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研究生:李舜淵
研究生(外文):Shun-Yuan Lee
論文名稱:Nolz-1及其交互作用蛋白的生化功能探討
論文名稱(外文):Biochemical study of Nolz-1 protein and its interacting proteins
指導教授:劉福清劉福清引用關係
指導教授(外文):Fu-Chin Liu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:神經科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:86
中文關鍵詞:Nolz-1蛋白
外文關鍵詞:Nolz-1
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Nolz-1 是一個大量表現於發育中前腦LGE的一個核蛋白,並且是NET蛋白質家族 (包括: Noc, Nlz, Elbow 及 Tlp-1)的其中一員。在過去的研究發現,Nolz-1在神經細胞株ST14A和Neuro2A中為一個轉錄抑制因子;然而在腎臟細胞株中,卻扮演轉錄活化因子的角色。而Nolz-1在其他種的同源系基因,Nlz-1和Elbow,也都看到了Nolz-1於神經系統發育上是做為轉錄抑制因子的證據。此外,Nolz-1也已知可以和共同抑制因子Grg3,Grg4及Grg5有交互作用的能力。在我的研究中,我利用了共同免疫沉降法證明了,這種交互作用是經由FKPY motif及Buttonhead box 這兩個位於Nolz-1蛋白上的功能性區域來完成。再者,我們也利用報導基因的方式去證明Nolz-1的確是經由和此類共同抑制因子中的Grg4交互作用而達到抑制轉錄的效果。
在我論文的第二部分,是探討Nolz-1和典型Wnt訊息傳遞鏈的關聯性。這個研究是在P19 細胞株的系統下去實行的。在報導基因的實驗中,我們發現了在Nolz-1啟動子,位於Nolz-1轉錄起始點上游200 bps至2400 bps的區域可能有Tcf/Lef1反應原件。利用西方墨點法,我們卻發現不論是持續活化典型Wnt訊息傳遞鏈,或是利用DKK1這個典型Wnt訊息傳遞鏈的抑制蛋白去抑制傳遞鏈的活性,都不會改變Nolz-1蛋白質的表現量。而當我們抑制Nolz-1蛋白質表現時,透過報導基因的方法,觀察到典型Wnt訊息傳遞鏈的活性增加了。由以上的實驗結果,可以知道Nolz-1並非是由典型Wnt訊息傳遞鏈所調控的下游基因,相反的,Nolz-1反而能逆向調控此訊息傳遞鏈的活性。此外,我們也認為Nolz-1可以和典型Wnt訊息傳遞鏈中的受動器,Tcf/Lef1,的蛋白家族成員(包括: TCF7, LEF1, TCF7L1 及 Tcf7l2 )有交互作用,因為他們同時都可以和Groucho這類的共同抑制因子做結合的動作。然而,我們卻無法利用免疫沉降法看到預期的結果。因為由已知的結果,我們知道Nolz-1可以調控典型Wnt訊息傳遞鏈,而這個訊息鏈最廣為人知的功能便是調節細胞周期的進行;基於這個原因,我們更進一步的去觀察當Nolz-1失去功能後,細胞的周期會不會因為典型Wnt訊息傳遞鏈產生變化而有所改變。從西方墨點法對於細胞增殖標識,PCNA,觀察的結果看來,抑制了Nolz-1的作用,並不會對細胞增殖有什麼影響;而,細胞存活實驗卻發現抑制了Nolz-1後,細胞的存活率卻上升了。
我論文的最後一個部分是利用蛋白質體學的分析方式去尋找其他可以和Nolz-1交互作用的蛋白質。我唯一分析到的蛋白質是Btbd11,然而,從它座落於HEK293T細胞的細胞質中,不像Nolz-1是一個核蛋白。此外,Btbd11在發育中大腦的表現區域來看,主要是表現在大腦皮質的島葉,丘腦,及後腦區域;在Nolz-1大量表現的前腦LGE中,並沒有發現其明顯地存在。因此,Btbd11可能是偽陽性的候選蛋白。經由免疫沉降法,我們證實這個猜想。
總括來說,我研究主要的發現就是確定了Nolz-1是利用FKPY motif及Buttonhead box這兩個區域,來和Groucho共同抑制因子結合。再者,我也發現了Nolz-1可以在神經誘發時期逆向地調控典型Wnt訊息傳遞鏈的活性,因此認為Nolz-1可能可以經由此調控來影響神經的發育。
Nolz-1 is a nuclear protein enriched in the subventricular zone (SVZ) of lateral ganglionic eminence (LGE) in developing ventral telencephalon, and is one of the members of NET family (Noc, Nlz, Elbow, and Tlp-1). Previous studies have shown that Nolz-1 is a transcriptional repressor in ST14A and Neuro2A neural cell lines, but Nolz-1 is a transcriptional activator in non-neural HEK293T cells. Studies of Nolz-1 homologues in zebrafish, Nlz-1, and in Drosophalia, Elbow, also indicate that Nolz-1 is a transcriptional repressor in neurons. Nolz-1 could interact with Groucho (Grg), a transcriptional co-repressor, as other members of NET family do. By co-immunoprecipitation assay, I found that Nolz-1 interacted with Grg5 via the protein domains containing putative Groucho binding motif, FKPY, and the Buttonhead box. Furthermore, using reporter gene assay, I demonstrated that over-expression of Grg4 could convert Nolz-1 from transcriptional activator into repressor in HEK293T cells, suggesting the functional significance of the interaction between Nolz-1 and Grg4.
The second part of my thesis is to study the interaction between Nolz-1 and β-catenin/Tcf-mediated Wnt signaling (canonical Wnt signal pathway). The investigation was carried out in P19 cells. By reporter gene assays, the promoter activities of Nolz-1 were analyzed, and these results showed that the putative Tcf/Lef1 response elements might be located in the promoter region between 200 bps to 2400 bps upstream from the Nolz-1 transcriptional start site. By immunoblotting assays, neither activation of Wnt signaling by constitutively active β-catenin or inhibition of Wnt signaling by DKK1, which is a secreted antagonist of the canonical Wnt signaling, did not change the protein levels of Nolz-1 in P19 cells. Further, knockdown of Nolz-1 leads to the increases of canonical Wnt signaling in neural induction of RA-aggregated P19 cells. These results suggested that Nolz-1 is not the downstream target gene of the canonical Wnt signal pathway in neural induction of P19 cells, but Nolz-1 could suppress canonical Wnt signaling activity. In addition, I hypothesized that Nolz-1 protein might interact with Tcf/Lef1 protein family, which are the transcription factors and effectors of canonical Wnt signaling, because both of them were associated with Groucho protein. Unfortunately, this hypothesis could not be verified by the co-immunoprecipitation assay of Nolz-1 and TCF7, LEF1, TCF7L1 or Tcf7l2 proteins. I studied the cell proliferation of P19 cells while knocking down Nolz-1 with Nolz-1 shRNAs. Knocking down Nolz-1 did not affect the expression levels of PCNA, a marker for cell proliferation, but the cell number assayed by cell viability test was increased. Taken together, Nolz-1 could regulate the canonical Wnt signaling activity in neural induction of P19 cells.
The last part of my thesis is to identify other interacting proteins of Nolz-1 by proteomic approach using MALDI-TOF. One protein that I found is Btbd11. However, double immunostaining shows that Btbd11 is localized in cytoplasma whereas Nolz-1 is localized in the nucleus, and the results of in situ hybridization and RT-PCR indicate that Btbd11 is enriched in the insular cortices, posterior thalamus and the hindbrain region but not in the ventral telencephalon of E13.5 mouse embryo. Because the Nolz-1 and Btbd11 protein interaction could not be verified by co-immunoprecipitation assay, Btbd11 might be a false-positive protein identified by proteomic approach.
In summary, my studies demonstrate that Nolz-1 is a transcriptional repressor, and it interacts with Groucho protein through the protein domains containing Buttonhead box and the FKPY Groucho binding motif. In addition, Nolz-1 could suppress the canonical Wnt signal pathway during neural induction of P19 clls, suggesting that Nolz-1 may control neural development by regulating canonical Wnt signal activity.
CONTENTS


ABSTRACT IN CHINESE••••••••••••••••••••••1

ABSTRACT••••••••••••••••••••••••••••3

INTRODUCTION•••••••••••••••••••••••••5

MATERIALS AND METHODS•••••••••••••••••••9

RESULTS••••••••••••••••••••••••••••24

DISCUSSION•••••••••••••••••••••••••••36

REFERENCES••••••••••••••••••••••••••45

FIGURES AND TABLES••••••••••••••••••••••52
Reference List

Backman M, Machon O, Mygland L, van den Bout CJ, Zhong W, Taketo MM, Krauss S (2005) Effects of canonical Wnt signaling on dorso-ventral specification of the mouse telencephalon. Dev Biol 279:155-168.
Bain G, Ray WJ, Yao M, Gottlieb DI (1994) From embryonal carcinoma cells to neurons: the P19 pathway. Bioessays 16:343-348.
Brantjes H, Roose J, van De Wetering M, Clevers H (2001) All Tcf HMG box transcription factors interact with Groucho-related co-repressors. Nucleic Acids Res 29:1410-1419.
Brown JD, Dutta S, Bharti K, Bonner RF, Munson PJ, Dawid IB, Akhtar AL, Onojafe IF, Alur RP, Gross JM, Hejtmancik JF, Jiao X, Chan WY, Brooks BP (2009) Expression profiling during ocular development identifies 2 Nlz genes with a critical role in optic fissure closure. Proc Natl Acad Sci U S A 106:1462-1467.
Campbell K (2003) Dorsal-ventral patterning in the mammalian telencephalon. Curr Opin Neurobiol 13:50-56.
Castelo-Branco G, Rawal N, Arenas E (2004) GSK-3beta inhibition/beta-catenin stabilization in ventral midbrain precursors increases differentiation into dopamine neurons. J Cell Sci 117:5731-5737.
Castelo-Branco G, Wagner J, Rodriguez FJ, Kele J, Sousa K, Rawal N, Pasolli HA, Fuchs E, Kitajewski J, Arenas E (2003) Differential regulation of midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a. Proc Natl Acad Sci U S A 100:12747-12752.
Chang CW, Tsai CW, Wang HF, Tsai HC, Chen HY, Tsai TF, Takahashi H, Li HY, Fann MJ, Yang CW, Hayashizaki Y, Saito T, Liu FC (2004) Identification of a developmentally regulated striatum-enriched zinc-finger gene, Nolz-1, in the mammalian brain. Proc Natl Acad Sci U S A 101:2613-2618.
Cheah PY, Meng YB, Yang X, Kimbrell D, Ashburner M, Chia W (1994) The Drosophila l(2)35Ba/nocA gene encodes a putative Zn finger protein involved in the development of the embryonic brain and the adult ocellar structures. Mol Cell Biol 14:1487-1499.
Chenn A, Walsh CA (2002) Regulation of cerebral cortical size by control of cell cycle exit in neural precursors. Science 297:365-369.
Chung KH, Hart CC, Al-Bassam S, Avery A, Taylor J, Patel PD, Vojtek AB, Turner DL (2006) Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155. Nucleic Acids Res 34:e53.
Costoya JA (2007) Functional analysis of the role of POK transcriptional repressors. Brief Funct Genomic Proteomic 6:8-18.
Coyle-Rink J, Del Valle L, Sweet T, Khalili K, Amini S (2002) Developmental expression of Wnt signaling factors in mouse brain. Cancer Biol Ther 1:640-645.
Dorfman R, Glazer L, Weihe U, Wernet MF, Shilo BZ (2002) Elbow and Noc define a family of zinc finger proteins controlling morphogenesis of specific tracheal branches. Development 129:3585-3596.
Gasperowicz M, Otto F (2005) Mammalian Groucho homologs: redundancy or specificity? J Cell Biochem 95:670-687.
Graybiel AM (2000) The basal ganglia. Curr Biol 10:R509-511.
Guillemot F (2005) Cellular and molecular control of neurogenesis in the mammalian telencephalon. Curr Opin Cell Biol 17:639-647.
Gulacsi AA, Anderson SA (2008) Beta-catenin-mediated Wnt signaling regulates neurogenesis in the ventral telencephalon. Nat Neurosci 11:1383-1391.
Haegele L, Ingold B, Naumann H, Tabatabai G, Ledermann B, Brandner S (2003) Wnt signalling inhibits neural differentiation of embryonic stem cells by controlling bone morphogenetic protein expression. Mol Cell Neurosci 24:696-708.
Hirabayashi Y, Itoh Y, Tabata H, Nakajima K, Akiyama T, Masuyama N, Gotoh Y (2004) The Wnt/beta-catenin pathway directs neuronal differentiation of cortical neural precursor cells. Development 131:2791-2801.
Hoyle J, Tang YP, Wiellette EL, Wardle FC, Sive H (2004) nlz gene family is required for hindbrain patterning in the zebrafish. Dev Dyn 229:835-846.
Iuchi S (2001) Three classes of C2H2 zinc finger proteins. Cell Mol Life Sci 58:625-635.
Ji SJ, Periz G, Sockanathan S (2009) Nolz1 is induced by retinoid signals and controls motoneuron subtype identity through distinct repressor activities. Development 136:231-240.
Katoh M (2002) Regulation of WNT signaling molecules by retinoic acid during neuronal differentiation in NT2 cells: threshold model of WNT action (review). Int J Mol Med 10:683-687.
Kawano Y, Kypta R (2003) Secreted antagonists of the Wnt signalling pathway. J Cell Sci 116:2627-2634.
Kelly KF, Daniel JM (2006) POZ for effect--POZ-ZF transcription factors in cancer and development. Trends Cell Biol 16:578-587.
Kicman AT, Parkin MC, Iles RK (2007) An introduction to mass spectrometry based proteomics-detection and characterization of gonadotropins and related molecules. Mol Cell Endocrinol 260-262:212-227.
Lange C, Mix E, Rateitschak K, Rolfs A (2006) Wnt signal pathways and neural stem cell differentiation. Neurodegener Dis 3:76-86.
Li J, Mahajan A, Tsai MD (2006) Ankyrin repeat: a unique motif mediating protein-protein interactions. Biochemistry 45:15168-15178.
Luque CM, Milan M (2007) Growth control in the proliferative region of the Drosophila eye-head primordium: the elbow-noc gene complex. Dev Biol 301:327-339.
Lyu J, Costantini F, Jho EH, Joo CK (2003) Ectopic expression of Axin blocks neuronal differentiation of embryonic carcinoma P19 cells. J Biol Chem 278:13487-13495.
Maretto S, Cordenonsi M, Dupont S, Braghetta P, Broccoli V, Hassan AB, Volpin D, Bressan GM, Piccolo S (2003) Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors. Proc Natl Acad Sci U S A 100:3299-3304.
McBurney MW (1993) P19 embryonal carcinoma cells. Int J Dev Biol 37:135-140.
McBurney MW, Jones-Villeneuve EM, Edwards MK, Anderson PJ (1982) Control of muscle and neuronal differentiation in a cultured embryonal carcinoma cell line. Nature 299:165-167.
Merrill RA, Ahrens JM, Kaiser ME, Federhart KS, Poon VY, Clagett-Dame M (2004) All-trans retinoic acid-responsive genes identified in the human SH-SY5Y neuroblastoma cell line and their regulated expression in the nervous system of early embryos. Biol Chem 385:605-614.
Molotkova N, Molotkov A, Duester G (2007) Role of retinoic acid during forebrain development begins late when Raldh3 generates retinoic acid in the ventral subventricular zone. Dev Biol 303:601-610.
Montcouquiol M, Crenshaw EB, 3rd, Kelley MW (2006) Noncanonical Wnt signaling and neural polarity. Annu Rev Neurosci 29:363-386.
Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, Kinzler KW (1997) Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 275:1787-1790.
Muroyama Y, Kondoh H, Takada S (2004) Wnt proteins promote neuronal differentiation in neural stem cell culture. Biochem Biophys Res Commun 313:915-921.
Nakamura M, Runko AP, Sagerstrom CG (2004) A novel subfamily of zinc finger genes involved in embryonic development. J Cell Biochem 93:887-895.
Oosterwegel M, van de Wetering M, Timmerman J, Kruisbeek A, Destree O, Meijlink F, Clevers H (1993) Differential expression of the HMG box factors TCF-1 and LEF-1 during murine embryogenesis. Development 118:439-448.
Otero JJ, Fu W, Kan L, Cuadra AE, Kessler JA (2004) Beta-catenin signaling is required for neural differentiation of embryonic stem cells. Development 131:3545-3557.
Perez-Torrado R, Yamada D, Defossez PA (2006) Born to bind: the BTB protein-protein interaction domain. Bioessays 28:1194-1202.
Pishvaian MJ, Byers SW (2007) Biomarkers of WNT signaling. Cancer Biomark 3:263-274.
Runko AP, Sagerstrom CG (2003) Nlz belongs to a family of zinc-finger-containing repressors and controls segmental gene expression in the zebrafish hindbrain. Dev Biol 262:254-267.
Runko AP, Sagerstrom CG (2004) Isolation of nlz2 and characterization of essential domains in Nlz family proteins. J Biol Chem 279:11917-11925.
Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH (2004) Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 10:55-63.
Schagger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368-379.
Schuurmans C, Guillemot F (2002) Molecular mechanisms underlying cell fate specification in the developing telencephalon. Curr Opin Neurobiol 12:26-34.
Smolich BD, Papkoff J (1994) Regulated expression of Wnt family members during neuroectodermal differentiation of P19 embryonal carcinoma cells: overexpression of Wnt-1 perturbs normal differentiation-specific properties. Dev Biol 166:300-310.
Stogios PJ, Downs GS, Jauhal JJ, Nandra SK, Prive GG (2005) Sequence and structural analysis of BTB domain proteins. Genome Biol 6:R82.
Tseng AS, Hariharan IK (2002) An overexpression screen in Drosophila for genes that restrict growth or cell-cycle progression in the developing eye. Genetics 162:229-243.
Watanabe K, Kamiya D, Nishiyama A, Katayama T, Nozaki S, Kawasaki H, Watanabe Y, Mizuseki K, Sasai Y (2005) Directed differentiation of telencephalic precursors from embryonic stem cells. Nat Neurosci 8:288-296.
Weihe U, Dorfman R, Wernet MF, Cohen SM, Milan M (2004) Proximodistal subdivision of Drosophila legs and wings: the elbow-no ocelli gene complex. Development 131:767-774.
Yan JX, Wait R, Berkelman T, Harry RA, Westbrook JA, Wheeler CH, Dunn MJ (2000) A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21:3666-3672.
Yoda A, Kouike H, Okano H, Sawa H (2005) Components of the transcriptional Mediator complex are required for asymmetric cell division in C. elegans. Development 132:1885-1893.
Zechner D, Fujita Y, Hulsken J, Muller T, Walther I, Taketo MM, Crenshaw EB, 3rd, Birchmeier W, Birchmeier C (2003) beta-Catenin signals regulate cell growth and the balance between progenitor cell expansion and differentiation in the nervous system. Dev Biol 258:406-418.
Zhao X, Yang Y, Fitch DH, Herman MA (2002) TLP-1 is an asymmetric cell fate determinant that responds to Wnt signals and controls male tail tip morphogenesis in C. elegans. Development 129:1497-1508.
Zhu CC, Dyer MA, Uchikawa M, Kondoh H, Lagutin OV, Oliver G (2002) Six3-mediated auto repression and eye development requires its interaction with members of the Groucho-related family of co-repressors. Development 129:2835-2849.
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