| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 100 === PAX3, a transcription factor, regulates many development-related gene expression to affect vertebrate development. When PAX3 protein is mutated, a differentiation disease, called Waardenburg syndrome happen. From previous study in Waardenburg syndrome, researchers has found that two kind of mutant Pax3 will cause disease, one is missense mutation, the other is truncating mutation. Previous study of PAX3 mutant mostly focus on missense mutation. The detail mechanism of how truncated mutant Pax3 leads to Waardenburg syndrome remains unclear. In this thesis we discover that Pax3(Q282X) has chromatin changing ability, through mimicry mutant Pax3 happen in Waardenburg syndrom patient. This suggests that truncated-form Pax3 might use its chromatin-changing ability to affect occurrence of Waardenburg syndrome. Therefore, in this thesis we try to investigate the role of truncated-form mutant Pax3 in Waardenburg syndrome.
To explore the role of truncated mutant Pax3 in Waardenburg syndrome, we use site-directed mutagenesis to generate Pax3 mutants which has been reported in WS patient. The result shows that Pax3(Q282X) has chromatin changing ability, but other truncated-mutants having loger Pax3CTD region have decreased chromatin changing ability. This suggets that Pax3CTD might play an important role in truncated-form mutant Pax3 in changing chromatin structure. In Waardenburg syndrome Pax3 is single allele mutant, in that a wild type Pax3 and mutant Pax3 exist in WS patient’s cell. We further mimic real condition in WS patient by investigating whether wild Pax3 and mutant can affect each other. Results show that wild type can disrupt mutant Pax3 chromatin change ability; mutant Pax3 can makes wild type Pax3 having chromatin changing ability. Further, we mapped the functional domain for mutant to Pax3 change chromatin structure and found that Pax3PDHD possesses chromatin changing ability that forms DAPI-dots. To characterize Pax3PDHD induced-DAPI dots, we stain various chromatin markers. Results show that Pax3PDHD induced-DAPI dots can associated with heterochromatin marker: HP1α、HP1β、 HP1γ、H3K9me3 and H3K27me2; and partially associated with euchromatin marker H3K4me3, implying that Pax3PDHD-induced DAPI dots associates with repressive status chromatin. To investigate whether Pax3(Q282X) and Pax3PDHD possesses the ability to compact chromatin structure, we demonstrate that Pax3(Q282X) and Pax3PDHD can decrease the accessibility of chromatin by MNase digestion assay.
To explore the mechanism of Pax3PDHD in altering chromatin structure, we demonstrate that DNA binding activity of PD and HD domain is necessary for Pax3PDHD change chromatin structure and HD domain is also important for Pax3PDHD to form protein dots. And we further use ChIP assay shows that Pax3PDHD might use non-specific DNA binding ability to change chromatin structure. On the other hand, we proposed that Pax3PDHD might through interact with histone modifier to change chromatin structure, and we find out PARP1 may be a candidate histone modifier. Although experiment results show that Pax3PDHD can interact with PARP1, however knockdown PARP1 did not disrupt Pax3PDHD’s chromatin changing ability. This suggests PARP1 might not required for Pax3PDHD to change chromatin structure.
My thesis shows that Pax3(Q282X) and Pax3PDHD can change chromatin structure and suggests that truncated-form Pax3 might use its chromatin-changing ability to affect occurrence of Waardenburg syndrome. We envisage that by disrupting Pax3(Q282X)’s chromatin changing ability could provide a potential therapeutic, and result shows that Pax3CTD can disrupts Pax3(Q282X)’s chromatin changing ability.
Taken together, the results demonstrate that truncate-form Pax3 in Waardenburg syndrome can change chromatin structure create a repressive status chromatin.
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