Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 104 === 1. The study of PAX3 Waardenburg syndrome mutants in causing S phase cellular effects
PAX3 is a transcription factor involved in regulating genes expression during embryonic development. Mutation in the PAX3 gene cause Waardenburg syndrome (WS). However, how PAX3 mutants cause Waardenburg syndrome remains unclear. Previously, it has been found that PAX3 loads onto DAPI dense regions (known as heterochromatin regions) during mid-to-late S phase, while the PAX3 WS mutants cannot. Moreover, the mutants block wildtype PAX3 from loading onto the DAPI dense region, implying that PAX3 WS mutants have undiscovered functions. To uncover the potential role of PAX3 WS mutants during S phase, we investigated whether PAX3 WS mutants could load onto replication forks and what effects PAX3 WS mutants caused during DNA replication. By immunostaining, we showed that PAX3 WS mutants could not load onto replication foci. To further understand the cellular effect caused by PAX3 WS mutants, we used flow cytometry to examine cell cycle progression and found that PAX3 HD mutants arrested cell in S phase. Because replication stress can induce micronuclei, by calculating the number of the micronuclei, we demonstrated that PAX3 WS mutants increased the cells containing micronuclei. Taken together, our results demonstrate that PAX3 WS mutants cannot load onto replication foci; they may cause cell cycle arrest and micronuclei production.
2. The study of STAF65γ abundance in regulating HDAC5 subcellular localization and the migration ability of lung adenocarcinoma cell lines
STAF65γ is a structural subunit of the GCN5 transcriptional co-activator complex under physiological conditions. Recently, a novel co-repressor function of STAF65γ was identified. STAF65γ represses transcription in a dose-dependent manner. When present at high levels, STAF65γ increases nuclear fraction of class IIa HDACs (HDAC5 and HDAC7) and cooperates with them in the nucleus to mediate c-Myc repression. Despite of the molecular function of STAF65γ is revealed, the biological relevance of STAF65γ-mediated transcriptional repression remains largely unknown. In our preliminary studies, we found that elevated STAF65γ protein expression occurs in lung adenocarcinoma, suggesting the involvement of co-repressor STAF65γ in tumorigenesis. However, the role of STAF65γ in lung tumor development has not been investigated. Here, we found that the STAF65γ expression is correlated to the enhancement of the invasiveness of lung carcinoma cell lines by RT-PCR. The nucleocytoplasmic localization of HDAC5 in lung adenocarcinoma cell line has been detected by immunostaining, and it correlated with highly STAF65γ expression in lung adenocarcinoma cell line. Finally, by the use of in vitro scratch assay, we found that STAF65γ might contribute to the migration ability of lung adenocarcinoma cell line.
|