類泛素化是很重要的後轉譯修飾，並調節各樣的生物途徑。Daxx蛋白在過去的文獻發現它可以辨認經類泛素化修飾後蛋白並且去抑制基因轉錄活性以及其在細胞中的分佈。Daxx蛋白透過其SUMO interacting motif (SIM)去辨認類泛素化修飾後的蛋白，但這樣不足以提供Daxx專一性的辨認。因此，在此論文中我們要探討除了SIM之外，是否存在其他區域或模組使Daxx可以專一性的辨認其交互作用的蛋白。已知Daxx蛋白傾向與Lys159類泛素化Smad4蛋白有交互作用並且抑制由Smad4轉錄因子調控的基因轉錄，因此首先，我們在細胞外的實驗中發現Smad4 會與Daxx625-740 有交互作用但Daxx660-740則無，接著利用點突變實驗，我們發現在細胞外與細胞內的實驗都驗證當Daxx 631到633位置突變後會降低與Lys159類泛素化Smad4蛋白的交互作用，並且失去抑制Smad4轉錄的活性。此外我們也探討Daxx 631到633位置對於Daxx與Promyelocytic leukaemia protein (PML)的交互作用，在細胞內外實驗也發現到突變的Daxx與PML有較弱的交互作用，並且影響其進入PML-NB (PML-nuclear body)，綜而言之，Daxx631-633對於其能專一辨認作用的蛋白是重要的，在未來我們將更廣泛去看Daxx這段區域與其他類泛素化蛋白的交互作用是否有保留性。

Sumoylation is an essential post-translational modification regulating diverse cellular functions. Daxx has been reported to associate with several sumoylated proteins, which regulates the transcriptional activity and in certain cases affects their subnuclear compartmentalization. While Daxx can bind to specific sumoylated factors by its SUMO interacting motif (SIM), such binding cannot provide the specificity for substrate recognition. Thus, we hypothesized that additional region or motif within Daxx may contribute to its substrate recognition. In order to identify such substrate recognition motif within Daxx, we first selected its interacting substrate Smad4 for study. Our lab has shown that sumoylation of Smad4 Lys159 is critical for its interaction with Daxx, leading to Smad4 sumoylation-elicited transcriptional repression. By GST pull-down assay, I demonstrated that sumoylated Smad4 at Lys159 can be pulled down by Daxx625-740 rather than by Daxx660-740. Via site-directed mutagenesis, we found that Daxx631-633 is critical for sumoylated Smad4 binding. Similarly, in vivo co-immunoprecipitation experiment presented that Daxx631-633 mutant cannot interact with SUMO-modified Smad4. Additionally, reporter gene assays indicated that Daxx631-633 mutant can mildly repress Smad4-mediated transcriptional activation. Furthermore, we also explored another Daxx interacting substrate, PML. GST pull-down and immunoprecipitation experiments revealed that Daxx631-633 mutant failed to interact with sumoylated PML. Immunofluorescence analysis indicated that the association of Daxx mutant with PML nuclear body was also reduced. Taken together, these results suggested that Daxx 631-633a.a. may play an important role in sumoylated Smad4 and PML recognition. In the future, we will examine whether Daxx631-633 is a conserved substrate recognition motif for other substrates or only specific to Smad4 and PML.

口試委員會審定書 I
誌謝 II
中文摘要 III
Abstract IV
Chapter I Introduction 1
SUMOylation 2
SUMO machinery 3
SUMO interacting motif (SIM) 4
The role of Daxx 4
Overview of TGF-b signaling 6
Smad4 as a co-Smad 7
PML and PML nuclear bodies 8
Specific Aims 11
Chapter II Materials and Methods 12
Plasmid constructs and site-directed mutagenesis 13
GST Pull-down Assay 13
Cell Culture, Transient Transfection, and Luciferase Reporter Assay 14
Cell lysis, Immunoprecipitation and Immunoblotting 15
Cell Culture, Transient Transfection and Immunofluorescence staining 16
Bacterial Expression and Purification of Daxx 16
Bacterial Expression and Purification of Sumoylated Smad4 17
Circular dichroism (CD) spectroscopy 18
Chapter III Results 19
Purification of sumoylated Smad4-delta-MH1 at K159 20
Identification of the substrate recognition motif on Daxx for interaction with sumoylated Smad4 20
Identification of the substrate recognition motif on Daxx for interaction with sumoylated PML 22
Secondary structure comparison between Daxx625-740 and DaxxK631-633A mutant 22
Identification of the substrate recognition motif on Daxx for interacting with sumoylated Smad4 and PML in vivo 23
DaxxK631-633A mutant abolished Daxx-induced transcriptional repression of Smad4 25
The association of PML nuclear body formation and the recruitment of Daxx 26
Chapter IV Discussion 28
Chapter V Figures 35
Chapter VI Tables 49
References 52

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