Pax3 是一個在正常生長發育上扮演重要角色的轉錄因子。Pax3 蛋白共有兩個
DNA 結合區域： 在N端的是 paired domain ( 簡稱PD) ， 在C端的是paired-type
homeodomain (簡稱 HD)。目前已知 Pax3 的變異會造成瓦登博革氏症候群與橫紋肌肉
瘤。在瓦登博革氏症候群中常見 Pax3 的點變異，而這些點變異常發生在 PD 或 HD
的部位。在肺泡型橫紋肌肉瘤中 Pax3 則和 FKHR 因染色體轉位而形成新的融合蛋
白。我們的實驗發現 Pax3 利用 PD 和 HD 與其他結合在染色體上或調控染色體功能
的蛋白相結合。Pax3 也能與異染色質以及有絲分裂染色體結合。此外，我們還發現一
個瓦登博革氏症候群病患特有的突變 Pax3 不會與異染色質或有絲分裂染色體結合。近
來文獻顯示與異染色質或有絲分裂染色體結合的蛋白能參與染色體之間的交互作用。因
此，我們提出以下的假說：Pax3 能與異染色質以及有絲分裂染色體結合，造成特定的
染色體之間交互作用，而這些交互作用在瓦登博革氏症候群與橫紋肌肉瘤中被破壞。下
列三項實驗目標將用來驗證這個假說：
目標一：利用功能區劃定、蛋白之間交互作用分析以及染色體分散分析來決定
Pax3 如何與異染色質以及有絲分裂染色體結合
目標二：利用共軛焦顯微鏡法、蛋白之間交互作用分析以及染色體分散分析來確
定瓦登博革氏症候群與橫紋肌肉瘤患者所帶有的突變 Pax3 是否還能與異染色質以及
有絲分裂染色體結合
目標三：利用染色體構型捕捉與染色質免疫沉澱法探討 Pax3 是否具有染色體之
間交互作用的功能
本計劃將闡明 Pax3 與異染色質及有絲分裂染色體結合的機制以及 Pax3 在染色
體之間交互作用中的意義。本計劃的完成不僅能讓我們了解 Pax3 正常的生理功能，更
將幫助我們解釋瓦登博革氏症候群與橫紋肌肉瘤的成因，並且為將來研發治療的方法鋪
路。
Pax3 is a transcription factor crucial for normal development. It contains two
DNA-binding domains, a paired domain (PD) at the N-terminus and a paired-type
homeodomain (HD) at the C-terminus. It has been shown that point mutations, which
mostly located in the PD or HD, cause Waardenburg syndrome, and chromosomal
translocations that create Pax3-FKHR fusion proteins cause alveolar
rhabdomyosarcoma. We found that Pax3 used its PD and HD to interact with proteins
known to associate with or to regulate the function of chromatin, and it associated
with heterochromatin during interphase and with mitotic chromosomes during mitosis.
Furthermore, a mutant Pax3 from Waardenburg syndrome patients was de-localized
from heterochromatin and the mitotic chromosome. Recent studies show that proteins
localized to heterochromatin and the mitotic chromosome participate in
interchromosomal interactions. Therefore, I hypothesize that Pax3 associates with
heterochromatin and the mitotic chromosome, leading to interchromosomal
interactions that are de-regulated in Waardenburg syndrome and rhabdomyosarcoma.
The following three aims are proposed to test this hypothesis:
Aim 1: to determine how Pax3 associates with heterochromatin and the mitotic
chromosome using domain-mapping, protein-protein interaction assays, and
chromosome spreading assays.
Aim 2: to determine if Pax3 mutants from patients with Waardenburg syndrome
and rhabdomyosarcoma are associated with heterochromatin and the mitotic
chromosome using confocal microscopy, protein-protein interaction assays, and
chromosome spreading assays.
Aim 3: to determine if Pax3 functions in interchromosomal interactions through
chromosome conformation capture (3C) and chromatin immunoprecipitation (ChIP)
assays.
This proposed study will demonstrate the mechanism by which Pax3 interacts
with heterochromatin and the mitotic chromosome and the significance of Pax3 in
interchromosomal interactions, which might explain how mutations in Pax3 cause
Waardenburg syndrome and alveolar rhabdomyosarcoma. Results from this study will
help us further understand the normal function of Pax3 and lead to possible
therapeutic interventions in the future.
