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University of California, San Diego UC San Diego UC San Diego Electronic Theses and Dissertations Title Analysis of pluripotent mouse stem cell proteomes : insights into post- transcriptional regulation of pluripotency and differentiation Permalink https://escholarship.org/uc/item/5wb983jr Author O'Brien, Robert Norman Publication Date 2010 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Analysis of pluripotent mouse stem cell proteomes: insights into post- transcriptional regulation of pluripotency and differentiation A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biology by Robert Norman O’Brien Committee in charge: Professor Steven P. Briggs, Chair Professor Lawrence Goldstein Professor Cornelius Murre Professor Karl Willert Professor Yang Xu 2010 Copyright Robert Norman O’Brien, 2010 All rights reserved. The Dissertation of Robert Norman O’Brien is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2010 iii DEDICATION To all of my friends and family who made this possible. I can’t thank all of you enough. I especially dedicate this to Chi, who has been my companion, friend and love throughout my time in graduate school: your unwavering support was essential in getting me to this point. iv EPIGRAPH My feet tug at the floor And my head sways to my shoulder Sometimes when I watch trees sway, From the window or the door. I shall set forth for somewhere, I shall make the reckless choice Some day when they are in voice And tossing so as to scare The white clouds over them on. I shall have less to say, But I shall be gone. Robert Frost The sound of the trees v TABLE OF CONTENTS Signature Page ..............................................................................iii Dedication .....................................................................................iv Epigraph .......................................................................................v Table of Contents .........................................................................vi List of Abbreviations .....................................................................xiii List of Figures ...............................................................................xiv List of Tables .................................................................................xvii Acknowledgements........................................................................xix Vita.................................................................................................xxi Abstract of the dissertation ............................................................xxii Chapter 1: Introduction .................................................1 1.1 Introduction ..............................................................................1 1.2 Overview of early mammalian development ............................1 1.3 Stem cell biology......................................................................2 1.4 Therapeutic uses of stem cells ................................................2 1.5 Cancerous stem cells: embryonal carcinoma ..........................3 1.6 Embryonic stem cells...............................................................3 1.7 Molecular basis of pluripotency: transcription factors ..............4 1.8 Molecular basis of pluripotency: chromatin remodeling ...........4 vi 1.9 Molecular basis of pluripotency: signaling pathways ...............5 1.10 Reprogramming somatic cells................................................5 1.11 Neural differentiation..............................................................6 1.12 Systems biology approach to understanding stem cells ........7 1.13 Proteomics approach to systems biology ..............................8 1.14 Focus of the thesis.................................................................11 Chapter 2: Large-scale analysis of the proteome of pluripotent mouse stem cells identifies protein markers of the pluripotent state and cases of post- transcriptional regulation...............................................13 2.1 Summary .................................................................................13 2.2 Introduction ..............................................................................14 2.3 Results.....................................................................................18 2.3.1 Differentiation of ES and P19 cells by aggregation and retinoic acid treatment leads to exit from the pluripotent state .............................18 2.3.2 Proteome analysis results in deep datasets with low measured false discovery rates................................................................................26 2.3.3 Single peptide hits make up the majority of false discoveries.........28 2.3.4 Incorporation of iTRAQ reagent allows for relative quantification of proteins enriched in undifferentiated EC and ES cells.........................34 vii 2.3.5 Quantitative proteome profiles identify conserved protein markers of pluripotency and differentiation............................................................36 2.3.6 GO annotation identifies biological processes enriched before and after differentiation in all three datasets............................................49 2.3.7 Comparison of GO terms enriched in proteome and transcriptome data .........................................................................................................51 2.3.8 Identification and confirmation of cases of post-transcriptional regulation in pluripotent cells ...................................................................53 2.3.9 Confirmation and functional analysis of a protein displaying evidence of post-transcriptional regulation during RA mediated ES differentiation...........................................................................................59 2.4 Discussion ...............................................................................65 2.4.1 Peptide identification and relative quantitation by iTRAQ ...............65 2.4.2 Proteins associated with the undifferentiated state.........................65 2.4.3 Proteins associated with RA-mediated differentiation.....................65 2.4.4 Putative cases of post-transcriptional regulation ............................66 2.4.5 Role of Racgap1 in ES cell self-renewal.........................................67 2.4.6 Protein level changes in pathways during differentiation of pluripotent cells. ......................................................................................68 2.4.6.1 Ribosome assembly complexes ...........................................68 2.4.6.2 Metabolism ...........................................................................69 2.4.6.3 Retinoic acid signaling..........................................................70 2.4.6.4 Signaling pathways activated during differentiation..............70 viii 2.4.6.5 Adhesion ..............................................................................71 2.4.6.6 Transcriptional regulators and chromatin remodeling...........71 Chapter 3: Phosphoproteome analysis identifies phosphorylation of pluripotency associated proteins............................................................................73 3.1 Summary .................................................................................73 3.2 Introduction ..............................................................................74 3.3 Results.....................................................................................75 3.3.1 TiO2 columns enrich phosphopeptides for deep phosphoproteome profiling on pluripotent cells before and after differentiation ....................75 3.3.2 Phosphorylation of markers of pluripotency and differentiation ......78 3.3.3 Mimicry or ablation of phosphorylation sites of UTF1 or mimicry of phosphorylation at those sites does not affect protein binding to the chromatin.................................................................................................82 3.3.4 Ablation of phosphorylation sites of UTF1 or mimicry of phosphorylation at those sites weakly alters UTF1 mediated repression of a TK-luciferase construct in ES, EC and HepG2 cells.........................86 3.3.5 Wild-type and phosphomutant UTF1 constructs fail to repress Oct4 in ES cells .......................................................................................91 3.3.6 Phosphomutant and wild-type UTF1 proteins all posses half-lives of greater than 24 hours ..........................................................................95 ix 3.4 Discussion ...............................................................................99 3.4.1 Use of comparators for semi-quantitative phosphoproteomes .......99 3.4.2 Phosphorylation of ES specific proteins .........................................99 3.4.3 UTF1 phosphorylation ....................................................................100 Chapter 4: Analysis of the phosphoproteome of mouse ES cells identifies increases in phosphorylation of mRNA splicing factors during differentiation ..................................................................103 4.1 Summary .................................................................................103 4.2 Introduction ..............................................................................104 4.3 Results.....................................................................................105
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