Yale University EliScholar – A Digital Platform for Scholarly Publishing at Yale Yale Medicine Thesis Digital Library School of Medicine 12-2012 Pumilio Proteins Regulate Translation in Embryonic Stem Cells and are Essential for Early Embryonic Development Katherine Elizabeth Uyhazi Yale University. Follow this and additional works at: http://elischolar.library.yale.edu/ymtdl Part of the Medicine and Health Sciences Commons Recommended Citation Uyhazi, Katherine Elizabeth, "Pumilio Proteins Regulate Translation in Embryonic Stem Cells and are Essential for Early Embryonic Development" (2012). Yale Medicine Thesis Digital Library. 2189. http://elischolar.library.yale.edu/ymtdl/2189 This Open Access Dissertation is brought to you for free and open access by the School of Medicine at EliScholar – A Digital Platform for Scholarly Publishing at Yale. It has been accepted for inclusion in Yale Medicine Thesis Digital Library by an authorized administrator of EliScholar – A Digital Platform for Scholarly Publishing at Yale. For more information, please contact [email protected]. ABSTRACT Pumilio Proteins Regulate Translation in Embryonic Stem Cells and are Essential for Early Embryonic Development Katherine E. Uyhazi 2012 Embryonic stem (ES) cells are defined by their dual abilities to self-renew and to differentiate into any cell type in the body. This vast potential is precisely controlled by spatial and temporal gene regulation at transcriptional, post-transcriptional, and epigenetic levels. Recent studies have revealed several transcription factors that are essential for stem cell self-renewal and pluripotency, but the role of translational control in ES cells is poorly understood. Translational control is a fundamental mechanism of gene regulation during early development, and likely explains the discrepancies between the transcriptome and proteome profiles of stem cells and their differentiated progeny. Pumilio proteins are well-characterized RNA-binding translational repressors that are required for germline stem cell maintenance in Drosophila. However, relatively little is known about the two mammalian Pumilio proteins, Pumilio 1 (Puml) and Pumilio 2 (Pum2). In this dissertation I characterize the mRNA targets, protein partners, and in vitro and in vivo function of Puml and Pum2. Puml - and Pum2-deficient mouse embryonic stem cell (mES) lines and conditional knockout mice were generated as a means to unravel the function of Pumilio proteins in ES cells and during early development. Puml -/- and Pum2 -/- ES cells grow more slowly than wild type ES cells but remain self-renewing and pluripotent. Puml-/- 1 and Pum2 -/- mice are fertile and viable. Puml-/- mice are smaller than their littermates, have a hunched appearance that becomes more prominent with age, frequently develop ulcerative dermatitis, and have disorganized, blunted intestinal villi compared to wild type mice. Puml+/-;Pum2-/- mice are viable, Puml-/-; Pum2 +/- mice are born alive but have no oral intake and die within 24 hours, and Pum 1-/-; Pum2 -/- double knockout animals are embryonic lethal by e8.5. Puml and Pum2 are highly expressed in the cytoplasm of mES cells. RNA Immunoprecipitation-Microarray (RIP-Chip) analysis of mES lysate reveals that Puml binds to 1947 mRNAs and Pum2 binds to 437 mRNAs that comprise almost a complete subset of Puml targets. Transcription factors, genes involved in cell cycle control, and genes involved in embryonic patterning are significantly enriched among the mRNA targets of both Puml and Pum2. Several targets including Cyclin E, Cyclin Bl, and Pum2 are translationally repressed by Pum 1, as indicated by changes in protein level without corresponding changes in mRNA level. In mES cells, Puml is part of a -450 kDa protein complex and Pum2 is part of a -350 kDa complex as shown by size exclusion chromatography. Co-immunoprecipitation and mass spectrometry were used to identify three novel binding partners of Puml: Anaphase-promoting complex subunit 1 (APC1), Regulator of nonsense transcripts 1 (RENT1), and Zinc Finger Protein 198 (ZNF198). Overall, this study reveals an essential function of mammalian Pumilio proteins during early embryogenesis, identifies mRNA targets that are translationally controlled by Puml and Pum2 in mES cells, and suggests novel protein-protein interactions that lend insight into the mechanism of action of Pumilio-mediated translational repression. 2 Pumilio Proteins Regulate Translation in Embryonic Stem Cells and are Essential for Early Embryonic Development A Dissertation Presented to the Faculty of the Graduate School of Yale University In Candidacy for the Degree of Doctor of Philosophy by Katherine Elizabeth Uyhazi Dissertation Director: Haifan Lin December 2012 3 © 2012 by Katherine E. Uyhazi All rights reserved. 4 Table of Contents List of Figures 7 List of Abbreviations 9 Acknowledgements 10 Chapter 1: Introduction 12 Embryonic Stem Cells and Translational Control 12 Pumilio is a Well-Characterized Translational Repressor in Drosophila 16 Pumilio Proteins Bind Directly to RNA 19 Evolutionary and Functional Conservation of Pumilio Proteins 22 Stem Cell Maintenance: An Ancestral Function of Pumilio Proteins 25 Chapter 2: The Function of Pumilio Proteins in Embryonic Stem Cells and Early Embryogenesis 29 Introduction 29 Mouse Embryogenesis 29 Murine Pumilio Proteins 32 Results 35 Puml and Pum2 are cytoplasmic in mouse embryonic stem cells 35 Pum-deficient mES cells grow more slowly but are self-renewing and pluripotent 35 Puml -/- mice are viable but are smaller than their littermates 47 Puml and Pum2 are partially functionally redundant 54 5 Puml -/-; Pum2 -/- mice axe embryonic lethal by e8.5 58 Discussion 65 Chapter 3: mRNA Targets and Protein Partners of Pumilio in mES Cells 71 Introduction 71 mRNA Targets of Pumilio Proteins 71 Protein Partners of Pumilio Proteins 73 Results 77 Puml binds to the mRNAs of 1947 genes in mES cells 77 Puml translationally represses Cyclin Bl, Cyclin El, Cyclin E2, and Pum2 80 Pum2 binds to the mRNAs of 437 genes in mES cells 84 Puml is a component of a 450 kDa complex and Pum2 is a component of a 350 kDa protein complex in mES cells 84 Puml directly binds to APC1, Rentl, and ZNF198 88 mRNA Targets of Puml and Pum2 are present in the co-IP of Rent 1 88 Discussion 91 Chapter 4: Conclusions 98 Chapter 5: Experimental Procedures 104 Appendix A: Puml RIP-Chip Microarray Analysis Ill Appendix B: Pum2 RIP-Chip Microarray Analysis 120 Appendix C: Overlapping mRNA targets of Puml and Pum2 130 Literature Cited 132 6 List of Figures Figure 1: Translational regulation in mouse embryonic stem cells 14 Figure 2: Structure and function of Pumilio proteins 18 Figure 3: Evolutionary conservation of the PUM-HD 23 Figure 4: Pumilio is required for the self-renewing asymmetric divisions of germline stem cells during Drosophila oogenesis 27 Figure 5: Murine embryogenesis 30 Figure 6: Subcellular localization of Puml and Pum2 36 Figure 7: Puml knockdown results in fewer mES cells 38 Figure 8: Derivation of Puml-deficient ES cell lines 39 Figure 9: Puml -/- cells are viable but grow more slowly than Puml+/1 cells 41 Figure 10: Cell doubling time of WT, Puml+/-, and Puml-/- ES cells 42 Figure 11: Cell proliferation is not impaired in Puml-/- ES cells 43 Figure 12: Puml-deficient cells have an increased rate of apoptosis 45 Figure 13: Embryoid body generation and lineage marker expression analysis 46 Figure 14: Generation of Puml knockout mice 48 Figure 15: Puml-/- mice are smaller than Puml+/- and WT littermates 49 Figure 16: Puml-/- mice have smaller organs than Puml+/- and WT littermates 50 Figure 17: Aged Puml-/- mice are smaller, have a prominent hunched appearance, and have an increased incidence of ulcerative dermatitis 52 Figure 18: Puml-/- mice have blunted, disorganized villi in the small intestine 53 Figure 19: Generation of Pum2 knockout mice 55 Figure 20: Pum2 -/- mice weigh the same as Pum2+/- and WT littermates 56 7 Figure 21: Puml -/-; Pum2 +/- ES cells have an increased cell doubling time 57 Figure 22: Puml-/-; Pum2 -/- double knockout mice are not viable at birth 59 Figure 23: Puml -/-; Pum2 -/- embryos are viable at e3.5 60 Figure 24: Puml -/- ;Pum2 -/- double knockout embryos are viable at e8.5 but appear developmentally delayed and have reduced tissue mass 62 Figure 25: Puml-deficient embryos are smaller at e9.5 63 Figure 26: Phenotype of el2.5 embryos 64 Figure 27: Pumilio proteins bind to functionally-related mRNAs in many species 72 Figure 28: Puml RNA Immunoprecipitation-Microarray (RIP-Chip) 78 Figure 29: Gene set enrichment analysis of Puml mRNA targets 79 Figure 30: Puml translationally represses mRNA targets in ES cells 81 Figure 31: The 3'UTR of Puml mRNA targets is sufficient for translational regulation in vitro 82 Figure 32: Pum2 binds to a subset of Puml mRNA targets 85 Figure 33: Puml elutes as part of a 450-660 kDa complex, and Pum2 elutes as part of a 350 kDa complex in mES cells 86 Figure 34: Puml directly binds to APC1, RENT1, and ZNF198 in ES cells 89 Figure 35: mRNA targets of Puml are present in the co-immunoprecipitate ofRENTl 90 Figure 36: A potential model of Pumilio-mediated mRNA repression 96 8 List of Abbreviations 3'UTR 3' Untranslated Region 5'UTR 5'Untranslated Region APCl Anaphase-Promoting Complex Subimit 1 ES cells Embryonic stem cells LIF Leukemia inhibitory factor MEF mouse embryonic fibroblasts mRNA messenger RNA miRNA microRNA Nos Nanos NRE Nanos Response Element PUF PUmilio and FBF Puml Pumilio 1 Pum2 Pumilio 2 Pum-HD Pumilio- Homology Domain RENTl Regulator of Nonsense Transcripts 1 (UPFl) ZNF198 Zinc Finger Nuclease 198 9 Acknowledgements First and foremost, I would like to thank my advisor Dr. Haifan Lin for his constant support and encouragement throughout my graduate training. Experiments didn't always work, but he always had helpful suggestions and advice to steer me in the right direction. His outgoing personality and wonderful sense of humor made the lab a fun place to be, and I hope that even a tiny fraction of his scientific knowledge and clarity of thinking have rubbed off on me over the years.