Identifying protein co factors of oct4, an essential stemness transcription factor, by affinity purification and mass spectrometry

IDENTIFYING PROTEIN PARTNERS OF OCT4, AN ES-CELL SPECIFIC TRANSCRIPTION FACTOR, BY GENE-TAGGING & PROTEOMICS APPROACHES CHEONG YUJING CLARA B.A. (Cum Laude), Cornell University A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2008 ACKNOWLEDGEMENTS Is there anything of which one can say, "Look! This is something new"? It was here already, long ago; It was here before our time. Ecclesiastes 1:10 To finally complete this work takes a village. Many thanks to my committee, especially Dr Thomas Lufkin, my main supervisor, for research direction, patience and keeping things in perspective while I was exploring and scrabbling things together. Drs Tsai Heng Hang, Patricia Ng & Rhonda Ponnampalam have been instrumental in various ways, especially with protein work, mass spectrometry, and random jokes. To members of the Lufkin lab past and present, especially Mathia, Sumantra, Serene, Song Jie, Yun, Gerry, Val, Max, Sook Peng, thank you for the scientific and non-scientific talk, coffee and muffins, and for making the past few years go by in a flash. I‟ve really enjoyed myself at lab. Others in GIS have provided timely assistance and guidance. I‟d like to thank Steph for handling the MS samples, Yun for the microinjections and Vega for walking us patiently through various data analyses methods. To my family, for loving me and dealing with my ups and downs, and believing that I could finish more than I myself thought I could. Same to DP, for love, patience, and all those fun surprises that kept me sane. Thanks God, for keeping me very well and alive. Clara Cheong Page ii PhD Thesis, National University of Singapore, 2008 TABLE OF CONTENTS Acknowledgements ii Table of Contents iii Summary . ix List of Tables & Figures . xi Abbreviations xiii Introduction 15 1.1 Transcriptional Regulation in Mammalian Development 15 1.1.1 Gene-Specific Transcription Factors . 15 1.1.1.1 Mechanism of Action . 15 1.1.1.2 Helix-Turn-Helix Domain Transcription Factors & Octamer Proteins . 16 1.1.2 1.1.2.1 1.2 DNA Binding Sites of Transcription Factors . 17 Analysis of Transcription Factor Binding Sites . 17 Early Mouse Development 19 1.2.1 ES Cells Are a Model for ICM Pluripotency . 19 1.2.2 Pluripotency is controlled on multiple fronts . 21 1.2.2.1 Known Signaling networks 21 1.2.2.2 Protein-Protein Interactions . 24 1.2.2.3 Epigenetic Regulation of Pluripotency 24 1.2.3 1.2.3.1 Oct4/Pou5f1 . 27 1.2.3.2 Sox2 . 28 1.2.3.3 Nanog . 28 1.2.4 1.3 Key Transcription Factors Regulate Pluripotency . 27 Large scale studies on pluripotency . 29 Role of Oct4 in Maintenance of Pluripotency 34 1.3.1 Regulation of Oct4 Expression 35 1.3.2 Oct4 Structure & Domains . 36 1.3.3 Known Protein Interaction Partners of Oct4 37 1.4 Finding protein-protein interactions . 44 1.4.1 Early Affinity Chromatography . 44 1.4.2 Immunoprecipitation and Co-Immunoprecipitation 45 1.4.3 Affinity Purification/Mass Spectrometry . 46 Clara Cheong Page iii PhD Thesis, National University of Singapore, 2008 1.4.3.1 Tandem Affinity Purification . 49 Project Goals 52 Chapter: Materials & Methods . 53 2.1 DNA Manipulation 53 2.1.1  Plasmids . 53 Targeting Plasmids . 53 2.1.2 Bacterial Strains & Antibiotics 54 2.1.3 Genomic DNA Extraction 54  2.1.4 2.2 ES Cell & Mouse Tail Tip Genomic DNA 54 Southern Blotting . 55  DIG Probe Design 55  Hybridization & Washing 55  Detection 56 RNA Manipulation . 57 2.2.1 RNA Extraction from ES Cells 57 2.2.2 RNA to cDNA Reverse Transcription . 57 2.2.3 siRNA Knockdown 57 2.2.4 Illumina Bead Chip Gene Expression Assay . 58 2.3  RNA Amplification 58  Illumina Bead Chip Hybridization & Data Analysis . 58 Protein Manipulation . 59 2.3.1 Protein Extraction 59  Total Protein Extraction . 59  Nuclear/Cytoplasmic Protein Extraction . 59 2.3.2 Affinity Purification of Protein Complexes . 60  His6 Tag . 60  Flag Tag . 60  S Tag 61  CBP Tag . 61  BAP Tag . 63 2.3.3 Buffer Exchange & Desalting of Proteins 64 2.3.4 Acetone Precipitation . 64 2.3.5 Detection of Proteins 64 Clara Cheong Page iv PhD Thesis, National University of Singapore, 2008  Western Blotting 64  Primary Antibodies 65  Secondary Antibodies 65  Coomassie Blue and Silver Staining 66 2.3.6 2.4  Precautions Against Keratin Contamination 66  1D Gel Separation 66  In Gel Digestion . 67  LC-MS/MS 68  Peptide and Protein Identification 68  Co-Immunoprecipitation 68 Tissue Culture 69 2.4.1 ES Cell Maintenance 69  Electroporation of ES cells 69  Homologous Recombination Targeting Vectors 70  Cre-expressing Vector 70  Alkaline Phosphatase (AP) Staining 70  lacZ Staining Protocol 70  Embryonic Stem Cells . 69  2.4.2 2.5 Mass Spectrometry . 66 HEK-293 Cells . 71 Cell Culture & Transfection . 71 Animal Work . 71 2.5.1 Blastocyst Microinjection 71 2.5.2 Genotyping by PCR . 72 Epitope Tagged-Oct4 Embryonic Stem Cells 73 3.1 Introduction 74 3.2 Design and Generation of Tagged Mouse Embryonic Stem Cells . 74 3.2.1 Choice of Affinity Purification Tags . 74 3.2.1.1 Tag Size . 76 3.2.1.2 Binding affinity and elution . 77 3.2.1.3 Localization of tags 77 3.2.1.4 Orthogonal Tandem Purifications 81 3.2.2 Clara Cheong Design and construction of targeting vectors . 81 Page v PhD Thesis, National University of Singapore, 2008 3.2.2.1 Endogenous tagging of bait by homologous recombination in mouse ES cells 81 3.2.2.2 Design of N and C-terminal targeting cassettes . 83 3.2.2.3 BAC Recombineering for generation of targeting constructs 84 3.2.2.4 Generation of tagged-Oct4 ES cell lines 86 3.2.2.5 Tagged-Oct4 Expression on Removal of Antibiotic Selection Cassette 88 3.2.2.6 NBH-Oct4 ES Cells require an additional targeting step . 91 3.2.2.7 Generation of Tagged-Oct4 Mice Evaluate the Effect of Tag on Oct4 Function 93 3.2.3 Discussion 96 Affinity Purification . 100 4.1 Basis of Affinity Purification . 100 4.2 Protein Extraction 102 4.3 Histidine6 (His) Purification . 103 4.4 FLAG (F) Purification . 108 4.5 Biotin Acceptor Peptide (BAP) Purification 110 4.6 Calmodulin Binding Peptide (CBP) Purification . 114 4.7 S-Tag (S) Purification 116 4.8 Tandem Affinity Purifications . 117 Preparing Proteins for Mass Spectrometry 121 5.1 Identification of Purified Proteins by Mass Spectrometry . 121 5.1.1 How MS and MS/MS work . 121 5.1.2 Preparing Proteins for Mass Spectrometry 122 5.1.3 Peptide Ionization Methods . 122 5.1.4 Protein Identification . 123 5.2 Improving Peptide Identification by Sample Preparation - Strategies for protein complex preparation . 124 5.2.1 Enriched Oct4 complexes: Multiple Friends and Freeloaders . 124 5.2.2 In solution tryptic digestion & LC-Separation . 125 5.2.3 Gel Separation and In-gel tryptic digestion . 126 5.2.4 1D SDS-PAGE and in-gel digestion is appropriate for low abundance complexes 127 5.2.5 Future improvements at parallel processing require less labor intensive approaches 128 5.2.6 Mass Spectrometry by LC-ESI-MS/MS 129 Clara Cheong Page vi PhD Thesis, National University of Singapore, 2008 Identifying Proteins by Tandem Mass Spectrometry . 130 6.1 6.1.1 Raw Spectra to Peptide Identification 130 6.1.2 Peptide Identification by de novo sequencing . 130 6.1.3 Peptide Identification by hybrid approaches 131 6.1.4 Peptide Identification by database searches . 132 6.2 From Peptides to Proteins 132 6.2.1 Choice of Sequence Database and Peptide Modifications . 133 6.2.2 Two heads are better than one 133 6.2.3 Initial Search – SEQUEST . 134 6.2.4 Moving on – Scaffold 134 6.3 Introduction 130 Assessing Purifications by Mass Spectrometry . 139 6.3.1 Problem of common proteins . 139 6.3.2 Semi-Quantitative Proteomics by Spectral Counting 141 Discovering Putative Interactors of Oct4 . 143 7.1 Identification of Proteins 144 7.2 Semi-quantitative Proteomics Revisited 145 7.3 Proteins Unique to the Tagged-Oct4 Samples are Putative Oct4 Interactors 149 7.4 Proteins Common to Tagged-Oct4 and Wildtype ES Cell Samples 153 7.4.1 Finding a normalizer 153 7.4.1.1 Normalization of Total Spectral Counts 154 7.4.1.2 Normalization with a Known Non-interactor Protein 155 7.4.2 Alternatively Speaking - Finding Proteins That Trend with Oct4 . 156 7.4.3 Additional Known Oct4 Interactors are Found by Correlation 158 7.4.4 New Oct4 Interactors are Discovered by the Correlation Method . 160 7.5 Co-Immunoprecipitation Analyses 164 7.5.1 Krueppel-like Factor (Klf5) 164 7.5.2 Estrogen-related receptor beta (Esrrb) . 165 7.5.3 Lysine-specific Histone Demethylase (Lsd1)/Amine Oxidase (Flavin-containing) Domain (Aof2) 168 7.6 Oct4 is associated with transcriptional regulators 170 7.6.1 7.6.1.1 Clara Cheong Oct4 as a repressor . 170 Oct4 is involved in transcriptional silencing through BHC complexes . 170 Page vii PhD Thesis, National University of Singapore, 2008 7.6.1.2 7.6.2 7.7 Oct4 and members of NuRD complexes 172 Oct4 as an Activator of Gene Expression 173 Discussion 174 Conclusion and Future Perspectives 180 8.1 Conclusion . 180 8.2 Future Applications 182 8.2.1 Extended Application 1: Examining the DNA bound protein complex . 182 8.2.2 Extended Application 2: Chromatin Immunoprecipitation using Epitope Tag Antibodies 183 8.2.3 10 Extended Application 3: Finding Oct4 partners in different cellular contexts 184 Appendices . 186 References 197 Clara Cheong Page viii PhD Thesis, National University of Singapore, 2008 SUMMARY The demands of embryonic development require tight transcriptional regulation in order to manage multiple signals and outcomes in the organism. Sequence-specific transcription factors play central roles in this coordinative procedure, acting as a molecular switchboard for the control of expression. While ongoing developments have facilitated extensive studies of protein-DNA interactions, the protein-protein interactions that surround these central players can provide fundamental clues to the recruitment of factors necessary for transcriptional regulation. Oct4 is a key factor governing the pluripotency of embryonic stem cells, which are feted for their capacity to both self-renew and differentiate to all cell lineages of the embryo proper. Part of this feature of ES cells is dependent upon the expression levels of Oct4 within the cell – beyond a given range; ES cells not retain their pluripotency, but begin to display signs of differentiation to specific lineages. To gain perspective on the role of Oct4 through its protein interactions, I have engineered ES cell lines expressing epitope tagged Oct4 from the endogenous locus at biologically relevant levels. These tagged-Oct4 cell lines were used for affinity purification to enrich for Oct4 complexes via the epitope tag, and isolated proteins then identified by mass spectrometry. Known partners of Oct4 were reaffirmed in this work; in addition, new interactions were established which pointed towards a spectrum of roles for Oct4 in transcriptional regulation. The establishment of this serves a dual purpose – one is the realization of protein interactions mediated by a transcription factor central to the maintenance of pluripotency. This study is the first to demonstrate an extensive breadth of interactions, which are validated in part by other experimental approaches both in this work, and in published studies, and serve as a platform for further insights into each of the complexes served by Oct4. Clara Cheong Page ix PhD Thesis, National University of Singapore, 2008 Additionally, the prevailing knowledge surrounding Oct4 provided this study with guiding posts in developing this technique as a generic technology suitable for the discovery of protein-protein interactions from less abundant proteins such as transcription factors. Epitope tagged proteins generated by the knock-in of tags to the endogenous locus are versatile in purpose, and higherthroughput studies can be made with a common optimized protocol for multiple proteins with similar tags. The biological context of these interactions is not sacrificed because of endogenous expression levels. Furthermore, tagged cells derived by this approach can be used for the generation of transgenic mice, to obtain previously inaccessible cell and tissue samples for the discovery of protein-protein interactions. Clara Cheong Page x PhD Thesis, National University of Singapore, 2008 binding, mrna transport, nuclear pore complex, nucleus, phosphoprotein, protein transport, translocation, transport, ubl conjugation, zinc, zinc-finger Rbm10 Rbm12 nucleus, phosphoprotein, rna-binding Rbm17 mrna processing, mrna splicing, nucleus, phosphoprotein, rnabinding, spliceosome Rbm22 metal-binding, mrna processing, mrna splicing, nucleus, rna-binding, spliceosome, zinc, zinc-finger Rbm26 Coiled coil, alternative splicing, metal-binding, phosphoprotein, rnabinding, zinc, zinc-finger Rfc2 Rfc3 atp-binding, dna replication, nucleotide-binding, nucleus atp-binding, dna replication, dna-binding, nucleotide-binding, nucleus, phosphoprotein Rrp1b Sall4 Transcription, Transcription regulation, alternative splicing, dnabinding, metal-binding, nucleus, zinc, zinc-finger Senp3 Ubl conjugation pathway, hydrolase, nucleus, phosphoprotein, protease, thiol protease Smarcd1 Chromatin regulator, Coiled coil, Direct protein sequencing, alternative splicing, nucleus Smc1a Smc3 Snrpb Ssrp1 Clara Cheong Coiled coil, DNA damage, DNA repair, Meiosis, atp-binding, cell cycle, cell division, chromosome partition, mitosis, nucleotidebinding, nucleus, phosphoprotein Coiled coil, DNA damage, DNA repair, Meiosis, atp-binding, cell cycle, cell division, chromosome partition, mitosis, nucleotidebinding, nucleus, phosphoprotein Methylation, mrna processing, mrna splicing, nucleus, ribonucleoprotein, rna-binding, spliceosome Chromosomal protein, DNA binding, DNA damage, DNA repair, Transcription, Transcription regulation, alternative splicing, dna replication, dna-binding, nucleus, phosphoprotein, ubl conjugation Page 194 PhD Thesis, National University of Singapore, 2008 Suz12 Chromatin regulator, Transcription, Transcription regulation, metalbinding, nucleus, repressor, zinc, zinc-finger Taf7 Coiled coil, Transcription, Transcription regulation, nucleus, phosphoprotein Tex10 Tox4 Twistnb U2af2 dna-binding, nucleus, phosphoprotein Transcription, dna-directed rna polymerase, nucleus, phosphoprotein acetylation, mrna processing, mrna splicing, nucleus, phosphoprotein, ribonucleoprotein, rna-binding, viral nucleoprotein Vil2 Wapal Wdr18 Wdr33 Wdr82 Coiled coil, phosphoprotein wd repeat wd repeat nucleus, phosphoprotein, wd repeat Xpo1 cytoplasm, mrna transport, nucleus, phosphoprotein, protein transport, rna-binding, transport Ywhaz Direct protein sequencing, acetylation, cytoplasm, monooxygenase, phosphoprotein Zmym2 Transcription, Transcription regulation, metal-binding, nucleus, phosphoprotein, zinc, zinc-finger Clara Cheong Page 195 PhD Thesis, National University of Singapore, 2008 APPENDIX Peptides identified by MS for Klf5 MPTRVLTMSARLGPLPQPPAAQDEPVFAQLKPVLGAANPARDAALFSGDD LKHAHHHPPAPPPAAGPRLPSEELVQTRCEMEKYLTPQLPPVPIISEHKK YRRDSASVVDQFFTDTEGIPYSINMNVFLPDITHLRTGLYKSQRPCVTQI KTEPVTIFSHQSESTAPPPPPAPTQALPEFTSIFSSHQTTAPPQEVNNIF IKQELPIPDLHLSVPSQQGHLYQLLNTPDLDMPSSTNQTAVMDTLNVSMA GLNPHPSAVPQTSMKQFQGMPPCTYTMPSQFLPQQATYFPPSPPSSEPGS PDRQAEMLQNLTPPPSYAATIASKLAIHNPNLPATLPVNSPTLPPVRYNR RSNPDLEKRRIHFCDYNGCTKVYTKSSHLKAHLRTHTGEKPYKCTWEGCD WRFARSDELTRHYRKHTGAKPFQCMVCQRSFSRSDHLALHMKRHQN Peptides identified by MS for Esrrb MSSEDRHLGSSCGSFIKTEPSSPSSGIDALSHHSPSGSSDASGGFGIALS THANGLDSPPMFAGAGLGGNPCRKSYEDCTSGIMEDSAIKCEYMLNAIPK RLCLVCGDIASGYHYGVASCEACKAFFKRTIQGNIEYNCPATNECEITKR RRKSCQACRFMKCLKVGMLKEGVRLDRVRGGRQKYKRRLDSENSPYLNLP ISPPAKKPLTKIVSNLLGVEQDKLYAMPPNDIPEGDIKALTTLCELADRE LVFLINWAKHIPGFPSLTLGDQMSLLQSAWMEILILGIVYRSLPYDDKLA YAEDYIMDEEHSRLVGLLDLYRAILQLVRRYKKLKVEKEEFMILKALALA NSDSMYIENLEAVQKLQDLLHEALQDYELSQRHEEPRRAGKLLLTLPLLR QTAAKAVQHFYSVKLQGKVPMHKLFLEMLEAKV Peptides identified by MS for Lsd1/Aof2 MATGAAGERTPRKKEPPRASPPGGLAEPPGSAGPQAGPTAGPGSATPMET GIAETPEGRRTSRRKRAKVEYREMDESLANLSEDEYYSEEERNAKAEKEK KLPPPPPQAPPEEENESEPEEPSGVEGAAFQSRLPHDRMTSQEAACFPDI ISGPQQTQKVFLFIRNRTLQLWLDNSKIQLTFEATLQQLEAPYNSDTVLV HRVHSYLERHGLINFGIYKRIKPLPIKKTGKVIIIGSGVSGLAAARQLQS FGMDVTLLEARDRVGGRVATFRKGNYVADLGAMVVTGLGGNPMAVVSKQV NMELAKIKQKCPLYEANGQAVPKEKDEMVEQEFNRLLEATSYLSHQLDFN VLNNKPVSLGQALEVVIQLQEKHVKDEQIEHWKKIVKTQEELKELLNKMV NLKEKIKELHQQYKEASEVKPPRDITAEFLVKSKHRDLTALCKEYDELAE TQGKLEEKLQELEANPPSDVYLSSRDRQILDWHFANLEFANATPLSTLSL KHWDQDDDFEFTGSHLTVRNGYSCVPVALAEGLDIKLNTAVRQVRYTASG CEVIAVNTRSTSQTFIYKCDAVLCTLPLGVLKQQPPAVQFVPPLPEWKTS AVQRMGFGNLNKVVLCFDRVFWDPSVNLFGHVGSTTASRGELFLFWNLYK APILLALVAGEAAGIMENISDDVIVGRCLAILKGIFGSSAVPQPKETVVS RWRADPWARGSYSYVAAGSSGNDYDLMAQPITPGPSIPGAPQPIPRLFFA GEHTIRNYPATVHGALLSGLREAGRIADQFLGAMYTLPRQATPGVPAQQS PSM Clara Cheong Page 196 PhD Thesis, National University of Singapore, 2008 10 REFERENCES Adjaye, J., Bolton, V., and Monk, M. (1999). Developmental expression of specific genes detected in high-quality cDNA libraries from single human preimplantation embryos. Gene 237, 373-383. Aebersold, R., and Mann, M. (2003). Mass spectrometry-based proteomics. Nature 422, 198-207. Aggarwal, K., Choe, L.H., and Lee, K.H. (2006). Shotgun proteomics using the iTRAQ isobaric tags. Brief Funct Genomic Proteomic 5, 112-120. Agresti, A., and Bianchi, M.E. (2003). HMGB proteins and gene expression. Curr Opin Genet Dev 13, 170-178. Ahmed, S., and Brickner, J.H. (2007). Regulation and epigenetic control of transcription at the nuclear periphery. Trends Genet 23, 396-402. Ambrosetti, D.C., Basilico, C., and Dailey, L. (1997). Synergistic activation of the fibroblast growth factor enhancer by Sox2 and Oct-3 depends on protein-protein interactions facilitated by a specific spatial arrangement of factor binding sites. Mol Cell Biol 17, 6321-6329. Andrews, P.W. (2002). From teratocarcinomas to embryonic stem cells. Philos Trans R Soc Lond B Biol Sci 357, 405-417. Aravind, L., Anantharaman, V., Balaji, S., Babu, M.M., and Iyer, L.M. (2005). The many faces of the helix-turn-helix domain: transcription regulation and beyond. FEMS Microbiol Rev 29, 231262. Ashburner, M., Ball, C.A., Blake, J.A., Botstein, D., Butler, H., Cherry, J.M., Davis, A.P., Dolinski, K., Dwight, S.S., Eppig, J.T., et al. (2000). Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 25, 25-29. Avilion, A.A., Nicolis, S.K., Pevny, L.H., Perez, L., Vivian, N., and Lovell-Badge, R. (2003). Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev 17, 126-140. Babaie, Y., Herwig, R., Greber, B., Brink, T.C., Wruck, W., Groth, D., Lehrach, H., Burdon, T., and Adjaye, J. (2007). Analysis of Oct4-dependent transcriptional networks regulating self-renewal and pluripotency in human embryonic stem cells. Stem Cells 25, 500-510. Baharvand, H., Fathi, A., Gourabi, H., Mollamohammadi, S., and Salekdeh, G.H. (2008). Identification of mouse embryonic stem cell-associated proteins. J Proteome Res 7, 412-423. Bartel, D.P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281297. Barthelery, M., Salli, U., and Vrana, K.E. (2007). Nuclear proteomics and directed differentiation of embryonic stem cells. Stem Cells Dev 16, 905-919. Barthelery, M., Salli, U., and Vrana, K.E. (2008). Enhanced nuclear proteomics. Proteomics 8, 1832-1838. Bauer, A., and Kuster, B. (2003). Affinity purification-mass spectrometry. Powerful tools for the characterization of protein complexes. Eur J Biochem 270, 570-578. Beckett, D., Kovaleva, E., and Schatz, P.J. (1999). A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci 8, 921-929. Beddington, R.S., and Robertson, E.J. (1989). An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo. Development 105, 733-737. Ben-Shushan, E., Pikarsky, E., Klar, A., and Bergman, Y. (1993). Extinction of Oct-3/4 gene expression in embryonal carcinoma x fibroblast somatic cell hybrids is accompanied by changes Clara Cheong Page 197 PhD Thesis, National University of Singapore, 2008 in the methylation status, chromatin structure, and transcriptional activity of the Oct-3/4 upstream region. Mol Cell Biol 13, 891-901. Bernstein, B.E., Mikkelsen, T.S., Xie, X., Kamal, M., Huebert, D.J., Cuff, J., Fry, B., Meissner, A., Wernig, M., Plath, K., et al. (2006). A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, 315-326. Bertwistle, D., Sugimoto, M., and Sherr, C.J. (2004). Physical and functional interactions of the Arf tumor suppressor protein with nucleophosmin/B23. Mol Cell Biol 24, 985-996. Bhatt, H., Brunet, L.J., and Stewart, C.L. (1991). Uterine expression of leukemia inhibitory factor coincides with the onset of blastocyst implantation. Proc Natl Acad Sci U S A 88, 11408-11412. Bibikova, M., Laurent, L.C., Ren, B., Loring, J.F., and Fan, J.B. (2008). Unraveling epigenetic regulation in embryonic stem cells. Cell Stem Cell 2, 123-134. Blumenthal, D.K., Takio, K., Edelman, A.M., Charbonneau, H., Titani, K., Walsh, K.A., and Krebs, E.G. (1985). Identification of the calmodulin-binding domain of skeletal muscle myosin light chain kinase. Proc Natl Acad Sci U S A 82, 3187-3191. Botquin, V., Hess, H., Fuhrmann, G., Anastassiadis, C., Gross, M.K., Vriend, G., and Scholer, H.R. (1998). New POU dimer configuration mediates antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and Sox-2. Genes Dev 12, 2073-2090. Bouwmeester, T., Bauch, A., Ruffner, H., Angrand, P.O., Bergamini, G., Croughton, K., Cruciat, C., Eberhard, D., Gagneur, J., Ghidelli, S., et al. (2004). A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway. Nat Cell Biol 6, 97-105. Boyer, L.A., Lee, T.I., Cole, M.F., Johnstone, S.E., Levine, S.S., Zucker, J.P., Guenther, M.G., Kumar, R.M., Murray, H.L., Jenner, R.G., et al. (2005). Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947-956. Bradley, A., Evans, M., Kaufman, M.H., and Robertson, E. (1984). Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309, 255-256. Brehm, A., Ohbo, K., and Scholer, H. (1997). The carboxy-terminal transactivation domain of Oct4 acquires cell specificity through the POU domain. Mol Cell Biol 17, 154-162. Brehm, A., Ohbo, K., Zwerschke, W., Botquin, V., Jansen-Durr, P., and Scholer, H.R. (1999). Synergism with germ line transcription factor Oct-4: viral oncoproteins share the ability to mimic a stem cell-specific activity. Mol Cell Biol 19, 2635-2643. Brehm, A., Ovitt, C.E., and Scholer, H.R. (1998). Oct-4: more than just a POUerful marker of the mammalian germline? Apmis 106, 114-124; discussion 124-116. Brickner, D.G., Cajigas, I., Fondufe-Mittendorf, Y., Ahmed, S., Lee, P.C., Widom, J., and Brickner, J.H. (2007). H2A.Z-mediated localization of genes at the nuclear periphery confers epigenetic memory of previous transcriptional state. PLoS Biol 5, e81. Brizzard, B. (2008). Epitope tagging. Biotechniques 44, 693-695. Brook, F.A., and Gardner, R.L. (1997). The origin and efficient derivation of embryonic stem cells in the mouse. Proc Natl Acad Sci U S A 94, 5709-5712. Burckstummer, T., Bennett, K.L., Preradovic, A., Schutze, G., Hantschel, O., Superti-Furga, G., and Bauch, A. (2006). An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells. Nat Methods 3, 1013-1019. Butteroni, C., De Felici, M., Scholer, H.R., and Pesce, M. (2000). Phage display screening reveals an association between germline-specific transcription factor Oct-4 and multiple cellular proteins. J Mol Biol 304, 529-540. Campbell, P.A., Perez-Iratxeta, C., Andrade-Navarro, M.A., and Rudnicki, M.A. (2007). Oct4 targets regulatory nodes to modulate stem cell function. PLoS ONE 2, e553. Carr, S., Aebersold, R., Baldwin, M., Burlingame, A., Clauser, K., and Nesvizhskii, A. (2004). The need for guidelines in publication of peptide and protein identification data: Working Group on Clara Cheong Page 198 PhD Thesis, National University of Singapore, 2008 Publication Guidelines for Peptide and Protein Identification Data. Mol Cell Proteomics 3, 531533. Carr, S.A., Hemling, M.E., Bean, M.F., and Roberts, G.D. (1991). Integration of mass spectrometry in analytical biotechnology. Anal Chem 63, 2802-2824. Chaga, G., Hopp, J., and Nelson, P. (1999). Immobilized metal ion affinity chromatography on Co2+-carboxymethylaspartate-agarose Superflow, as demonstrated by one-step purification of lactate dehydrogenase from chicken breast muscle. Biotechnol Appl Biochem 29 ( Pt 1), 19-24. Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S., and Smith, A. (2003). Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643-655. Chandhoke, T.K., Huang, Y.F., Liu, F., Gronowicz, G.A., Adams, D.J., Harrison, J.R., and Kream, B.E. (2008). Osteopenia in transgenic mice with osteoblast-targeted expression of the inducible cAMP early repressor. Bone 43, 101-109. Chant, A., Kraemer-Pecore, C.M., Watkin, R., and Kneale, G.G. (2005). Attachment of a histidine tag to the minimal zinc finger protein of the Aspergillus nidulans gene regulatory protein AreA causes a conformational change at the DNA-binding site. Protein Expr Purif 39, 152-159. Chen, X., Xu, H., Yuan, P., Fang, F., Huss, M., Vega, V.B., Wong, E., Orlov, Y.L., Zhang, W., Jiang, J., et al. (2008). Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133, 1106-1117. Chew, J.L., Loh, Y.H., Zhang, W., Chen, X., Tam, W.L., Yeap, L.S., Li, P., Ang, Y.S., Lim, B., Robson, P., et al. (2005). Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells. Mol Cell Biol 25, 6031-6046. Chodosh, L.A., and Buratowski, S. (2001). Purification of DNA-binding proteins using biotin/streptavidin affinity systems. Curr Protoc Protein Sci Chapter 9, Unit 7. Cleary, M.A., and Herr, W. (1995). Mechanisms for flexibility in DNA sequence recognition and VP16-induced complex formation by the Oct-1 POU domain. Mol Cell Biol 15, 2090-2100. Cormier, S., Vandormael-Pournin, S., Babinet, C., and Cohen-Tannoudji, M. (2004). Developmental expression of the Notch signaling pathway genes during mouse preimplantation development. Gene Expr Patterns 4, 713-717. de Boer, E., Rodriguez, P., Bonte, E., Krijgsveld, J., Katsantoni, E., Heck, A., Grosveld, F., and Strouboulis, J. (2003). Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc Natl Acad Sci U S A 100, 7480-7485. Dennis, G., Jr., Sherman, B.T., Hosack, D.A., Yang, J., Gao, W., Lane, H.C., and Lempicki, R.A. (2003). DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol 4, P3. Denslow, S.A., and Wade, P.A. (2007). The human Mi-2/NuRD complex and gene regulation. Oncogene 26, 5433-5438. Dillon, N. (2008). The impact of gene location in the nucleus on transcriptional regulation. Dev Cell 15, 182-186. Drakas, R., Prisco, M., and Baserga, R. (2005). A modified tandem affinity purification tag technique for the purification of protein complexes in mammalian cells. Proteomics 5, 132-137. Driegen, S., Ferreira, R., van Zon, A., Strouboulis, J., Jaegle, M., Grosveld, F., Philipsen, S., and Meijer, D. (2005). A generic tool for biotinylation of tagged proteins in transgenic mice. Transgenic Res 14, 477-482. Eggan, K., Akutsu, H., Loring, J., Jackson-Grusby, L., Klemm, M., Rideout, W.M., 3rd, Yanagimachi, R., and Jaenisch, R. (2001). Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc Natl Acad Sci U S A 98, 6209-6214. Clara Cheong Page 199 PhD Thesis, National University of Singapore, 2008 Eggers, D.K., and Valentine, J.S. (2001). Molecular confinement influences protein structure and enhances thermal protein stability. Protein Sci 10, 250-261. Einhauer, A., and Jungbauer, A. (2001). The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. J Biochem Biophys Methods 49, 455-465. Elias, J.E., and Gygi, S.P. (2007). Target-decoy search strategy for increased confidence in largescale protein identifications by mass spectrometry. Nat Methods 4, 207-214. Elliott, S.T., Crider, D.G., Garnham, C.P., Boheler, K.R., and Van Eyk, J.E. (2004). Two-dimensional gel electrophoresis database of murine R1 embryonic stem cells. Proteomics 4, 3813-3832. Eminli, S., Utikal, J.S., Arnold, K., Jaenisch, R., and Hochedlinger, K. (2008). Reprogramming of Neural Progenitor Cells into iPS Cells in the Absence of Exogenous Sox2 Expression. Stem Cells. Evan, G.I., Lewis, G.K., Ramsay, G., and Bishop, J.M. (1985). Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol 5, 3610-3616. Evans, M.J., and Kaufman, M.H. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154-156. Ezashi, T., Ghosh, D., and Roberts, R.M. (2001). Repression of Ets-2-induced transactivation of the tau interferon promoter by Oct-4. Mol Cell Biol 21, 7883-7891. Falvo, J.V., Thanos, D., and Maniatis, T. (1995). Reversal of intrinsic DNA bends in the IFN beta gene enhancer by transcription factors and the architectural protein HMG I(Y). Cell 83, 11011111. Field, J., Nikawa, J., Broek, D., MacDonald, B., Rodgers, L., Wilson, I.A., Lerner, R.A., and Wigler, M. (1988). Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol 8, 2159-2165. Forler, D., Kocher, T., Rode, M., Gentzel, M., Izaurralde, E., and Wilm, M. (2003). An efficient protein complex purification method for functional proteomics in higher eukaryotes. Nat Biotechnol 21, 89-92. Forneris, F., Binda, C., Adamo, A., Battaglioli, E., and Mattevi, A. (2007). Structural basis of LSD1CoREST selectivity in histone H3 recognition. J Biol Chem 282, 20070-20074. Fox, V., Gokhale, P.J., Walsh, J.R., Matin, M., Jones, M., and Andrews, P.W. (2008). Cell-cell signaling through NOTCH regulates human embryonic stem cell proliferation. Stem Cells 26, 715723. Fuhrmann, G., Chung, A.C., Jackson, K.J., Hummelke, G., Baniahmad, A., Sutter, J., Sylvester, I., Scholer, H.R., and Cooney, A.J. (2001). Mouse germline restriction of Oct4 expression by germ cell nuclear factor. Dev Cell 1, 377-387. Gao, J., Friedrichs, M.S., Dongre, A.R., and Opiteck, G.J. (2005). Guidelines for the routine application of the peptide hits technique. J Am Soc Mass Spectrom 16, 1231-1238. Gao, J., Opiteck, G.J., Friedrichs, M.S., Dongre, A.R., and Hefta, S.A. (2003). Changes in the protein expression of yeast as a function of carbon source. J Proteome Res 2, 643-649. Gardner, R.L. (1983). Origin and differentiation of extraembryonic tissues in the mouse. Int Rev Exp Pathol 24, 63-133. Gates, D.M., and Bekhor, I. (1979). DNA-binding activity of tightly-bound nonhistone chromosomal proteins in chicken liver chromatin. Nucleic Acids Res 6, 3411-3426. Gavin, A.C., Bosche, M., Krause, R., Grandi, P., Marzioch, M., Bauer, A., Schultz, J., Rick, J.M., Michon, A.M., Cruciat, C.M., et al. (2002). Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141-147. Giguere, V. (1999). Orphan nuclear receptors: from gene to function. Endocr Rev 20, 689-725. Giguere, V., Yang, N., Segui, P., and Evans, R.M. (1988). Identification of a new class of steroid hormone receptors. Nature 331, 91-94. Clara Cheong Page 200 PhD Thesis, National University of Singapore, 2008 Gilchrist, A., Au, C.E., Hiding, J., Bell, A.W., Fernandez-Rodriguez, J., Lesimple, S., Nagaya, H., Roy, L., Gosline, S.J., Hallett, M., et al. (2006). Quantitative proteomics analysis of the secretory pathway. Cell 127, 1265-1281. Gingras, A.C., Aebersold, R., and Raught, B. (2005). Advances in protein complex analysis using mass spectrometry. J Physiol 563, 11-21. Gocke, C.B., and Yu, H. (2008). ZNF198 stabilizes the LSD1-CoREST-HDAC1 complex on chromatin through its MYM-type zinc fingers. PLoS ONE 3, e3255. Godmann, M., Auger, V., Ferraroni-Aguiar, V., Di Sauro, A., Sette, C., Behr, R., and Kimmins, S. (2007). Dynamic regulation of histone H3 methylation at lysine in mammalian spermatogenesis. Biol Reprod 77, 754-764. Gudmundsson, K.O., Thorsteinsson, L., Sigurjonsson, O.E., Keller, J.R., Olafsson, K., Egeland, T., Gudmundsson, S., and Rafnar, T. (2007). Gene expression analysis of hematopoietic progenitor cells identifies Dlg7 as a potential stem cell gene. Stem Cells 25, 1498-1506. Guo, Y., Costa, R., Ramsey, H., Starnes, T., Vance, G., Robertson, K., Kelley, M., Reinbold, R., Scholer, H., and Hromas, R. (2002). The embryonic stem cell transcription factors Oct-4 and FoxD3 interact to regulate endodermal-specific promoter expression. Proc Natl Acad Sci U S A 99, 3663-3667. Haqqani, A.S., Kelly, J.F., and Stanimirovic, D.B. (2008). Quantitative protein profiling by mass spectrometry using label-free proteomics. Methods Mol Biol 439, 241-256. Hayward, P., Kalmar, T., and Arias, A.M. (2008). Wnt/Notch signalling and information processing during development. Development 135, 411-424. Head, J.F. (1992). A better grip on calmodulin. Curr Biol 2, 609-611. Heinrich, P.C., Behrmann, I., Haan, S., Hermanns, H.M., Muller-Newen, G., and Schaper, F. (2003). Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J 374, 1-20. Higgs, R.E., Knierman, M.D., Freeman, A.B., Gelbert, L.M., Patil, S.T., and Hale, J.E. (2007). Estimating the statistical significance of peptide identifications from shotgun proteomics experiments. J Proteome Res 6, 1758-1767. Higgs, R.E., Knierman, M.D., Gelfanova, V., Butler, J.P., and Hale, J.E. (2005). Comprehensive label-free method for the relative quantification of proteins from biological samples. J Proteome Res 4, 1442-1450. Hochuli, E., Dobeli, H., and Schacher, A. (1987). New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr 411, 177-184. Hosler, B.A., Rogers, M.B., Kozak, C.A., and Gudas, L.J. (1993). An octamer motif contributes to the expression of the retinoic acid-regulated zinc finger gene Rex-1 (Zfp-42) in F9 teratocarcinoma cells. Mol Cell Biol 13, 2919-2928. Inamoto, S., Segil, N., Pan, Z.Q., Kimura, M., and Roeder, R.G. (1997). The cyclin-dependent kinase-activating kinase (CAK) assembly factor, MAT1, targets and enhances CAK activity on the POU domains of octamer transcription factors. J Biol Chem 272, 29852-29858. Ishihama, Y., Oda, Y., Tabata, T., Sato, T., Nagasu, T., Rappsilber, J., and Mann, M. (2005). Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics 4, 1265-1272. Ivanova, N., Dobrin, R., Lu, R., Kotenko, I., Levorse, J., DeCoste, C., Schafer, X., Lun, Y., and Lemischka, I.R. (2006). Dissecting self-renewal in stem cells with RNA interference. Nature 442, 533-538. Jeong, Y., El-Jaick, K., Roessler, E., Muenke, M., and Epstein, D.J. (2006). A functional screen for sonic hedgehog regulatory elements across a Mb interval identifies long-range ventral forebrain enhancers. Development 133, 761-772. Clara Cheong Page 201 PhD Thesis, National University of Singapore, 2008 Jiang, J., Chan, Y.S., Loh, Y.H., Cai, J., Tong, G.Q., Lim, C.A., Robson, P., Zhong, S., and Ng, H.H. (2008). A core Klf circuitry regulates self-renewal of embryonic stem cells. Nat Cell Biol 10, 353360. Kanatsu-Shinohara, M., Inoue, K., Lee, J., Yoshimoto, M., Ogonuki, N., Miki, H., Baba, S., Kato, T., Kazuki, Y., Toyokuni, S., et al. (2004). Generation of pluripotent stem cells from neonatal mouse testis. Cell 119, 1001-1012. Kanellopoulou, C., Muljo, S.A., Kung, A.L., Ganesan, S., Drapkin, R., Jenuwein, T., Livingston, D.M., and Rajewsky, K. (2005). Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing. Genes Dev 19, 489-501. Karpeisky, M., Senchenko, V.N., Dianova, M.V., and Kanevsky, V. (1994). Formation and properties of S-protein complex with S-peptide-containing fusion protein. FEBS Lett 339, 209212. Kato, I., and Anfinsen, C.B. (1969). On the stabilization of ribonuclease S-protein by ribonuclease S-peptide. J Biol Chem 244, 1004-1007. Kehler, J., Tolkunova, E., Koschorz, B., Pesce, M., Gentile, L., Boiani, M., Lomeli, H., Nagy, A., McLaughlin, K.J., Scholer, H.R., et al. (2004). Oct4 is required for primordial germ cell survival. EMBO Rep 5, 1078-1083. Keller, A., Eng, J., Zhang, N., Li, X.J., and Aebersold, R. (2005). A uniform proteomics MS/MS analysis platform utilizing open XML file formats. Mol Syst Biol 1, 2005 0017. Keller, A., Nesvizhskii, A.I., Kolker, E., and Aebersold, R. (2002). Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 74, 5383-5392. Kim, J., Chu, J., Shen, X., Wang, J., and Orkin, S.H. (2008). An extended transcriptional network for pluripotency of embryonic stem cells. Cell 132, 1049-1061. Klemm, J.D., Rould, M.A., Aurora, R., Herr, W., and Pabo, C.O. (1994). Crystal structure of the Oct-1 POU domain bound to an octamer site: DNA recognition with tethered DNA-binding modules. Cell 77, 21-32. Kloosterman, W.P., and Plasterk, R.H. (2006). The diverse functions of microRNAs in animal development and disease. Dev Cell 11, 441-450. Knuesel, M., Wan, Y., Xiao, Z., Holinger, E., Lowe, N., Wang, W., and Liu, X. (2003). Identification of novel protein-protein interactions using a versatile mammalian tandem affinity purification expression system. Mol Cell Proteomics 2, 1225-1233. Koike, M., Sakaki, S., Amano, Y., and Kurosawa, H. (2007). Characterization of embryoid bodies of mouse embryonic stem cells formed under various culture conditions and estimation of differentiation status of such bodies. J Biosci Bioeng 104, 294-299. Korutla, L., Degnan, R., Wang, P., and Mackler, S.A. (2007). NAC1, a cocaine-regulated POZ/BTB protein interacts with CoREST. J Neurochem 101, 611-618. Kuo, C.T., Veselits, M.L., Barton, K.P., Lu, M.M., Clendenin, C., and Leiden, J.M. (1997). The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilization during murine embryogenesis. Genes Dev 11, 2996-3006. Lagarkova, M., Volchkov, P., Lyakisheva, A., Philonenko, E., and Kiselev, S. (2006). Diverse epigenetic profile of novel human embryonic stem cell lines. Cell Cycle 5, 416-420. Laitinen, O.H., Marttila, A.T., Airenne, K.J., Kulik, T., Livnah, O., Bayer, E.A., Wilchek, M., and Kulomaa, M.S. (2001). Biotin induces tetramerization of a recombinant monomeric avidin. A model for protein-protein interactions. J Biol Chem 276, 8219-8224. Lee, J., Kim, H.K., Han, Y.M., and Kim, J. (2008). Pyruvate kinase isozyme type M2 (PKM2) interacts and cooperates with Oct-4 in regulating transcription. Int J Biochem Cell Biol 40, 10431054. Clara Cheong Page 202 PhD Thesis, National University of Singapore, 2008 Lee, J., Rhee, B.K., Bae, G.Y., Han, Y.M., and Kim, J. (2005a). Stimulation of Oct-4 activity by Ewing's sarcoma protein. Stem Cells 23, 738-751. Lee, M.G., Wynder, C., Cooch, N., and Shiekhattar, R. (2005b). An essential role for CoREST in nucleosomal histone lysine demethylation. Nature 437, 432-435. Lengner, C., Welstead, G., and Jaenisch, R. (2008). The pluripotency regulator Oct4: A role in somatic stem cells? Cell Cycle 7. Li, J.Y., Pu, M.T., Hirasawa, R., Li, B.Z., Huang, Y.N., Zeng, R., Jing, N.H., Chen, T., Li, E., Sasaki, H., et al. (2007). Synergistic function of DNA methyltransferases Dnmt3a and Dnmt3b in the methylation of Oct4 and Nanog. Mol Cell Biol 27, 8748-8759. Li, S., Weidenfeld, J., and Morrisey, E.E. (2004). Transcriptional and DNA binding activity of the Foxp1/2/4 family is modulated by heterotypic and homotypic protein interactions. Mol Cell Biol 24, 809-822. Liang, J., Wan, M., Zhang, Y., Gu, P., Xin, H., Jung, S.Y., Qin, J., Wong, J., Cooney, A.J., Liu, D., et al. (2008). Nanog and Oct4 associate with unique transcriptional repression complexes in embryonic stem cells. Nat Cell Biol 10, 731-739. Lim, C.Y., Tam, W.L., Zhang, J., Ang, H.S., Jia, H., Lipovich, L., Ng, H.H., Wei, C.L., Sung, W.K., Robson, P., et al. (2008). Sall4 regulates distinct transcription circuitries in different blastocystderived stem cell lineages. Cell Stem Cell 3, 543-554. Lim, L.S., Loh, Y.H., Zhang, W., Li, Y., Chen, X., Wang, Y., Bakre, M., Ng, H.H., and Stanton, L.W. (2007). Zic3 is required for maintenance of pluripotency in embryonic stem cells. Mol Biol Cell 18, 1348-1358. Link, A.J., Fleischer, T.C., Weaver, C.M., Gerbasi, V.R., and Jennings, J.L. (2005). Purifying protein complexes for mass spectrometry: applications to protein translation. Methods 35, 274-290. Liu, H., Sadygov, R.G., and Yates, J.R., 3rd (2004). A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal Chem 76, 4193-4201. Liu, P., Wakamiya, M., Shea, M.J., Albrecht, U., Behringer, R.R., and Bradley, A. (1999). Requirement for Wnt3 in vertebrate axis formation. Nat Genet 22, 361-365. Loh, Y.H., Ng, J.H., and Ng, H.H. (2008). Molecular framework underlying pluripotency. Cell Cycle 7, 885-891. Loh, Y.H., Wu, Q., Chew, J.L., Vega, V.B., Zhang, W., Chen, X., Bourque, G., George, J., Leong, B., Liu, J., et al. (2006). The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 38, 431-440. Looijenga, L., Stoop, H., de Leeuw, H., de Gouveia Brazao, C., Gillis, A., van Roozendaal, K., van Zoelen, E., Weber, R., Wolffenbuttel, K., van Dekken, H., et al. (2003). POU5F1 (OCT3/4) identifies cells with pluripotent potential in human germ cell tumors. Cancer Res 63, 2244-2250. Lubec, G., and Afjehi-Sadat, L. (2007). Limitations and pitfalls in protein identification by mass spectrometry. Chem Rev 107, 3568-3584. Luo, J., Sladek, R., Bader, J.A., Matthyssen, A., Rossant, J., and Giguere, V. (1997). Placental abnormalities in mouse embryos lacking the orphan nuclear receptor ERR-beta. Nature 388, 778-782. Masui, S., Ohtsuka, S., Yagi, R., Takahashi, K., Ko, M.S., and Niwa, H. (2008). Rex1/Zfp42 is dispensable for pluripotency in mouse ES cells. BMC Dev Biol 8, 45. Matoba, R., Niwa, H., Masui, S., Ohtsuka, S., Carter, M.G., Sharov, A.A., and Ko, M.S. (2006). Dissecting Oct3/4-regulated gene networks in embryonic stem cells by expression profiling. PLoS ONE 1, e26. Merika, M., and Thanos, D. (2001). Enhanceosomes. Curr Opin Genet Dev 11, 205-208. Clara Cheong Page 203 PhD Thesis, National University of Singapore, 2008 Metzger, E., Wissmann, M., Yin, N., Muller, J.M., Schneider, R., Peters, A.H., Gunther, T., Buettner, R., and Schule, R. (2005). LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription. Nature 437, 436-439. Mikkelsen, T.S., Ku, M., Jaffe, D.B., Issac, B., Lieberman, E., Giannoukos, G., Alvarez, P., Brockman, W., Kim, T.K., Koche, R.P., et al. (2007). Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448, 553-560. Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., Maruyama, M., Maeda, M., and Yamanaka, S. (2003). The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113, 631-642. Mohamed, O.A., Clarke, H.J., and Dufort, D. (2004). Beta-catenin signaling marks the prospective site of primitive streak formation in the mouse embryo. Dev Dyn 231, 416-424. Murchison, E.P., Partridge, J.F., Tam, O.H., Cheloufi, S., and Hannon, G.J. (2005). Characterization of Dicer-deficient murine embryonic stem cells. Proc Natl Acad Sci U S A 102, 12135-12140. Muyrers, J.P., Zhang, Y., Benes, V., Testa, G., Rientjes, J.M., and Stewart, A.F. (2004). ET recombination: DNA engineering using homologous recombination in E. coli. Methods Mol Biol 256, 107-121. Nagano, K., Taoka, M., Yamauchi, Y., Itagaki, C., Shinkawa, T., Nunomura, K., Okamura, N., Takahashi, N., Izumi, T., and Isobe, T. (2005). Large-scale identification of proteins expressed in mouse embryonic stem cells. Proteomics 5, 1346-1361. Nagy, A., and Rossant, J. (2001). Chimaeras and mosaics for dissecting complex mutant phenotypes. Int J Dev Biol 45, 577-582. Nakagawa, M., Koyanagi, M., Tanabe, K., Takahashi, K., Ichisaka, T., Aoi, T., Okita, K., Mochiduki, Y., Takizawa, N., and Yamanaka, S. (2008). Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 26, 101-106. Nakatake, Y., Fukui, N., Iwamatsu, Y., Masui, S., Takahashi, K., Yagi, R., Yagi, K., Miyazaki, J., Matoba, R., Ko, M.S., et al. (2006). Klf4 cooperates with Oct3/4 and Sox2 to activate the Lefty1 core promoter in embryonic stem cells. Mol Cell Biol 26, 7772-7782. Nesvizhskii, A.I., Keller, A., Kolker, E., and Aebersold, R. (2003). A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 75, 4646-4658. Nesvizhskii, A.I., Vitek, O., and Aebersold, R. (2007). Analysis and validation of proteomic data generated by tandem mass spectrometry. Nat Methods 4, 787-797. Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe-Nebenius, D., Chambers, I., Scholer, H., and Smith, A. (1998). Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95, 379-391. Nilsson, B., Moks, T., Jansson, B., Abrahmsen, L., Elmblad, A., Holmgren, E., Henrichson, C., Jones, T.A., and Uhlen, M. (1987). A synthetic IgG-binding domain based on staphylococcal protein A. Protein Eng 1, 107-113. Niwa, H., Miyazaki, J., and Smith, A.G. (2000). Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24, 372-376. Niwa, H., Toyooka, Y., Shimosato, D., Strumpf, D., Takahashi, K., Yagi, R., and Rossant, J. (2005). Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation. Cell 123, 917929. Nordhoff, V., Hubner, K., Bauer, A., Orlova, I., Malapetsa, A., and Scholer, H.R. (2001). Comparative analysis of human, bovine, and murine Oct-4 upstream promoter sequences. Mamm Genome 12, 309-317. O'Callaghan C, A., Byford, M.F., Wyer, J.R., Willcox, B.E., Jakobsen, B.K., McMichael, A.J., and Bell, J.I. (1999). BirA enzyme: production and application in the study of membrane receptor-ligand interactions by site-specific biotinylation. Anal Biochem 266, 9-15. Clara Cheong Page 204 PhD Thesis, National University of Singapore, 2008 Ogawa, T., Ohmura, M., Tamura, Y., Kita, K., Ohbo, K., Suda, T., and Kubota, Y. (2004). Derivation and morphological characterization of mouse spermatogonial stem cell lines. Arch Histol Cytol 67, 297-306. Okita, K., and Yamanaka, S. (2006). Intracellular signaling pathways regulating pluripotency of embryonic stem cells. Curr Stem Cell Res Ther 1, 103-111. Old, W.M., Meyer-Arendt, K., Aveline-Wolf, L., Pierce, K.G., Mendoza, A., Sevinsky, J.R., Resing, K.A., and Ahn, N.G. (2005). Comparison of label-free methods for quantifying human proteins by shotgun proteomics. Mol Cell Proteomics 4, 1487-1502. Ong, S.E., Blagoev, B., Kratchmarova, I., Kristensen, D.B., Steen, H., Pandey, A., and Mann, M. (2002). Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1, 376-386. Palmieri, S.L., Peter, W., Hess, H., and Scholer, H.R. (1994). Oct-4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. Dev Biol 166, 259-267. Pang, J.X., Ginanni, N., Dongre, A.R., Hefta, S.A., and Opitek, G.J. (2002). Biomarker discovery in urine by proteomics. J Proteome Res 1, 161-169. Panne, D. (2008). The enhanceosome. Curr Opin Struct Biol 18, 236-242. Panne, D., Maniatis, T., and Harrison, S.C. (2007). An atomic model of the interferon-beta enhanceosome. Cell 129, 1111-1123. Panning, B., and Jaenisch, R. (1996). DNA hypomethylation can activate Xist expression and silence X-linked genes. Genes Dev 10, 1991-2002. Parisi, S., Passaro, F., Aloia, L., Manabe, I., Nagai, R., Pastore, L., and Russo, T. (2008). Klf5 is involved in self-renewal of mouse embryonic stem cells. J Cell Sci. Park, I.H., Lerou, P.H., Zhao, R., Huo, H., and Daley, G.Q. (2008). Generation of human-induced pluripotent stem cells. Nat Protoc 3, 1180-1186. Pesce, M., Wang, X., Wolgemuth, D.J., and Scholer, H. (1998). Differential expression of the Oct4 transcription factor during mouse germ cell differentiation. Mech Dev 71, 89-98. Pestov, N.B., and Rydstrom, J. (2007). Purification of recombinant membrane proteins tagged with calmodulin-binding domains by affinity chromatography on calmodulin-agarose: example of nicotinamide nucleotide transhydrogenase. Nat Protoc 2, 198-202. Poser, I., Sarov, M., Hutchins, J.R., Heriche, J.K., Toyoda, Y., Pozniakovsky, A., Weigl, D., Nitzsche, A., Hegemann, B., Bird, A.W., et al. (2008). BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat Methods 5, 409-415. Puig, O., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm, M., and Seraphin, B. (2001). The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218-229. Ramalho-Santos, M., Yoon, S., Matsuzaki, Y., Mulligan, R.C., and Melton, D.A. (2002). "Stemness": transcriptional profiling of embryonic and adult stem cells. Science 298, 597-600. Rappsilber, J., and Mann, M. (2002). What does it mean to identify a protein in proteomics? Trends Biochem Sci 27, 74-78. Ratner, D. (1974). The interaction bacterial and phage proteins with immobilized Escherichia coli RNA polymerase. J Mol Biol 88, 373-383. Remenyi, A., Lins, K., Nissen, L.J., Reinbold, R., Scholer, H.R., and Wilmanns, M. (2003). Crystal structure of a POU/HMG/DNA ternary complex suggests differential assembly of Oct4 and Sox2 on two enhancers. Genes Dev 17, 2048-2059. Remenyi, A., Pohl, E., Scholer, H.R., and Wilmanns, M. (2001). Crystallization of redox-insensitive Oct1 POU domain with different DNA-response elements. Acta Crystallogr D Biol Crystallogr 57, 1634-1638. Clara Cheong Page 205 PhD Thesis, National University of Singapore, 2008 Resing, K.A., Meyer-Arendt, K., Mendoza, A.M., Aveline-Wolf, L.D., Jonscher, K.R., Pierce, K.G., Old, W.M., Cheung, H.T., Russell, S., Wattawa, J.L., et al. (2004). Improving reproducibility and sensitivity in identifying human proteins by shotgun proteomics. Anal Chem 76, 3556-3568. Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., and Seraphin, B. (1999). A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17, 1030-1032. Rizzino, A. (2008). Transcription factors that behave as master regulators during mammalian embryogenesis function as molecular rheostats. Biochem J 411, e5-7. Rohila, J.S., Chen, M., Cerny, R., and Fromm, M.E. (2004). Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants. Plant J 38, 172-181. Rosfjord, E., and Rizzino, A. (1994). The octamer motif present in the Rex-1 promoter binds Oct1 and Oct-3 expressed by EC cells and ES cells. Biochem Biophys Res Commun 203, 1795-1802. Rosner, M.H., Vigano, M.A., Ozato, K., Timmons, P.M., Poirier, F., Rigby, P.W., and Staudt, L.M. (1990). A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature 345, 686-692. Rossant, J., Bernelot-Moens, C., and Nagy, A. (1993). Genome manipulation in embryonic stem cells. Philos Trans R Soc Lond B Biol Sci 339, 207-215. Ruzinova, M.B., and Benezra, R. (2003). Id proteins in development, cell cycle and cancer. Trends Cell Biol 13, 410-418. Sadygov, R.G., Liu, H., and Yates, J.R. (2004). Statistical models for protein validation using tandem mass spectral data and protein amino acid sequence databases. Anal Chem 76, 16641671. Saha, K., Bender, F., and Gizeli, E. (2003). Comparative study of IgG binding to proteins G and A: nonequilibrium kinetic and binding constant determination with the acoustic waveguide device. Anal Chem 75, 835-842. Sato, N., Meijer, L., Skaltsounis, L., Greengard, P., and Brivanlou, A.H. (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. Schmidt, T.G., and Skerra, A. (2007). The Strep-tag system for one-step purification and highaffinity detection or capturing of proteins. Nat Protoc 2, 1528-1535. Scholer, H.R., Ciesiolka, T., and Gruss, P. (1991). A nexus between Oct-4 and E1A: implications for gene regulation in embryonic stem cells. Cell 66, 291-304. Scholer, H.R., Dressler, G.R., Balling, R., Rohdewohld, H., and Gruss, P. (1990). Oct-4: a germlinespecific transcription factor mapping to the mouse t-complex. Embo J 9, 2185-2195. Schon, C., Wochnik, A., Rossner, A., Donow, C., and Knochel, W. (2006). The FoxP subclass in Xenopus laevis development. Dev Genes Evol 216, 641-646. Schulz, W.A., and Hoffmann, M.J. (2007). Transcription factor networks in embryonic stem cells and testicular cancer and the definition of epigenetics. Epigenetics 2, 37-42. Schuster, M., Wasserbauer, E., Einhauer, A., Ortner, C., Jungbauer, A., Hammerschmid, F., and Werner, G. (2000). Protein expression strategies for identification of novel target proteins. J Biomol Screen 5, 89-97. Segre, J.A., Bauer, C., and Fuchs, E. (1999). Klf4 is a transcription factor required for establishing the barrier function of the skin. Nat Genet 22, 356-360. Shi, Y., Lan, F., Matson, C., Mulligan, P., Whetstine, J.R., Cole, P.A., and Casero, R.A. (2004). Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 119, 941-953. Shi, Y.J., Matson, C., Lan, F., Iwase, S., Baba, T., and Shi, Y. (2005). Regulation of LSD1 histone demethylase activity by its associated factors. Mol Cell 19, 857-864. Clara Cheong Page 206 PhD Thesis, National University of Singapore, 2008 Shindo, T., Manabe, I., Fukushima, Y., Tobe, K., Aizawa, K., Miyamoto, S., Kawai-Kowase, K., Moriyama, N., Imai, Y., Kawakami, H., et al. (2002). Kruppel-like zinc-finger transcription factor KLF5/BTEB2 is a target for angiotensin II signaling and an essential regulator of cardiovascular remodeling. Nat Med 8, 856-863. Sims, R.J., 3rd, and Reinberg, D. (2006). Histone H3 Lys methylation: caught in a bind? Genes Dev 20, 2779-2786. Singh, S.K., Kagalwala, M.N., Parker-Thornburg, J., Adams, H., and Majumder, S. (2008). REST maintains self-renewal and pluripotency of embryonic stem cells. Nature 453, 223-227. Sinkkonen, L., Hugenschmidt, T., Berninger, P., Gaidatzis, D., Mohn, F., Artus-Revel, C.G., Zavolan, M., Svoboda, P., and Filipowicz, W. (2008). MicroRNAs control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells. Nat Struct Mol Biol 15, 259-267. Smith, A.G., Heath, J.K., Donaldson, D.D., Wong, G.G., Moreau, J., Stahl, M., and Rogers, D. (1988). Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336, 688-690. Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21, 70-71. Southern, J.A., Young, D.F., Heaney, F., Baumgartner, W.K., and Randall, R.E. (1991). Identification of an epitope on the P and V proteins of simian virus that distinguishes between two isolates with different biological characteristics. J Gen Virol 72 ( Pt 7), 1551-1557. Stewart, C.L., Kaspar, P., Brunet, L.J., Bhatt, H., Gadi, I., Kontgen, F., and Abbondanzo, S.J. (1992). Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor. Nature 359, 76-79. Surani, M.A., Hayashi, K., and Hajkova, P. (2007). Genetic and epigenetic regulators of pluripotency. Cell 128, 747-762. Swigut, T., and Wysocka, J. (2007). H3K27 demethylases, at long last. Cell 131, 29-32. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872. Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676. Takao, Y., Yokota, T., and Koide, H. (2007). Beta-catenin up-regulates Nanog expression through interaction with Oct-3/4 in embryonic stem cells. Biochem Biophys Res Commun 353, 699-705. Tantin, D., Gemberling, M., Callister, C., and Fairbrother, W. (2008). High-throughput biochemical analysis of in vivo location data reveals novel distinct classes of POU5F1(Oct4)/DNA complexes. Genome Res 18, 631-639. Terpe, K. (2003). Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60, 523-533. Teufel, A., Wong, E.A., Mukhopadhyay, M., Malik, N., and Westphal, H. (2003). FoxP4, a novel forkhead transcription factor. Biochim Biophys Acta 1627, 147-152. Tolkunova, E., Malashicheva, A., Parfenov, V.N., Sustmann, C., Grosschedl, R., and Tomilin, A. (2007). PIAS proteins as repressors of Oct4 function. J Mol Biol 374, 1200-1212. Trinkle-Mulcahy, L., Boulon, S., Lam, Y.W., Urcia, R., Boisvert, F.M., Vandermoere, F., Morrice, N.A., Swift, S., Rothbauer, U., Leonhardt, H., et al. (2008). Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J Cell Biol 183, 223-239. Tsou, A.P., Yang, C.W., Huang, C.Y., Yu, R.C., Lee, Y.C., Chang, C.W., Chen, B.R., Chung, Y.F., Fann, M.J., Chi, C.W., et al. (2003). Identification of a novel cell cycle regulated gene, HURP, overexpressed in human hepatocellular carcinoma. Oncogene 22, 298-307. Clara Cheong Page 207 PhD Thesis, National University of Singapore, 2008 Valenzuela, D.M., Murphy, A.J., Frendewey, D., Gale, N.W., Economides, A.N., Auerbach, W., Poueymirou, W.T., Adams, N.C., Rojas, J., Yasenchak, J., et al. (2003). High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat Biotechnol 21, 652-659. van den Berg, D.L., Zhang, W., Yates, A., Engelen, E., Takacs, K., Bezstarosti, K., Demmers, J., Chambers, I., and Poot, R.A. (2008). Estrogen-related receptor beta interacts with Oct4 to positively regulate Nanog gene expression. Mol Cell Biol 28, 5986-5995. Van Hoof, D., Passier, R., Ward-Van Oostwaard, D., Pinkse, M.W., Heck, A.J., Mummery, C.L., and Krijgsveld, J. (2006). A quest for human and mouse embryonic stem cell-specific proteins. Mol Cell Proteomics 5, 1261-1273. Veraksa, A., Bauer, A., and Artavanis-Tsakonas, S. (2005). Analyzing protein complexes in Drosophila with tandem affinity purification-mass spectrometry. Dev Dyn 232, 827-834. Vigano, M.A., and Staudt, L.M. (1996). Transcriptional activation by Oct-3: evidence for a specific role of the POU-specific domain in mediating functional interaction with Oct-1. Nucleic Acids Res 24, 2112-2118. Wang, J., Rao, S., Chu, J., Shen, X., Levasseur, D.N., Theunissen, T.W., and Orkin, S.H. (2006). A protein interaction network for pluripotency of embryonic stem cells. Nature 444, 364-368. Wang, M., You, J., Bemis, K.G., Tegeler, T.J., and Brown, D.P. (2008). Label-free mass spectrometry-based protein quantification technologies in proteomic analysis. Brief Funct Genomic Proteomic. Wang, W., Zhou, H., Lin, H., Roy, S., Shaler, T.A., Hill, L.R., Norton, S., Kumar, P., Anderle, M., and Becker, C.H. (2003). Quantification of proteins and metabolites by mass spectrometry without isotopic labeling or spiked standards. Anal Chem 75, 4818-4826. Washburn, M.P., Wolters, D., and Yates, J.R., 3rd (2001). Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19, 242-247. Wegner, G.J., Lee, H.J., and Corn, R.M. (2002). Characterization and optimization of peptide arrays for the study of epitope-antibody interactions using surface plasmon resonance imaging. Anal Chem 74, 5161-5168. Whetton, A.D., Williamson, A.J., Krijgsveld, J., Lee, B.H., Lemischka, I., Oh, S., Pera, M., Mummery, C., and Heck, A.J. (2008). The time is right: proteome biology of stem cells. Cell Stem Cell 2, 215-217. Xiao, Z., and Veenstra, T.D. (2008). Comparison of protein expression by isotope-coded affinity tag labeling. Methods Mol Biol 428, 181-192. Yamniuk, A.P., and Vogel, H.J. (2004). Calmodulin's flexibility allows for promiscuity in its interactions with target proteins and peptides. Mol Biotechnol 27, 33-57. Yang, H.M., Do, H.J., Oh, J.H., Kim, J.H., Choi, S.Y., Cha, K.Y., and Chung, H.M. (2005). Characterization of putative cis-regulatory elements that control the transcriptional activity of the human Oct4 promoter. J Cell Biochem 96, 821-830. Yang, J., Chai, L., Fowles, T.C., Alipio, Z., Xu, D., Fink, L.M., Ward, D.C., and Ma, Y. (2008). Genome-wide analysis reveals Sall4 to be a major regulator of pluripotency in murine-embryonic stem cells. Proc Natl Acad Sci U S A 105, 19756-19761. Yang, P., Sampson, H.M., and Krause, H.M. (2006). A modified tandem affinity purification strategy identifies cofactors of the Drosophila nuclear receptor dHNF4. Proteomics 6, 927-935. Yates, A., and Chambers, I. (2005). The homeodomain protein Nanog and pluripotency in mouse embryonic stem cells. Biochem Soc Trans 33, 1518-1521. Yeom, Y.I., Fuhrmann, G., Ovitt, C.E., Brehm, A., Ohbo, K., Gross, M., Hubner, K., and Scholer, H.R. (1996). Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 122, 881-894. Clara Cheong Page 208 PhD Thesis, National University of Singapore, 2008 Ying, Q.L., Nichols, J., Chambers, I., and Smith, A. (2003). BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115, 281-292. Yuan, H., Corbi, N., Basilico, C., and Dailey, L. (1995). Developmental-specific activity of the FGF4 enhancer requires the synergistic action of Sox2 and Oct-3. Genes Dev 9, 2635-2645. Zambrowicz, B.P., Imamoto, A., Fiering, S., Herzenberg, L.A., Kerr, W.G., and Soriano, P. (1997). Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. Proc Natl Acad Sci U S A 94, 3789-3794. Zhang, B., VerBerkmoes, N.C., Langston, M.A., Uberbacher, E., Hettich, R.L., and Samatova, N.F. (2006). Detecting differential and correlated protein expression in label-free shotgun proteomics. J Proteome Res 5, 2909-2918. Zhang, L., Rayner, S., Katoku-Kikyo, N., Romanova, L., and Kikyo, N. (2007). Successful coimmunoprecipitation of Oct4 and Nanog using cross-linking. Biochem Biophys Res Commun 361, 611-614. Zhou, Q., Chipperfield, H., Melton, D.A., and Wong, W.H. (2007). A gene regulatory network in mouse embryonic stem cells. Proc Natl Acad Sci U S A 104, 16438-16443. Clara Cheong Page 209 PhD Thesis, National University of Singapore, 2008 [...]... assembly of basal transcription factors (TFII factors) with RNAPII on the promoter region of a gene, to allow the formation of a pre-initiation complex (PIC) and subsequent transcription of downstream sequence into messenger RNA for later translation into proteins 1.1.1 Gene-Specific Transcription Factors 1.1.1.1 Mechanism of Action To integrate and regulate the transcriptional activities at the many possible... (Baharvand et al., 2008) , the use of microscale twodimensional liquid chromatography and tandem mass spectrometry (2D LC-MS/MS) to identify ES cell-specific factors including Oct4, Sox2 and Utf1 which are at relative low abundance (Nagano et al., 2005), or a more comprehensive overview of the ES cell proteome (Van Hoof et al., 2006) Because of the huge dynamic range of the proteome, any one assay of the... loci by the differential expression of these target genes on RNAi mediated knockdown of Oct4 or Nanog (Loh et al., 2008) A similar study in human ES cells sought to find the binding sites of Oct4, Sox2 and Nanog, with 353 genes showing common binding of these 3 factors (Boyer et al., 2005) Genes occupied by Oct4 and Nanog or all three factors, included an overrepresentation of transcription factors comprised... been determined, and shows a close association of multiple factors along the DNA strand (Panne et al., 2007) Intriguingly, although there are overlapping DNA domains and tight binding of the transcription factors within ~50bp of the promoter region, direct protein- protein interactions between these transcription factors are few, and suggests that the shared exposed surfaces of these factors are available... expression profile and knockout phenotype of Nanog points to a degree of interconnectedness between the roles of Nanog and Oct4 – an Oct4 transgene knockdown results in differentiation of ES cells to the trophectoderm, and this can be rescued specifically with Oct4, but not Nanog (Chambers et al., 2003; Niwa et al., 2000), hence Clara Cheong Page 28 PhD Thesis, National University of Singapore, 2008 Nanog-mediated... of recruitment, assembly and processing of basal transcriptional machinery,counter various active or repressive marks left by other factors and to aid in chromatin remodeling activities that change the accessibility of DNA Clara Cheong Page 15 PhD Thesis, National University of Singapore, 2008 to the transcriptional machinery This can be done both directly, and through the recruitment of the relevant... resulted in a loss of methylation at the key promoters of Pou5f1 and Nanog examined (Li et al., 2007), and a concomitant lack of repression of these genes on differentiation Dnmt1 is a methyltransferase involved in the maintenance of methylation marks at CpG-rich regions of promoters, and Dnmt1-/- mice are also embryonic lethal, although Dnmt1/- ES cells are able to self-renew (Panning and Jaenisch, 1996)... these genes In light of this, there is an apparent need for protein and epigenetic level information to understand the assembly and roles of factors present in different enhanceosomes Clara Cheong Page 33 PhD Thesis, National University of Singapore, 2008 1.3 Role of Oct4 in Maintenance of Pluripotency A crucial link in the maintenance of pluripotency, Oct4 is a transcription factor of the POU (PitOct-Unc)... octamer proteins, and demonstrates HTH binding to a specific DNA sequence This is further elaborated upon by the presence of the POU-S domain which varies between octamer proteins, and adds a degree of specificity to the targets which the particular octamer protein may bind at 1.1.2 DNA Binding Sites of Transcription Factors 1.1.2.1 Analysis of Transcription Factor Binding Sites Clearly, a means of understanding... the types of genes potentially regulated by a single TF However, a couple of caveats remain First, TFs often work in tandem with other TFs and co- regulators to achieve a particular regulatory outcome, and the vast number of binding sites identified by global ChIP analyses represent an overestimate of the actual number that may be relevant to a particular regulatory network Hence, demonstration of TF binding . Heng Hang, Patricia Ng & Rhonda Ponnampalam have been instrumental in various ways, especially with protein work, mass spectrometry, and random jokes. To members of the Lufkin lab past and. me and dealing with my ups and downs, and believing that I could finish more than I myself thought I could. Same to DP, for love, patience, and all those fun surprises that kept me sane. Thanks. Identification of Purified Proteins by Mass Spectrometry 121 5.1.1 How MS and MS/MS work 121 5.1.2 Preparing Proteins for Mass Spectrometry 122 5.1.3 Peptide Ionization Methods 122 5.1.4 Protein