跳到主要內容

臺灣博碩士論文加值系統

(3.235.120.150) 您好!臺灣時間:2021/08/06 02:47
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:吳芷瑄
研究生(外文):Chih-Hsuan Wu
論文名稱:SIRT6調控轉錄作用之探討
論文名稱(外文):The studies of SIRT6 in transcriptional regulation
指導教授:楊文明楊文明引用關係
指導教授(外文):Wen-Ming Yang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:62
中文關鍵詞:輔因子
外文關鍵詞:SIRT6p53Pax3cofactor
相關次數:
  • 被引用被引用:1
  • 點閱點閱:189
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
在真核生物中,染色質的結構會影響基因的表現,去乙醯酵素(histone deacetylase) 是真核細胞的一種組蛋白修飾蛋白(histone modifier),它能使染色質結構變得緊密,抑制基因的表現。去乙醯酵素 廣泛的存在於各種生物中,而SIRT6是哺乳類動物HDAC classⅢ的成員之ㄧ。過去研究發現,Sirt6基因剔除小鼠會產生與老化相關的代謝性疾病,並在一個月之內死亡,顯示了SIRT6對生物體的重要性,但目前對於SIRT6的分子層次中的機制仍尚未清楚。由於SIRT6位於細胞核中,根據過去研究發現,許多位於細胞核中的去乙醯酵素能藉由與轉錄因子的交互作用參與在轉錄的調控,因此本篇將探討SIRT6是否可作為輔因子與轉錄因子共同調控轉錄作用,影響目標基因的表現。
為了找出SIRT6參與在轉錄調控中的證據,首先分析SIRT6的基本特性:一、由螢光顯微鏡觀察到SIRT6位於細胞核中的真染色質區域。二、使用Gal4 system進行reporter assay發現SIRT6具有抑制轉錄的功能,這些結果暗示著SIRT6與轉錄作用的相關性。此外,利用純化SIRT6蛋白複合體的方式發現在細胞中,SIRT6會和輔因子KAP1、PRMT5,存在於核仁中的蛋白質B23、C23,histone H1,以及tubulin形成複合體。
為了更進一步找出能夠與SIRT6有交互作用的轉錄因子,先前實驗室已經發現SIRT6和轉錄因子PAX3有交互作用,因此本篇假設SIRT6可作為PAX3在轉錄作用的輔因子,但reporter assay的結果顯示SIRT6並不會影響PAX3目標基因MITF啟動子的轉錄活性,而在ChIP assay也證明SIRT6不會被誘導結合到MITF啟動子上,但在實驗過程中意外地發現knock down SIRT6會降低PAX3的蛋白表現量。此外,過去實驗室還發現源自瓦登柏格氏症候群病患中的PAX3(R271G)突變能改變SIRT6原有的細胞分布,而在本篇證實除了PAX3(R271G)外,其它在PAX3 R271位置的突變也會造成此現象,並且發現SIRT6和PAX3 R271突變之間的交互作用較SIRT6和PAX3強。另一方面,根據SIRT6蛋白複合體中含有KAP1的線索,利用免疫沈澱法證實了和KAP1相關的轉錄因子p53與SIRT6有交互作用,並藉由reporter assay證明SIRT6能夠調控p53的下游基因p21啟動子的轉錄活性。而針對p21啟動子做更進一步的研究,ChIP assay顯示SIRT6的確能夠被誘導結合至p21啟動子上的p53結合位。
綜合上述實驗結果得知:一、SIRT6無法作為PAX3的輔因子,但能維持PAX3在細胞中的穩定性。二、PAX3的第271個胺基酸突變和SIRT6有較強的交互作用,並改變SIRT6在細胞中的分布,可能與致病機制有所關連。三、SIRT6可作為p53的輔因子抑制下游基因p21的轉錄活性。四、SIRT6在核仁中具有功能。


In eukaryotes, chromatin structure is important for gene expression. HDACs (histone deacetylases) are histone modifiers in eukaryotes, which can condense chromatin structure and cause gene silencing. HDACs exist in many organisms, and SIRT6 is a member of the classⅢ HDACs in mammals. Previous studies showed that Sirt6 knock out mice have many degenerative diseases related to aging and die early. This indicated the importance of SIRT6 in organisms, but the molecular mechanism of SIRT6 is still unclear. Since SIRT6 localizes to the nucleus, and some nuclear HDAC members can interact with transcription factors and be involved in transcriptional regulation. Therefore, here we want to understand if SIRT6 can function as a cofactor of transcription factors and regulates the expression of target genes.
In order to find the evidence of SIRT6 in transcriptional regulation, first, we analyzed the basic characteristics of SIRT6: 1. SIRT6 locates on euchromatin of nucleus. 2. SIRT6 represses the transcriptional activity in Gal4 system. These results revealed the relationship between SIRT6 and transcriptional regulation. In addition, we found KAP1, PRMT5, some nucleolar proteins, hitone H1, and tubulin can form a complex with SIRT6 by SIRT6 protein purification.
Moreover, because SIRT6 has been shown to interact with transcription factor PAX3, we suggested that SIRT6 functions as a cofactor of PAX3. But reporter assay showed SIRT6 has no effects on MITF promoter of PAX3’s target gene, and ChIP assay revealed that SIRT6 couldn’t be recruited to MITF promoter by PAX3. However, we found that knock down SIRT6 reduced the expression of PAX3. Furthermore, previous our lab has been showed that PAX3 (R271G) changes the distribution of SIRT6. In this study, we found other PAX3 (R271X) mutants also change SIRT6’s distribution, and the interaction between SIRT6 and PAX3 (R271X) is stronger than PAX3 wild type. On the other hand, from the clue of KAP1 is a component of SIRT6 protein complex; since KAP1 interacts with transcription factor p53, we found that SIRT6 can also interact with p53 by co-immunoprecipitation. Reporter assay indicated that SIRT6 represses p53 target promoter, p21. Focus on p21 promoter, we found that SIRT6 could be recruited to two p53 binding sites on p21 promoter.
In summary, these results demonstrated that: (1) SIRT6 cannot function as a cofactor of PAX3, but SIRT6 could maintain the stability of PAX3. (2) PAX3 (R271X) mutants interact with SIRT6 stronger and alter the distribution of SIRT6, this phenomenon might relate to disease mechanism of Waardenburg syndrome. (3) SIRT6 functions as a cofactor of p53 to regulate the transcription activity of p21. (4) SIRT6 has some function in the nucleolus.



壹、緒論 ----------------------------------------------------------------------------------------1
一、 前言-----------------------------------------------------------------------------------------1
二、 SIRT6的背景介紹------------------------------------------------------------------------1
(一) SIRT6的發現-------------------------------------------------------------------------1
(二) SIRT6的功能區域分析 ------------------------------------------------------------2
(三) SIRT6的生理功能-------------------------------------------------------------------2
(四) SIRT6在分子層次的調控機制----------------------------------------------------3
三、 真核生物的轉錄調控機制--------------------------------------------------------------4
四、 染色體結構對轉錄機制的影響--------------------------------------------------------5
(一) 染色質的結構 -----------------------------------------------------------------------5
(二) 染色體的重塑 ----------------------------------------------------------------------6
(三) 組蛋白N端的轉譯後修飾 ---------------------------------------------------------6
五、 研究目的-----------------------------------------------------------------------------------8
六、 研究策略-----------------------------------------------------------------------------------8

貳、材料與方法-------------------------------------------------------------------------------9
一、 構築質體 (Plasmid) DNA---------------------------------------------------------------9
二、 細胞培養 (Cell culture)-----------------------------------------------------------------10
三、基因轉移感染 (Transfection) ----------------------------------------------------------10
四、轉錄活性測定 (Transcriptional assay)------------------------------------------------11
(一) 螢光酵素報告基因檢測 (Luciferase assay)-----------------------------------11
1. GAL4 system--------------------------------------------------------------------11
2. Native system -------------------------------------------------------------------11
(二) 反轉錄聚合酵素鏈鎖反應(RT-PCR)--------------------------------------12
五、 西方墨點法 (Western Blot)------------------------------------------------------------12
六、 免疫沉澱法 (co-immunoprecipitation)-----------------------------------------------13
七、 免疫螢光染色法 (Immunofluorescence) --------------------------------------------13
八、 純化蛋白複合體 (Immuniaffinity) ---------------------------------------------------14
九、 染色質免疫沈澱法(Chromatin immunoprecipitation assay) ---------------------14

參、結果 ---------------------------------------------------------------------------------------16
一、 SIRT6的基本特性 ----------------------------------------------------------------------16
(一) SIRT6位於細胞核的真染色質區域 --------------------------------------------16
(二) SIRT6不會座落在有絲分裂時期的染色體上 --------------------------------16
(三) SIRT6在Gal4 system具有抑制轉錄的能力 -----------------------------------16
(四) SIRT6的蛋白複合體 --------------------------------------------------------------17
二、 SIRT6和轉錄因子PAX3的分子機制------------------------------------------------18
(一) SIRT6和轉錄因子PAX3有交互作用 ------------------------------------------18
(二) SIRT6和PAX3在細胞核中的分布情形不會互相影響 ---------------------18
(三) SIRT6不會影響PAX3目標基因MITF 啟動子的轉錄活性----------------18
(四) SIRT6不會藉由PAX3被誘導結合到MITF 啟動子-------------------------19
(五) SIRT6可以穩定PAX3在細胞中的蛋白表現量------------------------------19
(六) PAX3的Homeobox domain突變會改變SIRT6在細胞中的分布 ---20
(七) SIRT6和PAX3的Homeobox domain突變有較強的交互作用 ----------20
三、 SIRT6和轉錄因子p53的分子機制---------------------------------------------------21
(一) SIRT6和轉錄因子p53有交互作用---------------------------------------------21
(二) SIRT6和p53在細胞核中有共位的現象---------------------------------------21
(三) SIRT6具有抑制p53目標基因轉錄活性的能力------------------------------22
(四) SIRT6能夠被誘導結合到p21啟動子上的p53結合位 ---------------------23
(五) 在沒有p53結合位情況下SIRT6依舊能夠抑制p21啟動子的轉錄活性 -24

肆、討論---------------------------------------------------------------------------------------25
一、 SIRT6參與轉錄調控的機制 ---------------------------------------------------------25
(一) SIRT6與轉錄因子PAX3的分子機制-------------------------------------------26
(二) SIRT6可作為p53的輔因子參與在p21的轉錄調控--------------------------28
(三) SIRT6參與在特定轉錄因子的調控機制--------------------------------------30
二、 SIRT6在細胞中的其他功能 ---------------------------------------------------------31


伍、參考文獻---------------------------------------------------------------------------------33
陸、圖表 ---------------------------------------------------------------------------------------38
柒、附圖 ---------------------------------------------------------------------------------------57


Mostoslavsky, R., Chua, K.F., Lombard, D.B., Pang, W.W., Fischer, M.R.,
Gellon, L., Liu, P., Mostoslavsky, G., Franco, S., Murphy, M.M., et al. (2006).
Genomic instability and aging-like phenotype in the absence of mammalian
SIRT6. Cell 124, 315–329.
Fabrizio, P., Gattazzo, C., Battistella, L., et al. (2005). Sir2 blocks extreme lifespan extension. Cell 123, 655–67.
Michishita, E., Park, J.Y., Burneskis, J.M., Barrett, J.C., and Horikawa, I.
(2005). Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins. Mol. Biol. Cell. 16, 4623–4635.
Michishita, E., McCord, R.A., Berber, E., Kioi, M., Padilla-Nash, H., Damian, M., Cheung, P., Kusumoto, R., Kawahara, T.L., Barrett, J.C., et al. (2008). SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin. Nature 452, 492–496.
Kawahara T.L., Michishita E., Adler A.S., Damian M., Berber E., Lin M.,
McCord R.A., Ongaigui K.C., Boxer L.D., Chang H.Y., et al. (2009). SIRT6
links histone H3 lysine 9 deacetylation to NF-kappaB-dependent gene
expression and organismal life span. Cell 136, 19-21.
Zhong L., D''Urso A., Toiber D., Sebastian C., Henry R.E., et al. (2010). The
histone deacetylase Sirt6 regulates glucose homeostasis via Hif1alpha. Cell 140, 280-293
Lombard, D.B., Schwer, B., Alt, F.W., and Mostoslavsky, R. (2008). SIRT6 in DNA repair, metabolism and ageing. J. Intern. Med . 263, 128–141.
Haigis, M.C., and Guarente, L.P. (2006). Mammalian sirtuins—emerging roles in physiology, aging, and calorie restriction. Genes Dev. 20, 2913–2921.
Nagaich, A.K., V.B. Zhurkin, H. Sakamoto, A.A. Gorin, G.M. Clore, A.M.
Gronenborn, E. Appella, and R.E. Harrington. (1997). Architectural
accommodation in the complex of four p53 DNA binding domain peptides with the p21 / waf1 / cip1 DNA response element. J. Biol. Chem. 272, 14830–14841.
Deng, C., P. Zhang, J.W. Harper, S.J. Elledge, and P. Leder. (1995). Mice
lacking p21CIP1 / WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell 82, 675–684.
Resnick-Silverman L., St Clair S., Maurer M., Zhao K., Manfredi JJ. (1998).
Identification of a novel class of genomic DNA-binding sites suggests a
mechanism for selectivity in target gene activation by the tumor suppressor
protein p53. Genes Dev. 1, 2102-2107.
Liszt, G., Ford, E., Kurtev, M., and Guarente, L. (2005). Mouse Sir2 homolog SIRT6 is a nuclear ADP-ribosyltransferase. J. Biol. Chem. 280, 21313–21320.
Frye, R. A. (2000). Phylogenetic classification of prokaryotic and eukaryotic
Sir2-like proteins. Biochem. Biophys. Res. Commun. 273, 793–798.
Heilbronn, L. K., and Ravussin, E. (2003). Calorie restriction and aging: review of the literature and implications for studies in humans. Am. J. Clin. Nutr. 78, 361–369.
Imai, S., Armstrong, C. M., Kaeberlein, M., and Guarente, L. (2000). Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403, 795– 800.
North, B. J., and Verdin, E. (2004). Sirtuins: Sir2-related NAD-dependent
protein deacetylases. Genome Biol. 5, 224.
Michishita, E., Park, J.Y., Burneskis, J.M., Barrett, J.C., and Horikawa, I.
(2005). Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins. Mol. Biol. Cell 16, 4623–4635.
Lombard, D.B., Chua, K.F., Mostoslavsky, R., Franco, S., Gostissa, M., and
Alt, F.W. (2005). DNA repair, genome stability, and aging. Cell 120, 497–512.
Kaeberlein, M., McVey, M., and Guarente, L. (1999). The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms. Genes Dev. 13, 2570–2580.
Riley, T., Sontag, E., Chen, P., and Levine, A. (2008). Transcriptional control of human p53-regulated genes. Nat Rev Mol Cell Biol 9, 402-412.
Mayanil, C. S., George, D., Freilich, L., Miljan, E. J., Mania-Farnell, B.,
McLone, D. G., and Bremer, E. G. (2001). Microarray analysis detects novel Pax3 downstream target genes. J Biol Chem. 276, 49299-49309.
Luger, K., and Richmond, T. J. (1998a). DNA binding within the nucleosome core. Curr Opin Struct Biol 8, 33-40.
Luger, K., and Richmond, T. J. (1998b). The histone tails of the nucleosome. Curr Opin Genet Dev. 8, 140-146.
Kouzarides, T. (2007). Chromatin modifications and their function. Cell 128, 693-705.
Hsieh, M. J., Yao, Y. L., Lai, I. L., and Yang, W. M. (2006). Transcriptional
repression activity of PAX3 is modulated by competition between corepressor KAP1 and heterochromatin protein 1. Biochem Biophys Res Commun 349, 573-581.
Chalepakis, G., Jones, F. S., Edelman, G. M., and Gruss, P. (1994). Pax-3
contains domains for transcription activation and transcription inhibition. Proc Natl Acad Sci USA. 91, 12745-12749.
Wegner, M., and Goossens, M. (2000). Interaction among SOX10, PAX3 and MITF, three genes altered in Waardenburg syndrome. Hum Mol Genet. 9, 1907-1917.
Balczarek, K. A., Lai, Z. C., and Kumar, S. (1997). Evolution of functional
diversification of the paired box (Pax) DNA-binding domains. Mol Biol Evol. 14, 829-842.
Koryakov, D. E. (2006). Histone modification and regulation of chromatin
function. Genetika. 42,1170-1185.
Kouzarides, T. (2007). Chromatin modifications and their function. Cell 128, 693-705.
Gray, S.G., and Ekstrom, T.J. (2001). The human histone deacetylase family. Exp Cell Res. 262, 75-83.
Grunstein, M. (1997). Histone acetylation in chromatin structure and
transcription. Nature 389, 349-352.
Turner, B.M. (2002). Cellular memory and the histone code. Cell 111, 285-291.
Global histone acetylation and deacetylation in yeast. Nature 408, 495-498.
Workman, J.L., and Kingston, R.E. (1998). Alteration of nucleosome structure as a mechanism of transcriptional regulation. Annu Rev Biochem. 67, 545-579.
Wolffe, A.P., and Hayes, J.J. (1999). Chromatin disruption and modification. Nucleic Acids Res. 27, 711-720.
Vaziri H., Dessain S.K., Ng Eaton E., Imai S.I., Frye R.A., Pandita T.K.,
Guarente L. and Weinberg R.A. (2001). hSIRT1 (SIRT1) functions as an NADdependent p53 deacetylase. Cell 107, 149-159.
Lang D., Lu M.M., Huang L., Engleka K.A., Zhang M., Chu E.Y., Lipner S.,
Skoultchi A., Millar S.E. and Epstein J.A. (2005). Pax3 functions at a nodal
point in melanocyte stem cell differentiation. Nature 433, 884-887.
Jang H, Choi S. Y., Cho E. J., Youn H.D. (2009). Cabin1 restrains p53 activity on chromatin. Nat Struct Mol Biol. 16, 910-915.
Hsieh Mei-Ju. (2003). Mechanism of PAX3 transcription repression activity.
Insitue of Molecular Biology/National Chung Hsing University Master Thesis.
Lin Tung-Ping. (2004). Studies of HDAC10 and its associated factors. Institue of Molecular Biology/National Chung Hsing University Master Thesis.

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top