| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 94 === 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.
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