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The Identification of Proteins That Interact with the N-Terminal Domain of Pitx2c

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The Identification of Proteins That Interact with the N-Terminal Domain of Pitx2c The identification of proteins that interact with the N-terminal domain of Pitx2c Dora Siontas A thesis submitted to McGill University in partial fulfillment of the requirement of the degree of Master of Science Department of Experimental Medicine McGill University Montreal, Quebec Canada August, 2011 © Dora Siontas Table of Contents Page ABSTRACT……………………………………………………………………..… i RÉSUMÉ………………….…………………………………………………...….. ii ACKNOWLEDGMENTS……………….……………………...…………...…... iii ABBREVIATIONS……………………...……………………………………..… iv LIST OF FIGURES ………………………………………………………..…... viii LIST OF TABLES……………………..……………………………………......... x 1. INTRODUCTION…………………………………………………………….... 1 1.1 Left-right asymmetric patterning……………………………………...…. 1 1.2 The left-right asymmetric molecular cascade………………………..…... 2 1.2.1 The left-right molecular cascade in the chick embryo..……………. 2 1.2.2 The left-right molecular cascade in the mouse embryo……….…… 7 1.3 The Pitx2 gene and protein isoforms………………………………….… 10 1.3.1 Identification of PITX2 as a mutated gene in ARS…………......… 11 1.3.2 Pitx2 function….……………………………..………………........… 11 1.3.3 Pitx2 isoforms…………………………………………..………..….. 13 1.3.4 Expression patterns of the different Pitx2 isoforms………............. 15 1.3.5 Pitx2 is important for asymmetric morphogenesis………………... 17 1.4 The Pitx2c N-terminal domain………………………………………...… 19 1.4.1 The Pitx2c N-terminal domain………………………………..…..… 19 1.4.2 Functional experiments with the Pitx2c N-terminal domain….….. 20 1.4.3 Pitx2-interacting proteins…………….……………………………... 23 2. HYPOTHESIS AND OBJECTIVES……………………………………..….. 25 2.1 Rationale…………………………………………………………………. 25 2.2 Hypothesis…………………………………………………………......… 25 2.3 Objectives……………………………………………………….……….. 26 3. MATERIALS AND METHODS………………………………………….…… 27 3.1 Cloning of the m2cN into pGBKT7………………………………………. 27 3.1.1 Midipreparation of pGBKT7…………………………………...…… 27 3.1.2 RT-PCR of m2cN cDNA…………………………………………..…. 27 3.1.3 Purification and digestion of the m2cN and the pGBKT7 vector…. 29 3.1.4 The ligation reaction and cloning………………………………....… 30 3.1.5 Minipreparation of pGBKT7-m2cN………………………………... 31 3.2 Control experiments testing the bait constructs…………………….…... 32 3.2.1 Bait transformation………………………………………………….. 32 3.2.2 Test for toxicity and autoactivation of the bait constructs…..……. 33 3.3 The two-hybrid library screen using yeast mating…………...………… 35 3.4 The identification of prey candidates that interact with the 2cN..…….. 37 3.5 GST pull-down experiments for the interacting proteins………..…….. 38 3.5.1 Preparation of the GST fusion bait proteins………………….…… 38 3.5.1.1 Cloning the m2cN into the pET14b-GST vector………...…… 38 3.5.1.2 Purification of bacterially expressed GST fusion proteins..…. 40 3.5.2 Preparation of prey proteins……………………………………….... 43 3.5.3 GST pull-down experiments…………………………………..…….. 43 4. RESULTS……………………………………………………………………… 45 4.1 The yeast two-hybrid library screen…………………………….………. 45 4.1.1 Preparation of the bait constructs…………………………….……. 45 4.1.2 Control experiments testing the bait constructs ………………….. 48 4.1.3 The yeast two-hybrid library screen of the c2cN against an adult mouse cDNA library………………………………………………… 50 4.1.3.1 Diploid yeast cells resulting from the mating of Y2HGold containing c2cN and Y187 containing the adult mouse cDNA library……………………………………………………...…… 50 4.1.3.2 Selection of interacting prey candidates on DDO/X/A and QDO/X/A……………………………………..……………..….. 56 4.1.3.3 The identification of candidate interaction partners………... 59 4.1.4 The yeast two-hybrid library screen of the m2cN against a E11 mouse cDNA library……………………….………………….…..… 61 4.1.4.1 Diploid yeast cells resulting from the mating of Y2HGold containing m2cN and Y187 containing the E11 mouse cDNA library…………………………………………………….…..… 61 4.1.4.2 Selection of interacting prey candidates on DDO/X/A and QDO/X/A……………………………………………………….. 62 4.1.4.3 The identification of candidate interaction partners……........ 62 4.1.5 Interacting protein candidate classification……………………..….. 63 4.1.5.1 Prioritization based on frequency…………………………...…. 63 4.1.5.2 Classification based on functional group…………………….... 64 4.1.5.3 Npc2, Ubc, Ube2n and Ube2e1 as 2cN candidate interaction partners……………………………………………………….…. 72 4.2 Confirmation of candidate protein interaction with the 2cN using a GST pull-down approach………………………………………………………. 79 4.2.1 Preparation of the GST fusion proteins……………………….……. 79 4.2.2 In vitro transcription/translation of prey candidate interaction partners……………………………………………………………….. 83 4.2.3 GST pull-down experiments…………………………………..…….. 83 5. DISCUSSION……………………………………………………………..…… 90 6. CONCLUSION……………………………………………….…………..….. 101 7. FUTURE DIRECTIONS………………………………………..………..…. 103 7.1 Confirming the interactions………………………………..…….….….. 103 7.2 Characterizing the co-expression of the candidate interacting proteins with Pitx2c……………………………………………………….…..….. 104 7.3 Functional experiments with the candidate interacting proteins…..… 105 7.3.1 Gain-of-function experiments…………………………………….. 105 7.3.2 Loss-of-function experiments…………………………….……….. 106 8. REFERENCES………………………………………………………….…… 108 9. APPENDIX………………………………………………………………….... 130 ABSTRACT During embryogenesis, a number of key events regulate the correct patterning of an organism, one of them being the correct positioning of internal organs. One of the factors important for this process is the homeodomain transcription factor Pitx2c, which is asymmetrically expressed in the left lateral plate mesoderm. Previous work has shown that the N-terminal domain of Pitx2c is important for left-right patterning, in that overexpression of the N-terminus randomizes the direction of heart tube looping. It is believed that the overexpressed N-terminus is antagonizing the activity of endogenous Pitx2c by competing for binding to one or more critical interaction partners. In order to better understand the role of the N-terminal domain, I have performed two different yeast two-hybrid library screens to identify proteins that interact with this domain. In the first, I used the chick Pitx2c N-terminal domain, cloned into the pGBKT7 vector in-frame with the DNA binding domain of the GAL4 transcription factor as bait, against a mouse adult cDNA library cloned in-frame with the GAL4 activation domain in the pGADT7 vector. In the second, I used a mouse Pitx2c N-terminal domain against an embryonic E11 mouse cDNA library. The resulting interacting candidates were analyzed and four were chosen based on the number of times they appeared in the screens and their function. The four chosen were Npc2, Ubc, Ube2n and Ube2e1. GST pull-down experiments were performed to confirm their interaction with the mouse and chick Pitx2c N-terminal domains, and proteins for the interacting candidates were generated using a coupled transcription- translation system. Only Ube2n and Ube2e1 were confirmed as interacting candidates, suggesting that Pitx2 may be ubiquitinated and this may be a regulatory mechanism. Future experiments will provide more insight on their role in interacting to the N- terminal domain of Pitx2c. i RÉSUMÉ Durant l'embryogenèse, un certain nombre d'événements clés réglementent la structuration correcte d'un organisme, l'un d'eux étant le positionnement correct des organes internes. Un des facteurs importants impliqué dans ce processus est le facteur de transcription homéodomaine Pitx2c, qui est asymétriquement exprimé dans le mésoderme de la plaque latérale gauche. Des études antérieures ont démontrées que le domaine N-terminal de Pitx2c est important pour la structuration gauche-droite, puisque la surexpression de l'extrémité N-terminale randomise la direction de la courbure du cœur. Nous croyons que la surexpression de la portion N-terminale de Pitx2c antagonise son activité endogène en faisant concurrence pour la liaison à un ou plusieurs partenaires d'interaction critique. Afin de mieux comprendre le rôle du domaine N-terminal de Pitx2c, j'ai effectué deux différentes sélections en utilisant la méthode de double hybride dans le but d’identifier les protéines qui interagissent avec ce domaine. Lors de la première sélection, j'ai utilisé le domaine N-terminal Pitx2c du poulet, cloné dans le vecteur pGBKT7 avec le domaine liant l'ADN du facteur de transcription GAL4 comme appât, contre une librairie adulte d’ADNc de souris avec le domaine d'activation GAL4 dans le vecteur pGADT7 comme proie. Dans la seconde, j'ai utilisé le domaine N-terminal Pitx2c de la souris contre une librairie d’ADNc d’embryon de souris E11. Les candidats qui interagissent ont été analysés et quatre ont été choisis en fonction du nombre de fois qu'ils apparaissent dans les sélections et selon leur function: Npc2, Ubc, Ube2n et Ube2e1. Des immunoprécipitations utilisant le glutathion-S-transférase comme étiquette ont été réalisées afin de confirmer l’interaction entre les partenaires potentiels et les domaines N-terminaux de Pitx2c de la souris et du poulet. Des protéines pour les candidats potentiels d’interactions avec Pitx2c ont été générées en utilisant un système de transcription-traduction. Ube2n et Ube2e1 sont les seuls candidats qui ont été confirmés comme protéines d’interaction, mettant en avant l'ubiquitination comme étant un mécanisme putatif par lequel Pitx2c agit dans la structuration gauche-droite durant le développement embryonnaire. ii ACKNOWLEDGMENTS I would like to thank my supervisor, Dr. Aimee Ryan, for giving me the opportunity to pursue my graduate research project in her lab. I am grateful for the time and effort she put into helping me through my project and guiding
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