PAX3-FKHR 的轉錄調控機制

Mechanism of PAX3-FKHR in transcriptional regulation

林靜宜(Ching-Yi Lin)

指導教授 ： 姚雅莉

亞洲大學/健康學院/生物科技學系碩士班/碩士(2007年)

摘要

當染色體 t(2；13) 發生轉位突變產生 PAX3-FKHR 融合蛋白，其生理功能為一轉錄因子，具有活化轉錄之活性，在癌症研究的臨床檢測中，PAX3-FKHR可當作橫紋肌肉瘤 (Rhabdomyosarcoma) 的致病標記。

肌肉發育的過程，基因表現主要受控於轉錄作用，需要轉錄因子參與轉錄調控。在橫紋肌肉瘤的相關研究，發現有染色體轉位現象，導致 PAX3轉錄因子與 FKHR 轉錄因子結合，產生 PAX3-FKHR 融合蛋白。PAX3-FKHR與 PAX3 在蛋白結構及功能上相似，然而 PAX3-FKHR 相較於 PAX3 具有更為強大的活化轉錄能力。目前，對於 PAX3-FKHR 是如何進行調控轉錄活性之機制，尚不清楚。前人研究已知 PAX3 可藉由蛋白間交互作用，調控轉錄之活性，本論文將以此為出發點，探討 PAX3-FKHR 調控轉錄的機制。

根據報導 corepressor KAP1和 histone modifiers 與 PAX3 有交互作用，並影響 PAX3 的轉錄活性，推論是否 PAX3-FKHR 也會藉由蛋白間交互作用調控轉錄，因此利用 co-immunoprecipitation 找尋與 PAX3-FKHR 有交互作用的蛋白，結果顯示 PAX3-FKHR 與 corepressor KAP1 有交互作用，並藉由 transcriptional assay 發現 KAP1減弱 PAX3-FKHR 的活化轉錄活性。進一步發現 PAX3-FKHR 與 histone modifiers：HDAC10、SIRT1、SIRT6、SIRT7 以及 JMJD2A 有交互作用，且 JMJD2A 增強 PAX3-FKHR 的活化轉錄活性。在了解PAX3-FKHR 與轉錄調控蛋白的交互作用之後，另一方面，已知PAX3-FKHR 與PAX3 具有相同 DNA 結合區域，功能皆為轉錄因子，想要進一步探討，真實情況中 PAX3-FKHR 是否會競爭 PAX3 的 target promoters，影響 PAX3-FKHR的調控轉錄活性，利用 transcriptional assay 分別於 PAX3 的 target promoters：MITF、TRP-1 及 TRP-2 promoters 同時表現 PAX3-FKHR 與 PAX3，結果顯示當增加 PAX3 或 PAX3-FKHR 的表現量，會影響 PAX3-FKHR 與 PAX3原本的轉錄活性。

本篇論文主要發現 corepressor KAP1 及 histone modifier JMJD2A 參與PAX3-FKHR 的轉錄調控機制，並且 PAX3-FKHR 會競爭 PAX3 的 target promoters 而影響轉錄作用。

關鍵字

轉錄調控

並列摘要

The PAX3-FKHR fusion protein is formed as a result of t(2;13) chromosomal translocations and functions as a transcription factor to activate transcription. In the clinical sense of cancer research, PAX3-FKHR is the pathogenetic marker for alveolar rhabdomyosarcoma.

During the development cycle of skeletal muscles, gene expression is controlled by transcription that requires regulation of transcription factors. PAX3-FKHR is characterized by the t(2;13) chromosomal translocation, which results in the fusion of two transcription factors, PAX3 and FKHR, in rhabdomyosarcoma. The protein structure and function of PAX3-FKHR is similar to those of PAX3, with a much greater transcriptional activity ability. However, the mechanism of PAX3-FKHR in transcriptional regulation is still unknown. It is reported that PAX3 regulates transcriptional activity through protein-protein interactions. In this thesis we want to investigate the mechanism of PAX3-FKHR in transcriptional regulation.

It has been shown that corepressor KAP1 and histone modifiers, through interacting with PAX3, affect the transcriptional activity of PAX3. We hypothesize that PAX3-FKHR modulates its transcriptional activity through protein-protein interactions. We tried to find proteins interacting with PAX3-FKHR by co-immunoprecipitation. Our results show that PAX3-FKHR, through interacting with KAP1, decreases its activation transcriptional activity in transcriptional assay. Further we find that PAX3-FKHR interacts with histone modifiers, including HDAC10, SIRT1, SIRT6, SIRT7 and JMJD2A. We also find that JMJD2A, through interacting with PAX3-FKHR, increases the activation transcriptional activity of PAX3-FKHR. We already know that PAX3-FKHR and PAX3 have the same DNA binding domains and they both function as transcription factors. After understanding the relationship between PAX3-FKHR and its transcriptional associated proteins, we want to determine if PAX3-FKHR competes with PAX3’s target promoters, MITF, TRP-1 and TRP-2 promoters, and affects its activation activity. Our results show that PAX3-FKHR and PAX3 affect each other’s transcriptional activity.

These results suggest that corepressor KAP1 and histone modifier JMJD2A affect the mechanism of PAX3-FKHR in transcriptional regulation. PAX3-FKHR competes with PAX3 on PAX3 target promoters and affects the transcriptional activity of PAX3.

並列關鍵字

transcriptional regulation

參考文獻

Anderson, J., Ramsay, A., Gould, S., and Pritchard-Jones, K. (2001a). PAX3-FKHR induces morphological change and enhances cellular proliferation and invasion in rhabdomyosarcoma. Am J Pathol 159, 1089-1096.

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