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 protein 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 genes 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 histone H3 acetylation. 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 gene expression 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 isozymes, 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 enolase 2 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- chromosome 3, 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 proteins, 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. 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 enzyme 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
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