臺灣博碩士論文加值系統

中文摘要
轉錄因子PAX3為調控胚胎神經與體節發育的重要蛋白，當PAX3基因發生突變會導致分化上的疾病瓦登伯格式症(Waardenburg syndrome)產生。PAX3不僅具有轉錄調控的功能，也參與在異染色質的形成機制中。本實驗室的研究發現，PAX3座落異染色質的時間為S期中晚期，並且實驗室進一步證明PAX3在S期座落於複製叉中，然而，瓦登伯格式症突變型PAX3於S期晚期無法座落複製叉，並且會延滯S期進程，使細胞產生微核,這些研究意味著PAX3能否座落複製叉將會影響基因穩定性。然而，目前對於PAX3座落複製叉的機制與功能並不清楚。本篇研究以DNA複製位標誌技術 (EdU labeling) 證明PAX3藉由其LHD domain座落於異染色質區的複製叉，並且以新生成染色質捕捉技術 (Nascent chromatin capture assay, NCC assay) 確認PAX3LHD具有結合新生成染色質能力。此外，我們以NCC assay證明PAX3結合新生成染色質需要PARP1蛋白酶(Poly [ADP-ribose] polymerase 1)，並以共免疫沈澱法找出PAX3與PARP1交互作用的功能性區域在LHD domain。接著，我們進一步證明PAX3LHD會被PARP1的酵素活性poly ADP-ribosylation(parylation)修飾，並且我們發現PAX3在細胞週期不同時期皆會被修飾。另外，我們探討突變型PAX3在S期晚期無法座落複製叉是否與PARP1交互作用有關，我們以共免疫沈澱法證明PAX3PD突變蛋白會與 PARP1 交互作用，然而，大部份PAX3HD突變蛋白不會與PARP1交互作用。最後，我們探討PAX3在S期是否有維持基因穩定的能力，我們構築含有S期降解決定子的PAX3載體，使PAX3蛋白在S期被降解，並以顯微鏡觀察細胞發現，當PAX3在S期降解會導致基因不穩定性，使微核產生。綜合以上，我們的研究證明PAX3LHD會與PARP1交互作用，藉此座落複製叉，並且修飾PAX3LHD。另外，我們發現大部份PAX3HD突變蛋白不會與PARP1交互作用。最後，我們證明PAX3在S期PAX3具有維持基因穩定，避免細胞產生微核的功能。

Abstract
Transcription factor PAX3 is important for embryonic development, and is previously known to function in gene regulation and heterochromatin formation. Mutation in PAX3 gene causes inherited disease--Waardenburg syndrome. In previous researches done in our laboratory, we found PAX3 loads onto heterochromatin and replication forks during S phase. In contrast, PAX3 mutants in Waardenburg syndrome cannot load onto replication forks during late S phase, which results in S-phase delay and micronucleus formation. These results imply that the ability of PAX3 loading onto replication fork is important for genome stability. However, despite its importance, the mechanism and function of PAX3 loading onto replication forks remain unknown. Here, we demonstrate that PAX3LHD is sufficient for PAX3 loading onto replication foci, and the location is associated with heterochromatin region. The ability of PAX3LHD to bind on nascent chromatin is further confirmed by Nascent Chromatin Capture assay (NCC assay). Moreover, we find that PAX3 loading onto replication forks requires PARP1（Poly [ADP-ribose] polymerase 1）by NCC assay. And, co-immunoprecipitation result shows that PAX3 LHD is the interaction domain with PARP1. Furthermore, through PARylation assay, we demonstrate that PAX3 can be poly ADP-ribosylated (PARylated) at LHD domain via PARP1-PAX3 interaction. And PAX3 is PARylated through the whole cell cycle. On the other hand, we investigate whether PARP1 is related to the inability of PAX3 mutants to load onto replication forks. In co-immunoprecipitation, we find that PAX3 PD mutants interact with PARP1, while most PAX3 HD mutants cannot. Finally, to validate whether PAX3 maintains genome stability in S phase, we use PAX3 with S phase degron to analyze if degradation of PAX3 in S phase leads to micronucleus formation. The result shows increased cells with micronucleus in the absence of PAX3 during S phase. To conclude, we prove that PAX3 loads onto replication forks through LHD domain, and it requires interaction with PARP1. Moreover, we find that PAX3 is PARylated at LHD domain through interaction with PARP1. On the other hand, PAX3 PD mutants can interact with PARP1, but most PAX3 HD mutants cannot. In addition, we prove that PAX3 has the ability to prevent micronucleus formation and therefore maintain genome stability during S phase.

中文摘要 i
Abstract ii
目錄 iii
緒論 1
一、前言 1
二、研究背景 1
（一）PAX3的基因與蛋白結構 1
（二）PAX3的生理功能 2
（三）PAX3 參與異染色質形成 2
（四）瓦登伯格氏症候群與突變型 PAX3 蛋白 4
（五）PAX3 與突變型 PAX3 對 S 期的影響 5
（六）細胞週期S期 5
（七）PARP蛋白的背景介紹 10
三、研究目的 12
四、研究策略 12
（一）確認PAX3是否會結合到複製叉，並探討PAX3座落複製叉是否位在異染色質中 12
（二）探討PAX3是否是藉由PARP1交互作用結合到複製叉 13
（三）探討PARP1是否會對PAX3進行PARylation修飾作用 13
（四）探討瓦登伯格氏症突變型PAX3是否會與PARP1 交互作用 14
（五）探討PAX3在S期降解後是否會導致基因不穩定性 14
貳、材料與方法 16
一、質體（plasmid）DNA 16
二、細胞培養 (Cell culture) 16
三、基因轉移感染 (Transfection) 17
四、細胞同步化（Cell synchronization） 17
（一）細胞週期G0/G1期同步化 17
（二）細胞週期S期同步化 18
（三）細胞週期G2/M期同步化 18
五、免疫沉澱法 (co-immunoprecipitation) 18
六、西方墨點法 (Western Blot) 19
七、免疫螢光染色法（Immunofluorescence） 20
八、DNA 複製位標誌技術（EdU labeling） 20
九、ADP-核醣基化修飾分析技術（PARylation assay） 21
十、新生成染色質捕捉技術(Nascent Chromatin Capture assay, NCC assay) 21
十一、細胞週期分析技術（Flow cytometry） 22
參、結果 23
一、PAX3 主要於S期中晚期利用其LHD domain座落於DAPI dots中的 replication foci 23
二、於 S 期中期 PAX3 藉由與 PARP1 交互作用結合到新生成染色質中 25
三、PAX3在全細胞週期皆會受到PARylation，並且其PARylation site位於LHD domain中 26
四、突變型PAX3與PARP1的交互作用 27
五、PAX3在S期降解會導致微核產生 27
肆、討論 29
一、PAX3座落異染色質區上的複製叉的意義 29
二、探討PAX3藉由PARP1的交互作用結合到新生成染色質的意義 29
三、PAX3在全細胞週期皆會受到PARylation，並且其PARylation site位於LHD domain 30
四、突變型PAX3與PARP1的交互作用 31
五、探討PAX3在S期降解如何導致基因不穩定而導致微核 32
伍、參考文獻 33
陸、圖表 37
柒、附圖 57

伍、參考文獻
1.Alabert, C, JC Bukowski-Wills et al. “Nascent Chromatin Capture Proteomics Determines Chromatin Dynamics During DNA Replication and Identifies Unknown Fork Components.” Nat Cell Biol 16.3 (2014): 281-93.
2.Alabert, C, and A Groth. “Chromatin Replication and Epigenome Maintenance.” Nat Rev Mol Cell Biol 13.3 (2012): 153-67.
3.Annunziato, AT, and RL Seale. “Chromatin Replication, Reconstitution and Assembly.” Mol Cell Biochem 55.2 (1983): 99-112.
4.Baldwin, CT, CF Hoth et al. “Mutations in Pax3 That Cause Waardenburg Syndrome Type I: Ten New Mutations and Review of the Literature.” Am J Med Genet 58.2 (1995): 115-22.
5.Beneke, S. “Regulation of Chromatin Structure By Poly(adp-Ribosyl)ation.” Front Genet 3 (2012): 169.
6.Bulut-Karslioglu, A, V Perrera et al. “A Transcription Factor-Based Mechanism for Mouse Heterochromatin Formation.” Nat Struct Mol Biol 19.10 (2012): 1023-30.
7.Burhans, WC, and M Weinberger. “DNA Replication Stress, Genome Instability and Aging.” Nucleic Acids Res 35.22 (2007): 7545-56.
8.Choi, J, M Xu et al. “A Common Intronic Variant of Parp1 Confers Melanoma Risk and Mediates Melanocyte Growth Via Regulation of Mitf.” Nat Genet (2017)
9.Conway, SJ, DJ Henderson et al. “Development of a Lethal Congenital Heart Defect in the Splotch (Pax3) Mutant Mouse.” Cardiovasc Res 36.2 (1997): 163-73.
10.Darmon, E, and DR Leach. “Bacterial Genome Instability.” Microbiol Mol Biol Rev 78.1 (2014): 1-39.
11.De Vos, M, R El Ramy et al. “Poly(adp-Ribose) Polymerase 1 (Parp1) Associates With E3 Ubiquitin-Protein Ligase Uhrf1 and Modulates Uhrf1 Biological Functions.” J Biol Chem 289.23 (2014): 16223-38.
12.Epstein, JA, P Lam et al. “Pax3 Inhibits Myogenic Differentiation of Cultured Myoblast Cells.” J Biol Chem 270.20 (1995): 11719-22.
13.Fragkos, M, and V Naim. “Rescue From Replication Stress During Mitosis.” Cell Cycle 16.7 (2017): 613-33.
14.Fragkos, Michalis, Olivier Ganier et al. “DNA Replication Origin Activation in Space and Time.” Nat Rev Mol Cell Biol 16.6 (2015): 360-74.
15.Guetg, C, F Scheifele et al. “Inheritance of Silent Rdna Chromatin is Mediated By Parp1 Via Noncoding RNA.” Mol Cell 45.6 (2012): 790-800.
16.Hakme, A, HK Wong et al. “The Expanding Field of Poly(adp-Ribosyl)ation Reactions. ‘Protein Modifications: Beyond the Usual Suspects’ Review Series.” EMBO Rep 9.11 (2008): 1094-100.
17.Herce, HD, M Rajan et al. “A Novel Cell Permeable DNA Replication and Repair Marker.” Nucleus 5.6 (2014): 590-600.
18.Hsieh, MJ, YL Yao et al. “Transcriptional Repression Activity of Pax3 is Modulated By Competition Between Corepressor Kap1 and Heterochromatin Protein 1.” Biochem Biophys Res Commun 349.2 (2006): 573-81.
19.Kioussi, C, MK Gross, and P Gruss. “Pax3: A Paired Domain Gene as a Regulator in Pns Myelination.” Neuron 15.3 (1995): 553-62.
20.Kirsch-Volders, M, G Plas et al. “The in Vitro Mn Assay in 2011: Origin and Fate, Biological Significance, Protocols, High Throughput Methodologies and Toxicological Relevance.” Arch Toxicol 85.8 (2011): 873-99.
21.Kliszczak, AE, MD Rainey et al. “DNA Mediated Chromatin Pull-Down for the Study of Chromatin Replication.” Sci Rep 1 (2011): 95.
22.Kubic, JD, KP Young et al. “Pigmentation Pax-Ways: The Role of Pax3 in Melanogenesis, Melanocyte Stem Cell Maintenance, and Disease.” Pigment Cell Melanoma Res 21.6 (2008): 627-45.
23.Leung, KH, M Abou El Hassan, and R Bremner. “A Rapid and Efficient Method to Purify Proteins At Replication Forks Under Native Conditions.” Biotechniques 55.4 (2013): 204-6.
24.Machado, AF, LJ Martin, and MD Collins. “Pax3 and the Splotch Mutations: Structure, Function, and Relationship to Teratogenesis, Including Gene-Chemical Interactions.” Curr Pharm Des 7.9 (2001): 751-85.
25.Martienssen, RA, A Kloc et al. “Epigenetic Inheritance and Reprogramming in Plants and Fission Yeast.” Cold Spring Harb Symp Quant Biol 73 (2008): 265-71.
26.Mazouzi, A, G Velimezi, and JI Loizou. “DNA Replication Stress: Causes, Resolution and Disease.” Exp Cell Res 329.1 (2014): 85-93.
27.Mechali, M. “Eukaryotic DNA Replication Origins: Many Choices for Appropriate Answers.” Nat Rev Mol Cell Biol 11.10 (2010): 728-38.
28.Medic, Sandra, and Melanie Ziman. “Pax3 Across the Spectrum: From Melanoblast to Melanoma.” Critical Reviews in Biochemistry and Molecular Biology 44.2-3 (2009): 85-97.
29.Nishitani, H, S Taraviras et al. “The Human Licensing Factor for DNA Replication Cdt1 Accumulates in G1 and is Destabilized After Initiation of S-Phase.” J Biol Chem 276.48 (2001): 44905-11.
30.Pingault, V, D Ente et al. “Review and Update of Mutations Causing Waardenburg Syndrome.” Hum Mutat 31.4 (2010): 391-406.
31.Rhind, N., and D. M. Gilbert. “DNA Replication Timing.” Cold Spring Harbor Perspectives in Biology 5.8 (2013): a010132.
32.Sabharwal, R, P Verma et al. “Emergence of Micronuclei as a Genomic Biomarker.” Indian J Med Paediatr Oncol 36.4 (2015): 212-18.
33.Saksouk, N, E Simboeck, and J Dejardin. “Constitutive Heterochromatin Formation and Transcription in Mammals.” Epigenetics Chromatin 8 (2015): 3.
34.Schmitt, MW, MJ Prindle, and LA Loeb. “Implications of Genetic Heterogeneity in Cancer.” Ann N Y Acad Sci 1267 (2012): 110-16.
35.Sirbu, BM, FB Couch, and D Cortez. “Monitoring the Spatiotemporal Dynamics of Proteins At Replication Forks and in Assembled Chromatin Using Isolation of Proteins on Nascent DNA.” Nat Protoc 7.3 (2012): 594-605.
36.Swindall, AF, JA Stanley, and ES Yang. “Parp-1: Friend or Foe of DNA Damage and Repair in Tumorigenesis.” Cancers (Basel) 5.3 (2013): 943-58.
37.Walther, C, JL Guenet et al. “Pax: A Murine Multigene Family of Paired Box-Containing Genes.” Genomics 11.2 (1991): 424-34.
38.Wu, TF, YL Yao et al. “Loading of Pax3 to Mitotic Chromosomes is Mediated By Arginine Methylation and Associated With Waardenburg Syndrome.” J Biol Chem 290.33 (2015): 20556-64.
39.Wu, TF, YL Yao et al. “Pax3 Loads Onto Pericentromeric Heterochromatin During S Phase Through Parp1.” Anticancer Res 34.9 (2014): 4717-22.
40.Xu, B, Z Sun et al. “Replication Stress Induces Micronuclei Comprising of Aggregated DNA Double-Strand Breaks.” PLoS One 6.4 (2011): e18618.
41.Zeman, MK, and KA Cimprich. “Causes and Consequences of Replication Stress.” Nat Cell Biol 16.1 (2014): 2-9.
42.劉筱君. “瓦登伯格氏症突變型 Pax3 在DNA 複製期對細胞功能影響之探討.” 國立中興大學分子生物研究所碩士論文 (2016)
43.王漢宇. “瓦登伯格氏症候群中突變型 Pax3 在轉錄調控上及dapi 密集區之機制探討.” 國立中興大學分子生物研究所碩士論文 (2015)