The TFG-TEC fusion gene created by the t(3;9) translocation in human extraskeletal myxoid chondrosarcomas encodes a more potent transcriptional activator than TEC

Abstract

The t(3;9)(q11-q12;q22) translocation associated with human extraskeletal myxoid chondrosarcomas results in a chimeric molecule in which the N-terminal domain (NTD) of the TFG (TRK-fused gene) is fused to the TEC (Translocated in Extraskeletal Chondrosarcoma) gene. Little is known about the biological function of TFG-TEC. Because the NTDs of TFG-TEC and TEC are structurally different, and the TFG itself is a cytoplasmic protein, the functional consequences of this fusion in extraskeletal myxoid chondrosarcomas were examined. The results showed that the chimeric gene encoded a nuclear protein that bound DNA with the same sequence specificity as the parental TEC protein. Comparison of the transactivation properties of TFG-TEC and TEC indicated that the former has higher transactivation activity for a known target reporter containing TEC-binding sites. Additional reporter assays for TFG (NTD) showed that the TGF (NTD) of TFG-TEC induced a 12-fold increase in the activation of luciferase from a reporter plasmid containing GAL4 binding sites when fused to the DNA-binding domain of GAL4, indicating that the TFG (NTD) of the TFG-TEC protein has intrinsic transcriptional activation properties. Finally, deletion analysis of the functional domains of TFG (NTD) indicated that the PB1 (Phox and Bem1p) and SPYGQ-rich region of TFG (NTD) were capable of activating transcription and that full integrity of TFG (NTD) was necessary for full transactivation. These results suggest that the oncogenic effect of the t(3;9) translocation may be due to the TFG-TEC chimeric protein and that fusion of the TFG (NTD) to the TEC protein produces a gain-of-function chimeric product.

Molecular strategies for detecting chromosomal translocations in soft tissue tumors (Review)

“…Gene TCF12, also known as HTF4, presents different isoforms by alternative splicing and the breakpoint affects the region of intron 5 (158,159). Further molecular analyses have revealed additional chromosomal aberrations that can aid in the diagnosis of EMC, identifying other chimeric variants, such as the fused trascript TFG (TRK-fused gene)/CHN associated with t(3;9)(q11-q12;q22) (156,160,161). …”

A TFG–TEC nuclear localization mutant forms complexes with the wild-type TFG–TEC oncoprotein and suppresses its activity

“…To generate pEF-BOS/GST-TFG-TEC, pCMV-Tag2A/TFG-TEC [19] was digested with NotI and the digested fragment was cloned into the same site of pEF-BOS/GST. To generate pcDNA4-HisMaxA/TFG (NTD), pCMV-Tag2A/TFG (NTD) [19] was digested with NotI and the digested fragment was cloned into the same site of pcDNA4-HisMaxA. To generate pcDNA4-HisMaxA/TFG (NTD), pCMV-Tag2A/TFG (NTD) [19] was digested with NotI and the digested fragment was cloned into the same site of pcDNA4-HisMaxA.…”

“…The plasmids pcDNA3/EGFP and pcDNA3/EGFP-TFG-TEC have been described previously [19]. To generate pcDNA3-mCherry, an mCherry gene was PCR-amplified from pmCherry-N1 (Clontech), using primers 5-HindIIImCherry (5 -GATC-AAGCTTATGGTGAGCAAGGGCGAG-3 ; HindIII site underlined) and 3 -BamHImCherry (5 -GATCGGATCCGCTTA-CTTGTACAGCTCGTC-3 , BamHI site underlined).…”

“…We previously reported that the KRRR sequence within the DBD targets the TFG-TEC oncoprotein to the nucleus [19]. To determine whether the NLS mutant TFG-TEC (AAAA) protein interacts with TFG-TEC in vivo, we performed co-affinity precipitations using extracts of HEK-293T cells transfected with an expression vector containing the NLS mutant TFG-TEC (AAAA) protein ( Figure 4A).…”

“…To determine whether the NLS mutant TFG-TEC (AAAA) protein interacts with TFG-TEC in vivo, we performed co-affinity precipitations using extracts of HEK-293T cells transfected with an expression vector containing the NLS mutant TFG-TEC (AAAA) protein ( Figure 4A). Specifically, mutations were introduced into the highly conserved basic amino sequence, K 612 RRR 615 , within the DBD (which is predicted to function as a NLS for TFG-TEC) [19]; these amino acids were mutated to A 612 AAA 615 ( Figure 4A The data in Figure 3(C) indicate that the DBD of TFG-TEC is capable of forming a heterodimer with TEC. Specifically, mutations were introduced into the highly conserved basic amino sequence, K 612 RRR 615 , within the DBD (which is predicted to function as a NLS for TFG-TEC) [19]; these amino acids were mutated to A 612 AAA 615 ( Figure 4A The data in Figure 3(C) indicate that the DBD of TFG-TEC is capable of forming a heterodimer with TEC.…”