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Characterization of EWS Family Fusion Oncogenes Generated by Chromosomal Translocation

  • 김솔 (Kim, Sol, 서강대학교 생명과학과 분자 세포 생물학)

초록/요약

골 및 육아 종양의 발병은 유전적이고 환경적인 조건에 기인한다. 암을 유발하는 조건들 중 염색체 전좌 (chromosomal translocation)는 다양한 융합 단백질을 생성하거나 발암 유전자를 활성화시키고 항암 유전자를 억제시킬 수 있다. 염색체 전좌로 인해 생성된 EWS-Oct-4와 hTAFII68-TEC 융합 단백질이 골 및 육아 종양에서 발견되었으나 이 단백질의 생물학적인 기능에 대해선 잘 알려져 있지 않다. hTAFII68-TEC은 TEC에 hTAFII68의 아미노 말단 활성 도메인과의 융합으로 생성된다. 이 융합 유전자는 TEC과 동일한 DNA 순서를 인식하는 핵 내 단백질을 encoding하고 있으며 hTAFII68-TEC의 핵 내 위치는 DNA 결합 도메인에 있는, KRRR 아미노산 서열에 의존적이라는 것을 밝혔다. hTAFII68-TEC의 전사활성은 TEC보다 높으며, 이런 전사 활성에는 hTAFII68의 아미노 말단 활성 도메인과 hTAFII68-TEC의 AF1, AF2 도메인이 필요하다. 한편 hTAFII68-TEC 결합 단백질인 glyceraldehyde-3-phosphate dehydrogenase (GAPDH)를 matrix assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF) 방법을 통해 찾아 냈다. GAPDH는 핵 내에서 세포 예정사, nuclear tRNA export, DNA 복제, DNA 복구, 그리고 전사작용 등 세포 내에서 다양한 기능을 수행한다. 본 연구에서는 GAPDH가 hTAFII68-TEC의 아미노 말단 도메인의 106-159 아미노산 부분과 결합하며 hTAFII68-TEC에 의한 전사 활성을 증가시킬 수 있음을 밝혔다. EWS-Oct-4 융합 단백질의 경우, EWS의 아미노 말단 지역 (70-163 아미노산)과 POU DNA 결합 도메인이 자가 복합체를 형성하는데 중요하며 EWS-Oct-4 (V351P)와 이형 복합체를 형성할 수 있었다. EWS-Oct-4 (V351P) 돌연변이체는 야생형 EWS-Oct-4의 DNA 결합을 방해하고, EWS-Oct-4에 의한 전사 활성을 억제하여 dominant negative 단백질로 작용한다. 이러한 결과들을 통해 염색체 전좌에 의해 새로운 기능을 얻은 hTAFII68-TEC 단백질은 결합 파트너보다 전사 활성이 높으며 GAPDH에 의해 전사 활성 능력이 증가될 수 있다는 것을 나타나며, EWS-Oct-4의 생물학적인 기능은 EWS-Oct-4 (V351P) 돌연변이체의 dominant negative 효과에 의해 조절될 수 있음을 나타낸다.

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초록/요약

Pathogenesis of the bone and soft tissue tumors are based on various inherited and environmental factors. One of these factors, chromosomal translocation makes various fusion proteins or activates oncogene. EWS-Oct-4 and hTAFII68-TEC fusion proteins were little known about the biological function. I have examined the functional consequences of the fusion of the amino terminal domain (NTD) of hTAFII68 to TEC. The chimeric gene encodes a nuclear protein that binds DNA with the same sequence specificity as parental TEC. Nuclear localization of hTAFII68-TEC was dependent on the DNA binding domain, and I identified a cluster of basic amino acids in the DNA binding domain, KRRR, that specifically mediate the nuclear localization of hTAFII68-TEC. The transactivation activity of hTAFII68-TEC was higher than TEC towards a known target promoter that contained several TEC binding sites. Finally, deletion analysis of hTAFII68-TEC indicated that the hTAFII68 NTD, and the AF1 and AF2 domains of hTAFII68-TEC are necessary for full transactivational potential. As well as, I identified a novel hTAFII68-TEC-interacting protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), by affinity chromatography utilizing immobilized hTAFII68 (NTD) and matrix assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF). GAPDH functions as a glycolytic enzyme within cytoplasm, but beside its metabolic function it is involved in early steps of apoptosis, nuclear tRNA export, DNA replication, DNA repair, and transcription in the nucleus. Co-immunoprecipitation experiments are consistent with an in vivo association between hTAFII68-TEC and GAPDH. Glutathione S-transferase pull-down assays revealed that at least three region of GAPDH is required for interaction with the hTAFII68 (NTD). In addition, C-terminal part (amino acids 106-159) of hTAFII68 (NTD) is involved in binding to GAPDH. Functionally, GAPDH stimulates transcriptional activation by hTAFII68-TEC. Moreover, a chimeric protein generated by fusion of GAPDH to the GAL4 DNA-binding domain significantly increases promoter activity of a reporter containing GAL4 DNA-binding sites, suggesting the presence of an active transactivation domain(s) within GAPDH. Using in vitro and in vivo systems, I found that the EWS-Oct-4 protein self-associates. The major domains required for self-association mapped to the EWS NTD (amino acids 70–163) and the POU DNA-binding domain. EWS-Oct-4 protein also associated with EWS-Oct-4 (V351P), which contains a mutation in the POU DNA-binding domain. Using electrophoretic mobility shift assays, I found that the EWS-Oct-4 (V351P) mutant interfered with wild-type EWS-Oct-4 DNA-binding activity. In addition, we found that EWS-Oct-4-mediated transcriptional activation was inhibited by EWS-Oct-4 (V351P) protein in vivo. Thus, this mutation in the POU DNA-binding domain results in a dominant negative protein. These results represent that fusion of the hTAFII68 NTD to the TEC protein produces a gain of function chimeric product and GAPDH stimulates the transactivation potential of hTAFII68-TEC oncogene. Also, the biological functions of the EWS-Oct-4 oncogene can be modulated by the dominant negative mutant EWS-Oct-4 (V351P).

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