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標題: PAX3與染色質及其交互作用蛋白之分析
Studies of PAX3 on chromatin association and its associated proteins
作者: 曾逸欣
Tzeng, Yee-Hsing
關鍵字: 染色質;PAX3;抑制轉錄;hSIRT1;Ku70;Ku80;PARP1;chromatin;M phase;transcription factor
出版社: 分子生物學研究所
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PAX3 regulates gene transcription during normal development of neurons and somatic muscles. However, how PAX3 regulates gene expression remains unknown. It is known that chromatin structure influences gene expression and can be altered by post-translational modification of histones, such as deacetylation or methylation. In this thesis, we focused on how PAX3 repressional activity is regulated through deacetylation or methylation, the possible function of the PAX3's stable complex with Ku80 and PARP1, and the mitotic chromosome association of PAX3.

(1) Histone modification: Co-immunoprecipition assays showed that PAX3 interacted with hSIRT1, EZH1 and SETDB1 but not with EZH2, M33, hPC2 and LSD1. hSIRT1 also interacted with acetylated PAX3 regardless of the presence or absence of NAM, which was an inhibitor of hSIRT1 enzymatic activity. Transcriptional assays showed that knocking-down hSIRT1 expression by siRNA could derepress PAX3's repressional activity. Over expressed hSIRT1, EZH1, SETDB1 or NAM treatment had no effect in PAX3's repressional activity. hSIRT1 couldn't affect PAX3 cellular localization. (2) PAX3 stable complex: PAX3 interacted with Ku80 and also Ku70 via PD and HD regions. PAX3, Ku80 and Ku70 form a ternary complex. Overexpression of Ku did not affect in PAX3's transcriptional activity or localization. Overexpression of PARP1 could slightly derepress PAX3 on the N-CAM promoter. PARP activation by NAD+ or inhibition by 3AB did not affect PAX3 on its transcriptional activity or localization. ChIP assays revealed that PARP1 was targeted to the N-CAM promoter through PAX3, but Ku80 bound consistently. (3) Chromatin association: DAPI staining revealed that PAX3 co-localized with chromosomes during M phase, but PD, HD or PAX3-FKHR partially did. PAX3 existed in isolated chromatin fraction. GST pull-down assays showed that PAX3 interacted with histones. PARP1, which interacted with PAX3, showed similar co-localization with chromatin during M phase. hSIRT1, Ku70 and Ku80 did not show such localization pattern.

These results suggest that (1) PAX3 interacts with hSIRT1 and represses transcription without deacetylation activity of hSIRT1. (2) PAX3 interacts with Ku, but Ku does not affect PAX3's transcription or cellular localization. (3) PAX3 shows a specific localization patterns in M phase which is different from that of PAX3-FKHR.


(1) 組蛋白修飾作用: Co-immunoprecipition (Co-IP) 實驗證明PAX3可和hSIRT1、EZH1、SETDB1作用,而不會與EZH2、M33、hPC2、LSD1作用。hSIRT1酵素活性並不影響與PAX3的分子作用。以hSIRT1 siRNA降低hSIRT1表現量可減弱PAX3抑制轉錄能力,但hSIRT1不影響PAX3在細胞中的分佈位置。 (2) PAX3蛋白複合體: Co-IP實驗和GST pull down assay可證明PAX3確實與Ku作用,並且PAX3、Ku70和Ku80可形成ternary complex。在PAX3轉錄功能方面,增加Ku70、Ku80的表現量不影響PAX3的轉錄活性,而增加PARP1則可稍微減弱PAX3在N-CAM promoter的抑制作用,並發現PARP酵素活性與PAX3抑制轉錄的能力無關。此外,以UV irradiation或Doxorubicin造成DNA damage則可分別降低或增強Gal4-PAX3的抑制活性,但不影響PAX3在N-CAM promoter的抑制活性。在PAX3細胞分佈功能,發現PAX3和Ku70、Ku80、PARP1不會互相影響細胞中的分佈位置,以 UV irradiation、Doxorubicin處理也不影響PAX3在細胞中的位置。在promoter結合能力,以chromatin immunoprecipition assay證明PAX3可増加PARP1在N-CAM promoter的結合能力,Ku80則不受影響。 (3) 染色質作用關係: PAX3在細胞分裂時可發現與染色體有相同的分佈,並利用GST pull down實驗證明PAX3可與組蛋白直接作用,PAX3-FKHR融合蛋白與染色質分佈以及與組蛋白作用的能力明顯降低。觀察其它PAX3作用蛋白在細胞分裂時的分佈位置,發現PARP1也可分佈於染色質區域,hSIRT1、Ku70、Ku80則無此分佈。

本篇研究主要發現PAX3可藉由hSIRT1與PARP1作用調控轉錄活性,而此作用與hSIRT1和PARP1的酵素活性無關。另外發現PAX3在M phase時期與染色體有共同分佈以及PAX3和組蛋白的分子作用並且也發現PAX3-FKHR喪失此功能。
其他識別: U0005-1807200622392100
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