Post-Translational Regulatory Mechanisms Acting on KLF4 Mediate Pluripotency Exit in Naïve Mouse Embryonic Stem Cells

Post-Translational Regulatory Mechanisms acting on KLF4 mediate Pluripotency Exit in Naïve Mouse Embryonic Stem Cells

Navroop Kaur Dhaliwal

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate department of Cell and Systems Biology University of Toronto

Post-Translational Regulatory Mechanisms acting on KLF4 mediate Pluripotency Exit in Naïve Mouse Embryonic Stem Cells

Navroop Kaur Dhaliwal

Doctor of Philosophy

Graduate Department of Cell and Systems Biology University of Toronto

2019 Abstract

Pluripotent embryonic stem (ES) cells have the potential to self-renew and differentiate to generate all adult tissues. Pluripotency is regulated by an interconnected network of transcription factors including OCT4, SOX2, NANOG and KLF4. This transcriptional network is integrated with extracellular signaling pathways regulating pluripotency and differentiation. Mouse ES cells can be maintained in a state known as naïve pluripotency by culture in leukemia inhibitory factor (LIF) and two signaling pathway inhibitors (LIF/2i). A previous study observed that a reduction in Klf4 transcript, due to LIF withdrawal, was the first change in pluripotency gene expression during differentiation. Based on this finding I hypothesized that removal of KLF4 protein from transcriptional network would be required for exit from pluripotency. To investigate this I examined the levels of pluripotency associated transcription factors in the nucleus of ES cells as they exit the pluripotent state. This investigation revealed that a reduction in KLF4 protein levels did not occur immediately following the reduction in Klf4 transcription; instead I identified nuclear export of KLF4 protein was required for the reduction in Klf4 transcription and pluripotency exit.

Next, I investigated Klf4 gene and protein regulation and found that LIF/2i maintains Klf4 expression through both transcriptional and post-translational mechanisms. Specifically, KLF4 protein is highly stable in LIF/2i and this stability is maintained by physical interaction with active

ii transcriptional complexes which ensure nuclear anchoring of the KLF4 protein. Surprisingly,

KLF4 protein stability is so high (t½ >24 hr) that protein levels change by <2 fold when RNA levels are reduced by 17 fold. LIF/2i removal causes both nuclear export of KLF4 and reduced

KLF4 stability which together lead to reduced gene expression of the other pluripotency transcription factors and exit from the pluripotent state. These mechanisms regulating KLF4 protein can inform the design of reprogramming and differentiation approaches for cellular therapies.

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Acknowledgments

First and foremost, I would like to express my most sincere appreciation and gratitude to my supervisor, Professor Jennifer Mitchell for giving me opportunity to be a part of her lab. She is a true visionary representing a combination of leadership, scientific innovation and humanity from which I continue to learn. I am extremely obliged for receiving her tremendous support and guidance throughout the course of my research and especially during my maternity leave of absence. She has become a role model for me as a scientist, guide and working mother. I am grateful to her for providing me the opportunity to expand my academic horizons by presenting at national and international conferences. It has been my absolute honor to have been supervised by her over the past few years.

I would also like to express my enduring gratitude to my advisory committee members, Professor Tony Harris and Professor Tim Westwood, for their insightful advice and suggestions throughout my PhD. I am very thankful to Professor Sue Varmuza for her guidance to carry out in vivo experiments. I sincerely acknowledge the support of Professor Eva Sapi by providing me letters of recommendation that helped me achieve success in my 2014, 2016 and 2017 OGS applications.

I owe special thanks to Dr. Kamelia Miri for training and helping me out with mice handling, dissections and embryo staining. Thanks to Henry Hong for training me in confocal microscopy and for further assistance while imaging. I would also like to thank Yulia Katsman for being a good friend and guiding me through CRISPR/Cas9 protocols. I also thank Dr. Scott Davidson and Hala Tamim for their great help in carrying out site directed mutagenesis while I was away. Finally, I acknowledge the support of all of my previous and current lab members.

I would like to thank my parents, Dr. Kulwant Dhillon and Dr. Guntejinderinderjit Dhillon for their unconditional love, support and guidance. Without their incredible inspiration, I would not be here today. Next, I would like to thank my favourite engineer and entrepreneur, my husband Himmat Dhaliwal who has brought joy, happiness and endless love in my life ever since we met. I thank him and my father in law, Harbans Dhaliwal, for understanding and allowing me to go back to school after marriage and one kid. I look forward to everything Himmat will help me accomplish and owe him more thanks that I can ever put in words. I would like to thank my mother in law, Ranjit Dhaliwal, the most. I admire her positive attitude. I could not have carried out my

iv research with peaceful mind without her constant support, love, care and dedication at home. She has been the strongest pillar on which I ever leaned on during my hardest times. I would also like to thank rest of my family and friends to be patient and bearing with me during my PhD. Last but certainly not the least, I would like to thank my two little angels, Guransh and Jitkarn for being so supportive and patient during the whole course of my research.

“If you wish to succeed in life, make perseverance your bosom friend, experience your wise counselor, caution your elder brother, and hope your guardian genius.”

― Joseph Addison

September 2018

Table of Contents

Acknowledgments ...... iv

Table of Contents ...... vi

List of Tables ...... xi

List of Figures ...... xii

List of Abbreviations ...... xiv

List of Appendices ...... xvii

Statement of publications ...... xviii

Chapter 1: Literature Review ...... 1

1.1 Cell potency ...... 2

1.2 Early stages of mammalian embryogenesis ...... 3

1.2.1 The mouse as mammalian development model system ...... 3

1.2.2 Preimplantation phase ...... 3

1.2.3 Postimplantation phase ...... 6

1.2.4 Regulative nature of early mammalian embryo ...... 6

1.3 Embryonic stem (ES) cell derivation ...... 7

1.4 In vitro culturing of mouse and human ES cells ...... 8

1.5 States of pluripotent ES cells ...... 10

1.5.1 Naïve pluripotent state ...... 11

1.5.2 Primed pluripotent state ...... 12

1.6 Regulation of naïve pluripotent ES cells ...... 14

1.6.1 Transcriptional regulation ...... 14

1.6.1.1 RNA polymerase ...... 14

1.6.1.2 Transcription factors, enhancers and the regulatory landscape for gene transcription ...... 17

1.6.1.3 Nuclear transport mechanisms ...... 19

1.6.1.4 Transcriptional networks regulating pluripotency ...... 20

1.6.2 Epigenetic regulation of pluripotency ...... 22

1.6.3 Signaling pathways modulating naïve pluripotent state ...... 24

1.6.3.1 LIF and JAK/STAT3 signaling ...... 24

1.6.3.2 BMP4 signaling ...... 25

1.6.3.3 ERK signaling ...... 26

1.6.3.4 Wnt/β-Catenin signaling ...... 27

1.6.3.5 PI3 kinase/AKT signaling ...... 27

1.6.3.6 Crosstalk among different signaling pathways for pluripotency maintenance ...... 28

1.6.3.7 Integration of signaling pathways with transcriptional circuitries ...... 29

1.6.4 Post transcriptional and pst translational regulation of pluripotency ...... 32

1.7 KLFs in pluripotency ...... 33

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1.8 Thesis objective and hypothesis...... 35

1.8.1 Major research questions ...... 35

1.8.2 Thesis aims...... 36

Chapter 2: KLF4 Nuclear Export Requires ERK Activation and Initiates Exit from Naive Pluripotency ...... 37

2.1 Abstract ...... 38

2.2 Introduction ...... 39

2.3 Results ...... 41

2.3.1 KLF4 nuclear export occurs as ES cells exit naive pluripotency ...... 41

2.3.2 ERK Activation and Interaction with KLF4 Is Coincident with KLF4 Nuclear Export ...... 51

2.3.3 KLF4 Nuclear Export Occurs through an XPO1-Mediated Nuclear Export Mechanism ...... 57

2.3.4 KLF4 Nuclear Export Requires Both Nuclear Export Sequences and Phosphorylation of S132 ...... 61

2.3.5 Inhibiting KLF4 Nuclear Export Delays Differentiation of ES cells ...... 67

2.3.6 MEK-ERK Signaling during Embryogenesis Downregulates KLF4 and NANOG ...... 73

2.4 Discussion ...... 79

2.5 Materials and methods ...... 81

2.5.1 Embryonic stem cell culture ...... 81

2.5.2 Immunoflourescence and proximity ligation amplification ...... 82

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2.5.3 Immunoblot and Co-Immunoprecipitation ...... 83

2.5.4 Blastocyst collection, Immunostaining and Gene expression ...... 84

2.5.5 Real time qPCR...... 87

2.5.6 Expression of KLF4 mutants ...... 87

Chapter 3: KLF4 protein is stabilized by interaction with pluripotency transcription factors and mutants with increased stability block embryonic stem cell differentiation ...... 87

3.1 Abstract ...... 91

3.2 Introduction ...... 92

3.3 Results ...... 94

3.3.1 Klf4 transcript and protein levels are uncoupled in embryonic stem cells maintained in LIF/2i ...... 94

3.3.2 KLF4 protein is stable in LIF/2i cultured ES cells ...... 101

3.3.3 KLF4 protein stability is regulated by LIF and MAPK signaling pathways ...... 104

3.3.4 Nuclear localization maintains KLF4 protein stability in LIF/2i maintained ES cells ...... 109

3.3.5 KLF4 association with DNA and RNAPII is required to maintain protein stability through nuclear anchoring ...... 112

3.3.6 Ubiquitination of KLF4 is required for nuclear export and degradation during differentiation...... 112

3.3.7 The loss of KLF4 protein stability is required for ES cell differentiation. ..119

3.3.8 Activated STAT3, NANOG and SOX2 stabilize KLF4 protein through its involvement in nuclear complexes...... 122

3.4 Discussion ...... 138

3.5 Materials and methods ...... 144

3.5.1 Embryonic stem cell culture ...... 144

3.5.2 Cellular fractionation, co-Immunoprecipitation and western blotting ...... 144

3.5.3 Proximity ligation amplification (PLA) ...... 147

3.5.4 CRISPR/Cas9 deletion ...... 147

3.5.5 Real time qPCR ...... 148

3.5.6 Expression of KLF4 mutants ...... 149

3.5.7 Transient transfections ...... 150

Chapter 4: Global discussion and future directions ...... 152

4.1 Summary and discussion...... 153

4.2 Future directions ...... 160

References ...... 163

List of Tables

Table 2.1: Antibody list ...... 85

Table 2.2: Gene expression primer list ...... 87

Table 2.3: Site directed mutagenesis primer list ...... 88

Table 3.1: Antibody list ...... 145

Table 3.2: List of CRISPR/Cas9 guide sequences ...... 148

Table 3.3: List of gene expression primers ...... 149

Table 3.4: List of primers for site directed mutagenesis ...... 150

List of Figures

Figure 1.1: The preimplantation phase of mouse embryogenesis ...... 5

Figure 1.2: Major signaling pathways involved in modulation of pluripotency...... 31

Figure 2.1: Changes in pluripotency factor mRNA and protein levels during the first 24 hr of differentiation...... 43

Figure 2.2: KLF4 nuclear exit occurs as ES cells exit naïve pluripotency...... 45

Figure 2.3: Changes in nuclear protein complexes associated with pluripotency exit...... 47

Figure 2.4: The removal of MEK inhibition phosphorylates and accumulates KLF4 to the cytoplasm...... 49

Figure 2.5: Active ERK2 interacts with KLF4 initiating KLF4 nuclear export ...... 53

Figure 2.6: Signaling mechanisms regulating KLF4 nuclear export...... 55

Figure 2.7: KLF4 nuclear export occurs through an XPO1-mediated mechanism ...... 58

Figure 2.8: KLF4 interacts with XPO1 independently of MEK ...... 60

Figure 2.9: The KLF4 ERK phosphorylation site S132, NES1, and NES2 are required for KLF4 nuclear export ...... 63

Figure 2.10: Experimental controls and supplementary data ...... 65

Figure 2.11: KLF4 nuclear export inhibition delays exit from naive pluripotency and differentiation of embryoid bodies ...... 69

Figure 2.12: Expression of KLF4(S132A) blocks ES cell differentiation ...... 71

Figure 2.13: KLF4 downregulation in the Inner Cell Mass of mouse blastocysts depends on MEK activation ...... 75

Figure 2.14: Proximity ligation amplification and immunostaining controls in mouse embryos...... 77

Figure 2.15: Principles of proximity ligation amplification...... 83

Figure 2.16: The model of KLF4 nuclear export and naïve pluripotency exit...... 89

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Figure 3.1: CRISPR deletions of two Klf4 enhancer regions showed a 17 fold decrease in Klf4 transcript but a <2 fold decrease in KLF4 protein levels...... 96

Figure 3.2: Klf4 enhancer deletion does not affect transcript or protein levels for other pluripotency transcription factors...... 98

Figure 3.3: Interaction between KLF4/RNAPII is disrupted by reduced KLF4 protein levels ...100

Figure 3.4: KLF4 protein stability is reduced after 24 hr differentiation ...... 102

Figure 3.5: Removal of LIF or the MEK inhibitor reduces KLF4 half-life...... 105

Figure 3.6: An integrated KLF4-GFP expression construct showed a similar reduction in protein stability after removal of LIF or the MEK inhibitor...... 107

Figure 3.7: Nuclear localization and anchoring maintain KLF4 stability ...... 110

Figure 3.8: KLF4 protein undergoes degradation by ubiquitination...... 115

Figure 3.9: Monoubiquitination at K249 of nuclear KLF4 protein causes nuclear export and degradation...... 117

Figure 3.10: The loss of KLF4 protein stability is required for ES cell differentiation...... 120

Figure 3.11: Activated STAT3 stabilizes KLF4 protein ...... 125

Figure 3.12: Activated STAT3 stabilizes KLF4 by recruiting it to transcriptional complexes. ..128

Figure 3.13: NANOG stabilizes KLF4 protein...... 130

Figure 3.14: NANOG stabilizes KLF4 protein by recruiting KLF4 to transcriptional complexes...... 132

Figure 3.15: SOX2 increases the stability of KLF4 protein in ES cells maintained in LIF/2i. ...134

Figure 3.16: SOX2 stabilizes KLF4 protein by stabilizing its association with transcriptional complexes...... 136

Figure 3.17: Summary of KLF4 mutations and their effects ...... 140

Figure 3.18: The summary figure showing transcription factor status and complex after 24 hr differentiated ES cell ...... 143

Figure 4.1: The naïve pluripotency exit model for ES cells ...... 157

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List of Abbreviations iPSC Induced pluripotent stem cell 2i Double inhibition 3i Triple inhibition 5i/L/A(F) 5 signaling inhibitors/LIF/Activin (FGF) 5SRNA 5S ribonucleic acid APC Adenomatous polyposis coli BDNF Brain-derived neurotrophic factor bFGF Basal fibroblast growth factor BMP4 Bone morphogenetic potein-4 BMPR Bone morphogenetic potein receptor bp Base pairs CBP Catenin binding protein ChIP Chromatin immunoprecipitation ChIP- Chromatin immunoprecipitation followed by paired-end tag PET sequencing Co-IP Co immunoprecipitation COMPASS COMplex of Proteins ASsociated with Set1 CpG 5'-Cytosine-phosphate-Guanine-3' CRISPR Clustered Regularly Interspaced Short Palindromic Repeats CTCF CCCTC-Binding factor CTD Carboxy terminal domain CTDK-1 Carboxy terminal domain kinase-1 DIA Differentiation-inhibiting activity DMEM Dulbecco’s modified minimal essential medium DNA Deoxyribonucleic acid DRE Distant regulatory elements DVL1 Dishevelled EC Embryonal carcinoma EPI Epiblast ERK Extracellular signal-regulated kinases ES Embryonic stem FACS Fluorescence assisted cell sorting FBXW8 F-box/WD repeat-containing protein 8 FGF2 Fibroblast growth factor-2 FGF4 Fibroblast growth factor-4 FOXD3 Forkhead box D3 FRAP fluorescent recovery after photobleaching Grb2 Growth factor receptor-bound protein 2 GSK3 Glycogen synthase kinase-3 GTF General transcription factors H3K27ac Histone 3 acetylated at 27 lysine

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H3K27me Histone 3 methylated at 27 lysine H3K27me3 Histone 3 trimethylated at 27 lysine H3K4 Histone 3 lysine 4 H3K9 Histone 3 lysine 9 Hbb haemoglobin beta HMG High mobility group HP1 heterochromatin protein 1 HS1 hypersensitive sites ICM Inner cell mass IF Immunoflourescence IVF In vitro fertilization JAK Janus kinase Kb Kilobases kDa kilodalton KLF2 Kruppel-like factor 2 KLF4 Kruppel-like factor 4 KLF5 Kruppel-like factor 5 LCR Locus control region LEF Lymphoid enhancer-binding factor 1 LIF Leukemia inhibitory factor Lrh-1 Liver receptor homolog-1 LRP Lipoprotein receptor-related protein MAPK Mitogen-activated protein kinase MB Megabases MEF Mouse embryonic fibroblast MEK MAPK/ERK kinase miRNA Micro ribonucleic acid mRNA Messenger ribonucleic acid MYC Myelocytomatosis NANOG Tír na nÓg (Scottish origin) Nr5a2 Nuclear receptor subfamily 5, group A, member 2 Nr6a1 Nuclear receptor subfamily 6, group A, member 1 NT3 Neurotrophin-3 NT4 Neurotrophin-4 OCT4 Octamer-binding transcription factor 4 OSN OCT4/SOX2/NANOG PCR Polymerase chain reaction PDGF Platelet-derived growth factor PE Primitive endoderm PEST proline (P), glutamic acid (E), serine (S), and threonine (T) PI3K Phosphoinositide 3-kinase PIC Preinitiation complex PKB Protein kinase B

PKC Protein kinase C PLA Proximity ligation assay p-TEFb Positive transcription elongation factor b qPCR Quantitative polymerase chain reaction rhbFGF Recombinant human basal fibroblast growth factor rhTGF-β Recombinant human transforming growth factor RNA Ribonucleic acid RNAPII RNA polymerase II RPB RNA polymerase II subunits rRNA Ribosomal riboneic acid S1P Sphingosine-1-phosphate SCR Sox2 controlling region SDM Site directed mutagenesis SH2 Src-homology 2 domain SHP1 Src-homology 2 domain containing protein tyrosine phosphatases -1 SiRNA Silencing ribonucleic acid SMAD human homologue of Mad and Sma genes = Smad SnRNA Small nuclear ribonucleic acid SOX2 (sex determining region Y)-box 2 SRR Sox2 regulating region SRY Sex determining region Y STAT3 Signal transducer and activator of transcription 3 SUMO Small ubiquitin-related modifier Syk Spleen tyrosine kinase t2iL+PKCi transgenes/double inhibition (MAPK and GSK3)/ LIF/PKC inhibitor TAD Topology associated domain Tbx3 T-box transcription factor TCF T cell factor TCR T-Cell receptor TF Trancription factor TGF-β Transforming growth factor β TNF-2 Tumor necrosis factor tRNA Transfer ribonuleic acid USP21 Ubiquitin-specific protease 21 WB Western blotting Zap70 Zeta-chain T-Cell receptor associated protein kinase β-TRCP Beta-transducin repeat containing

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List of Appendices

Appendix1

Figure A1.1: The schematic of mouse KLF4 and mutated/deleted sites ...... 191

Figure A1.2: KLF4 sequence mouse...... 192

Figure A1.3: KLF4 sequence human...... 193

Figure A1.4: conserved sites S132, K249 and K275 among mouse and human...... 194

Appendix2

Table A2.1: Half life summary table ...... 195

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Statement of publications

Navroop K. Dhaliwal, Kamelia Miri, Scott Davidson, Hala Tamim El Jarkass, Jennifer A. Mitchell (2018) KLF4 Nuclear Export Requires ERK Activation and Initiates Exit from Naive Pluripotency. Stem Cell Reports. Apr 10;10(4):1308-1323. https://doi.org/10.1016/j.stemcr.2018.02.007 J.A.M. and N.K.D. conceived and designed the experiments. N.K.D. performed all experiments with the following assistance: K.M. collaborated on the embryo experiments, and N.K.D., S.D., and H.T.E.J. generated KLF4-GFP mutant constructs. N.K.D. and J.A.M. analyzed the data and wrote the manuscript, which was approved by all co-authors.

This is chapter 2 of my thesis.

Navroop K. Dhaliwal and Jennifer A. Mitchell (2016) Nuclear RNA Isolation and Sequencing. Published book chapter in Long Non-Coding RNAs: Methods and Protocols, Methods in Molecular Biology, vol. 1402. N.K.D. conducted experiments and wrote the first draft of book chapter.

The protocol described in this book chapter was used to perform cell fractionation experiments mentioned in chapters 2 and 3.

Virlana M. Shchuka, Nakisa Malek-Gilani, Gurdeep Singh, Lida Langroudi, Navroop K. Dhaliwal, Sakthi D. Moorthy, Scott Davidson, Neil N. Macpherson and Jennifer A. Mitchell (2015) Chromatin Dynamics in Lineage Commitment and Cellular Reprogramming. Genes, 6(3), 641-661. All authors participated in the writing of the manuscript.

Harry Y. Zhou, Yulia Katsman, Navroop K. Dhaliwal, Scott Davidson, Neil N. Macpherson, Moorthy Sakthidevi, Felicia Collura and Jennifer A. Mitchell (2014) A Sox2 distal enhancer cluster regulates embryonic stem cell differentiation potential. Genes & Dev., 28: 2699-2711. doi:10.1101/gad.248526.114 N.K.D. conducted the immunofluorescence and immunoblot analysis.

xviii

The Sox2 control region (SCR) deleted clones made in this publication were used for investigation in chapter 3.

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Chapter 1

Literature review

1.1 Cell potency

A cell’s ability to differentiate into other cell types is referred to as the cell’s potency. During the process of development cells proceed toward more highly differentiated and specialized states with more limited cellular potency. A cell’s potential to generate all cell types of the organism is known as totipotency and cells with such ability are called totipotent cells. Totipotent cells have the greatest differentiation potential and they are capable of giving rise to the complete organism. An example of a totipotent cell is the mammalian zygote. In mouse and humans, the zygote is formed when the oocyte is fertilized by a sperm cell. The zygote undergoes multiple divisions in just a few hours to give rise to the totipotent cells of the morula stage that can give rise to all three embryonic germ layers, as well as trophoblast cells and later the extra embryonic tissues.

Pluripotency refers to a cell’s ability to differentiate into many different types of cells. In the context of mammalian development pluripotent cells are able to form all of the cell types of the embryo proper but not the extra embryonic tissues. The earliest emergence of pluripotency in the embryo are the cells of the inner cell mass (ICM) of the blastocyst (as reviewed by Nichols et al, in 2012). Cells from the ICM of the blastocyst can be isolated and cultured in vitro and are capable of forming all three germ layers of the embryo proper. Reprogramming of an adult cell back to a state resembling an embryonic pluripotent state in vitro produces cells referred to as induced pluripotent stem cells (iPSCs). Advances in understanding the molecular mechanisms maintaining pluripotency and differentiation made this reversion possible. An iPSC has the potential to differentiate to any adult cell type in response to given environmental cues. The discovery of the process through which cells can be reprogrammed to induced pluripotency is the basis for regenerative medicine which aims to produce mature cells from patient derived iPSCs for cellular therapies to treat numerous diseases.

As cells advance through development, they further lose their potential to differentiate and become lineage-restricted. Although pluripotent stem cells do not persist beyond early development in mammals, multipotent stem cells exist which are able to differentiate into all cell types of one specific lineage; for example, hematopoietic stem cells required for the production of blood cells.

1.2 Early stages of mammalian embryogenesis

Early embryogenesis in mammals is divided into two major phases: preimplantation and postimplantation. In mouse, the preimplantation phase begins with formation of the zygote and proceeds through a number of intermediary stages defined by an increasing number of cells, known as blastomeres, and concludes with formation of the blastocyst, capable of uterine implantation (Aiken et al., 2004). Two major cell fate decisions occur during the preimplantation period. The first decision involves segregation of trophoblast cells and the ICM. The second decision involves further segregation of the ICM into epiblast and primitive endoderm cells. The postimplantation period in mouse begins after uterine attachment of the blastocyst and proceeds through various lineage commitment events to produce the developing embryo and the extra embryonic tissues.

1.2.1 The mouse as a mammalian development model system

Most of what is known about the regulation of early embryonic development in mammals comes from research on the mouse model system. Mice are evolutionarily separated from humans by about 100 million years and have a genome that is similar to the human genome in size and sequence; mouse and human proteins are generally 80–90% identical in their amino-acid sequence (Makalowski et al., 2011). Genetic modification to the mouse genome has been carried out for several years providing a rich source of information about the molecular genetics of mammalian development (Alberts, Johnson and Lewis, 2002).

1.2.2 Preimplantation phase

Embryogenesis begins with fertilization of the ovum by sperm resulting in a single diploid cell termed the zygote. The zygote undergoes mitotic divisions known as cleavage and cellular differentiation, leading to development of a multicellular embryo. In mouse, the switch from maternal to zygotic control of gene expression occurs at the 2-cell stage (Piko et al., 1982; Prather 1989). Mouse blastomeres are loosely arranged until the 8-cell stage. After the 8-cell stage, blastomeres form a compact ball of cells that are stabilized by adherens first and then tight junctions (Ducibella et al., 1979; Hyafil et al., 1980; Vestweber et al., 1987). By embryonic developmental day 3.0 (E3.0), the 16-cell morula begins to segregate into a small group of internal cells surrounded by a larger group of external cells (Barlow et al. 1972). The descendants of the

3 external cells of the morula become trophoblast cells giving rise to the extra embryonic tissues whereas the descendants of the inner cells will form the ICM that goes on to form the embryo proper (Pedersen et al. 1986; Fleming 1987). The cells of the ICM are referred to as pluripotent as they have the ability to form all the cell types of the embryo. Trophoblast cells of the 32-cell late morula at E3.25 begin to create an osmotic gradient across them, which drives the transport of water, forming a small fluid-filled cavity inside the 32-cell early blastocyst by E3.5 (Wiley et al., 1984). The ICM of the E3.5, 32-cell early blastocyst expresses Nanog and Gata6 in a random “salt and pepper” pattern and in a mutually exclusive manner (Chazaud et al., 2006). In 2017 Posfai et al., reported that the developmental potential of trophectoderm cells is terminally restricted by the 32-cell early blastocyst, whereas cells of the ICM only fully commit during the 32- to 64-cell-stage transition. This distinction between trophoblast and pluripotent ICM cells represents the first lineage segregation event in mammalian development (Dyce et al. 1987; Fleming 1987). The ICM cells secrete proteins including fibroblast growth factor 4 (FGF4) that cause the trophoblast cells to divide as the blastocyst continues to grow (Tanaka et al. 1998). As the blastocyst grows bigger the blastocoel enlarges, pushing the ICM to one side of the embryo. The 64-cell mid blastocyst formed by E4.0 is marked by the enlarged blastocoel, outer trophectoderm layer and mosaic ICM with heterogeneous Nanog and Gata6 expression containing progenitors of epiblast (EPI) and primitive endoderm (PE), whose distinction marks the second lineage segregation event. In 2006, Chazaud et al, suggested that (Grb2)-Ras- mitogen-activated protein kinase (MAPK) signaling, activated by an unknown mechanism, induce Gata6 and repress Nanog in the PE progenitors, whereas the absence of Grb2-dependent signals allows for Nanog expression in EPI progenitors. In PE progenitors, higher expression of Gata6 induces target genes such as Laminin and Dab2, which modulate cellular adhesive functions to initiate sorting out of the two lineages. The basal lamina consisting of laminin is formed by the late blastocyst stage at E4.5, and separates the EPI from the PE cells (Chazaud et al, 2006). Implantation of the blastocyst occurs about 5 days after fertilization of the mouse embryo when the blastocyst makes direct contact with the uterine epithelium marking the end of the preimplantation phase.

Figure 1.1

Figure 1.1: The preimplantation phase of mouse embryogenesis

The temporal sequence of events that occur during the preimplantation phase of mouse embryogenesis. Embryonic stages and days post fertilization (E) are indicated. The cell lineages generated as a result of the first and the second cell-fate decisions are marked. In E3.0 and 3.25- day embryos the inner cells which will become the pluripotent cells of the inner cell mass (ICM) are indicated in purple.

1.2.3 Postimplantation phase

The pluripotent cells of the epiblast persist after uterine attachment before these cells ultimately undergo further lineage commitment to the three germ layers; endoderm, mesoderm and ectoderm (Gilbert, 2000). The epiblast grows and begins to differentiate to an extraembryonic structure called yolk sac and embryo proper by gastrulation and neurulation (Gilbert, 2000). Gastrulation is the process involving cellular movement by which the single-layered blastula is reorganized into a multilayered structure known as the gastrula. The primary function of gastrulation is to correctly place precursor tissues for subsequent morphogenesis (Gilbert, 2000). Neurulation refers to the folding process by which neural plate formed in the gastrula is transformed into the neural tube (Gilbert, 2000). Embryogenesis is followed by organogenesis which includes the development of specific organs based on the growth rate of fetus till birth (Gilbert, 2000).

1.2.4 Regulative nature of early mammalian embryo

Mammalian development is a robust system capable adjusting to disturbances; hence the process is referred to as highly regulative (as reviewed in Klimczewska et al, 2017). The mammalian zygote has the natural potential to give rise to full viable organism and referred as totipotent, but the occurrence of monozygotic multiplets, either after experimental intervention or spontaneously in nature, proves that totipotency is maintained at later developmental stages (Mullen et al, 1970; Papaioannou et al, 1995; Papaioannou et al, 1989; Tsunoda et al, 1983; Tsunoda et al, 1985). Blastomeres produced after the first few cell divisions are also totipotent; if the early embryo is split in two, it can generate a pair of identical twins (Matsumoto et al, 1989). Similarly, if one of the cells in a 2-cell mouse embryo is destroyed and rest of the embryo is placed in the uterus of a foster mother, it can develop into a normal viable mouse (Tarkowski, 1959). Moreover, a single giant morula generated by fusing two 8-cell stage embryos develops into a normal mouse and is known as a chimeric mouse (Johnson et al., 1981). Chimeras are formed from the aggregates of genetically different groups of cells. Chimeras can also be made by injecting cells from an early embryo of one genotype into a blastocyst of another genotype. The injected cells are incorporated into the ICM of the host blastocyst and go on to form the embryo proper (Rossant, 1975a and b).

1.3 Embryonic stem (ES) cell derivation

The plasticity of early mammalian blastomeres can also be reflected in their ability to give rise to embryonic stem (ES) cells. Individual cells of the ICM are pluripotent and these cells can be isolated and cultured in vitro due to their ability to self-renew under the correct conditions. The foundation of ES cell derivation began with studies of malignant tumors known as teratocarinomas (Dixon & Moore 1952). Stevens & Hummel (1957) showed the induction of these tumors by explanting the genital ridges of fetuses between 11 and 13.5 days of development to ectopic sites in strain 129 mice indicating the embryonal origin of teratomas. Later, in 1959, Pierce and Dixon generated ascitic mouse teratocarinomas containing embryoid bodies in their ascitic fluid and these embryoid bodies could be passaged. Pierce and Verney (1961) demonstrated differentiation of these embryoid bodies by explanting. In order to understand the difference between the biological behavior of benign and malignant tumors, Pierce and his colleagues carried out many studies and demonstrated that tumor cells can be grown in tissue culture and most importantly Kleinsmith and Pierce (1964) showed that even a single tumor cell has the full potential to generate teratocarinomas, containing a wide range of differentiated cells, in vivo after transplantation. This observation laid the idea of pluripotent tumor stem cells, later named as embryonal carcinomas (ECs).

To further understand the concept of differentiation and the biology of pluripotent tumor stem cells, clonal cultures of ECs isolated from embryoid bodies were established (Rosenthal et al, 1970; Kahan et al., 1970). These clonal populations of ECs were able to generate a teratocarcinoma tumor upon reinjection into mice (Rosenthal et al, 1970; Kahan et al., 1970). The potency of these cells, however, diminished severely upon in vitro passaging (Rosenthal et al, 1970; Kahan et al., 1970). Cell lines generated by disaggregating cells of well-differentiated solid teratocarinomas, cultured with irradiated chicken fibroblast feeder layers, had improved differentiation potential after prolonged in vitro passaging (Evans et al., 1972). Furthermore, EC cells isolated originally from embryoid bodies were shown to participate in mouse embryonic development after being transferred to blastocysts but these cells displayed very low chimerism (Brinster et al, 1974). Afterwards, normal chimeric mice were produced by injecting EC cells isolated from core of embryoid bodies (Mintz et al, 1975; Papaioannou et al., 1975). As EC cells in tissue culture were not karyotypically normal, none of the chimeric mice generated were germline chimeras

(Papaioannou et al., 1975). Around the same time, in vitro differentiation of an EC cell line to layers of differentiated cells such as beating cardiac myocytes and nerve like processes, was reported (Nicholas et al, 1975; Martin et al, 1975). Martin et al (1975) also showed that EC cells can be differentiated in vitro making embryoid bodies. To further evaluate the differentiation of embryoid bodies derived in vitro from ECs, embryoid bodies were allowed to reattach to the tissue culture plastic surface. PE was the first differentiated cell type observed after attachment (Martin et al, 1975). This was similar to the mechanism of differentiation reported in the early mouse embryo; Richard Gardner in 1968 and Janet Rossant in 1975 showed that an isolated inner cell mass (ICM) from a mouse blastocyst formed a complete outer layer of extra-embryonic endoderm (Gardener, 1968; Rossant, 1975; Rossant 1975a).

Identification of biochemical and cell surface markers further confirmed the homology between ECs and mouse early embryonic cells (Adamson et al, 1977; Stern et al, 1978; Lovell-Badge et al, 1980; Stinnakre et al, 1981; Kapadia et al, 1981). In 1981, Evans and Kaufman isolated pluripotent cells from the ICM of delayed blastocysts. These cells possess all the features of previously established EC cells and a normal karyotype. Later in 1981 Gail Martin gave the term “embryonic stem cells” to these cells and described the maintenance of mouse ES cell culture in teratocarcinoma conditioned medium. Nagy et al., in 1993 showed that upon injecting cultured mouse ES cells into a recipient blastocyst, ES cells participate in forming all tissue types of a chimeric mouse.

1.4 In vitro culturing of mouse and human ES cells

Although the cells of the ICM only retain pluripotency for a short time during embryogenesis, ES cells retain pluripotency and the ability to self-renew through many passages in culture. Different conditions have been used to culture mouse ES cells and these affect various aspects of their cellular phenotype. Conditions established for maintaining mouse ES cells in a pluripotent state laid the foundation for later isolation and culture of human ES cells.

The first isolated ES cells were from mouse by Evans and Kauffman in 1981. They were cultured on gelatin coated petri plates containing mitomycin C-inactivated fibroblasts feeder layers in Dulbecco’s modified minimal essential medium (DMEM) supplemented with 10% fetal calf serum and newborn calf serum. Later one study described the use of teratocarcinoma conditioned media

8 which was thought to contain a growth factor to promote pluripotent mouse ES cell maintenance and inhibit their differentiation in culture for a more prolonged time (Martin et al, 1981). In the late 1980’s several studies focused on identifying the different components of media required for pluripotency maintenance of ES cells in feeder free conditions.

Austin Smith and Martin Hooper in 1987 reported that differentiation-inhibiting activity (DIA) polypeptides isolated from buffalo rat liver cell conditioned media could prevent the spontaneous differentiation of ECs and mouse ES cells cultured without feeders. DIA polypeptides were shown to be structurally and functionally similar to leukemia inhibitory factor (LIF) (Williams et al., 1988). Purified recombinant LIF can substitute for DIA even when culturing mouse ES cells in the absence of a feeder layer (Williams et al., 1988). Mouse ES cells cultured in vitro for longer times or number of passages with LIF and serum on feeder layers were reported to have a stable karyotype, higher telomerase levels and could make chimeras and teratomas (Rossant et al, 2001; reviewed in Burdon et al., 2002; Edwards et al, 2002; Gardner et al, 2002; Prelle et al., 2002).

The first successfully derived human ES cells were isolated from blastocyst ICM of in vitro fertilization (IVF) derived embryos and cultured on plates having irradiated mitotically inactivated mouse embryonic fibroblast (MEF) feeders by Thompson et al, in 1998. They established five human ES cell lines. The culture conditions used for human ES cell cultures were similar to as described for mouse and primate ES cells (Robertson et al, 1987; Thompson et al, 1995; Thomson et al., 1996). The human ES cell lines were also derived and characterized for their differentiation potential later by Reubinoff et al, (2000). Human recombinant leukemia inhibitory factor (hLIF) was used while isolating and culturing human ES cells; however, studies in primate ES cell culture showed that LIF is not required for their propagation (Thomson et al. 1998). Unlike mouse ES cells it was not possible to grow human ES cells by addition of LIF in feeder-free conditions (Thomson et al., 1998; Reubinoff et al., 2000). Later in 2001, Xu et al. reported a system to grow human ES cells on Matrigel matrix with 100% MEF-conditioned medium supplemented with basal fibroblast growth factor (bFGF). These serum free growth conditions led to the clonal derivation of human ES cells (Amit et al. 2000); however, in feeder- free culture systems, human ES cells often generate fibroblast or stromal-like cells which serve as feeders for the maintenance of their undifferentiated state (Stojkovic et al., 2005; Wang et al., 2005). Eventually these types of cultures become variable in terms of their pluripotent state which

9 makes them not suitable for further downstream therapeutic purposes; thus, additional efforts were required to completely eliminate the need for feeder cells and establish a chemically defined culture system for the growth of both mouse and human ES cells.

Knowledge about how external signaling pathways regulate pluripotency maintenance and lineage commitment led to the discovery of novel synthetic molecules inhibiting these signaling pathways and better culture strategies for both mouse and human ES cells. The use of chemically defined media (N2B27) with selective small molecule inhibitors for the mitogen- activated protein kinase (MAPK) pathway (PD0325901), glycogen synthase kinase-3 (GSK3, CHIR99021) and LIF (LIF/2i) was sufficient to maintain the undifferentiated state of mouse ES cells (Silva et al, 2008). Furthermore, mouse ES cells cultivated in LIF/2i exhibit greater pluripotent gene expression than mouse ES cells cultivated in serum with LIF (Silva et al, 2008).

Unlike mouse ES cells, which can be maintained by only LIF and serum, human ES cell pluripotency maintenance requires additional factors. Fibroblast growth factor (FGF)-2 was the first factor found to be critical for human ES cell maintenance (Amit et al., 2000). Ligands from the transforming growth factor β (TGFβ) family (such as TGFβ, activin, and nodal) (Vallier et al., 2005), FGF (Amit et al., 2000), neurotrophins (BDNF, NT3, and NT4), Sphingosine-1-phosphate (S1P), Platelet-derived growth factor (PDGF), and heregulin (Pébay et al., 2005), have been identified as modulating human ES cell maintenance. Apart from Matrigel, recombinant Vitronectin could provide for stem cell maintenance in defined medium, via engagement of the alpha V beta 5 integrin expressed on ES cells (Braam et al., 2008). The most widely used commercially available feeder free defined culture medium for human ES cells is mTeSR™1 (Ludwig et al, 2006). Complete mTeSR™1 medium (Basal Medium + 5X Supplement) contains recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor β (rh TGFβ). The human ES cells have been cultured with complete mTeSR™1 media on either Matrigel or Vitronectin XF™.

1.5 States of pluripotent ES cells

The characterization of mouse and human ES cells cultured in the above described conditions showed that they were different from each other in phenotype and gene expression. Microscopic studies revealed that human ES cells grew as large flattened colonies whereas mouse ES cells grew

10 in smaller, rounder clumps of cells. Human ES cells formed 2-4 cell layers over the feeder cells while mouse ES cells formed 4–10 cell layers over the feeder layer. Cell surface marker expression comparison revealed that mouse ES cells expressed SSEA-1 but not SSEA-4, by contrast human ES cells expressed SSEA-4 but not SSEA-1 (Reubinoff et al., 2000, Thomson et al., 1998, Humphrey et al., 2004, Ginis et al., 2004). Human ES cells express TRA-1-60 and TRA-1-81 antigens while mouse ES cells do not (Reubinoff et al., 2000, Thomson et al., 1998, Humphrey et al., 2004, Ginis et al., 2004). Expression of forkhead box D3 (FOXD3) and Kruppel-like factor 4 (KLF4) transcription factors was detected only in mouse ES cells and not in human ES cells indicating the fundamental differences in the transcriptional regulation of these cells (Reubinoff et al., 2000, Thomson et al., 1998, Humphrey et al., 2004, Ginis et al., 2004; Takashima et al., 2014; Theunissen et al., 2014).

Different signaling pathways operate in both mouse and human ES cells. For example, the p53 pathway, known to be responsible for growth arrest and cell death in response to DNA damage (Gottifredi et al., 2000; Schultz et al., 2000) is active in mouse ES cells but not human ES cells (Ginis et al., 2004). Human ES cells express tumor necrosis factor (TNF) receptor 2 (Ginis et al., 2004), implicated in a cell survival signaling cascade (Peschon et al., 1998) through nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) activation (Dopp et al., 2002). A striking difference between mouse and human ES cells occurs at the X chromosome in female cells. Human ES cells have inactivated one of their X chromosomes whereas female mouse ES cells retain the activity of both X chromosomes (Heard et al., 2004). This observation suggested that human ES cells may exist in a more differentiated state than mouse ES cells as X-inactivation occurs during differentiation of mouse ES cells (Takagi et al., 1975; Huynh et al., 2003; Okamoto et al., 2004). Furthermore, genome wide gene expression studies showed that gene expression profiles and transcription factor networks of mouse ES cells were closer to early blastocyst ICM cells whereas that of human ES cells were more similar to postimplantation epiblast cells (as reviewed in Nichols et al., 2009) indicating the existence of more than one distinct state of pluripotency.

1.5.1 Naïve pluripotent state

In 1998, Gardner et al. demonstrated that 10-20 undifferentiated cells of the early stage blastocyst ICM can give rise to all the three embryonic lineages. This preimplantation pluripotent population

11 was later termed the ground state or naïve pluripotent state (as reviewed in Nicholas et al., 2009). ES cells isolated and cultured in vitro at this stage exhibit molecular and epigenetic properties similar to early blastocyst ICM and if these cultured cells are injected back to the blastocysts they incorporate themselves to the ICM and enter the developmental program. Mouse ES cells maintained in vitro by culture in LIF/2i media are a closer representation to early blastocyst ICM cells that mouse ES cells cultured in LIF and serum (Tosolini et al., 2016). These observations established mouse LIF/2i maintained ES cells in the state of naive pluripotency.

Naïve ES cells display domed colony morphology and high single cell survival rate indicating clonogenicity (Ginis et al., 2004). The molecular framework characteristic of naïve ES cells includes the expression of Oct4, Nanog, Sox2, Klf4 and specific naïve markers namely: Rex1, NrOb1 and Fgf (as reviewed in Nicholas et al., 2008; Ginis et al.,2004). These naïve cells self- renew in response to LIF/ Signal transducer and activator of transcription 3 (STAT3) signaling and differentiate in response to FGF/ Extracellular signal-regulated kinases (ERK) signaling (as reviewed in Nicholas et al., 2008; Ginis et al., 2004). Naïve ES cells have lower global CpG methylation, lower H3K27me3 at promoters and female cells have two active X chromosomes (Heard et al., 2004; Ginis et al., 2004). Human ES cells, in contrast to mouse ES cells, exist in a state known as the primed pluripotent state which is more similar to the epiblast cells of the mouse postimplantation embryo (as reviewed in Nicholas et al., 2009). Although naïve pluripotency is more difficult to induce in human ES cells, two approaches have successfully transitioned human ES cells from the primed state to a state that resembles the mouse naïve state (Takashima et al, 2014; Theunissen et al., 2016).

1.5.2 Primed pluripotent state

In mouse, postimplantation epiblast cells can be isolated and cultured without LIF but in presence of activin and FGF (Brons et al., 2007, Tesar et al., 2007). These cells generated cell lines called epiblast stem cells (EPiSCs) and their differentiation potential is restricted or primed and hence referred to as the primed pluripotent state. These cells do not generate mouse chimeras when injected into a blastocyst but do form teratomas (Guo et al., 2009; Tesar et al., 2007). EPiSCs can efficiently generate mouse chimera upon injection into post implantation epiblast demonstrating functional equivalence to that stage of mouse development (Huang et al., 2012). EPiSCs self-

12 renew in response to FGF/ERK signaling and unlike naïve ES cells they either differentiate or die when cultured in 2i media (as reviewed in Nicholas et al., 2008). Even though both naïve and primed mouse ES cells can self-renew and differentiate to multiple cell types, they differ from each other with respect to transcriptional, epigenetic and metabolic makeup that contribute to the classification of pluripotent states.

EPiSCs still express pluripotency factors Oct4, Sox2 and Nanog but lack the expression of naïve markers such as Rex1, NrOb1, Fgf and Klf4 (as reviewed in Nicholas et al., 2008). EPiSCs express specification markers not expressed in naïve ES cells such as Fgf5, T (as reviewed in Nicholas et al., 2008). EPiSCs have low clonogenicity and in female cells one of the X chromosomes is inactive (as reviewed in Nicholas et al., 2008; Ginis et al., 2004). Furthermore, global CpG methylation and H3K27me3 at promoters is higher in primed state (Ginis et al., 2004). The primed pluripotent state has active glycolytic metabolism compared to the oxidative metabolism of the naïve state (Ginis et al., 2004). EpiSCs can be reprogrammed to naïve pluripotency by transfection with Klf4 (Guo et al., 2009). The resulting cells show X chromosome reactivation in female cells, a naïve ES cell specific transcriptional profile, and contribute highly to somatic chimera production with germline transmission (Guo et al., 2009). As human ES cells were determined to be molecularly more similar to postimplantation epiblast derived mouse EpiSCs it has been argued that the use of “human ES cells” should be reserved for cells in the naïve state (Brons et al., 2007, Tesar et al., 2007 and Rossant, 2008).

Alternative pluripotent states can be acquired by preimplantation stage ICM cells in response to different culture conditions. Mouse ES cells maintained in LIF/2i acquire a naïve state whereas when derived in FGF and Activin A they acquire a primed EpiSC state which can also be obtained from the postimplantation epiblast (Nicholas et al., 2008; Ying et al., 2008). It was suggested that during human blastocyst culture, embryo cells may continue to progress to postimplantation epiblast status making it more difficult to isolate naïve human ES cells (Theunissen et al, 2016). Based on the hallmarks of naïve pluripotency guided by mouse studies, careful isolation of a naïve human ES cell population was achieved from blastocysts (Nakamura et al., 2016, Huang et al., 2014, Pastor et al., 2016, Theunissen et al., 2016). Human ES cells maintained in two different media formulations t2iL+PKCi (Takashima et al, 2014) and 5i/L/A(F) (Theunissen et al, 2016) showed characteristics similar to naïve state (Collier et al., 2018).

1.6 Regulation of naive pluripotent ES cells

Naïve pluripotent embryonic stem cells have been proposed to exist in a delicate balance between an undifferentiated and a differentiated state. Multiple gene regulatory mechanisms are integrated to maintain the naïve pluripotent state in ES cells. Minor disturbances caused to this integrated network often lead to exit from naïve pluripotent state and results in differentiation.

1.6.1 Transcriptional regulation

Mouse ES cells are transcriptionally hyperactive on a genome-wide scale and this transcriptional hyperactivity is down regulated rapidly following the initiation of differentiation (Effroni et al., 2008). Specifically, after only 24 hours of LIF withdrawal there is a significant down-regulation in global transcription of intergenic, intronic and exonic transcription (Effroni et al., 2008). This mechanism of ES cell regulation includes the expression of encoded information in DNA required for maintenance of the pluripotent state and progression through differentiation. Transcriptional regulation is a way that enables cell to regulate the conversion of DNA to RNA (transcription) and thus modulating a gene’s activity. Gene activity can be regulated by altering the number of transcribed copies of RNA or regulating when the gene is transcribed. This control enables cells to respond to a variety of intra and extracellular signals. Eukaryotes have complex transcriptional machinery undertaking the whole process that can be controlled at broadly three different levels; regulating promoter access to transcribing enzyme RNA polymerase, productive elongation of the transcript and transcript termination. All the three levels work in concert to integrate signals to and from the cell in order to change the transcriptional program accordingly.

1.6.1.1 RNA Polymerase

RNA polymerase is the enzyme that is required to produce RNA that is either coding for protein namely messenger RNA (mRNA) or non-coding RNA such as transfer RNA (tRNA) that transfers specific amino acids to growing polypeptide chains, ribosomal RNA (rRNA) that is a component of ribosomes, micro RNA (miRNA) that regulates gene activity and catalytic RNA called ribozyme. There are three major RNA polymerase enzymes namely: RNA polymerase I, RNA polymerase II (RNAPII) and RNA polymerase III; each responsible for synthesis of a distinct subset of RNA. All three RNA polymerases are structurally and mechanistically similar. RNA

14 polymerase I is located in the nucleolus of the cell and transcribes non-coding rRNA. RNA polymerase III transcribes tRNA, 5S rRNA and other small RNAs. RNAPII is 550 kDa complex of 12 subunits and transcribes the precursors of mRNAs and most snRNA and microRNAs. Both RNAPII and III are located in the nucleoplasm.

RNAPII transcribes protein coding genes and is the most studied of the eukaryotic RNA polymerases. The core of this enzyme is composed of 12 interacting DNA-directed RNA polymerase II subunits (RPB1-12). Soon after their translation RPB2 and RPB3 makes a complex which interacts with RPB1 (Kolodziej et al., 1991). After the association of RPB1 other subunits, namely RPB5 and RPB7, can enter further allowing the association of RPB6, 8, 10, 11, 12 (Acker et al., 1997; Kolodziej et al., 1991). RPB4 and RPB9 enter once most of the complex is assembled. RPB4 forms a complex with RPB7 (Acker et al., 1997). RBP1 is the largest subunit of the core RNAPII. In combination with several other polymerase subunits, the RPB1 subunit forms the DNA binding domain of the polymerase, a groove in which the DNA template is transcribed into RNA (Acker et al., 1997; Kolodziej et al., 1991). The RPB1 subunit is known to strongly interact with RPB8 (Kolodziej et al., 1999). RPB1 contains a carboxy terminal domain (CTD) composed of up to 52 heptapeptide repeats (YSPTSPS) that are essential for polymerase activity (Meinhart et al., 2004).

The CTD was first discovered by C.J. Ingles at the University of Toronto and by JL Corden at Johns Hopkins University in 1985. This domain is involved in the initiation of transcription, the capping of the RNA transcript, and attachment to the spliceosome for RNA splicing (Brickey et al., 1995). The CTD is only specific to RNAPII, it is absent in the other two polymerases. The CTD serves as a binding scaffold which coordinates transcription with a variety of nuclear processes via the recruitment of specific factors during the appropriate stage of transcription. The domain stretches from the core of the RNAPII enzyme to the exit channel. The bound nuclear factors to this CTD of RNAPII activate polymerase activity.

The binding specificity of the CTD for particular factors is determined by a series of site-specific phosphorylation/dephosphorylation events. As a consequence of phosphorylation, RNAPII can exist in a form with a highly phosphorylated CTD (subunit II0; designated as RNAPII0) and a form with a nonphosphorylated CTD (subunit IIa; designated as RNAPIIA) (Cadena and Dahmus

1987; Payne et al. 1989). The best studied of these events occur at Serine 2 and 5 of the heptapeptide repeats. As reviewed by Phatnani et al., in 2006, at initiation, Ser5 phosphorylation predominates followed by increased Ser2 phosphorylation during elongation, yielding a CTD containing both phosphorylated Ser5 and 2 as polymerase progresses to the middle of the gene. Ser5 phosphorylation levels decrease approaching the 3’ end of the gene leading the Ser2 phosphorylated polymerase to terminate transcription. In mammals, the RPB1 CTD can be phosphorylated at Ser2, Ser5 and Ser7, as well as Tyr1 and Thr4, acetylated, glycosylated and methylated (as reviewed in Chapmann et al., 2008; as reviewed in Meinhart et al., 2005). The prolyl bonds within the CTD can also be isomerized by the peptidyl prolyl cis-trans isomerase (Wu et al., 2000; as reviewed in Hanes et al., 2014). These dynamic modifications constitute a ‘CTD code’ facilitating the recruitment of co factors required for enhancing transcription and RNA processing (Buratowski, 2003).

RNAPII forms the holoenzyme with a subset of general transcription factors, and regulatory proteins recruited to the promoters of protein-coding genes. The assembly of this holoenzyme occurs on a gene promoter before transcription begins, thus the process of its assembly is referred to as the preinitiation complex (PIC). The PIC positions RNAPII over gene transcription start sites, denatures the DNA, and positions the DNA in the RNAPII active site for transcription. A gene- specific combination of transcription factors will recruit TFIID and/or TFIIA to the core promoter, followed by the association of TFIIB, creating a stable complex onto which the rest of the General Transcription Factors (GTFs) and RNAPII can assemble. This assembly is further dictated by CTD phosphorylation patterns in order to complete a productive transcription cycle. Early in the transition from preinitiation to elongation, the CTD is phosphorylated on Ser5 residues to create the second CTD phosphorylation state by subunits of TFIIH (helicase that remains associated with Pol II throughout transcription) having kinase activity. After initiation, an elongation-phase kinase (Carboxy terminal domain kinase-1 (CTDK-I) in yeast; positive transcription elongation factor b (P-TEFb) in metazoa) (Marshall et al. 1996; Lee and Greenleaf 1997) phosphorylates Ser2 residues which facilitates the binding of elongation-related factors leading to the third CTD phosphorylation state. Finally, near the 3′ end of the gene it is widely believed that CTD phosphorylation is dominated by Ser2 phosphorylation enhancing the binding specificity and localization of 3′-end processing factors (Licatalosi et al. 2002; Ahn et al. 2004). Unlike the other

16 two mammalian polymerases RNAPII does not have promoter specificity; therefore, a wide range of transcription factors are required to bind upstream of gene promoters and begin transcription.

As previously mentioned RNA polymerase I is restricted to the nucleolus whereas RNAPII activity is distributed through the nucleoplasm. RNAPII activity does appear to be localized to distinct sites in the nucleoplasm where nascent transcripts are observed (Jackson et al., 1993; Wansink et al., 1993). These compartments in the nucleus are enriched in active CTD-phosphorylated RNAPII, components of the mediator complex, and transcription factors referred as transcription factories (Iborra et al., 1996; as reviewed in Cook 1999). Expressed protein coding genes are located at these RNAPII rich regions of the nucleus whereas silent genes appear to be excluded (Osborne et al., 2004).

1.6.1.2 Transcription factors, enhancers and the regulatory landscape for gene transcription

Transcription factors are proteins which regulate tissue specific gene expression by binding to specific DNA sequences. Transcription factors can be divided in two main categories: activators and repressors based on their ability to activate and/or repress downstream target genes. Only a small subset of an organism's genome encodes transcription factors and these factors usually work in a combinatorial manner. Transcription factors function through a wide variety of mechanisms. Transcription is regulated by a transcription factor’s localization to the nucleus and affinity to bind DNA. Transcription factor function can be controlled by signal transduction pathways which can alter subcellular localization or activity of the transcription factor. Post-translational modifications known to regulate the functional state of transcription factors are phosphorylation, acetylation, SUMOylation and ubiquitylation. Activators can interact directly or indirectly with the core machinery of transcription. Repressors predominantly recruit co-repressor complexes leading to transcriptional repression by chromatin condensation of enhancer regions. Overlapping DNA- binding motifs for both activators and repressors induce a physical competition to occupy the site of binding.

Enhancers are non-coding DNA sequences, range from 200 bp to 1 kb in length, containing multiple activator and repressor binding sites. Enhancers can be either proximal, upstream to the promoter or within the first intron of the regulated gene, or distal, intergenic regions far away from

17 the locus. Genome wide binding of transcription factors and co-activators has predicted that enhancers bind combinations of transcription factors that then interact with components of the mediator complex which recruits RNAPII (as reviewed in Young, 2011). Transcription factor bound enhancers then form a loop with the promoter region of the gene they regulate while excluding the intervening sequences. Several chromatin loops have been extensively characterized in the β-globin locus. The mouse β-globin locus consists of four genes (Hbb-y, -hb1, -b1, and – b2) differentially expressed during erythroid development. Two 5’ hypersensitive sites (HS) located 60-62kb upstream of the Hbb-b1gene, the LCR (locus control region) located 50kb upstream, and a 3’ HS1 located 20kb downstream (Palstra et al., 2003). All of these HS and the LCR interact spatially to establish a unique three-dimensional chromatin architecture specific to erythroid cells (Palstra et al., 2003). At each developmental stage, the LCR forms chromatin loops with specific β-globin genes that are actively expressed, suggesting that chromatin looping is associated with transcriptional activation (Palstra et al., 2003; Tolhuis et al., 2002). In erythroid cells, the β-globin LCR can enhance the expression of nearby genes when it is integrated into a different chromosome locus by relocating the locus out of its chromosome territory to associate with transcription factories (Noormerdeer et al., 2010). Distal regulatory elements (DREs) are capable of regulating both gene positioning and expression in a cell-type specific manner. It had been shown that regulation of one gene might include the interaction of its promoter with multiple enhancers. In mouse ES cells, Sox2 expression is regulated by two gene-proximal enhancers, SRR1 and SRR2 (Zappone et al. 2000; Tomioka et al. 2002; Miyagi et al. 2004) and three distal regulatory elements SRR18, SRR107, and SRR111 (Chen et al. 2012) within 130 kb of Sox2 gene. The distal enhancers SRR107 and SRR111 contact the gene-proximal enhancers SRR1, SRR2, and SRR18 through the formation of a large chromatin loop in mouse ES cells (Zhou et al., 2014). Furthermore, deletion revealed this distal Sox2 control region (SCR) region, containing SRR107 and SRR111, is required for Sox2 transcription in mouse ES cells.

Chromosome conformation capture techniques have provided evidence that active chromatin regions are “compacted” in nuclear domains or bodies where transcriptional regulation is facilitated. Three-dimensional genome organization is essential for enhancer-promoter proximity and gene regulation. Metazoan genomes have been observed to be physically partitioned into structural and functional units termed topological association domains (TADs) (Dixon et al., 2012). TADs are megabase regions of DNA usually containing several genes and their associated 18 enhancers. Previously identified interacting enhancers and gene promoters generally fall within one single large functional domain or TAD (Smallwood et al, 2013). However, studies of mouse development demonstrated that two adjacent TADs may regulate the same gene cluster (Woltering et al., 2014). TADs are megabase long and their boundaries are often composed of housekeeping genes, tRNAs, other highly expressed sequences, Short Interspersed Elements (SINE) and insulators like CTCF and TFIIIC that recruit structural proteins cohesins and condensins (Wang et al., 2012). TAD boundaries are usually conserved and static between cell types whereas intra-TAD interaction sites, or subTADs are dynamic according to the state of the cell or cell type. Mediator proteins act as architectural proteins cooperating with cohesin when subTADs are at less than 100 kb from each other. For subTADs larger than 100 kb and TAD boundaries, CTCF is the typical insulator found to interact with cohesin (Phillips-Cremins et al., 2013). The highly dynamic genome reorganizations at interphase can switch on or off entire gene regulatory networks through short to long range chromatin rearrangements and these can be involved in cell fate decisions (Gómez-Díaz et al., 2014).

1.6.1.3 Nuclear transport mechanism

Although small molecules can simply diffuse through the nuclear membrane to enter the nucleus, the entry and exit of large molecules, such as RNA and protein, is regulated by nuclear pore complexes (NPCs) (Watson, 2004). RNA and protein molecules require association with transport factors called importins to enter the nucleus and exportins to exit the nucleus (Alberts, 2002). The ability of both importins and exportins (together called as karyopherins) to transport their cargo is regulated by the small Ras related GTPase, Ran (Alberts, 2002). GTPases are enzymes that bind to a molecule called guanosine triphosphate (GTP), hydrolyze bound GTP to guanosine diphosphate (GDP) and release energy (Alberts, 2002). NPC-mediated transport does not require energy, but depends on concentrations gradients associated with the Ran cycle (Alberts, 2002). The conformation of Ran changes depending on whether it is bound to GTP or GDP. In its GDP bound state, Ran is capable of binding karyopherins (Alberts, 2002). Importins release cargo upon binding to RanGTP, whereas exportins must bind RanGTP to form a ternary complex with their export cargo (Alberts, 2002). The dominant nucleotide binding state of Ran depends on whether it is located in the nucleus (RanGTP) or the cytoplasm (RanGDP) (Alberts, 2002).

Proteins that must be imported to the nucleus from the cytoplasm carry nuclear localization signals (NLS) (Alberts, 2002). An NLS is most commonly composed of hydrophilic amino acids and acts an importins binding motif. Importins bind their cargo molecule in the cytoplasm, after which they are able to interact with the nuclear pore complex to transport cargo into the nucleus (Alberts, 2002). Once inside the nucleus, a conformational change in the importin after GTP interaction causes it to dissociate from its cargo (Alberts, 2002). The resulting complex of importin and RanGTP then returns to the cytoplasm (Alberts, 2002). Ran Binding Protein (RanBP) separates RanGTP from importin in the cytoplasm (Alberts, 2002). GTPase activating protein (GAP) then binds RanGTP and induces the hydrolysis of GTP to GDP. The RanGDP produced from this process now binds the nuclear transport factor 2 (NUTF2) which returns it to the nucleoplasm (Alberts, 2002). Once in the nucleus, RanGDP interacts with a guanine nucleotide exchange factor (GEF) that replaces the GDP with GTP (Alberts, 2002). The resulting RanGTP can then begin a new cycle (Alberts, 2002).

Proteins, transfer RNA, and assembled ribosomal subunits are exported from the nucleus due to their association with exportins, which bind signal sequences called nuclear export signals (NES) (Pemberton et al., 2005). Nuclear export requires exportins, for example XPO1, to bind the cargo and RanGTP for transport through the NPC to the cytoplasm (Pemberton et al., 2005). The complex dissociates in the cytoplasm where RanGTP binds GAP and hydrolyzes GTP to GDP. The resulting RanGDP complex returns to the nucleus where it exchanges its bound ligand for GTP (Pemberton et al., 2005). In summary, importins depend on RanGTP to dissociate from their cargo, whereas exportins require RanGTP in order to bind to their cargo (Pemberton et al., 2005). Many proteins have both NES and NLS and thus shuttle constantly between the nucleus and the cytoplasm (Pemberton et al., 2005). Post-translational modifications often regulate both nuclear import and nuclear export of specific proteins (Pemberton et al., 2005).

1.6.1.4 Transcriptional networks regulating pluripotency

The pluripotent state is governed by a highly interconnected pluripotency gene regulatory network that is orchestrated by a set of core pluripotency transcription factors namely, OCT4 (POU5F1), SOX2 and NANOG. These transcription factors bind to the promoters of the Oct4, Sox2 and Nanog genes as a group, suggesting the existence of auto regulatory feedback loops (Kim et al., 2008).

All three of these when studied separately have been observed to regulate different aspects of differentiation and pluripotency.

Oct4 (octamer-binding transcription factor 4) is a POU transcription factor family member expressed in blastomeres, pluripotent early embryo cells and the germ cell lineage. Homozygous deletion of Oct4 in mouse generates embryos that develop to the blastocyst stage, but their ICM is not pluripotent and therefore differentiates to the extra-embryonic trophectoderm lineage (Nichols et al., 1998). In accordance with this observation, Oct4 inactivation in mouse ES cells causes differentiation to the trophectoderm lineage; however, over expression of Oct4 causes differentiation to mesoderm and primitive endoderm lineages (Niwa et al., 2000). Oct4 siRNA injection into the ICM impairs cardiogenesis (Zeineddine et al., 2006). In vitro, for ES cells that lack Oct4, the presence of LIF does not maintain pluripotency (Niwa et al., 2000).

Nanog is a highly divergent homeodomain-containing transcription factor (Chambers et al., 2003), expressed only in the ICM, mouse ES cells and in the proximal epiblast at embryonic day 6 (Hart et al., 2004). Nanog null embryos develop an ICM by embryonic day 3.5. However, the ICM does not proliferate when cultured on gelatinized plastic, instead differentiated into parietal endoderm- like cells (Mitsui et al., 2003). Nanog expression is heterogenous in LIF/serum cultured mouse ES cells but becomes homogeneous in LIF/2i conditions. In contrast to Oct4 and Sox2, forced expression of the Nanog cDNA was able to maintain ES cell self-renewal in the absence of LIF signaling (Chambers et al., 2003).

Sox2 ((sex determining region Y)-box 2) is a small gene with only one exon, which is an SRY- related high-mobility-group box family member (Lefebvre et al., 2007). Unlike Oct4 and Nanog, the expression of Sox2 is widespread, including tissues such as extraembryonic ectoderm (Avilion et al., 2003) and a variety of endodermal and ectodermal tissue progenitor cells in both the developing and adult mouse (Arnold et al., 2011). Sox2-null embryos die shortly after implantation (Avilion et al., 2003). Sox2 is necessary for pluripotency in mouse ES cells as inducible Sox2-null cell lines differentiate mainly into trophectoderm-like cells. This elimination of Sox2 also causes the expression of Oct4 and Nanog to drop by 50% 72 hours after induction (Masui et al., 2007). Over-expression of Sox2 causes an increase in expression of genes associated with ectodermal and mesodermal lineages, even in the presence of LIF. Mouse ES cells with reduced SOX2 expression

21 levels showed impaired differentiation to neuroectoderm and increased mesendoderm formation (Zhou et al., 2014).

Chromatin immunoprecipitation followed by paired-end tag sequencing (ChIP-PET) has shown that around 70% of OCT4 clusters also contain an adjacent SOX2 binding site. (Loh et al., 2006). SOX2 has been found to regulate expression of multiple transcription factors which in turn affect Oct4 expression (Masui et al., 2007), indicating the OCT4/SOX2 heterodimer as a functional binding unit (Chen et al., 2008). ChIP-seq analysis has shown that over 600 sites in the mouse genome are co-bound by OCT4/SOX2/NANOG (OSN) and at least one other transcription factor. Only 2% of all OSN bound genes are not bound by any other pluripotency associated transcription factors, indicating extended co-ordination of the OSN circuitry with other pathways implicated in pluripotency (as reviewed in Young, 2011). Kruppel-like factor 4 (KLF4), a member of Kruppel- like factor (KLF) family of conserved zinc finger transcription factors, has been known to interact with the core network of pluripotency transcription factors in order to regulate genes required for the maintenance of pluripotency and reprogramming (Zhang et al., 2010; Wei et al., 2009; Wei et al., 2013). SMAD1 and STAT3 also frequently co-bind to regions occupied by OSN (Chen et al., 2008). Multiple transcription factor binding loci (MTLs) containing OSN binding sites and bound by p300 have robust mouse ES cell specific enhancer activity (Chen et al., 2008). Interestingly, many Polycomb Group proteins known to epigenetically silence genes, have also been shown to co-bind with OSN at repressed lineage specific genes, indicating connection between chromatin remodeling and transcriptional control of pluripotency (Boyer et al., 2006).

1.6.2 Epigenetic regulation of pluripotency

The eukaryotic genome is organized into gene activation permissive chromatin state domains called euchromatin and gene-poor or transcriptionally silenced chromatin state domains called heterochromatin (as reviewed in Kornberg et al., 1992). Transcriptional activities can be influenced by chromatin compaction which regulates accessibility to transcription factors and other DNA interacting proteins (as reviewed in Beato et al., 1997). Chromatin in pluripotent ES cells is less condensed compared to differentiated cell types (as reviewed in Francastel et al., 2000 and Arney et al., 2004). Pluripotency is associated with open chromatin structure and differentiation is associated with less transcriptionally permissive chromatin (Ahmed et al., 2010).

8-cell stage, 3.5 epiblast cells and mouse ES cells have largely dispersed chromatin as a mesh of 10 nm fibres but in post-implantation embryonic day 5.5 epiblast cells chromatin compaction of various sizes occurs and 10 nm fibers were rarely observed (Ahmed et al., 2010). Oct4 knock out early epiblast cells showed significant condensation of the chromatin suggesting the role of ES cell transcriptional program in global chromatin organization (Ahmed et al., 2010).

For the purposes of this discussion, the term epigenetics is used to refer to those heritable modifications that can activate or silence the expression of a gene without altering the DNA sequence (Jaenisch et al., 2003). These mechanisms enable cells in a specific state to express only the genes that are necessary for maintenance of their cellular phenotype. The core histone proteins modified by post translational modifications such as phosphorylation, acetylation and methylation at numerous residues have been correlated with chromatin states and the transcriptional status of genes (Jenuwein,et al., 2001; as reviewed in Fischle et al., 2003). Repressed genes are associated with the di- and tri-methylation of H3K9, tri methylation of H3K27 whereas actively transcribed gene promoters are associated with mono methylation of H3K9 (Lachner et al., 2001; Rosenfeld et al., 2009), tri methylation of H3K4 (Liang et al., 2004) and acetylation of H3K27 (H3K27ac) (Tie et al., 2009). H3K27ac is often used to find active enhancers and poised enhancers (Creyghton et al., 2010). Regions called “bivalent domains” containing both active and inactive histone modifications (H3K27ac and H3K27me respectively) are widespread in ES cells (Bernstein et al., 2006). These appear to mark the silenced genes in ES cells while allowing their activation upon differentiation concurrently with the loss of the inactive histone modifications (Bernstein et al., 2006). For example, in mouse ES cells genes associated with neural differentiation were determined to have such bivalent markings, but when subjected to differentiation to neural precursor cells, the repressive H3K27me3 signatures were erased and genes expressed (Bernstein et al., 2006).

In addition to the differences in genome wide chromatin accessibility in pluripotent cells chromatin associated proteins are more dynamic in pluripotent mouse ES cells as compared to cells 24 hr after LIF withdrawal. This has been shown by FRAP (fluorescent recovery after photobleaching) assays in which Histone H2B and heterochromatin protein family protein HP1 replaced bleached proteins more rapidly in pluripotent mouse ES cells compared to the 24 hr LIF withdrawal cells

(Meshorer et al., 2006). It was suggested that these proteins have increased mobility in pluripotent cells allowing them to quickly remodel chromatin in response to differentiation.

Signaling pathways are known to relay extracellular signals to modify chromatin by modulation of epigenetic machinery. In mouse ES cells, Janus kinase (JAK) signaling has been reported to phosphorylate histone H3 on tyrosine 41 (H3Y41) in a LIF independent manner. This event leads to a reduction in the binding of heterochromatin protein 1α (HP1α) on pluripotency genes (Dawson et al., 2009). MAP kinase signaling has been shown to phosphorylate H3 Ser10 (H3S10) of its target genes through the JNK pathway, favoring mouse ES cell differentiation (as reviewed in Fagnocchi et al., 2016). The ERK pathway regulates polycomb repressive complex 2 (PRC2) deposition at developmental genes, by phosphorylating RNA polymerase II at Ser5 and establishing poised domains in mouse ES cells (as reviewed in Fagnocchi et al., 2016). In human ES cells, the Activin A/Smad pathway has been demonstrated to be involved in the correct deposition of H3K4me3 on key developmental genes, through its effectors SMAD2/3, which cooperate with NANOG to recruit DPY30, a subunit of the COMplex of Proteins ASsociated with Set1 (COMPASS) histone methyltransferase complex, contributing to the capacity of stem cells to differentiate into specific lineages (as reviewed in Fagnocchi et al., 2016).

1.6.3 Signaling pathways modulating the naïve pluripotent state

Naïve pluripotency in mouse ES cells is modulated by various signaling pathways integrated with transcriptional programs and with cross talk among themselves (Figure 1.2). The extra embryonic tissues of the embryo are known to produce growth factors and other signaling molecules that dictate the fate of embryonic cells of the ICM. Moreover, the cells of ICM themselves secrete proteins including FGF4 that cause the trophoblast cells to divide as the blastocyst continues to grow (Tanaka et al. 1998). The Grb2-Ras-MAP kinase signaling activated in ICM cells had been reported to induce early differentiation events resulting in the formation of epiblast and primitive endoderm (Chazaud et al., 2006).

1.6.3.1 LIF and JAK/STAT3 signaling

In 1988, Smith et al, demonstrated the use of LIF and serum to maintain the undifferentiated state of mouse ES cells in vitro. The binding of LIF to the LIF receptor activates JAK and

24 phosphorylates STAT3 at Tyr705 (Zhong et al., 1994; Niwa et al., 1998). Activated STAT3 had been shown to maintain the c-MYC and KLF4 levels integrating LIF/JAK/STAT3 signaling to the transcriptional circuitry in order to maintain pluripotency in naïve mouse ES cells (Cartwright et al., 2005; Hall et al., 2009). Additionally, STAT3 induced Pramel7 blocks the phosphorylation of ERK which is associated with differentiation of naïve ES cells (Casanova et al., 2011). A contradictory study, however, reported LIF signaling to also negatively regulate pluripotency and cause differentiation induction in mouse ES cells. The zeta-chain T-Cell receptor (TCR) associated protein kinase (Zap70), a spleen tyrosine kinase (Syk) family tyrosine kinase, has been found to negatively regulate the JAK/STAT3/c-MYC pathway by interacting with Src-homology 2 domain (SH2)-containing protein tyrosine phosphatases -1 (SHP1) and inhibits the phosphorylation of JAK, which in turn downregulates the STAT3 dependent c-MYC induction (Cha et al., 2010).

1.6.3.2 BMP4 signaling

Bone morphogenic factor 4 (BMP4) is provided through serum supplementation in culture media for mouse ES cell maintenance. Mouse ES cells cannot retain pluripotency in the absence of serum and presence of LIF alone, but the addition of BMP4 and LIF does maintain pluripotency in the absence of serum (Ying et al., 2003). Dimeric BMP4 binds to type II receptor BMPR2 and facilitates the assembly of receptor heteromers which were then shown to phosphorylate type I receptors, BMPR1 (BMPR1A, also known as ALK3 or BMPR1B, also known as ALK6) and the type I receptors in turn phosphorylate either of the receptor SMADs (SMAD1or SMAD5 or SMAD8) (Heldin et al., 1997; Kawabata et al., 1998; Derynck et al.,, 2003; Yu at al., 2005). Furthermore, phosphorylated receptor-SMAD induced and repressed the expression of genes in order to facilitate the pluripotent mouse ES cell state (Ying et al., 2003). Ying et al. further demonstrated that BMP/SMAD signaling activation induces expression of inhibitor of differentiation (Id) family members to suppress neural differentiation by inhibiting pro-neural basic helix-loop-helix (bHLH) factors. BMP4 signaling also modulates the H3K27 demethylase, Kdm6b and Dihydropyrimidinase-like 2 or Dpysl2 in order to maintain pluripotency of mouse ES cells (Fei et al., 2010).

Furthermore, BMP4 could attenuate the ERK activity by localizing to the promoter region of dual specificity phosphatase 9 (DUSP9 gene) upregulating its expression through SMAD1/5 activation which then decreased phosphorylation of ERK (Li et al., 2012; Qi et al., 2004). DUSP9 is thus suggested to strengthen BMP4 signaling, by attenuating ERK activity and solidify the self-renewal status of mouse ES cells together with LIF.

1.6.3.3 ERK signaling

ERK signaling cascade can be activated by various stimuli, such as receptor tyrosine kinase (RTK) and G protein-coupled receptors. Once activated it can regulate the different cell processes such as proliferation, differentiation and apoptosis (Huynh et al., 2003). In mouse ES cells fibroblast growth factor 4 (FGF4) produced by the cells themselves or provided by serum in culture media could activate ERK1/2 causing the ES cells to differentiate spontaneously in culture (Kunath et al., 2007; Stavridis et al., 2007). Briefly, the cascade begins after binding of FGF4 to FGFR, Ras recruits and activates the protein kinase Raf; Raf, a serine/threonine protein kinase activates MEK1/2 (MAPK/ERK kinase) dual-specificity protein kinase and the activation of ERK1/2; and finally activated ERK1/2 phosphorylates several targets affecting gene expression (as reviewed in Thisse et al., 2005 and Burdon et al., 2002). In 2008, Ying et al, showed that inhibition of FGF4/ERK signaling pathway in culture could promote mouse ES cell self-renewal and naïve state but they eventually die suggesting the role of other pathways in maintaining metabolic activity, biosynthetic capacity and overall viability of mouse ES cells. They speculated GSK3-mediated negative regulation of biosynthetic pathways causing mouse ES cell degeneration in absence of FGF4/ERK signaling. Therefore, they suggested the use of two small synthetic inhibitor molecules in mES cell culture in order to maintain pluripotency namely ERK pathway inhibitor (PD0325901) and GSK-3 inhibitor (CHIR99021) in culture media supplemented with serum or BMP4 in feeder free conditions.

Moreover, ERK1/2 activation is known to integrate into the pluripotency specific transcription circuitry involving transcription factors such as OCT4, SOX2, NANOG and KLF4 in both ES cells and cancers (as reviewed in Dreesen et al., 2007). In mouse ES cells Kim et al, 2012 showed that ERK1/2 binds at the C-terminal domain of KLF4 and phosphorylates it at Ser132 (described as S123 in Kim et al. (2012) as in their construct the first 9 amino acids of the endogenous KLF4

26 protein were missing) residue leading to the ubiquitination and degradation of KLF4 affecting the KLF4 activity and inducing differentiation.

1.6.3.4 Wnt/β-catenin signaling

Wnt signaling involves the association of the destruction complex composed of a phosphoprotein drosophila dishevelled gene homolog (DVL1), a cytoplasmic protein which contains a regulation of G-protein signaling domain and a dishevelled domain (AXIN), adenomatous polyposis coli (APC), a serine/threonine protein kinase Glycogen synthase kinase 3 (GSK3) and phosphorylated β-catenin with phosphorylated transmembrane low-density lipoprotein receptor-related protein (LRP) after induction by Wnt (as reviewed by Nusse, 2005; Hans, 2006; Hans and Nusse, 2012). This complex further cannot phosphorylate β-catenin and hence blocks the ubiquitination by beta- transducin repeat containing (β-TrCP). As a result, newly synthesized β-catenin is accumulated in the cytosol and translocated into the nucleus and makes complex with C (CBP). The β-catenin/CBP complex binds T cell factor/lymphoid enhancer factor (TCF/LEF) and induces STAT3 expression (Miyabayashi et al., 2007; Hao et al., 2006). The stabilized β-catenin had also been reported to upregulate Oct4 expression for the maintenance of pluripotency in mouse ES cells (Kelly et al., 2011). Accumulated β-catenin after entering the nucleus, interacts with the TCF/LEF and binds to a consensus motif AGATCAAAGG to activate the transcription of target genes such as Axin2, Cdx1 and T. Therefore, the addition of GSK3 inhibitor in mouse ES cell culture media could maintain the required β-catenin levels to regulate downstream gene expression.

1.6.3.5 PI3 kinase/AKT signaling

Phosphoinositide 3-kinase (PI3K) pathway is important for proliferation, survival, and maintenance of pluripotency in ES cells. The activated PI3K relays signal through secondary messengers which further transmit their effect through a serine threonine kinase namely AKT. AKT had been implicated in different aspects of cellular metabolism and tumorigenesis. LIF- dependent activation of PI3K pathway was found to be responsible for regulating pluripotency in mouse ES cells which was described as LIF activated gp130 that then induces PI3K to phosphorylate PKB/AKT which in turn could influence its downstream effectors to maintain the pluripotency in mouse ES cells (Paling et al., 2004). LIF, BMP4 or Wnt/β-catenin cause independent activation of AKT by post translational modification named myristolation could

27 maintain ES cell self-renewal (Watanabe et al., 2006). They also suggested that activated PI3K/AKT might inhibit GSK3 signaling and hence could maintain the pluripotency in mouse as well as in primate (monkey) ES cells.

1.6.3.6 Crosstalk among different signaling pathways for pluripotency maintenance

The number of signaling pathways had been reported to be critical in the modulation of pluripotent states and differentiation. Even though all the individual signaling pathways have their own effectors and targets in order to modulate various cellular processes such as pluripotency, differentiation, metabolism, tumorigenesis, cell cycle and cell death, still it had been observed widely that all the operational signaling mechanisms establish a cross talk among themselves in order to relay specific cues to the cell from its external and internal environments. In ES cells such cross talks among signaling pathways had been widely reported for the pluripotency maintenance.

In naïve mouse ES cells, the cross talk among intracellular signaling pathways regulate pluripotency. For example, LIF/JAK/STAT3 signaling upregulates Klf4 to maintain pluripotency also the binding of LIF to LIFR and gp130 heterodimer had been shown to activate PI3K /AKT pathway targeting pluripotency associated genes such as Tbx3 (as reviewed in Burdon et al., 1999; Hirai et al., 2011). Both of these pathways could also get activated by FGF/Ras-Raf-MEK- MAPK/ERK pathway activated by FGF binding to FGFR. All of these pathways also share their final targets for pluripotency maintenance. These pathways either upregulate or downregulate their targets and often they have contradicting effects on the same target. The best explained example of this is Klf4, LIF/STAT3 signaling had been reported to upregulate its expression whereas ERK signaling had been reported to suppress its expression (as reviewed in Huang et al., 2015). Therefore, to maintain pluripotency in vitro mouse ES cells are cultured in media supplemented with serum, LIF and inhibitor of MEK and GSK3. GSK3 had been shown to be activated after Wnt binding to Frizzled which then phosphorylates β-catenins reinforcing their degradation by ubiquination. The GSK3 inhibitor in the culture media prevented this β-catenin degradation promoting the upregulation of target genes required for pluripotency maintenance. Similarly, BMP4 and Activin /Nodal pathways relay their signals through activation of series of SMAD proteins such as SMAD1/5/8/4 and SMAD2/3 respectively (as reviewed in Huang et al., 2015).

The downstream targets of SMAD4, Dsp9 had been reported to inhibit FGF/ERK signaling promoting pluripotency (as reviewed in Huang et al., 2015).

1.6.3.7 Integration of signaling pathways with transcriptional circuitries

The signaling pathways serve the role of relaying the external and internal environmental cues to the cell for the maintenance of its various cellular processes. The final targets or relay molecules of signaling pathways are transcription factors which can further relay the information to the nucleus for maintenance of pluripotent cell state or differentiation. This is how and why the signaling pathways merge with core transcriptional circuitries regulating the gene expression required for cell state maintenance.

Provided that mouse ES cells can be maintained by LIF/BMP enabling SMAD1 and STAT3 activation and binding to genomic sites; generates the direct evidence that LIF/BMP signaling supports self-renewal by strengthening core pluripotency circuitry. A number of studies identified downstream target genes of JAK/STAT3 signaling pathway as potential candidates for key pluripotency factors such as Myc, Klf4, Pim1/3, Prr13, Gbx2, Pramel7, Pem/Rhox5, Jmjd1a and Tfcp2l (Tai et al., 2013; Hall et al., 2009; Cartwright et al., 2005; Martello et al., 2013; Ye et al., 2013; Cinelli et al., 2008; Casanova et al., 2011; Li et al., 2005).

Similarly, the pluripotency circuitry associated downstream targets for Wnt-β Catenin signaling includes Axin2, Cdx1 and T. Genome-wide studies suggested TCF3, which is the part of GSK3/β- catenin/TCF3 axis, serves as a limiting factor for higher expression of Oct4, Nanog, Tfcp2l1 and Esrrb (Wray et al., 2011; Cole et al., 2008; Pereira et al., 2006). GSK3/β-catenin/TCF3 had been shown to directly activate the orphan nuclear receptor liver receptor homolog-1 (Lrh-1)/ nuclear receptor subfamily 5, group A, member 2 (Nr5a2) transcription and stabilizes Oct4, Nanog and Tbx3 expression. Even though Nr5a2 had been reported to replace Oct4 in iPSC reprogramming yet it is not required for the maintenance of ES cell self-renewal (Wagner et al., 2010; as reviewed in Tanaka et al., 2011). Another nuclear receptor, nuclear receptor subfamily 6, group A, member 1 (Nr6a1), had been shown to repress Oct4 expression during differentiation (Furhmann et al., 2001; Gu et al., 2005). The mouse ES cells are cultured in media containing GSK3 inhibitor, but over-inhibition of GSK3 increases β-catenin activity leading to differentiation induction. One possible explanation is that over-inhibition of GSK3 increases the expression of the canonical Wnt

29 pathway effector LEF1, and elevated LEF1 interacts with β-catenin and leads to activation of lineage specification genes Cdx2 and T (as reviewed in Huang et al., 2015).

The FGF/MEK/ERK signaling dictates mouse ES cells to exit pluripotency. By blocking MEK activation the expression of many pluripotency associated genes had been shown upregulated in mouse ES cells, such as Nanog, Tfcp2l1 and Klf4 (Ye et al., 2013; as reviewed in Silva et al., 2009; Kim et al., 2012). The downstream targets of RAS such as Gmnn, Psmb3 and Ifna14 had been shown to increase ERK activation by downregulating Dusp1/6, thus inducing mouse ES cell differentiation (Yang et al., 2012). The downstream effectors of BMP4 and TGFβ/Activin/Nodal are SMAD proteins. Genome-wide binding analysis revealed that SMAD1 shares many common targets with core pluripotency factors Oct4, Sox2 and Nanog (Chen et al., 2008). The ChIP-seq analysis had shown that TGFβ/Activin/Nodal/SMAD2/3 signaling cascade could sustain pluripotency via upregulating Nanog expression after binding SMAD2/3 directly to the proximal promoter of Nanog (Vallier et al., 2009; Xu et al., 2008).

Figure 1.2

Figure 1.2: Major signaling pathways involved in modulation of pluripotency.

1.6.4 Post transcriptional and post translational regulation of pluripotency

After transcription, the primary transcripts undergo a series of steps including processing, export, modification, translation, and degradation to complete their life cycles. These steps can be regulated to influence the amount of final protein product. The post transcriptional regulation involves the processing of transcribed RNA by RNA binding proteins, RNA processing machineries, and regulatory RNA molecules to maintain pluripotency. The large proportion of protein level changes during ES cell fate transition can be explained by post-transcriptional regulation studied on the genome and proteome wide scale (as reviewed in Chen et al., 2017). With the emerge of technologies such as high-throughput sequencing, large-scale screening, and systematic identification of protein-RNA interactions or RBPs, more and more post-transcriptional mechanisms have been identified which can fine-tune the gene expression program to maintain pluripotent state (as reviewed in Chen et al., 2017). The alternate splicing could generate splice variants of pluripotency associated genes required for different stages of pluripotency and differentiation, alternate polyadenylation activates an ES cell-specific pattern on a group of pluripotency associated genes to enhance their expression. RNA can be chemically modified and RNA modifications serve as another layer of post-transcriptional control in gene expression. In both naïve and primed ES cells, m6A modification of mRNAs regulate pluripotent stem cell fate by acting on cell-type specific transcripts (as reviewed in Chen et al., 2017).

Various studies have identified the important role of post-translational modifications such as ubiquitination, sumoylation, phosphorylation, methylation, and acetylation, in regulating the levels and activity of pluripotency factors to achieve a balance between pluripotency and differentiation. In mouse ES cells, OCT4 can be ubiquitinated by an HECT-type E3 ubiquitin ligase, WWP2, via the Lys63 during ES cell differentiation (Xu et al., 2004). In human ES cells, NANOG is regulated through ubiquitination of a proline (P), glutamic acid (E), serine (S), and threonine (T) (PEST) motif that lies in the N-terminal region of NANOG from amino acid 47 to 72, although the specific ubiquitin ligase E3 are still unknown (Ramakrishna et al., 2011). The small ubiquitin-related modifier (SUMO), which is structurally related to but functionally divergent from ubiquitin, can modify many nuclear proteins to affect their subcellular localization, thus altering their interaction with cooperative molecules. Studies show that OCT4 can be sumoylated at a single lysine, lysine 118, which is located at the end of the N-terminal transactivation domain and next to the POU

DNA-binding domain (Zhang et al., 2007). Sumoylation of OCT4 significantly increases its stability, DNA binding, and thus the transcriptional activity without altering its subnuclear localization (Wei et al., 2007). The phosphorylation of NANOG by ERK-1 reduces its stability by inducing the binding of F-box/WD repeat-containing protein 8 (FBXW8), hence leading to mouse ES cell differentiation (Kim et al, 2014). Conversely, the deubiquitination of NANOG by ubiquitin-specific protease (USP21) stabilizes NANOG and maintains the stemness of mouse ES cells (Liu et al, 2016). Furthermore, KLF4 is shown to be a target of ubiquitination and proteosomal degradation followed by ERK mediated phosphorylation (Kim et al, 2012). Another study identified the conserved Lysine residues critical for ubiquitination and degradation of KLF4 (Lim et al, 2014). Post-translational modifications play an important role in these processes by regulating the activity, stability, and cellular distribution of transcription factors that control pluripotency.

1.7 KLFs in pluripotency

Krüppel-like factors (Klfs) are evolutionarily conserved zinc finger-containing transcription factors implicated in many biological processes, including proliferation, apoptosis, differentiation and development. These factors have gained a lot of attention as a transfection cocktail of transcription factors needed for production of iPSCs include Klf4 as one of the factors. Klf factors, Klf2, Klf4 and Klf5, are known to be involved in maintaining pluripotent cells (Jiang et al., 2008). These Klf factors are highly expressed in naïve pluripotent mouse ES cells and this expression drops dramatically after induction of differentiation by withdrawal of LIF (Bourillot et al., 2009).

Klf2 is also called lung Klf due to its high expression in adult lung. Klf2 null mice died by embryonic day 14.5 from growth retardation, massive hemorrhage, and signs of anemia. The vasculature displayed defective morphology demonstrating the important role of KLF2 in blood vessel stabilization during embryogenesis (Kuo et al., 1997; Wani et al., 1999). Klf4, also known as gut Klf4 or GKlf, null mice survive embryonic development but die shortly after birth due to a skin defect that results in loss of fluids and dramatic decrease in number of goblet cells of the colon (Jiang et al., 2008, Segre et al., 1999). Klf5, known as colon or intestinal Klf, Klf5 null embryos fail to develop beyond the blastocyst stage in vivo or to produce mouse ES cell lines in vitro (Emma et al., 2008).

Inactivation of any one of these genes by RNA interference in mouse ES cells induces spontaneous differentiation whereas overexpression induces self-renewal and delays differentiation (Parisi et al., 2008; Jiang et al., 2008). The Klf2/Klf4/Klf5 triple knockdown induced differentiation was shown to be rescued by overexpression of any one of these three suggesting functional redundancy (Parisi et al., 2008; Jiang et al., 2008). However, upon Klf4 knock down by stable expression of Klf4 shRNA, the redundancy effects of Klf2 and Klf5 were insufficient to maintain ES cell identity (Zhang et al., 2010). Moreover, even though Klf5 has been reported to be required for ES cell growth in the ICM of early embryos, Klf5 cannot prevent ES cell differentiation in culture for extended period (Ema et al., 2008).

Although Klf2, Klf4, Klf5 are expressed in ES cells and are down-regulated during ES cell differentiation (Bruce et al., 2007), their regulation behavior on the naïve ES cell is different. Klf4 is a direct downstream target of LIF/STAT3 signaling loop but Klf5 responds to LIF in a STAT3- independent manner whereas Klf2 does not respond to LIF at all (Hall et al., 2009). Moreover, Klf4 regulate ES cell self-renewal and pluripotency by upregulating Nanog expression (Zhang et al., 2010), Klf5 maintains ES cell self-renewal through suppression of differentiation associated gene expression (Parisi et al., 2010). Klf2 and Klf4 can activate the Lefty1promoter to regulate ES cell but Klf5 cannot (Nakatake et al., 2006). Moreover, Klf2 is activated by Oct4; Klf4 and Klf5 are activated by Nanog (Jiang et al., 2008). These dissimilarities illustrate the distinct roles of independent Klfs in maintenance of pluripotency even though they exhibit some level of redundancy and cooperation.

The expression of Klf4 and Klf5 is downregulated very early, whereas expression of Klf2 is downregulated later upon induction of differentiation suggesting progressive deconstruction of the molecular circuitry controlling pluripotency during ES cell differentiation (Parisi et al., 2010). It has been shown that upon differentiation induction by LIF withdrawal, Klf4 acts as a fast responding mediator to LIF-STAT3 signal changes, eventually affecting the expression of Nanog that is required for maintenance of naïve ES cells (Zhang et al., 2010). Therefore, Klf4 is an important candidate gene to be investigated when trying to understand the underlying mechanisms for maintaining the naïve pluripotency state and the mechanisms involved in exit from naïve pluripotency.

1.8 Thesis Objectives and Hypothesis

The overarching goal of my project is to better understand how different layers of pluripotency regulation interact to maintain the balanced naïve pluripotent state of mouse ES cells and how these interactions change dynamically while mouse ES cells exit the pluripotent state. To be more precise, I have investigated the dynamics of gene and protein regulation that change and are required for naïve pluripotency exit of mouse ES cells.

Pluripotency is regulated by interconnected transcriptional network of OCT4, SOX2, NANOG and KLF4 under the influence of extracellular cues provided by interconnected signaling pathways. Klf4 had been reported among the first of the pluripotency associated transcription factor affected by differentiation induction after removal of LIF/2i (Zhang et al., 2010). I hypothesize that the nuclear environment changes dramatically within 24 hrs of LIF/2i removal causing naïve pluripotent mouse ES cells to exit naïve pluripotency. I also hypothesize that the removal of KLF4 protein from the pluripotency associated transcriptional complex would be required for exit from naïve pluripotent state of mouse ES cells by LIF/2i withdrawal.

1.8.1 Major research questions:

I will primarily focus on answering the following questions: 1. What are the dynamic changes occurring in the nuclear environment during pluripotency exit? 2. How do the changes in extracellular signaling relate to the function of transcriptional complexes regulating gene expression during the first 24 hrs of mouse ES cell differentiation? 3. How do signaling mechanisms affect gene and protein regulation of Klf4 as mouse ES cells exit naïve pluripotent state?

1.8.2 Thesis aims:

Aim1: To investigate changes in the nuclear environment as mouse ES cells begin to differentiate. Addressing this aim will likely identify novel mechanisms critical to exit from naïve pluripotency.

Aim2: To investigate the KLF4 gene and protein regulatory mechanisms in context to pluripotency maintenance and exit.

Chapter 2 KLF4 Nuclear Export Requires ERK Activation and Initiates Exit from Naive Pluripotency

The content of this chapter is published as: Dhaliwal NK, Miri K, Davidson S, Tamim El Jarkass H, Mitchell JA. KLF4 Nuclear Export Requires ERK Activation and Initiates Exit from Naive Pluripotency. Stem Cell Reports. 2018 Apr 10;10(4):1308-1323. DOI:https://doi.org/10.1016/j.stemcr.2018.02.007

2.1 Abstract

Cooperative action of a transcription factor complex containing OCT4, SOX2, NANOG, and KLF4 maintains the naïve pluripotent state; however, less is known about the mechanisms that disrupt this complex, initiating exit from pluripotency. I show that, as embryonic stem cells (ES cells) exit pluripotency, KLF4 protein is exported from the nucleus causing rapid decline in Nanog and Klf4 transcription; as a result, KLF4 is the first pluripotency transcription factor removed from transcription-associated complexes during differentiation. KLF4 nuclear export requires ERK activation, and phosphorylation of KLF4 by ERK initiates interaction of KLF4 with nuclear export factor XPO1, leading to KLF4 export. Mutation of the ERK phosphorylation site in KLF4 (S132) blocks KLF4 nuclear export, the decline in Nanog, Klf4, and Sox2 mRNA, and differentiation. These findings demonstrate that relocalization of KLF4 to the cytoplasm is a critical first step in exit from the naïve pluripotent state and initiation of ES cell differentiation.

2.2 Introduction

Kruppel-like factor 4 (Klf4) is a transcriptional regulator of the naive pluripotent state sufficient to revert epiblast stem cells to naïve embryonic stem cells (ES cells) (Bruce et al., 2007, Guo et al., 2009, Zhang et al., 2010). KLF4 forms a protein complex with the pluripotency master regulators OCT4 (POU5F1) and SOX2 and binds many of the same genomic regions as OCT4, SOX2, and NANOG (Chen et al., 2008, Gao et al., 2012, Wei et al., 2009). Leukemia inhibitory factor (LIF) can be used to maintain mouse ES cells in the pluripotent state, and Klf4 is a direct target of LIF signaling through activation of STAT3 (Bourillot et al., 2009, Hall et al., 2009, Niwa et al., 2009). In mouse ES cells, MAPK (mitogen-activated protein kinase) and GSK3 (glycogen synthase kinase 3) signaling promote differentiation; however, inhibition of both pathways through the use of two inhibitors (2i) supports self-renewal (Doble et al., 2007, Kunath et al., 2007, Ying et al., 2008). The mouse ES cells cultured in 2i media have been proposed to be the closest in vitro representation of pluripotent cells from pre-implantation embryos, referred to as naïve pluripotency (Nichols and Smith, 2009). Media supplemented with 2i can support ESC self- renewal even in the absence of LIF; however, LIF is often included in the media to further support pluripotency maintenance (Nichols et al., 2009, Theunissen et al., 2011). The mechanisms of pluripotency exit, specifically how the pluripotency transcription factors are downregulated during differentiation, remain poorly understood.

Although KLF4 is involved in pluripotency maintenance in vitro, its expression has not been investigated in the pre-implantation mouse embryo; however, analysis of rhesus macaque embryos revealed nuclear KLF4 in all cells of pre-implantation blastocysts, including the pluripotent cells of the inner cell mass (ICM) (Harvey et al., 2009). Klf4 null mice survive early development, indicating that Klf4 is not required for embryogenesis, due to compensatory mechanisms involving other Klf factors (Jiang et al., 2008, Segre et al., 1999). Although not required during early mouse development, there may be a need for Klf4 downregulation upon differentiation, as overexpression of Klf4 has been shown to prevent differentiation of ES cells even in the absence of LIF (Zhang et al., 2010).

The mechanisms of pluripotency induction during reprogramming have been widely investigated, but less effort has been expended in understanding the mechanisms of pluripotency exit. As ES cells exit pluripotency, Klf4 and Nanog are the first transcription factors to show a decrease in

39 transcript expression (Zhang et al., 2010). This, coupled with the observation that KLF4 protein binds the Nanog promoter and activates transcription, led to the idea that reduced Klf4 transcription is the trigger that causes exit from pluripotency (Zhang et al., 2010). To further investigate this mechanism, I monitored nuclear levels of KLF4 and NANOG protein in ES cells as they exit naive pluripotency. In contrast to the mechanism proposed above, our study revealed that pluripotency exit is initiated by an ERK-mediated interaction between KLF4 and the nuclear export factor XPO1 and the subsequent relocalization of KLF4 to the cytoplasm. Blocking KLF4 nuclear export by mutating the ERK phosphorylation site in the KLF4 protein or KLF4 nuclear export sequences (NES) required for interaction with XPO1 prevents exit from pluripotency. To investigate this mechanism in the embryo, I inhibited MEK signaling to prevent ERK activation; this resulted in a loss of KLF4/ERK2 and KLF4/XPO1 interaction and a disruption in KLF4 and NANOG downregulation in pre-implantation mouse blastocysts. These findings extend our proposed mechanism for controlling KLF4 nuclear levels to the pre-implantation embryo, demonstrating the significance of KLF4 nuclear export in regulating stem cell differentiation both in vitro and in vivo.

2.3 Results

2.3.1 KLF4 Nuclear Export Occurs as ES cells Exit Naive Pluripotency

Levels of Klf4 and Nanog mRNA decline within the first 24 hr of pluripotency exit, and it has been suggested that reduced Klf4 transcription after removal of external maintenance factors is the trigger that initiates ESC differentiation; however, protein levels have not been investigated during this time frame (Zhang et al., 2010). I first confirmed that the decline in both Klf4 and Nanog mRNA occurred for ES cells maintained in LIF/2i after removal of these components, and observed a reduction in both Klf4 and Nanog mRNA at 6 hr (Figure 2.1A). To investigate changes in nuclear protein levels as ES cells exit naive pluripotency, I monitored subcellular localization of KLF4, OCT4, SOX2, and NANOG for 24 hr after removal of LIF/2i. In undifferentiated ES cells KLF4, OCT4, SOX2, and NANOG are located in the nucleus (Figures 2.2 and 2.1B). Quantification of immunofluorescence images from different times after differentiation induction indicated that SOX2 and OCT4 nuclear protein levels were maintained over the first 24 hr of ESC differentiation (Figures 2.1C and 2.1D), whereas NANOG and KLF4 exhibited dynamic behavior (Figure 2.2B). At 6 hr, a significant portion of KLF4 protein was localized in the cytoplasm (arrows in Figure 2.2A), suggesting that KLF4 nuclear export occurs as ES cells exit naive pluripotency. Over this same time period, NANOG nuclear protein levels decreased with a significant reduction first observed at 6 hr; however, NANOG was not observed in cytoplasm (Figures 2.2A and 2.2B). To obtain a more quantitative measure of the amount of cytoplasmic and nuclear KLF4, I separated ES cells into nuclear and cytoplasmic fractions and assayed them by immunoblot. This analysis confirmed the subcellular redistribution of KLF4; nuclear KLF4 was significantly reduced at 6 hr and cytoplasmic KLF4 was increased (Figures 2.2C and 2.2D). Interestingly, 24 hr after LIF/2i removal KLF4 was again predominantly nuclear, although at half the levels observed in undifferentiated ES cells. Total protein was also monitored, which confirmed the 50% decrease in KLF4 protein at 24 hr compared with undifferentiated ES cells (Figure 2.3A).

As KLF4 was observed to exit the nucleus at 6 hr but accumulate in the nucleus at 24 hr, I monitored nuclear protein complexes to determine whether this dynamic behavior disrupted KLF4 function in the nucleus. Proximity ligation amplification (PLA) (Fredriksson et al., 2002) was conducted to quantify the interaction between KLF4/RNAPII-S5P or pluripotency transcription factors in single nuclei. In undifferentiated ES cells, KLF4 interacts with RNAPII-S5P at about 20

41 nucleoplasmic foci (Figures 2.4A and 2.4B). As ES cells differentiate this is reduced to about 6 foci per nucleus at 6 hr; at 24 hr, on average 10 foci were detected, indicating that the reduction in nuclear KLF4 by half affects participation of KLF4 in polymerase complexes. In addition, interaction between KLF4/NANOG and NANOG/RNAPII-S5P (Figures 2A, 2B, and 2.3B) was reduced at 24 hr. This was expected as nuclear NANOG levels were dramatically reduced at 24 hr. Although SOX2 levels were found to be unchanged over the first 24 hr of ESC differentiation, and interaction of SOX2/RNAPII-S5P was unchanged (Figures 2.3A and 2.3B), interaction between KLF4/SOX2 was also reduced at 24 hr (Figure 2.4). Similarly, interaction between KLF4/OCT4 was reduced at 24 hr (Figure 2.3B). Together these data indicate that KLF4 function in nuclear transcription complexes is disrupted at 6 hr and only partially restored at 24 hr of differentiation.

The observation that KLF4 protein became predominantly nuclear again at 24 hr led us to investigate whether this was newly synthesized KLF4 or the same pool of protein that was exported at 6 hr. To investigate this I treated ES cells undergoing differentiation for 12 hr, when KLF4 is almost exclusively cytoplasmic (Figure 2.2D), for a further 12 hr with the protein synthesis inhibitor cycloheximide (CHX) in the presence or absence of the proteasome inhibitor MG132. Immunoblotting revealed that KLF4 protein levels in the nuclear fraction are significantly reduced at 24 hr when cells are treated with CHX, indicating that KLF4 accumulation in the nucleus at 24 hr requires protein synthesis (Figures 2.4C and 2.4D). The significantly increased amount of KLF4 in the cytoplasmic fraction of the MG132-treated cells indicates that KLF4 exported to the cytoplasm undergoes proteosomal degradation.

Figure 2.1

Figure 2.1: Changes in pluripotency factor mRNA and protein levels during the first 24 hr of differentiation.

A) Real time RT-qPCR from three biological replicate samples reveals downregulation of Klf4 and Nanog mRNA 6 hr after LIF/2i withdrawal. Statistical differences are indicated by *** P < 0.001. Error bars represent standard deviation.

B) Immunofluorescence images of ES cells cultured with LIF/2i and ES cells 2, 6, 12 and 24 hr after LIF/2i removal. OCT4, SOX2, and RNAPII-S5P proteins were detected by immunofluorescence. Merged images display SOX2 or OCT4 in green, RNAPII-S5P in red and DAPI DNA stain in blue. Scale bar = 10μm.

C) Box and whisker plots display intensities per nucleus of SOX2 (left) and OCT4 (right) and RNAPII-S5P (bottom). Green line indicates the average intensity at each time point, black line indicates the median intensity. Boxes indicate interquartile range of intensity values, whiskers indicate the 10th and 90th percentiles, and outliers are shown as black dots. Images were collected from at least three biological replicate samples and ≥100 nuclei were quantified for each. No significant differences were observed in the intensity of these proteins during this time frame.

D) Immunoblot of the nuclear and cytoplasmic fractions confirmed that SOX2 and OCT4 are exclusively nuclear in ES cells.

Figure 2.2

Figure 2.2: KLF4 nuclear exit occurs as ES cells exit naïve pluripotency.

A) Immunofluorescence images of ES cells cultured with LIF/2i and ES cells 2, 6, 12 and 24 hr after LIF/2i removal display KLF4 nuclear exit starting at 6 hr after LIF/2i removal. NANOG, RNAPII-S5P and KLF4 proteins were detected by immunofluorescence. Merged images display NANOG or KLF4 in green, RNAPII-S5P in red and DAPI DNA stain in blue. Scale bar = 10 μm.

B) Box and whisker plots display intensities per nucleus of NANOG (left) and KLF4 (right). Green line indicates the average intensity at each time point, black line indicates the median intensity. Boxes indicate interquartile range of intensity values, whiskers indicate the 10th and 90th percentiles, and outliers are shown as black dots. Statistical differences are indicated by * P< 0.05, ** P < 0.01 and *** P < 0.001.

C) Immunoblot analysis from nuclear and cytoplasmic fractions of ES cells cultured with LIF/2i (0) and ES cells 2, 6, 12 and 24 hr after LIF/2i removal. Cyclophilin A (CYPA) and the nucleolar protein upstream binding factor (UBF) reveal purity of the cytoplasmic and nuclear fractions respectively. NANOG levels decrease in the nuclear fraction starting at 6 hr. KLF4 levels are increased in cytoplasmic fraction at 6 hr and correspondingly decreased in the nuclear fraction. At 24 hr KLF4 was mainly located in the nuclear fraction.

D) Quantification of relative intensity levels of KLF4 in immunoblots from three biological replicates. Statistical differences compared to the 0 hr values are indicated by ** P < 0.01 and *** P < 0.001.

Figure 2.3

Figure 2.3: Changes in nuclear protein complexes associated with pluripotency exit.

A) Immunoblot analysis of total protein isolated from ES cells cultured with LIF/2i (ES) and cells 2, 6, 12 and 24 hr after LIF/2i removal is shown on the left. GAPDH levels were monitored to evaluate protein loading. Mouse embryonic fibroblasts (MEF) were used as a negative control for the expression of the pluripotency transcription factors. On the right quantification from three biological replicates is shown as the average relative intensity for each protein compared to GAPDH. Whereas NANOG levels are dramatically reduced at 24 hr compared to undifferentiated cells KLF4 levels are only reduced to about 50% compared to undifferentiated cells. Statistical differences compared to the undifferentiated ES cell values are indicated by ** P < 0.01 and *** P < 0.001. Error bars represent standard deviation.

B) Proximity ligation amplification (PLA) indicating the amount of interaction between KLF4/OCT4, SOX2/RNAPII-S5P and NANOG/RNAPII-S5P in ES cells after LIF/2i withdrawal. Scale bar = 10μm. Quantification of the number of interaction foci per nucleus is shown to the right. Box and whisker plots display the number of PLA foci per nucleus. Boxes indicate interquartile range of intensity values, whiskers indicate the 10th and 90th percentiles, outliers in the 5th and 95th percentiles are shown as black dots. Images were collected from at least three biological replicate samples and ≥100 nuclei were quantified for each. Statistical differences are indicated by *** P < 0.001.

Figure 2.4

Figure 2.4: The removal of MEK inhibition phosphorylates and accumulates KLF4 to the cytoplasm.

A) Immunofluorescence images of ES cells cultured for 6 hr with combinations of the indicated components: LIF (Leukemia Inhibitory Factor), MEKi (MEK1 inhibitor, PD0325901), GSKi (GSK3 inhibitor, CHIR99021). Merged images display KLF4 in green, RNAPII-S5P in red and DAPI in blue. Scale bar = 10 μm.

B) Immunoblot for activated ERK1/2 (pTEpY) and total ERK1/2 in ES cells cultured with LIF/2i (0) and ES cells 15 and 30 min, 1, 2, 4, 6, 12, 24 and 48 hr after LIF/2i removal.

C) Proximity ligation amplification (PLA) for KLF4/p-ERK displays the interaction between p- ERK and KLF4 6 hr after LIF/2i withdrawal but not in ES cells maintained in LIF/2i. Images shown are single optical sections. Merged images display DAPI in blue and PLA in red. Scale bar = 10 μm.

D) Immunoblot of immunoprecipitated KLF4 protein from nuclear and cytoplasmic fractions of mouse ES cells cultured with LIF/2i and mouse ES cells 6 hr after LIF/2i removal with KLF4 and phospho-Serine antibody. Cyclophilin A (CYPA) and the nucleolar protein upstream binding factor (UBF) reveal purity of the cytoplasmic and nuclear fractions respectively.

2.3.2 ERK Activation and Interaction with KLF4 Is Coincident with KLF4 Nuclear Export

As KLF4 was exported to the cytoplasm 6 hr after removal of LIF/2i, I investigated the mechanisms involved by removal of individual media components (LIF, MEK inhibitor, GSK inhibitor) for 6 hr followed by KLF4 subcellular localization by immunofluorescence (Figure 2.5A). Removal of the MEK inhibitor resulted in relocalization of KLF4 to the cytoplasm; however, when the MEK inhibitor remained in the media, KLF4 was localized to the nucleus. Stimulation of the fibroblast growth factor receptor (FGFR) by FGF4 causes activation of the MEK-ERK pathway and is required for ESC differentiation (Kunath et al., 2007). KLF4 nuclear export after removal of MEK inhibition was blocked by an FGFR inhibitor, indicating that FGFR activation is required for this process (Figure 2.6A). Together these observations suggest that ERK activation plays a central role in mediating ESC differentiation by controlling the subcellular localization of KLF4.

To determine whether ERK activation was coincident with KLF4 nuclear export, I monitored total and active ERK1/2 by immunoblot (Figure 2.5B). Phosphorylated active ERK (pTEpY) was detected 4–6 hr after removal of LIF/2i when cytoplasmic KLF4 was observed. ERK phosphorylation was no longer observed at 24 hr when KLF4 again accumulates in the nucleus. Upon investigation of the subcellular localization of ERK2 in ES cells, I observed phosphorylated ERK in the nucleus and increased overlap of ERK2 with KLF4 at 6 hr (Figures 2.6B and 2.6C). The concurrence between KLF4 nuclear export, ERK2 phosphorylation, and nuclear accumulation suggested that active nuclear ERK2 promotes KLF4 nuclear export. To investigate whether ERK2 directly interacts with KLF4, I conducted PLA to identify proximity between KLF4 and ERK2. In undifferentiated ES cells, ERK2 remains inactive and KLF4/ERK2 PLA revealed no interaction between these two proteins; however, after 6 hr of differentiation, when phosphorylated ERK2 accumulates in the nucleus, interaction between KLF4 and ERK2 was observed (Figure 2.5C).

As ES cells maintained in LIF/2i are prevented from differentiating by inhibiting MEK-ERK signaling and the release of this inhibition may be linked to KLF4 nuclear export, I sought to determine whether KLF4 nuclear export would occur during pluripotency exit for ES cells maintained in LIF. I observed KLF4 nuclear export at 12 hr, which was delayed compared with cells maintained in LIF/2i (Figure 2.6D). MEK-ERK signaling is often rapid, peaking within

51 minutes. To determine whether MEK-ERK signaling and KLF4 nuclear export can occur more rapidly in ES cells, I treated undifferentiated cells with TPA (12-O-tetradecanoylphorbol-13- acetate), an upstream activator of the MEK-ERK pathway (Figure 2.5D). TPA treatment caused ERK phosphorylation and KLF4 nuclear export at 15–30 min, indicating that this response can be rapid. In addition, a more dramatic decrease in both Klf4 and Nanog transcript levels was observed 2 hr after LIF/2i removal in the presence of TPA (Figure 2.6E).

Figure 2.5

Figure 2.5: Active ERK2 interacts with KLF4 initiating KLF4 nuclear export

A) Immunofluorescence images of ES cells cultured for 6 hr with the indicated components: LIF, MEKi (MEK1 inhibitor, PD0325901), and GSKi (GSK3 inhibitor, CHIR99021). Merged images display KLF4 in green, RNAPII-S5P in red, and DAPI in blue. Scale bars, 10 μm.

B) Immunoblot for activated ERK1/2 (pTEpY) and total ERK1/2 in ES cells cultured with LIF/2i (0), 15 and 30 min, 1, 2, 4, 6, 12, 24, and 48 hr after LIF/2i removal.

C) Proximity ligation amplification (PLA) for KLF4/ERK2 displays the interaction between ERK2 and KLF4 6 hr after LIF/2i withdrawal but not in ES cells maintained in LIF/2i. Images shown are single optical sections. Merged images display DAPI in blue and PLA in red. Bottom images display grayscale PLA signal. Scale bars, 10 μm.

D) Immunoblot for ES cells treated with TPA and sampled at the indicated time in minutes. With TPA treatment, ERK phosphorylation and KLF4 nuclear export occur more rapidly starting at 15 min. Cyclophilin A (CYPA) and the nucleolar protein upstream binding factor (UBF1) reveal purity of the cytoplasmic and nuclear fractions, respectively.

Figure 2.6

Figure 2.6: Signaling mechanisms regulating KLF4 nuclear export.

A) Immunofluorescence images of ES cells cultured with LIF/2i and ES cells 6 hr after LIF/2i removal or after 6 hr of LIF/2i removal in the presence of the FGFR inhibitor (FGFRi, PD173074). Merged images display KLF4 in green, RNAPII-S5P in red and DAPI in blue. Scale bar = 10μm.

B) Removal of LIF/2i causes ERK2 to accumulate in the nucleus concurrently with KLF4 nuclear exit. Immunofluorescence images of ES cells cultured with LIF/2i and ES cells 6 hr after LIF/2i removal. Merged images display KLF4 in red, ERK2 in green and DAPI in blue. Scale bar = 10μm.

C) Immunoblot analysis from nuclear and cytoplasmic fractions of ES cells cultured with LIF/2i (0) and ES cells 4, 6, 12 and 24 hr after LIF/2i removal.

D) Immunoblot analysis from nuclear and cytoplasmic fractions of ES cells, and total protein from ES cells cultured with LIF (0) and ES cells 6, 12 and 24 hr after LIF removal.

E) TPA treatment causes greater decrease in Klf4 and Nanog mRNA 2hr after LIF/2i withdrawal. The average of three biological replicates are normalized to the levels observed in undifferentiated ES cells and GAPDH. Statistical differences compared to the undifferentiated cells or the 2hr differentiated cells are indicated by * P < 0.05, ** P < 0.01, *** P < 0.001. Error bars represent standard deviation.

2.3.3 KLF4 Nuclear Export Occurs through an XPO1-Mediated Nuclear Export Mechanism

To investigate the involvement of Xportin1 (XPO1) in KLF4 nuclear export, I differentiated ES cells in the presence or absence of the XPO1-mediated nuclear export inhibitor leptomycin B (LMB) (Figure 2.7A). LMB was found to block KLF4 nuclear export, as KLF4 remained nuclear. XPO1-mediated nuclear export occurs when XPO1 binds to a cargo protein NES. PLA for KLF4/XPO1 revealed an interaction between KLF4 and XPO1 only in differentiating ES cells (Figure 2.7B). ERK2 and XPO1 were also shown to interact by PLA; however, signals were observed in both ES cells and 6-hr cells. Interactions between KLF4/ERK2, KLF4/XPO1, and ERK2/XPO1 were validated by co-immunoprecipitation (Figure 2.7C). ERK2 is unphosphorylated in undifferentiated cells; however, I identified interaction with XPO1 under these conditions, suggesting that continuous export of ERK2 by XPO1 prevents nuclear accumulation of unphosphorylated ERK2; indeed this has been described in both Xenopus and rat cells where ERK nuclear export depends on XPO1 and the MEK NES (Adachi et al., 2000). In addition, I found that MEK interacts with XPO1 in both undifferentiated and 6-hr differentiated ES cells; however, KLF4 does not interact with MEK in either condition (Figure 2.8), despite the observation that both MEK and KLF4 interact with XPO1 in differentiating ES cells, indicating that export of KLF4 is independent of the MEK NES in ES cells.

Figure 2.7

Figure 2.7: KLF4 nuclear export occurs through an XPO1-mediated mechanism

A) Immunofluorescence images of ES cells cultured for 6 and 12 hr without LIF/2i in the presence of 5 μg/mL leptomycin B (LMB), which inhibits XPO1-mediated nuclear export. Merged images display KLF4 in green, RNAPII-S5P in red, and DAPI in blue. Scale bars, 10 μm.

B) Proximity ligation amplification (PLA) displays the interaction between KLF4/XPO1 and XPO1/ERK2 in ES cells and 6 hr after LIF/2i withdrawal. Images shown are single optical sections. Merged images display DAPI in blue and PLA in red. Bottom images display the grayscale PLA signal. Scale bars, 10 μm.

C) KLF4 and ERK2 immunoprecipitation (IP) for undifferentiated ES cells and ES cells 6 hr after LIF/2i removal, probed with anti-ERK2 and anti-XPO1 (PD, pull-down). GAPDH levels reveal equal loading of the input.

Figure 2.8

Figure 2.8: KLF4 interacts with XPO1 independently of MEK.

PLA in undifferentiated and 6 hr differentiated ES cells revealed interaction between ERK2/XPO1 and MEK/XPO1 in both conditions but no interaction between KLF4 and MEK in either condition. Scale bar = 25μm.

2.3.4 KLF4 Nuclear Export Requires Both Nuclear Export Sequences and Phosphorylation of S132

As KLF4 nuclear export was initiated by interaction of KLF4 with active ERK2, and KLF4 has been shown to be phosphorylated by ERK at S132 (described as S123 in Kim et al. (2012) as their construct lacked the first 9 amino acids of the endogenous KLF4 protein). Moreover, the S132 site is highly conserved among KLF4 orthologues in many primate and non-primate species but not among other KLF homologues expressed in ES cells such as KLF2 and KLF5 (Appendix 1, Figure A1.5 and A1.6). I next investigated the role of S132 in KLF4 nuclear export (Figure 2.9). To determine whether KLF4 is phosphorylated prior to nuclear export, I immunoprecipitated KLF4 from nuclear and cytoplasmic fractions of undifferentiated and 6-hr differentiated ES cells and subjected it to immunoblotting using an anti-phosphoserine antibody (Figure 2.9B). The presence of a phosphoserine band in both the cytoplasmic and nuclear fractions at 6 hr suggests that activated ERK2 interacts with KLF4 and phosphorylates KLF4 at a serine residue. PLA for ERK2/RNAPII-S5P revealed that at 6 hr, when active ERK2 enters the nucleus, it interacts with active RNAPII (Figure 2.10A) which could facilitate interaction with, and phosphorylation of, KLF4 associated with active RNAPII (Figure 2.4).

To investigate the role of S132 in KLF4 nuclear export, I generated ESC lines that stably express wild-type (WT) KLF4-GFP, an S132 mutant (KLF4(S132A)-GFP), or a phosphomimetic (KLF4(S132D)-GFP) with the assistance of a post doctorate fellow in our lab Scott Davidson and an undergraduate student Hala Tamim. Endogenous KLF4 and WT KLF4-GFP were observed to exit the nucleus at 6 hr (Figure 2.9C). By contrast, KLF4(S132A)-GFP remains in the nuclear fraction, indicating that S132 is required for nuclear export. In addition, the phosphomimetic KLF4(S132D)-GFP is exported to the cytoplasm in undifferentiated ES cells, suggesting that phosphorylation of S132 is sufficient for KLF4 nuclear export (Figure 2.10B). Nuclear export occurs when XPO1 binds a cargo protein NES; to investigate this we mutated four predicted NES in KLF4-GFP (Figure 2.9A). Ppredicted NES1, 2 and 3 are highly conserved among both primates and non-primates whereas NES 4 is conserved only in non-primates based on Clustal W sequence alignments (Appendix 1, Figure A1.4). Immunoblot with anti-GFP revealed that mutation of NES1 or NES2 inhibited KLF4 export to the cytoplasm, whereas mutation of NES3 or NES4 had no effect (Figure 2.9D).

I next investigated the effect of KLF4 mutation on interaction with ERK2 and XPO1 (Figure 2.9E). Similar to results for the interaction of endogenous KLF4 with ERK2 and XPO1, I found no interactions in undifferentiated ES cells (Figure 2.10C). Cells differentiated for 6 hr displayed interaction between WT KLF4-GFP and ERK2 as well as XPO1 (Figure 2.9E). Mutation of KLF4 at S132 or NES2, immediately upstream of S132, disrupted the interaction of KLF4-GFP with ERK2, whereas mutation of NES1, 3, or 4 had no effect. The interaction between KLF4-GFP and XPO1 was disrupted when S132, NES1, or NES2 was mutated, whereas mutation of NES3 or NES4 had no effect. To evaluate the effect of specific mutations on the phosphorylation of KLF4, I immunoprecipitated cell lysates with GFP-Trap and conducted immunoblotting using anti- phosphoserine (Figure 2.9F). Serine phosphorylation at 6 hr was disrupted by mutation of S132 or NES2. Together, these data indicate that NES1, NES2, and S132 are required for the interaction of KLF4 with XPO1 and KLF4 nuclear export.

To rule out the possibility that mutant KLF4-GFP was not able to participate in nuclear complexes, I investigated interaction with RNAPII-S5P by PLA, which revealed that all KLF4-GFP mutants participated in transcriptional complexes to a similar degree (Figure 2.10D). To determine whether KLF4 nuclear localization is important for interaction with ERK2, we generated an ESC line stably expressing a KLF4 nuclear localization mutant, KLF4(NLS)-GFP. GFP/ERK2 PLA in these cells revealed no interaction between KLF4(NLS)-GFP and ERK2 after 6 hr, indicating that localization to the nucleus is important for this interaction (Figure 2.10D). Together these data indicate the S132 ERK phosphorylation site, NES1, and NES2 are together required for KLF4 nuclear export. Furthermore, mutation of these residues does not appear to interfere with the participation of KLF4 in active RNAPII transcriptional complexes.

Figure 2.9

Figure 2.9: The KLF4 ERK phosphorylation site S132, NES1, and NES2 are required for KLF4 nuclear export

A) Schematic of mouse KLF4 depicting predicted nuclear export signals (NES), nuclear localization signal (NLS), ERK phosphorylation site S132, and zinc fingers (ZNFs).

B) Immunoprecipitated KLF4 protein from nuclear and cytoplasmic fractions of ES cells cultured with LIF/2i and 6 hr after LIF/2i removal, probed with anti-KLF4 and anti-phosphoserine (P-SER; PD, pull-down). Cyclophilin A (CYPA) and the nucleolar protein upstream binding factor (UBF1) reveal purity of the cytoplasmic and nuclear fractions, respectively.

C) Nuclear and cytoplasmic fractions prepared from KLF4-GFP ES lines indicate that wild-type (WT) KLF4-GFP (top band) but not KLF4(S132A)-GFP is exported to the cytoplasm 6 hr after LIF/2i removal. Endogenous KLF4 (bottom band) is exported to the cytoplasm in both cases.

D) Nuclear and cytoplasmic fractions prepared from WT KLF4-GFP and KLF4-GFP NES mutants (1–4). Anti-GFP immunoblot indicated that NES1 and NES2 are required for nuclear export of KLF4 after removal of LIF/2i.

Figure 2.10

Figure 2.10: Experimental controls and supplementary data

A) PLA indicating the interaction between ERK2 and RNAPII-S5P in ES cells and 6 or 24 hr after LIF/2i withdrawal. Images shown are maximum intensity projections. Scale bar = 10μm.

B) Nuclear and cytoplasmic fractions prepared from ES cells expressing WT KLF4-GFP or KLF4- GFP mutants maintained in LIF/2i. Anti-GFP immunoblot indicated the S132D phosphomimetic is exported to the cytoplasm in LIF/2i. Treatment with the proteasome inhibitor MG132 prevents degradation of cytoplasmic KLF4(S132D)-GFP.

C) PLA indicating no interaction between any KLF4-GFP (WT or indicated mutants) and ERK2 or XPO1 in ES cells maintained in LIF/2i. Scale bar = 10μm.

D) PLA controls in KLF4-GFP transfected cells. PLA for GFP/RNAPII-S5P in WT KLF4-GFP expressing cells and KLF4(S132A)-GFP, KLF4(NES1, 2, 3, 4)-GFP expressing cells indicates that these mutations do not affect participation of KLF4 in RNAPII complexes. NLS mutation does affect KLF4 nuclear localization and interaction with RNAPII. PLA for GFP/ERK in cells expressing mutant KLF4(NLS)-GFP 6 hr after LIF/2i removal indicates the NLS is required for KLF4/ERK interaction. Scale bar = 10μm.

E) Immunoblot from ES cell lines shown from left to right with stable integration of mutant KLF4(NES4)-GFP, KLF4(NES3)-GFP, KLF4(NES2)- GFP, KLF4(NES1)-GFP, KLF4(NLS)- GFP, KLF4(S132A)-GFP, or WT KLF4-GFP and untransfected ES cells. KLF4 detection identifies the endogenous and the transfected KLF4-GFP proteins. GAPDH levels indicate relative protein amounts in each lane.

2.3.5 Inhibiting KLF4 Nuclear Export Delays Differentiation of ES cells

Nanog downregulation upon ES cell differentiation has been suggested to occur due to a decrease in Klf4 transcription, which was assumed to be followed by a subsequent decrease in KLF4 protein levels (Zhang et al., 2010). By contrast, our data reveal that KLF4 is exported to the cytoplasm at this time, suggesting that this export causes Nanog and Klf4 downregulation at the transcriptional level. To determine whether nuclear export is required for the downregulation of Nanog and Klf4, I monitored transcript abundance by qRT-PCR in undifferentiated and 12 hr differentiated ES cells in the presence or absence of the nuclear export inhibitor LMB. The presence of LMB blocked KLF4 nuclear export and the early decrease in both Klf4 and Nanog transcripts observed after 12 hr of differentiation (Figure 2.11A). In the same samples, no change in the amount of Sox2 or Oct4 transcripts was observed (Figure 2.12A).

To investigate the effect of inhibiting nuclear export of KLF4 specifically, I analyzed relative transcript levels of Nanog and endogenous Klf4 in KLF4-GFP-expressing cells. Blocking KLF4 nuclear export by mutating S132, NES1, or NES2 prevents the decline in transcript levels for Nanog and endogenous Klf4 normally observed at 12 hr but does not affect Oct4 and Sox2 levels (Figures 2.11B and 2.12B). In addition, inhibiting KLF4 nuclear export by mutation of S132 prevents the reduction in KLF4/RNAPII-S5P interaction observed for WT KLF4 at both 6 and 24 hr (Figure 2.12C). By contrast, expression of WT KLF4-GFP or the NLS, NES3, and NES4 mutants, all of which exit the nucleus, or in the case of the NLS mutant are resident in the cytoplasm, did not prevent the decline in transcript levels for Nanog and endogenous Klf4.

Next I investigated the effect of blocking KLF4 nuclear export on ESC differentiation. I observed that alkaline phosphatase (AP) activity was maintained 5 days after withdrawal of LIF/2i in cells expressing KLF4(S132A)-GFP but not WT KLF4-GFP (Figure 2.12D). To evaluate the effect of blocking KLF4 nuclear export on the specification of germ layers, I differentiated ES cells to embryoid bodies (EBs) (Figure 2.11C). qRT-PCR revealed that expression of the pluripotency markers Nanog, Klf4, and Sox2 was maintained in KLF4(S132A)-GFP EBs for 5 days, whereas WT KLF4-GFP EBs displayed a reduction in the expression of these genes similar to the pattern displayed by untransfected ES cells. In addition, the onset of expression for all germ layer markers was delayed by 4–5 days in KLF4(S132A)-GFP EBs compared with WT KLF4-GFP and

67 untransfected embryonic day 14 (E14) ES cells. Together these data indicate that inhibiting KLF4 nuclear export in ES cells maintains naive pluripotency in the absence of external signals (LIF/2i) and delays differentiation to endoderm, mesoderm, and ectoderm by about 5 days.

Figure 2.11

Figure 2.11: KLF4 nuclear export inhibition delays exit from naive pluripotency and differentiation of embryoid bodies

A) Treatment with 5 μg/mL LMB prevents downregulation in Klf4 and Nanog transcripts that normally occurs 12 hr after LIF/2i withdrawal. Average data from three biological replicates are normalized to levels observed in undifferentiated ES cells. Statistical differences are indicated by ∗∗p < 0.01 and ∗∗∗p < 0.001. Error bars represent standard deviation.

B) Expression of endogenous Klf4 and Nanog is maintained after 12 hr of differentiation in KLF4(S132A)-GFP, KLF4(NES1)-GFP, and KLF4(NES2)-GFP mutants but not in wild-type (WT) KLF4-GFP, KLF4(NLS)-GFP, KLF4(NES3)-GFP, and KLF4(NES4)-GFP mutants. Average data from three biological replicates are normalized to the levels observed in undifferentiated ES cells expressing WT KLF4-GFP. Statistical differences from the undifferentiated ESC levels for endogenous Klf4 transcript levels are indicated by ∗∗∗p < 0.001, and Nanog transcript levels are indicated by ΔΔΔp < 0.001. Error bars represent standard deviation.

C) Relative gene expression analysis for pluripotency and germ layer markers in untransfected ES cells (E14), WT KLF4-GFP, and KLF4(S132A)-GFP transfected ES cells differentiated to embryoid bodies for 12 days reveals that expression of KLF4(S132A)-GFP delays ESC differentiation. Data shown are averages of three qRT-PCR replicates for each time point. Error bars represent standard deviation.

Figure 2.12

Figure 2.12: Expression of KLF4(S132A) blocks ES cell differentiation.

A) Treatment with 5µg/ml LMB does not affect Oct4 or Sox2 transcript abundance. Average data form three biological replicates are normalized to the levels observed in undifferentiated ES cells. Error bars represent standard deviation.

B) Relative transcript abundance of Oct4 and Sox2 is not affected by 12 hr of LIF/2i removal in WT KLF4-GFP, KLF4(NLS)-GFP, KLF4(S132A)- GFP, KLF4(NES1)-GFP, KLF4 NES2)-GFP, KLF4(NES3)-GFP, or KLF4(NES4)-GFP. Average data form three biological replicates are normalized to the levels observed in undifferentiated ES cells expressing WT KLF4-GFP. Error bars represent standard deviation.

C) The reduction in KLF4/RNAPII interaction during differentiation depends on KLF4 nuclear export. Proximity ligation amplification (PLA) indicating the interaction between WT KLF4- GFP and RNAPII-S5P or KLF4(S132)-GFP and RNAPII-S5P in ES cells and 6 or 24 hr after LIF/2i withdrawal is shown on the left. Images shown are maximum intensity projections. Scale bar = 10μm. On the right box and whisker plots display the number of PLA foci per nucleus. Boxes indicate interquartile range of intensity values, whiskers indicate the 10th and 90th percentiles, outliers in the 5th and 95th percentiles are shown as black dots. Images were collected from at least three biological replicate samples and ≥100 nuclei were quantified for each. Statistical differences are indicated by *** P < 0.001.

D) Alkaline phosphatase staining of untransfected E14, WT KLF4-GFP, or KLF4(S132A)-GFP colonies 5 days after LIF/2i removal. Scale bar = 50μm. Positive and negative colonies were counted form at least three replicates for each revealing that expression of WT KLF4-GFP does not block differentiation whereas expression of the S132A mutant does. Error bars represent standard deviation.

2.3.6 MEK-ERK signaling during embryogenesis downregulates KLF4 and NANOG

In the embryo, FGF-MEK-ERK signaling occurs between E3.25 and E4.5 and is required for embryo development (Yamanaka et al., 2010). To investigate the effect of MEK-ERK signaling on KLF4 and NANOG expression, I monitored protein and mRNA levels in mouse embryos (Figure 2.13A). In E3.5 blastocysts, immunofluorescence for KLF4 and NANOG revealed more intense signal in the OCT4-positive ICM (arrows). To evaluate changes in protein levels, I quantified images from at least 30 embryos in each group with the assistance of a post doctorate fellow Kamelia Miri (Figure 2.13B). At E4.5, KLF4 and NANOG protein levels were both significantly reduced compared with E3.5 levels, and this reduction was inhibited by treatment with the MEK inhibitor. TPA treatment, by contrast, caused a further reduction in the levels of both KLF4 and NANOG protein to nearly undetectable levels. Similar to the changes in protein levels, mRNA for both Klf4 and Nanog was reduced at E4.5 compared with E3.5, and this reduction was prevented by treatment with the MEK inhibitor (Figure 2.13B). No decrease in OCT4 protein or mRNA levels was detected in these experiments (Figures 2.13 and 2.14A).

As treatment with the MEK inhibitor blocked the downregulation of KLF4, I investigated the interaction of KLF4 with ERK2 or XPO1 in mouse blastocysts using the PLA assay. XPO1 and ERK2 are present throughout the embryo as confirmed by immunofluorescence (Figure 2.14B). The localization of XPO1 was not affected by the MEK inhibitor, whereas ERK2 shifted from nuclear localization under control conditions to a cytoplasmic localization for embryos maintained in the MEK inhibitor, consistent with a shift to inactive ERK2 under these conditions. Similar to our observations in cultured ES cells, PLA for KLF4/ERK2 or KLF4/XPO1 revealed that interaction between these proteins in blastocysts requires MEK-ERK signaling (Figure 2.13C). Interestingly I found that the signal was not restricted to the ICM but occurred throughout the blastocyst, indicating that active ERK2, KLF4, and XPO1 interact in the trophectoderm as well as the ICM. Although MEK inhibition does not cause as dramatic an increase in the levels of KLF4 in the trophectoderm cells as observed in the ICM, a notable increase was observed compared with untreated E4.5 embryos (Figure 2.13A). I did note that in some early blastocysts (E3), KLF4 protein was localized in all nuclei including the NANOG-positive ICM (arrow) and the NANOG- negative trophectoderm (asterisk in Figure 2.14C), suggesting that KLF4 is initially expressed in most cells and later downregulated in the trophectoderm. Considered together, these findings

73 indicate that, as observed in ES cells, ERK activation is required for the reduction in KLF4 nuclear protein levels in the embryo, which involves XPO1-mediated nuclear export. Furthermore, this drop in KLF4 protein is coincident with a reduction in Klf4 and Nanog transcript levels as well as NANOG protein levels.

Figure 2.13

Figure 2.13: KLF4 downregulation in the inner cell mass of mouse blastocysts depends on MEK activation

(A) Immunofluorescence images of mouse blastocysts at E3.5 and E4.5 maintained for 24 hr in control conditions, MEKi (MEK1 inhibitor, PD0325901) treated, or TPA treated. Blastocysts at E3.5 display nuclear KLF4 and NANOG in the OCT4-positive ICM (arrows). Blastocysts at E4.5 display more diffuse and lower KLF4 and NANOG levels in the ICM. Blastocysts at E4.5 maintained for 24 hr in MEKi display higher KLF4 and NANOG expression in the ICM. Blastocysts at E4.5 treated with TPA display low levels of KLF4 and NANOG, whereas OCT4 levels remain high. Images shown are maximum-intensity projections. Merged images display DAPI in blue, KLF4 or NANOG in green, and OCT4 in red. Scale bars, 25 μm.

(B) Quantification of immunofluorescence images from ≥30 embryos in each group reveal significant changes in KLF4 and NANOG protein levels (left). qRT-PCR from three separate pools of embryos at each stage reveals significant differences in Klf4and Nanog mRNA levels (right). Error bars represent standard deviation. Statistical differences (p < 0.05) between different treatments for each gene are indicated by different letters.

(C) Proximity ligation amplification (PLA) for KLF4/ERK2 and KLF4/XPO1 displays the interaction between KLF4, ERK2 and XPO1 in E4.5 blastocysts maintained in control conditions but not in blastocysts pre-treated with MEKi for 24 hr. The interactions were observed in both the ICM (arrows) and trophectoderm. Images shown are maximum-intensity projections. Merged images display DAPI in blue and PLA signal in red. Scale bars, 25 μm.

Figure 2.14

Figure 2.14: Proximity ligation amplification and immunostaining controls in mouse embryos.

A) Quantification of immunofluorescence images from at least 30 embryos in each group reveal no significant change in OCT4 protein levels.

B) XPO1 and ERK2 immunofluorescence at the indicated embryonic day and PLA control experiments. Scale = 25μm. As a positive control proximity ligation amplification (PLA) was conducted with antiRNAPII-S5P and anti-RNAPII-core. As a negative control PLA was conducted with the antiKLF4 antibody alone followed by both the anti-mouse and anti-rabbit oligo linked secondary antibodies. Embryos shown are at embryonic day 4.5.

C) Immunofluorescence image of an e3 mouse blastocyst. KLF4 is detectable in both NANOG positive cells (arrow) and NANOG negative outer cells (*). Images shown are maximum intensity projections. Merged images display DAPI in blue NANOG in red and KLF4 in green. Scale bar = 25μm.

2.4 Discussion

Although differentiation of ES cells occurs over several days, I identified significant changes in nuclear environment occur within 6 hr of differentiation, primarily KLF4 nuclear export, which I show initiates exit from the naive pluripotent state. The pluripotent state is regulated by an interconnected network of transcription factors of which OCT4, SOX2, NANOG, and KLF4 play a central role in pluripotency maintenance through binding to many of the same enhancer regions to regulate gene expression (Chen et al., 2008, Chen et al., 2012, Zhou et al., 2014). My study reveals that KLF4 followed by NANOG are the first proteins of this network to be removed from nuclear transcription complexes and that this is initiated by export of KLF4 from the nucleus, mediated by XPO1 in response to ERK-mediated phosphorylation of KLF4. Interaction between KLF4, ERK2, and XPO1 also occurs in pre-implantation blastocysts leading to decreased KLF4 and NANOG, and blocking this process during EB formation delays exit from naive pluripotency by 5 days. These findings reveal that XPO1-mediated KLF4 nuclear export initiates exit from the naive pluripotent state both in vitro and in the pre-implantation embryo.

KLF4 and NANOG display reduced occupancy in nuclear protein complexes associated with transcription within 24 hr of LIF/2i removal. In undifferentiated ES cells, NANOG functions as part of a complex with OCT4 and SOX2, and together these three proteins bind the same DNA consensus motif (Chen et al., 2008, Gao et al., 2012). KLF4 also binds many of the same enhancer regions and activates similar genes, although it binds a separate DNA consensus motif (Chen et al., 2008, Moorthy et al., 2017). It has been proposed that pluripotency is maintained by the precise balance of the pluripotency transcription factors (Loh and Lim, 2011); my data indicate that this balance is first disrupted by export of KLF4 from the nucleus, followed by reduced levels of nuclear NANOG, causing the removal of these two transcription factors from nuclear complexes.

Downregulation of Klf4 has been shown to precede Nanog downregulation and is the first observed change in expression of pluripotency-associated genes (Zhang et al., 2010); however, when investigating KLF4 protein we observed that KLF4 is exported from the nucleus concurrently with the downregulation in Klf4 and Nanog transcription and that blocking KLF4 nuclear export blocks this downregulation. KLF4 and NANOG bind to enhancer regions upstream of the Nanog promoter and have been shown to be required for Nanog transcription (Sanchez-Castillo et al.,

2014, Zhang et al., 2010). Similarly, there are a number of binding sites for KLF4 and NANOG at enhancers downstream of Klf4, required for Klf4 transcription (Pradeepa et al., 2016, Sanchez- Castillo et al., 2014). After 24 hr of differentiation, I observed accumulation of KLF4 in the nucleus at half of the levels observed in undifferentiated ES cells; however, Klf4 and Nanog transcript levels were not found to increase (Figure S1A). The inability of nuclear KLF4 at 24 hr to induce de novo transcription of Klf4 and Nanog could be due to the reduced interaction of KLF4 with other transcriptional complexes (RNAPII and OCT4/SOX2) and due to reduced NANOG in the nucleus at 24 hr, both of which may prevent cooperative action of the complex and subsequent activation of Klf4 and Nanog transcription.

XPO1 is often involved in constitutive nuclear export to maintain the cytoplasmic accumulation of proteins, which can enter the nucleus constitutively. In an ESC-specific context, ERK2 interacts with XPO1 irrespective of its activation state, consistent with the idea that continual export of the inactive MEK/ERK complex maintains the cytoplasmic levels of these proteins. KLF4, by contrast, only interacts with XPO1 after ERK activation occurs. Furthermore, I showed that highly conserved S132, the ERK phosphorylation site, as well as two predicted NES are required for this interaction and nuclear export, indicating that it is the S132 phosphorylated form of KLF4 that interacts with XPO1. In support of this, disruption of S132, either by direct mutation or mutation of NES2 that prevented phosphorylation of S132, disrupted the interaction of KLF4 with XPO1. NES1 was also required for the interaction of KLF4 and XPO1; however, mutation of this site did not affect KLF4 serine phosphorylation, suggesting that NES1 is the functional NES that supports the direct interaction of KLF4 and XPO1, whereas mutation of NES2 indirectly affects the interaction of KLF4 and XPO1 by preventing KLF4 phosphorylation at S132. These findings indicate that in the absence of S132 phosphorylation, NES1 is masked and not available for interaction with XPO1. As the critical residues involved in nuclear export are not found in the other KLF protein sequences this regulatory mechanism is likely to be unique to KLF4.

Differentiation impairment observed in ES cells expressing S132A mutant KLF4 revealed that nuclear export of KLF4 is critical for exit from the pluripotent state and subsequent differentiation of ES cells to specific germ layers. Our findings are different from those of Zhang et al. (2010) who found that overexpression of WT KLF4 prevents differentiation; this difference may be due to expression levels, as in our cells KLF4-GFP was expressed at levels comparable with

80 endogenous KLF4 and did not prevent differentiation. In mouse pre-implantation blastocysts, MEK-ERK signaling downregulates KLF4 and NANOG, similar to our observations in ES cells; this mechanism involves the interaction between KLF4/ERK2 and KLF4/XPO1. Although Klf4 is not required for early embryogenesis in the mouse, as embryos lacking Klf4 survive to birth due to compensatory mechanisms involving other Klf factors, my in vitro differentiation experiments revealed that expression of constitutively nuclear KLF4 in ES cells prevents pluripotency exit and differentiation. This suggests that a failure in KLF4 downregulation could result in phenotypic consequences during early development. My finding that KLF4 is the first of the pluripotency factors to be removed from transcription-associated nuclear complexes, through active ERK- and XPO1-dependent nuclear export, indicates KLF4 nuclear export as a critical first step in exiting the naive pluripotent state and committing to differentiation (Figure 2.15).

2.5 Materials and Methods

2.5.1 Embryonic stem cell culture

The mouse ES cells (E14TG2a; ATCC [CRL-1821]), were maintained in feeder-free conditions on 0.1% gelatin in DMEM supplemented with 15% (v/v) fetal bovine serum (FBS), 0.1 mM non- essential amino acids, 1 mM sodium pyruvate, 2 mM GlutaMAX, 0.1 mM 2-mercaptoethanol, 1,000 U/mL LIF, 3 μM CHIR99021 (GSK3β inhibitor, Biovision) and 1 μM PD0325901 (MEK inhibitor, Invivogen); referred to as LIF/2i medium. The differentiation medium contained the same components with the exception of LIF/2i. For FGFR inhibition, the MEK inhibitor was replaced with 25 nM PD173074 (Selleckchem). For TPA (NEB) treatment, cells were cultured overnight in low serum (0.2%) ESC medium, followed by treatment with 200 nM TPA in the absence of LIF/2i. For ES cells maintained in LIF alone, cells were cultured in ESC medium containing LIF without 2i for 8–10 passages before removal of LIF to induce differentiation. For evaluation of AP activity, cells were seeded at 250 cells/well in a 12-well plate and allowed to differentiate for 5 days. AP staining (Millipore) was performed following the manufacturer’s instructions. EBs were formed by the hanging-drop method; 1,000 cells were suspended in 20-μL droplets of ESC medium without LIF/2i (Ohnuki and Kurosawa, 2013). EBs were transferred to 0.1% gelatin on day 4, and collected until day 12; RNA was isolated for gene expression analysis by qRT-PCR.

2.5.2 Immunofluorescence and proximity ligation amplification

Cells were fixed for 20 min in neutral buffered 10% formalin, blocked, and permeabilized for 30 min with 10% FBS in 0.1% Triton X-100 in PBS, both at room temperature. Cells were incubated sequentially with primary antibodies in antibody buffer (0.2% FBS, 0.1% Triton X-100 in PBS). After three PBST (0.1% Tween 20 in PBS) washes, cells were incubated in secondary antibodies. Cells were counterstained in DAPI, then washed twice with PBS and once in distilled water. Coverslips were mounted onto glass microscope slides using Vectashield (Vector Labs). Images were collected using a 100× magnification objective lens and analyzed to determine the intensity per nucleus (area defined by the DAPI counterstain) using VOLOCITY 6.0.1. A t test was performed to analyze significant differences in the mean intensity data of differentiating ES cells as compared with undifferentiated ES cells (0 hr). Controls using no primary antibody were conducted for all secondary antibodies, which revealed that there was no non-specific binding of the secondary antibodies. All immunofluorescence experiments were carried out on at least three biological replicate samples. Antibodies used are listed in Table 2.1. PLA was conducted using Duolink (Sigma-Aldrich) following the manufacturer's instructions. Images were collected using a Leica TCS SP8 and a 63× magnification objective lens. The number of PLA foci per nucleus was quantified using Imaris 7.1 by manual 3D masking of nuclei in ESC colonies defined by the DAPI signal. The two primary antibodies against two different epitopes in same protein (RNAPII core (ARNA3) and RNAPII-S5P were used as positive control, whereas two primary antibodies against proteins in two different compartments (nuclear RNAPII and nucleolar UBF1) were used as negative control for PLA. Also, no primary antibody and single primary antibody controls were performed. All PLA experiments were carried out on at least three biological replicate samples. A t test was performed to evaluate significant differences in foci number per nucleus.

Figure2.15

Figure 2.15: Principles of proximity ligation assay

A) Two proteins in the close proximity (close enough to interact) are incubated with primary antibodies generated in two different animal species.

B) The primary antibody incubated proteins are then incubated with species specific PLA probes (plus and minus) linked to oligos.

C) The oligos are connected by hybridizing mediated by connecter oligo and are ligated to form complete circle.

D) The signal of proximity is amplified by rolling circle amplification.

E) The amplified signal is viewed by addition of fluorescently labelled probes.

2.5.3 Immunoblot and Co-Immunoprecipitation

Cells were separated into nuclear and cytoplasmic fractions as previously described (Dhaliwal and Mitchell, 2016, Mitchell et al., 2012). Protein was extracted using RIPA buffer containing protease inhibitor complete EDTA free (Roche) and phosphatase inhibitor cocktail (Millipore) and quantified using bicinchoninic acid (Thermo Fisher Scientific). Protein samples were analyzed by SDS-PAGE (15% Bis-Tris resolved with a 5% stacking gel). Blots were incubated with primary antibodies (Table 2.1) followed by horseradish peroxidase-conjugated secondary antibodies. Blots were quantified by relative intensity using background correction from adjacent regions. At least three biological replicates were analyzed for each experiment, and statistical differences were determined by t test.

For immunoprecipitation of protein, fractions or total lysates in RIPA were incubated overnight with the appropriate antibody and then incubated overnight with a 50:50 mixture of protein A and protein G Dynabeads. Beads were washed three times with non-denaturing lysis buffer (20 mM Tris-HCl, 137 mM NaCl, 10% glycerol, 1% NP-40, 2 mM EDTA, 1 mM PMSF, and proteinase inhibitors), twice with PBS, eluted in loading buffer, and analyzed by SDS-PAGE. To immunoprecipitate KLF4-GFP, I used GFP-Trap (Chromotek) according to the manufacturer's protocol.

2.5.4 Blastocyst collection, Immunostaining and Gene expression

CD-1 females 6–8 weeks old were injected intraperitoneally with 5 IU of pregnant mare serum gonadotropin (Prospec, HOR-272) at 4:30 pm followed by 5 IU of human chorionic gonadotropin (Prospec, HOR-250) 46 hr later and mated with CD-1 males. Females were euthanized by cervical dislocation and day 3–3.5 embryos were flushed out in M2 medium (Millipore, MR-015-D). Embryos were either subsequently fixed or cultured in a humidified incubator at 37°C with 5% CO2 in potassium-supplemented simplex optimized medium (KSOM) (Millipore, MR-106-D), KSOM with 0.5 μM MEK inhibitor (PD0325901, Invivogen) for 24 hr, or KSOM for 24 hr followed by 25 min with 200 nm TPA.

Following fixation in neutral buffered 10% formalin (Sigma-Aldrich) for 15 min, blastocysts were rinsed in PBS and permeabilized in 0.3% Triton X-100 for 15 min. Blocking was done in 10% goat serum and 0.1% Triton X-100 in PBS for 1 hr at room temperature. Antibodies (Table 2.1) were diluted in 5% goat serum and 0.1% Triton X-100 in PBS. Blastocysts were washed in PBS/0.05% Tween 20. Counterstaining was done with 0.1 μg/mL DAPI in PBS and embryos were mounted in 90% glycerol. All PLA experiments were carried out on at least ten embryos. PLA- positive control was detection of the core RNAPII RPB1 together with detection of the S5P CTD of RPB1; the negative control was the KLF4 primary antibody alone (Figure 2.14B). All immunofluorescence experiments were conducted on at least 30 embryos. Images were collected with a Leica TCS SP8 at 40×. The immunofluorescence images were analyzed for average staining intensities of KLF4, OCT4, and NANOG using IMARIS.

For gene expression analysis, ≥15 embryos were pooled for each condition. The cDNA was made using a SuperScript III CellsDirect cDNA Synthesis Kit (Thermo Fisher) and gene expression was

84 monitored by qPCR with genomic DNA used to generate standard curves. Gapdh expression was used to normalize expression values. Three biological replicates were analyzed for each experiment, and significant differences in expression were determined by ANOVA. Primers used are listed in Table 2.2.

All animal experiments were approved by the University Animal Care Committee (UACC) at the University of Toronto and the Bioscience Local Animal Care Committee (LACC).

Table 2.1: Antibody list

Name Company Experiment rabbit anti-KLF4 Abcam IF (1:1000), PLA (1:1000),WB (1:1000), Embryo IF (1:500) and PLA (1:500) mouse anti-KLF4 Santa Cruz IF (1:1000), PLA (1:1000), Embryo IF and PLA (1:500) rabbit anti-NANOG Cosmo Bio IF (1:1000), PLA (1:1000),WB (1:000) rabbit anti-NANOG Abcam Embryo IF (1:500) mouse anti-NANOG BD Biosciences Embryo IF and PLA (1:500) mouse anti-SOX2 R&D Systems IF (1:1000), PLA (1:1000) mouse anti-OCT3/4 Santa Cruz IF (1:1000), PLA (1:1000), Embryo IF (1:500) mouse anti-RNAPII-PS5 Abcam IF (1:1000), PLA (1:2000) rabbit anti-RNAPII-PS5 Abcam IF (1:1000), PLA (1:1000), Embryo PLA (1:1000)

85 mouse anti-RNAPII core Millipore PLA (1:1000), Embryo PLA (1:500) (ARNA3) rabbit anti-ERK1 Santa Cruz WB (1:1000) rabbit anti-ERK2 Santa Cruz IF (1:1000), PLA (1:1000),WB (1:1000), Embryo IF and PLA (1:1000) rabbit anti-ERKpTEpY Promega WB (1:500) mouse anti-XPO1 Santa Cruz IF (1:1000), PLA (1:1000), Embryo IF (CRM1) and PLA (1:1000) mouse anti-GFP Life technologies IF (1:1000), PLA (1:1000) chicken anti-GFP Abcam WB (1:2000) goat anti-rabbit A488 Abcam IF (1:1000) goat anti-mouse A594 Thermo Fisher IF (1:1000) Scientific rabbit anti-CYPA Abcam WB (1:1000) rabbit anti-UBF1 Santa Cruz WB (1:1000)

Rabbit anti- Abcam IP-WB (1:500) phosphoserine

2.5.5 Real time qPCR

Total RNA was purified using TRIzol (Thermo Fisher Scientific). Following a DNaseI digestion to remove DNA, total RNA was reverse transcribed with random primers using the iScript cDNA synthesis kit (Bio-Rad). Gene expression was monitored by qPCR using genomic DNA to generate standard curves. Gapdh expression was used to normalize expression values. Three biological replicates were analyzed for each experiment, and significant differences in expression were determined by t test. Primers used are listed in Table 2.2. All samples were confirmed not to have DNA contamination by generating a reverse transcriptase negative sample and monitoring Gapdh amplification.

Table 2.2: Gene expression primer list

Gene Forward primer Reverse primer Amplicon size

Klf4 GAAGACGAGGATGAAGCTGAC TGGACCTAGACTTTATCCTTTCC 94bp

Nanog TCCCAAACAAAAGCTCTCAAG ATCTGCTGGAGGCTGAGGTA 165bp

Sox2 ACGCCTTCATGGTATGGTC CGGACAAAAGTTTCCACTC 114bp

Oct4 ATGAGGCTACAGGGACACCTT GTGAAGTGGGGGCTTCCATA 100bp

Gapdh GCACCAGCATCCCTAGACC CTTCTTGTGCAGTGCCAGGTG 109bp

2.5.6 Expression of KLF4 mutants

The nuclear localization signal (NLS) was predicted using NLStradamus (Nguyen Ba et al., 2009) and NES were predicted using Wregex (Prieto et al., 2014). A mouse KLF4-GFP vector (RG206691) was obtained from Origene and subjected to site-directed mutagenesis (SDM) (QuikChange Lightning, Agilent Technologies). Primers for SDM are indicated in Table 2.3. Sequence-confirmed plasmids were transfected by electroporation and cells were selected with 400 μg/mL G418. The cells were sorted by fluorescence-activated cell sorting, and individual

87 clones selected and maintained in 50 μg/mL G418 to obtain KLF4-GFP-positive clones. Expression of each KLF4-GFP construct was confirmed by immunoblot (Figure 2.10E).

Table 2.3: Site directed mutagenesis primer list

Primer name Sequence

S132A forward CCACCTCGGCGTCAGCTTCATCCTCGTCTGCCCCAGCGAGCAGCGGCCCTGCC

S132A reverse GGCAGGGCCGCTGCTCGCTGGGGCAGACGAGGATGAAGCTGACGCCGAGGTGG

NLS forward CGGGGCCACGACCCGCTTCCGCTCTTTGGCTTGG

NLS reverse CCAAGCCAAAGAGCGGAAGCGGGTCGTGGCCCCG

NES1 forward AAAGGATAAAGTCTAGGTCCTGTTGGTCGTTGAACTCCTCGGTC

NES1 reverse GACCGAGGAGTTCAACGACCAACAGGACCTAGACTTTATCCTTT

NES2 forward GTGGTCACGGTGCCGCCCACCGATTCCT

NES2 reverse AGGAATCGGTGGGCGGCACCGTGACCAC

NES3 forward GCCAGGGGTGGTCTGAGACGCCTTCAG

NES3 reverse CTGAAGGCGTCTCAGACCACCCCTGGC

NES4 forward CAAATGGGCCTCTTGGGACCGGCTGAC

NES4 reverse GTCAGCCGGTCCCAAGAGGCCCATTTG

S132D forward TGCTCGCTGGGTCAGACGAGGATGAAGCTGACGC

S132D reverse GCGTCAGCTTCATCCTCGTCTGACCCAGCGAGCA

Figure 2.16

Figure 2.16: The model of KLF4 nuclear export and naïve pluripotency exit

Chapter 3 KLF4 protein is stabilized by interaction with pluripotency transcription factors and mutants with increased stability block embryonic stem cell differentiation

3.1 Abstract

Embryonic stem (ES) cells are regulated by an interconnected network of transcription factors which together maintain the pluripotent state. During differentiation KLF4 nuclear export is the first step in disrupting the function of this regulatory network. While investigating the regulation of Klf4 transcription I observed that homozygous deletion of a distal regulatory region caused a 17-fold decrease in Klf4 transcript levels but decreased protein levels by less than 2-fold suggesting high KLF4 protein stability. Indeed, the half-life of KLF4 protein was >24 hr in ES cells maintained in LIF/2i. This stability is context dependent as it was disrupted during differentiation, evidenced by a shift to a half-life of <2hr. KLF4 protein stability is maintained through interaction with other pluripotency transcription factors (NANOG, SOX2 and STAT3) that together facilitate KLF4 interaction with RNA polymerase II. In addition, the KLF4 DNA binding and transactivation domains are required for both KLF4 function and protein stability. Post- translational modification such as phosphorylation at S132 and ubiquitination at Lysine 249 of KLF4 destabilizes the protein as cells exit the pluripotent state. Mutations preventing these post translational modifications prevented both protein destabilization and differentiation. These data highlight a new way in which the core pluripotency transcription factors are integrated to function as transcription factor in order to maintain the pluripotent state.

3.2 Introduction

Kruppel-like factor 4 (KLF4), a member of the Kruppel-like factor family of conserved zinc finger transcription factors, is known to interact with the core network of pluripotency transcription factors namely OCT4, SOX2 and NANOG in order to regulate genes required for maintenance of pluripotency and reprogramming (Dhaliwal et al., 2018; Wei et al., 2013; Wei et al., 2009; Xie et al., 2017; Zhang et al., 2010). Differentiation of ES cells to specific cell types occurs over several days, during which Sox2 and Oct4 expression are maintained; however, disruption of Klf4 and Nanog transcription due to KLF4 nuclear export occurs within 24 hr and is critical for pluripotency exit (Dhaliwal et al., 2018; Zhang et al., 2010). Together these data indicate loss of KLF4 protein function is a critical early step in pluripotency exit and differentiation induction.

Genome wide binding of transcription factors and coactivators can identify enhancers required for gene transcription in a particular cellular context (Chen et al., 2012; Chen et al., 2008; Moorthy et al., 2017; Visel et al., 2009). These enhancers are often located at multi kb distances from the genes they regulate and form physical loops with their target gene promoters (Carter et al., 2002; Schoenfelder et al., 2015; Tolhuis et al., 2002). In pluripotent ES cells, Klf4 transcription is regulated by three enhancer elements (E1, E2 and E3) located 54-68kb downstream of the Klf4 transcription start site (TSS) (Xie et al., 2017). Transcription of Sox2 is regulated by the Sox2 control region (SCR), a distal enhancer region located >100kb downstream of the Sox2 gene (Zhou et al., 2014). In addition to these transcriptional mechanisms that regulate the expression of pluripotency transcription factors, the balance between maintaining the pluripotent state and inducing differentiation is also modulated by post-transcriptional regulatory mechanisms. LIF maintains the pluripotent state by activating JAK-STAT signaling causing phosphorylation and activation of STAT3 (Davis et al., 1993; Matsuda et al., 1999; Narazaki et al., 1994; Niwa et al., 1998). Activated STAT3 induces transcription of Klf4 through binding to the enhancers downstream of KLF4 (Hall et al., 2009; Xie et al., 2017; Zhang et al., 2010). In addition, dual inhibition (2i, GSK3 and MEK inhibition) maintains ES cells in a state closest to that of the precursor cells from the pluripotent epiblast of pre-implantation embryos (Nichols and Smith, 2009; Tosolini and Jouneau, 2016; Wray et al., 2010). MEK-ERK signaling post-translationally regulates KLF4 by phosphorylation which causes KLF4 nuclear export and degradation (Dhaliwal

92 et al., 2018; Kim et al., 2012). KLF4 is also regulated by ubiquitination leading to its proteosomal degradation in response to ERK phosphorylation (Kim et al., 2012). As MEK-ERK activation disrupts KLF4 function, MEK inhibition maintains pluripotency by preventing KLF4 inactivation and degradation (Dhaliwal et al., 2018; Kim et al., 2012). GSK3 inhibition prevents ES cell differentiation by facilitating nuclear accumulation of β-catenins that maintain the expression of pluripotency-associated genes (Cole et al., 2008; Okita and Yamanaka, 2006; Sato et al., 2004).

At a genome scale, evaluation of the correlation between mRNA abundance and protein abundance estimates that for cells in a steady state, for 50-80% of the genes being expressed, protein levels correlated with the levels of mRNA present (reviewed in (Liu et al., 2016). For cells undergoing dynamic transitions, for example during monocyte to macrophage differentiation, protein and mRNA levels become decoupled during early differentiation phase, mainly due to a delay in translation compared to transcription (Kristensen et al., 2013). In both cases, exceptions exist where mRNA and protein levels do not correlate even when delays in translation are taken into account. Transcription factors generally display low protein stability which allows rapid cell state transitions (Hochstrasser and Varshavsky, 1990; Jovanovic et al., 2015; Zhou et al., 2004); however, in this study, we show that KLF4 protein levels are highly decoupled from the mRNA levels due to the exceptional stability of the KLF4 protein in ES cells maintained in LIF/2i. Homozygous deletion of downstream Klf4 enhancer regions caused a 17 fold reduction in Klf4 transcript levels whereas KLF4 protein levels were reduced by <2 fold. Surprisingly, we observed a greater reduction of KLF4 protein levels (>3 fold) in ES cells with compromised SOX2 expression, despite the observation that Klf4 transcript levels are unchanged in these cells. These discrepancies in KLF4 protein and transcript levels are due to the modulation of KLF4 protein stability by interaction with SOX2, NANOG and activated STAT3 as well as domains within the KLF4 protein that retain KLF4 in the nucleus. During pluripotency exit, KLF4 protein becomes destabilized and preventing this destabilization through mutation of KLF4 stability motifs blocks this exit. The core pluripotency maintenance transcription factors are known to function in a highly integrated way to maintain transcriptional control of the pluripotent state. Here we show a new way in which these factors are also integrated in the post-translational control of KLF4 function.

3.3 Results 3.3.1 Klf4 transcript and protein levels are uncoupled in embryonic stem cells maintained in LIF/2i

During the first 24 hr of differentiation induction, by removal of LIF/2i from ES cells maintained in culture, Klf4 transcript levels decrease to less than 10% of the undifferentiated levels. At the same time the levels of KLF4 protein are only reduced to 50% indicating that Klf4 transcript and protein levels are uncoupled in LIF/2i conditions (Dhaliwal et al., 2018). In ES cells maintained in LIF/serum Klf4 has been shown to be regulated by three enhancers 54-68kb downstream of the gene; deletion of this region was found to reduce Klf4 transcription by 90%, greatly affecting KLF4 protein levels (Xie et al., 2017). As Klf4 transcript and protein levels are uncoupled in LIF/2i conditions, we evaluated the effect of Klf4 enhancer deletion in LIF/2i maintained ES cells. I used F1 (Mus musculus129 x Mus castaneus) ES cells, allowing allele-specific deletion screening and gene expression analysis (Moorthy and Mitchell, 2016; Zhou et al., 2014). As expected I observed similar results upon deletion of two (Δ1, 8 fold reduction), or all three enhancers (Δ2, 17 fold reduction, Figure 3.1A, B, C). Consistent with the idea that Klf4 transcript and protein levels are uncoupled, the drastically reduced transcript levels after each deletion caused only modest changes in KLF4 protein levels (Figure 3.1D, E). KLF4 protein levels are significantly reduced only in cells with the Δ2 homozygous deletion (Δ2129/Cast) and in these cells, which displayed a 17 fold reduction in total mRNA, protein was reduced by <2 fold (Figure 3.1C, E). To confirm this was not an effect of recent enhancer deletion I investigated Klf4 transcript and protein levels in cells maintained to later passages (P9); however, no significant differences were observed between early and late passages (Figure 3.1C, E). Transcript and protein levels of other pluripotency transcription factors, Oct4, Sox2 and Nanog, remained unchanged in Klf4 enhancer deleted clones (Figure 3.1D, 3.2A, B). To investigate the KLF4 protein that is actively involved in regulating transcription, we used proximity ligation amplification (PLA) to investigate the extent to which KLF4 interacts with the serine 5 phosphorylated elongating form of RNAPII (RNAPII-S5P). This approach detects the number of foci per nucleus where KLF4 and RNAPII-S5P are adjacent to each other and could reveal sub-populations of cells with differing KLF4 function. Similar to the results for total KLF4 protein levels, I found that only the larger enhancer deletion (Δ2129/Cast) had a significant effect on

94 interaction between KLF4 and RNAPII-S5P (Figure 3.3). ΔSCR129/Cast cells, with a deletion of the enhancer that regulates Sox2 in naïve ES cells, were included as a control line to illustrate the degree to which enhancer deletion can affect transcript and protein levels. Surprisingly KLF4 protein but not transcript levels were affected by homozygous SCR deletion. This finding will be explored further in section 3.3.8

Figure 3.1

Figure 3.1: CRISPR deletions of two Klf4 enhancer regions showed a 17 fold decrease in Klf4 transcript but a <2 fold decrease in KLF4 protein levels.

A) The genome browser view of enhancer regions for Klf4 54-68kb downstream of the Klf4 gene indicating the 1 and 2 enhancer deletions. The region shows the binding of transcription factors (black bars) involved in pluripotency maintenance obtained from Chen et al., 2012. All data are displayed on the mm9 assembly of the University of California at Santa Cruz (UCSC) Genome Browser.

B) Allele-specific primers detect Klf4 129 or Cast RNA in RT-qPCR from the indicated clones. Transcript levels are relative to Gapdh. Deletion of the region on the 129 allele is indicated by 129, Deletion of the region on both alleles is indicated by 129/Cast. Error bars represent standard deviation in at least three biological replicates. Statistical differences, calculated by one way ANOVA, from the F1Cast allele are indicated by *** p < 0.001, and from the F1129 allele by ΔΔΔ p < 0.001.

C) Total Klf4 transcript levels quantified relative to Gapdh from three biological replicates of both early and late passages of Klf4 enhancer deleted clones and the ΔSCR129/Cast clone. Error bars represent standard deviation. Statistical differences in two way ANOVA (p < 0.05) of indicated clones in both early and later passages are displayed by different letters.

D) Immunoblots showing levels of pluripotency transcription factors in both early and late passages of Klf4 enhancer deleted clones and ΔSCR129/Cast. GAPDH levels indicate equal sample loading.

E) The intensity of KLF4 protein immunoblot was quantified relative to GAPDH intensity from three biological replicates of both early and late passages of Klf4 enhancer deleted clones and ΔSCR129/Cast. Error bars represent standard deviation. Statistical differences determined by two way ANOVA (p < 0.05) are indicated by different letters.

Figure 3.2

Figure 3.2: Klf4 enhancer deletion does not affect transcript or protein levels for other pluripotency transcription factors.

A) Total transcript levels of Oct4, Sox2 and Nanog were quantified relative to Gapdh from three biological replicates of both early and late passages of Klf4 enhancer deleted clones and the ΔSCR clone. Error bars represent standard deviation. Statistical differences determined with two way ANOVA (p < 0.05) are displayed by different letters.

B) The intensities of immunoblots for OCT4, SOX2 and NANOG were quantified relative to the intensity of GAPDH from three biological replicates for both early and late passages of Klf4 enhancer deleted clones and the ΔSCR clone. Error bars represent standard deviation. Statistical differences determined by two way ANOVA (p < 0.05) are indicated by different letters.

Figure 3.3

Figure 3.3: Interaction between KLF4/RNAPII is disrupted by reduced KLF4 protein levels.

A) Proximity ligation amplification (PLA) displays the interaction between KLF4/RNAPII in Klf4 enhancer deleted and ΔSCR clones. Images shown are maximum-intensity projections. Merged images display DAPI in blue and PLA in red. Scale bar = 10 μm.

B) Box-and-whisker plots indicate the number of PLA foci per nucleus. Boxes indicate interquartile range of intensity values and whiskers indicate the 10th and 90th percentiles; outliers are shown as black dots. Images were collected from at least three biological replicates and ≥100 nuclei were quantified for each sample. Statistical differences determined by one-way ANOVA (p < 0.05) are indicated by different letters.

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3.3.2 KLF4 protein is stable in LIF/2i cultured ES cells

The finding that a dramatic reduction in Klf4 transcript levels does not greatly affect KLF4 protein levels suggested KLF4 protein may be highly stable in ES cells maintained in LIF/2i. Investigation of KLF4 protein stability in undifferentiated ES cells and cells differentiated for 24 hr revealed that KLF4 protein is more stable in undifferentiated cells with a t½ >24 hr; after removal of LIF/2i for 24 hr KLF4 becomes unstable with a t½ <2 hours (Figure 3.4). By contrast, the other pluripotency transcription factors, OCT4, SOX2 and NANOG are more unstable (t½ 2-4hr) in undifferentiated cells and their stability was not affected by differentiation (Figure 3.4). KLF4 protein stability was not affected by deletion of the downstream enhancers explaining the modest reduction in KLF4 protein in these clones (Figure 3.4C, D).

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Figure 3.4

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Figure 3.4: KLF4 protein stability is reduced after 24 hr differentiation.

A) Immunoblots for OCT4, SOX2, NANOG, KLF4 and GAPDH in ES cells cultured with LIF/2i and 24 hr after removal of LIF/2i, sampled at 0, 2, 4, 6, 8 and 12 hr after CHX treatment. GAPDH levels were used as the CHX chase assay control and displayed the expected protein half-life (t½ >30 hr).

B) The percent remaining (OCT4, SOX2, NANOG, KLF4 and GAPDH) protein at specific time point (0, 2, 4, 6, 8, and 12 hr) was calculated from the intensity of CHX treated immunoblots, measured in three biological replicates. Half-life was calculated for each time series replicate by best fit to exponential decay. Error bars represent standard deviation. Statistical differences determined by t test (p <0.001) are indicated as ***.

C) Immunoblots for KLF4 and GAPDH in Klf4 enhancer deleted clones and the ΔSCR129/cast clone, cultured with LIF/2i and 24 hr after removal of LIF/2i, sampled at 0, 2, 4, 6, 8 and 12 hr after CHX treatment. GAPDH levels were used as the CHX chase assay control and displayed the expected protein half-life (t½ >30 hr).

D) KLF4 protein half-life in the indicated clones maintained in the presence or absence of LIF/2i was calculated for each time series replicate by best fit to exponential decay. Error bars represent standard deviation of three biological replicates. Statistical differences in two way ANOVA (p < 0.05) are indicated by different letters.

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3.3.3 KLF4 protein stability is regulated by LIF and MAPK signaling pathways

Reduced KLF4 protein stability upon differentiation induction by removal of LIF/2i suggested extrinsic factors that activate signaling cascades could be involved in regulating KLF4 stability. Upon investigating the effect of individual signaling pathways on KLF4 stability, we observed that activation of the MAPK pathway (MEKi removal) for 24 hr has the most significant effect on KLF4 stability followed by inhibition of the JAK-STAT pathway by removing LIF (Figure 3.5A, B). Removing GSK3i, allowing activation of Wnt signaling for 24 hr, did not show any significant effect on KLF4 stability (Figure 3.5A, B). Previous studies have demonstrated that these external signaling pathways regulate gene transcription (Dhaliwal et al., 2018; Nichols and Smith, 2009; Theunissen et al., 2011; Zhang et al., 2010); therefore, I investigated the effect of individual signaling pathways on Klf4, Nanog, Oct4 and Sox2 transcription and observed that the removal of MEKi alone or in combination with LIF/GSK3i for 12 hrs significantly reduces the Klf4 and Nanog transcription but Oct4 and Sox2 transcription remains unaffected (Figure 3.5 C, 3.6 C). As changes to Klf4 transcription could confound the analysis of KLF4 stability during differentiation I investigated the stability of KLF4-GFP controlled by a CMV promoter upon removal of MEKi, LIF or GSK3i (Figure 3.6 A, B). The stability of KLF4-GFP in these conditions was similar to the stability of the endogenous protein with removal of MEKi and LIF reducing KLF4 stability more dramatically than removal of GSK3i (Figure 3.5 and 3.6).

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Figure 3.5

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Figure 3.5: Removal of LIF or the MEK inhibitor reduces KLF4 half-life.

A) Immunoblots for KLF4 and GAPDH in ES cells cultured with LIF/2i and 24 hr after removal of individual media components (LIF, GSK3i, or MEKi), sampled at 0, 2, 4, 6, 8 and 12 hr after CHX treatment. GAPDH levels were used as the CHX chase assay control and displayed the expected protein half-life (t½ >30 hr).

B) Percent remaining KLF4 and GAPDH protein at specific time points (0, 2, 4, 6, 8, and 12 hr) was calculated from the intensity of CHX treated immunoblots, measured in three biological replicates. Half-life was calculated for each time series replicate by best fit to exponential decay. The error bars represent standard deviation. Statistical differences between protein half-life in different culture conditions compared to ES cells maintained in LIF/2i were determined by t-test (p < 0.001) and are indicated by ***.

C) Transcript levels of Klf4 and Nanog were quantified relative to Gapdh levels, in three biological replicates of ES cells cultured with LIF/2i and 12 hr after removal of LIF/2i components. Error bars represent standard deviation. Statistical differences for each transcript were determined by one way ANOVA (p < 0.05) and displayed as lower case letters for Klf4 and upper case letters for Nanog.

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Figure 3.6

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Figure 3.6: An integrated KLF4-GFP expression construct showed a similar reduction in protein stability after removal of LIF or the MEK inhibitor.

A) Immunoblots for WT KLF4-GFP and GAPDH in ES cells cultured with LIF/2i and 24 hr after removal of individual media components (LIF, GSK3i, and MEKi), sampled at 0, 2, 4, 6, 8 and 12 hr after CHX treatment. GAPDH levels were used as the CHX chase assay control and displayed the expected protein half-life (t½ >30 hr).

B) Percent remaining WT KLF4-GFP and GAPDH protein at specific time points (0, 2, 4, 6, 8, and 12 hr) was calculated from the intensity of CHX treated immunoblots, measured in three biological replicates. Half-life was calculated for each time series replicate by best fit to exponential decay. The error bars represent standard deviation. Statistical differences between protein half-life in different culture conditions compared to ES cells maintained in LIF/2i were determined by t-test (p < 0.001) and are indicated by ***.

C) Total transcript levels of Sox2 and Oct4 were quantified relative to Gapdh levels, in three biological replicates of ES cells cultured with LIF/2i and 12 hr after removal of LIF/2i components. Error bars represent standard deviation. No significant differences were observed.

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3.3.4 Nuclear localization maintains KLF4 protein stability in LIF/2i maintained ES cells

The MAPK pathway has been shown to have a role in KLF4 nuclear localization, ubiquitination and degradation (Dhaliwal et al., 2018; Kim et al., 2012; Kim et al., 2014). As early as 6 hr after MEKi removal, KLF4 was observed to exit the nucleus, and the cytoplasmic KLF4 fraction undergoes proteosomal degradation (Dhaliwal et al., 2018). In addition, nuclear export was found to depend on the presence of both a KLF4 nuclear export signal (NES1 at 97-107) and the ERK phosphorylation site in KLF4 at S132 (Dhaliwal et al., 2018). KLF4 nuclear export after MEKi removal, which allows for ERK activation, could explain the observed reduction in KLF4 protein stability; to investigate this further I used mouse ES cells with stable integration of wild-type (WT) KLF4-GFP, the NES1 mutant (KLF4(NES1)-GFP), KLF4(S132A)-GFP and a nuclear localization sequence (NLS) mutant KLF4(NLS)-GFP (Figure 3.7A). WT KLF4-GFP displayed stability similar to endogenous KLF4 with a t½ >24 hr in undifferentiated cells which was reduced to <2hr after 24 hr differentiation (Figure 3.7B, C). Interestingly, both of the constitutively nuclear mutants, KLF4(NES1)-GFP and KLF4(S132A)-GFP, were highly stable proteins (t½ >51hr) and this stability was not affected by differentiation (Figure 3.7B, C). By contrast, the constitutively cytoplasmic KLF4(NLS)-GFP was unstable, with a t½ <2hr in both undifferentiated and differentiated cells (Figure 3.7B, C). Together these data indicate nuclear localization is critical for KLF4 protein stability.

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Figure 3.7

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Figure 3.7: Nuclear localization and anchoring maintain KLF4 stability.

A) Schematic of mouse KLF4 depicting nuclear export signals (NES), ERK phosphorylation site S132, ubiquitination site K249, sumoylation site K275, nuclear localization signal (NLS) and zinc fingers (ZNFs).

B) Immunoblots for WT KLF4-GFP, the indicated mutants, and GAPDH from ES cells cultured with LIF/2i and 24 hr after LIF/2i removal, sampled at 0, 2, 4, 6, 8 and 12 hr after CHX treatment. GAPDH levels were used as the CHX chase assay control and displayed the expected protein half- life (t½ >30 hr).

C) KLF4 protein half-life in the indicated mutants cultured with LIF/2i and 24 hr after LIF/2i removal. Half-life was calculated for each time series replicate by best fit to exponential decay. Error bars represent standard deviation of three biological replicates. Statistical differences determined by two-way ANOVA (p < 0.05) are indicated by different letters.

D) Nuclear and cytoplasmic fractions prepared from WT KLF4-GFP and the indicated mutants, immunoblots probed with anti-GFP and anti-KLF4, indicated the expression and localization of endogenous KLF4 and KLF4-GFP. UBF1 and CYPA were used to analyze the purity of nuclear and cytoplasmic fractions respectively.

E) Proximity ligation amplification (PLA) displays the interaction between KLF4-GFP/RNAPII in WT and the indicated mutants cultured with or without LIF/2i. Images shown are maximum- intensity projections. Merged images display DAPI in blue and PLA in red. Scale bar = 10 μm.

F) Box-and-whisker plots display the number of KLF4-GFP/RNAPII PLA foci per nucleus for WT and the indicated mutants. Boxes indicate interquartile range of intensity values and whiskers indicate the 10th and 90th percentiles; outliers are shown as black dots. Images were collected from at least three biological replicates and ≥100 nuclei were quantified for each sample. Statistical differences determined by two way ANOVA (p < 0.05) are indicated by different letters.

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3.3.5 KLF4 association with DNA and RNAPII is required to maintain protein stability through nuclear anchoring

As localization to both the nucleus and association with other pluripotency transcription factors in RNAPII rich transcription compartments increased the stability of KLF4 I next investigated protein stability of mutants with disrupted KLF4 transcription factor function. KLF4 contains two c-terminal zinc finger (ZNF) domains required for DNA binding (Schuetz et al., 2011; Wei et al., 2009) and a sumoylation site at K275 shown to be important for transactivation of target promoters in reporter assays (Du et al., 2010). The sumoylation consensus sequence is highly conserved among primate and non-primate species highlighting conservation of the K275 sumoylation site (Appendix 1, Figure A1.5 and A1.6). Stable expression of KLF4 loss of function mutants, with a mutated sumoylation site (KLF4(K275R)-GFP) or deleted zinc fingers (KLF4ΔZNF-GFP) (Figure 3.7A), were disrupted in their nuclear localization (Figure 3.7D) and were more unstable compared to WT KLF4-GFP protein with a t½ of ~3hr (Figure 3.7B, C). Mutation of S132 together with K275 or zinc finger deletion restored nuclear anchoring and increased the stability of KLF4 (Figure 3.7B, C). Interestingly, disruption of either KLF4 sumoylation (K275R) or DNA binding (ΔZNF) interfered with recruitment to RNAPII-S5P nuclear compartments as shown by the decrease in the number of PLA foci per nucleus compared to WT KLF4-GFP (Figure 3.7E, F). Introducing the S132 mutation into the KLF4 loss of function mutants did not restore RNAPII-S5P association indicating that sumoylation and DNA binding are required for KLF4 association with RNAPII- S5P.

3.3.6 Ubiquitination of KLF4 is required for nuclear export and degradation during differentiation

Upon phosphorylation by ERK and subsequent nuclear export, KLF4 has been shown to be ubiquitinated and degraded causing ES cell differentiation (Dhaliwal et al., 2018; Kim et al., 2012). As expected, treatment with the proteasome inhibitor (MG132) increased levels of KLF4 protein in cells differentiated for 24 hr (Figure 3.8 A, B). KLF4 has a predicted molecular weight of 54 kDa; in ES cells immunoblot for KLF4 protein identifies a prominent band at 56 kDa (Figure 3.8 C). Immunoprecipitation of KLF4 with and anti-KLF4 antibody followed by immunoblot with

112 anti-KLF4 identified a 56 kDa band from ES cell lysate (Figure 3.8C). This band was absent from the no antibody and IgG controls indicating that the precipitation of KLF4 was specific (Figure 3.8C). KLF4 immunoprecipitation with anti-KLF4 followed by immunoblot for ubiquitin identified prominent bands at 65 kDa, 75 kDa, 89 kDa, 100 kDa and 165 kDa. As ubiquitin is 8.5 kDa in size these could correspond to the addition of 1, 2, 4, 5 and 13 ubiquitin units to KLF4 (Figure 3.8 D). Alternatively some of these bands could represent proteins associated with KLF4 that are themselves ubiquitinated. A previous study showed K232 was the most critical residue in human KLF4 for ubiquitination and degradation (Lim et al., 2014). KLF4 is highly conserved among primate and non-primate species in region with K249 a predicted ubiquitination site by UbPred and NetChop (Kesmir et al., 2002; Radivojac et al., 2010) (Appendix 1, Figure A1.5 and A1.6). In order to further investigate the role of K249 in KLF4 function I generated stable ES lines expressing a KLF4 ubiquitination site mutant, KLF4(K249R)-GFP. KLF4(K249R)-GFP is nuclear in undifferentiated ES cells, similar to the WT protein (Figure 3.5D) but displayed an increased t½ of 53hr independent of culture conditions similar to the S132 and NES1 mutants (Figure 3.7B, C) indicating that blocking KLF4 ubiquitination prevents the loss of KLF4 stability upon differentiation. Blocking KLF4 ubiquitination by mutation of K249 does not disrupt the interaction with RNAPII-S5P in undifferentiated cells and prevents the loss of KLF4/RNAPII-S5P interaction associated with differentiation (Figure 3.7E, F). As this was similar to what occurs in the KLF4 S132 and NES1 mutants which showed disrupted nuclear export and interaction with Xportin 1 (XPO1)(Dhaliwal et al., 2018), I investigated the interaction between KLF4(K249R)-GFP and XPO1 in differentiating ES cells by PLA. Indeed, mutation of K249 did disrupt the interaction between KLF4 and XPO1 normally observed in differentiating cells (Figure 3.9A).

I next investigated the ubiquitination status of KLF4-GFP in nuclear and cytoplasmic fractions and the effect of K249 mutation on KLF4-GFP ubiquitination. KLF4-GFP protein has a predicted molecular weight of 81 kDa; in ES cells immunoblot for KLF4-GFP protein identifies a prominent band at 84 kDa (Figure 3.9B). Immunoprecipitation of KLF4-GFP with and anti-GFP antibody followed by immunoblot with anti-GFP identified an 84 kDa band from ES cell lysate (Figure 3.9B). This band was absent from the no antibody and IgG controls indicating that the precipitation of KLF4-GFP was specific (Figure 3.9B). WT KLF4-GFP immunoprecipitated from the nuclear fraction using anti-GFP antibody displayed a prominent ubiquitin band at 95 kDa (Figure 3.9C), which could correspond to monoubiquitation of KLF4-GFP. This band was only apparent after 113 removal of LIF/2i, and was absent from the ubiquitination mutant KLF4(K249R)-GFP revealing that ubiquitination depends on the presence of K249. WT KLF4-GFP immunoprecipitated from cytoplasmic fractions using anti-GFP antibody revealed more of a high molecular weight laddering pattern above 105 kDa which could correspond to polyubiquitination of cytoplasmic KLF4-GFP in MG132 treated, differentiated (-LIF/2i) cells. The presence of an 84 kDa band and the lower band at 72 kDa could represent partial KLF4-GFP degradation (Figure 3.9C). These ubiquitin immunoreactive bands were not detected in the absence of MG132 suggesting cytoplasmic polyubiquitinated KLF4 is rapidly degraded. In addition, ubiquitin immunoreactive bands were absent from the cytoplasmic fraction for the K249R mutant protein and furthermore KLF4-GFP was not observed in the input cytoplasmic fraction indicating that mutation of the ubiquitination site blocked nuclear export of KLF4-GFP.

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Figure 3.8

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Figure 3.8: KLF4 protein undergoes degradation by ubiquitination.

A) Immunoblots for KLF4 in whole cell lysate prepared from ES cells cultured with LIF/2i, and 24 hr after removal of LIF/2i, both treated and untreated with proteasome inhibitor MG132. GAPDH levels indicate equal loading.

B) Quantification of KLF4 protein intensity level relative to GAPDH and ES cell levels (+LIF/2i, - MG132), in three biological replicates of ES cells cultured with LIF/2i, and 24 hr after removal of LIF/2i, both treated and untreated with proteasome inhibitor MG132 demonstrated the role of proteosomal pathway in KLF4 regulation. Error bars represent standard deviation. Statistical differences determined by one way ANOVA (p < 0.05) are displayed by different letters.

C) The immunoblots for KLF4 antibody specific pull down. Individual lanes include protein G+A beads mixture incubated with lysate (no antibody control), pull down with same IgG as KLF4 antibody (IgG pull down control), pull down with respective antibody (positive control showing 55.84kDa band, shown in red, with KLF4), protein molecular weight marker, cell lysate prepared from LIF/2i cultured E14 immunoblotted with anti-KLF4.

D) KLF4 immunoprecipitation (IP) in ES cells cultured with LIF/2i, 24 hr after LIF/2i removal and both treated and untreated with proteasome inhibitor MG132, probed with anti-Ubiquitin (PD, pull down). The calculated molecular weight of the bands is marked in red. Molecular weight markers designate the corresponding band sizes. GAPDH reveals equal loading of the input.

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Figure 3.9

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Figure 3.9: Monoubiquitination at K249 of nuclear KLF4 protein causes nuclear export and degradation.

A) Proximity ligation amplification (PLA) displays the interaction between KLF4-GFP/XPO1 in WT and the K249R mutant cultured with LIF/2i and 24 hr after removal of LIF/2i. Images shown are maximum-intensity projections. Merged images display DAPI in blue and PLA in red. Scale bar = 10 μm.

B) The immunoblots for GFP antibody specific pull down. Individual lanes include protein G+A beads mixture incubated with lysate (no antibody control), pull down with same IgG as GFP antibody (IgG pull down control), pull down with respective antibody (positive control showing 83.98 kDa band, shown in red, with anti GFP), protein molecular weight marker, cell lysate prepared from LIF/2i cultured E14 immunoblotted with anti-GFP.

C) KLF4-GFP immunoprecipitation (IP) using anti-GFP from nuclear and cytoplasmic fractions of WT and the K249R mutant cultured with LIF/2i and 24 hr after removal of LIF/2i, treated and untreated with proteosomal inhibitor MG132. Input and pull-down (PD) samples were probed with anti-Ubiquitin. Molecular weight markers designate the corresponding band sizes. The calculated molecular weight of the bands is marked in red. Cyclophilin A (CYPA) and the nucleolar protein upstream binding factor (UBF1) were detected simultaneously for all samples and reveal purity of the cytoplasmic and nuclear fractions, respectively.

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3.3.7 The loss of KLF4 protein stability is required for ES cell differentiation.

To evaluate the role of KLF4 stability in pluripotency maintenance and exit from the pluripotent state we differentiated cells expressing KLF4(WT)-GFP or mutant proteins for 5 days in the absence of LIF/2i. Cells in the pluripotent state exhibit high alkaline phosphatase activity which is lost upon differentiation (Štefková et al., 2015). Investigation of alkaline phosphatase activity revealed that expression of KLF4 mutants with increased protein stability, KLF4(K249R)-GFP and KLF4(S132A)-GFP, maintains higher alkaline phosphatase activity after removal of LIF/2i suggesting expression of these mutants blocks differentiation (Figure 3.10A, B). In addition, expression of either KLF4(K249R)-GFP or KLF4(S132A)-GFP, maintained the expression of endogenous pluripotency transcription factors normally downregulated after removal of LIF/2i (Figure 3.10C).

To further investigate the importance of the role of KLF4 as a transcription factor in this context I evaluated the role of the sumoylation site we found was important for recruitment to the RNAPII- S5P compartment and which has been shown to be involved in transactivation (Du et al., 2010). Although expression of the K275 sumoylation site mutant does not affect differentiation, mutation of this site in an S132A mutant background abolished the ability of the compound mutant to maintain naïve pluripotency in the absence of LIF/2i (Figure 3.10). Similarly, I found that KLF4 requires the ability to bind DNA through its zinc finger domains as the compound mutant, KLF4(S132AΔZNF)-GFP was unable to maintain pluripotency in the absence of LIF/2i.

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Figure 3.10

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Figure 3.10: The loss of KLF4 protein stability is required for ES cell differentiation.

A) Alkaline phosphatase staining of untransfected E14 ES cells, WT and the indicated KLF4-GFP mutant colonies 5 days after LIF/2i removal. Scale bar = 50μm.

B) Positive and negative colonies were counted form at least three replicates for each indicated mutant, revealing that expression of WT KLF4-GFP does not block differentiation whereas expression of the S132A or K249R mutant does. Error bars represent standard deviation. Statistical differences of each type of colonies as compare to WT KLF4-GFP determined by t test are indicated by ΔΔΔ p < 0.001 for dark colonies, * p < 0.5, *** p < 0.001 for light colonies and + p < 0.5 for negative colonies.

C) Total transcript level of endogenous Klf4, Nanog, Oct4 and Sox2 were quantified relative to Gapdh levels, in three biological replicates of untransfected E14 ES cells, WT and the indicated KLF4-GFP mutant cells revealing that only KLF4(S132A)-GFP, KLF4(K249R)-GFP blocked differentiation whereas others do not. Error bars represent standard deviation.

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3.3.8 Activated STAT3, NANOG and SOX2 stabilize KLF4 protein through its involvement in nuclear complexes

LIF signaling activates the JAK-STAT pathway in ES cells causing phosphorylation and activation of STAT3 (pTyr705) (Hirai et al., 2011; Ohtsuka et al., 2015; Raz et al., 1999; Zhang et al., 2010). After removal of LIF/2i the levels of phosphorylated STAT3 (pTyr705) in the nucleus decrease rapidly and are undetectable after 6 hr of differentiation as demonstrated both by whole cell lysate analysis and by cell fractionation (Figure 3.11A). PLA for STAT3/KLF4 during early differentiation showed interaction between these two proteins occurs in undifferentiated cells but is lost during the first 24 hr of differentiation (Figure 3.11B and C). We next examined whether a short 1hr LIF induction starting after 24 hr of differentiation could restore STAT3/KLF4 interaction and KLF4 protein stability. The t½ of KLF4 increased form <2hr to 24 hr after a 1hr treatment with LIF indicating that LIF signaling can rapidly restore KLF4 protein stability even in cells differentiated for 24 hr (Figure 3.11I and J). This 1hr treatment with LIF also increased the levels of Klf4 transcript and protein in 24 hr differentiated cells (Figure 3.11D, E and F), and restored the interaction between KLF4 and STAT3 (Figure 3.11G, H). PLA revealed a significant increase in the number of interaction foci per nucleus for KLF4 with RNAPII-S5P after short LIF induction suggesting that pSTAT3 is able to stabilize KLF4 protein and recruit KLF4 to RNAPII complexes (Figure 3.12A, B).

As the levels of NANOG protein in the nucleus are drastically reduced by 24 hr of differentiation, whereas OCT4 and SOX2 levels remain high at this time (Dhaliwal et al., 2018), I hypothesized that loss of NANOG protein could be involved in reduced KLF4 stability after 24 hr of differentiation. To test this hypothesis, cells differentiated for 24 hr were transfected with Nanog- t2a-GFP to restore NANOG levels. After an additional 24 hr in differentiation media KLF4 protein levels were restored in Nanog-t2a-GFP transfected cells without any change in Klf4 transcript levels indicating a post-transcriptional regulatory mechanism (Figure 3.13A, B and C). Similar to what was observed for activated STAT3, NANOG expression in differentiated cells restores KLF4 protein stability from t½ = 1.5hr to t½ =25hr (Figure 3.13D and E). To investigate whether NANOG stabilizes KLF4 through association with KLF4, KLF4 was immunoprecipitated to determine whether KLF4 and NANOG associate in ES cells. Indeed, KLF4 and NANOG interact in

122 differentiated cells transfected with Nanog-t2A-GFP (Figure 3.14A). Restoring NANOG levels in differentiated cells increased not only the interaction between KLF4 and NANOG but also restored the interaction of KLF4 with RNAPII-S5P, SOX2 and OCT4 which is normally completely lost by 48hr of differentiation as revealed by PLA (Figure 3.14B, C and D). Together these data reveal that NANOG has a role in both stabilizing KLF4 protein and recruiting KLF4 to RNAPII-S5P compartments in the nucleus.

The levels of OCT4 and SOX2 protein and their association with RNAPII complexes does not change during first 24 hr of differentiation (Dhaliwal et al., 2018). In addition OCT4 and SOX2 are known to dimerize and bind the same DNA consensus motif to exert their function as transcription factors in ES cells (Chen et al. 2008). As SOX2 and OCT4 function as part of a dimer in ES cells I analyzed only the effect of SOX2 expression on KLF4 protein stability. I used cells in which Sox2 transcript and protein levels are stably downregulated by homozygous deletion of the Sox2 control region (ΔSCR129/Cast), the enhancer that regulates Sox2 in ES cells (Li et al., 2014; Zhou et al., 2014). I analyzed these cells for KLF4 protein levels and protein stability. Surprisingly, I observed that KLF4 protein levels were significantly reduced by >3 fold in clones with a homozygous deletion the Sox2 control region (ΔSCR129/Cast) in which SOX2 protein is reduced by >9 fold (Figure 3.1D, E and Figure 3.2B) despite the observation that Klf4 transcript levels are unaffected by SCR deletion (Figure 3.1C). Moreover, I again observed that the greatest reduction in the association of KLF4 protein with RNAPII-S5P occurred in cells with disrupted Sox2 transcript and protein expression rather than in cells with reduced KLF4 transcription due to the loss of the KLF4 enhancers (ΔSCR129/Cast) (Figure 3.3).

ΔSCR129/Cast cells express low levels of SOX2 (Zhou et al., 2014) and when maintained in LIF/2i,

KLF4 protein is unstable with a t½ <2hrs and stability is unaffected by removal of LIF/2i (Figure 3.4A, B). To evaluate the role of SOX2 in stabilizing KLF4 protein, ΔSCR129/Cast cells were transfected with Sox2-t2A-GFP. Transfection restored Sox2 mRNA and protein to wild type levels and significantly increased KLF4 protein but not transcript levels indicating the regulation occurs post-transcriptionally (Figure 3.15A, B and C). In addition, the stability of KLF4 protein increased significantly from t½=2hr to t½=17hr after SOX2 protein levels were restored (Figure 3.15D, E). To investigate whether SOX2 stabilizes KLF4 through association with KLF4, KLF4 was

123 immunoprecipitated to determine whether KLF4 and SOX2 associate in ΔSCR129/Cast cells with restored SOX2 expression. Indeed, KLF4 and SOX2 interact and this interaction is restored in Sox2-t2A-GFP transfected cells (Figure 3.16A). Similarly, increased nuclear interaction between KLF4 and SOX2 was observed by PLA in Sox2-t2A-GFP transfected cells (Figure 3.16B, C, and D). Interestingly, the number of PLA foci/nucleus for KLF4 with RNAPII-S5P was unchanged when SOX2 protein levels were restored, however, the signal intensity per focus was observed to increase significantly suggesting SOX2 protein is involved in stabilizing KLF4 protein in nuclear transcriptional complexes involving other pluripotency transcription factors such as OCT4 and NANOG (Figure 3.16E, F and G).

Together these data indicate that nuclear anchoring through interaction with RNAPII complexes, pluripotency transcription factors, SOX2, NANOG and STAT3 as wells as association with DNA prevents KLF4 nuclear export and degradation by the proteasome and maintains the stability of the KLF4 protein.

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Figure 3.11

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Figure 3.11: Activated STAT3 stabilizes KLF4 protein

A) Immunoblots for activated STAT3 (pTyr705) and total STAT3. Whole cell lysate (WCL) prepared from ES cells cultured with LIF/2i, and 6, 12 and 24 hr after removal of LIF/2i. GAPDH levels indicate equal loading. Nuclear and cytoplasmic fractions prepared from ES cells cultured with LIF/2i, 6, 12 and 24 after removal of LIF/2i. UBF1 and CYPA were used to validate the purity of nuclear and cytoplasmic fractions respectively.

B) Proximity ligation amplification (PLA) displays the interaction between KLF4/STAT3 in ES cells cultured with LIF/2i, 6 and 24 hr after removal of LIF/2i. Images shown are maximum- intensity projections. Merged images display DAPI in blue and PLA in red. Scale bar = 10 μm.

C) Box-and-whisker plots display the number of KLF4/STAT3 PLA foci per nucleus for ES cells cultured with LIF/2i, 6 and 24 hr after removal of LIF/2i. Boxes indicate interquartile range of intensity values and whiskers indicate the 10th and 90th percentiles; outliers are shown as black dots. Images were collected from at least three biological replicates and ≥100 nuclei were quantified for each sample. Statistical differences determined with one way ANOVA (p < 0.05) are indicated by different letters.

D) Immunoblots for p(Tyr705)-STAT3 and total STAT3 in whole cell lysate prepared from ES cells cultured with LIF/2i, 24 hr after LIF/2i removal and in cells 24 hr after LIF/2i removal followed by a 1 hr treatment with LIF. GAPDH levels indicate equal sample loading.

E) Quantification of KLF4 protein relative to GAPDH and ES levels, in three biological replicates of ES cells cultured with LIF/2i, 24 hr after LIF/2i removal and in cells 24 hr after LIF/2i removal followed by a 1 hr treatment with LIF. Error bars represent standard deviation. Statistical differences determined with one-way ANOVA (p < 0.05) are displayed by different letters.

F) Transcript levels for Klf4 were quantified relative to Gapdh and ES levels, in three biological replicates of ES cells cultured with LIF/2i, 24 hr after LIF/2i removal and in cells 24 hr after LIF/2i removal followed by a 1hr treatment with LIF. Error bars represent standard deviation. Statistical differences were determined by one-way ANOVA (p < 0.05) and displayed by different letters.

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G) KLF4 immunoprecipitation (IP) in ES cells cultured with LIF/2i, 24 hr after LIF/2i removal and in cells 24 hr after LIF/2i removal followed by a 1hr treatment with LIF, probed with anti- STAT3 (PD, pull down). GAPDH reveals equal loading of the input.

H) PLA displays the interaction between KLF4/STAT3 in ES cells cultured with LIF/2i, 24 hr after LIF/2i removal and in cells 24 hr after LIF/2i removal followed by a 1hr treatment with LIF. Images shown are maximum-intensity projections. Merged images display DAPI in blue and PLA in red. Scale bar = 10 μm.

I) Immunoblots for KLF4 and GAPDH in ES cells 24 hr after LIF/2i removal and in cells 24 hr after LIF/2i removal followed by a 1hr treatment with LIF, sampled at 0, 2, 4, 6, 8 and 12 hr after CHX treatment. GAPDH levels were used as the CHX chase assay control and displayed the expected protein half-life (t½ >30 hr).

J) The percent remaining KLF4 and GAPDH protein at specific time point (0, 2, 4, 6, 8, and 12 hr) was calculated from the intensity of CHX treated immunoblots, measured in three biological replicates. Half-life was calculated for each time series replicate by best fit to exponential decay. Error bars represent standard deviation. Statistical differences between protein half-life determined by t-test (p < 0.001) are indicated as ***.

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Figure 3.12

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Figure 3.12: Activated STAT3 stabilizes KLF4 by recruiting it to transcriptional complexes.

A) Proximity ligation amplification (PLA) displays the interaction between KLF4 and RNAPII, OCT4, SOX2, and NANOG in ES cells cultured with LIF/2i, 24 hr after LIF/2i removal and in cells 24 hr after LIF/2i removal followed by a 1hr treatment with LIF. Images shown are maximum-intensity projections. Merged images display DAPI in blue and PLA in red. Scale bar = 10 μm.

B) Box-and-whisker plots display the number of PLA foci per nucleus for ES cells cultured with LIF/2i, 24 hr after LIF/2i removal and in cells 24 hr after LIF/2i removal followed by a 1hr treatment with LIF. Boxes indicate interquartile range of intensity values and whiskers indicate the 10th and 90th percentiles; outliers are shown as black dots. Images were collected from at least three biological replicates and ≥100 nuclei were quantified for each sample. Statistical differences determined with one way ANOVA (p < 0.05) are indicated by different letters.

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Figure 3.13

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Figure 3.13: NANOG stabilizes KLF4 protein

A) Total transcript levels of Klf4 and Nanog were quantified relative to Gapdh, in three biological replicates of ES cells cultured with LIF/2i, 24 and 48hr after removal of LIF/2i, and in ES cells 48hr after removal of LIF/2i where Nanog-t2A-GFP was transfected 24 hr after removal of LIF/2i. Error bars represent standard deviation. Statistical differences in each transcript expression between different conditions were determined by one way ANOVA (p<0.05) and displayed as lower case letters for Klf4 and upper case letters for Nanog.

B) Immunoblots for KLF4 and NANOG in ES cells cultured with LIF/2i, 24 and 48hr after removal of LIF/2i, and in ES cells 48hr after removal of LIF/2i where Nanog-t2A-GFP was transfected 24 hr after removal of LIF/2i. GAPDH levels indicate equal sample loading.

C) Quantification of KLF4 and NANOG protein intensity levels relative to GAPDH in three biological replicates. Error bars represent standard deviation. Statistical differences for each protein were determined by one way ANOVA (p<0.05) and displayed as lower case letters for KLF4 and upper case letters for NANOG.

D) Immunoblot for KLF4 and GAPDH in ES cells 48hr after removal of LIF/2i, and in ES cells 48hr after removal of LIF/2i where Nanog-t2A-GFP was transfected 24 hr after removal of LIF/2i, sampled at 0, 2, 4, 6, 8 and 12 hr after CHX treatment. GAPDH levels were used as the CHX chase assay control and displayed the expected protein half-life (t½ >30 hr).

E) The percent remaining KLF4 and GAPDH protein at specific time point (0, 2, 4, 6, 8, and 12 hr) was calculated from the intensity of CHX treated immunoblots, measured in three biological replicates. Half-life was calculated for each time series replicate by best fit to exponential decay. Error bars represent standard deviation. Statistical differences between protein half-life determined by t-test (p < 0.001) are indicated as ***.

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Figure 3.14

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Figure 3.14: NANOG stabilizes KLF4 protein by recruiting KLF4 to transcriptional complexes.

A) KLF4 immunoprecipitation (IP) from ES cells cultured with LIF/2i, 24 and 48hr after removal of LIF/2i, and in ES cells 48hr after removal of LIF/2i where Nanog-t2A-GFP was transfected 24 hr after removal of LIF/2i, probed with anti-NANOG (PD, pull down). GAPDH levels reveal equal loading of the input.

B) Proximity ligation amplification (PLA) displays the interaction between KLF4/NANOG in ES cells cultured with LIF/2i, 24 and 48hr after removal of LIF/2i, and in ES cells 48hr after removal of LIF/2i where Nanog-t2A-GFP was transfected 24 hr after removal of LIF/2i. Images shown are maximum-intensity projections. Merged images display DAPI in blue and PLA in red. Scale bar = 10 μm.

C) PLA displays the interaction between KLF4 and RNAPII, OCT4 or SOX2 in ES cells cultured with LIF/2i, 24 and 48hr after removal of LIF/2i, and in ES cells 48hr after removal of LIF/2i where Nanog-t2A-GFP was transfected 24 hr after removal of LIF/2i. Images shown are maximum-intensity projections. Merged images display DAPI in blue and PLA in red. Scale bar = 10 μm.

D) Box-and-whisker plots indicate the number PLA foci per nucleus for the interaction between KLF4 and RNAPII, OCT4 or SOX2. Boxes indicate interquartile range of intensity values and whiskers indicate the 10th and 90th percentiles; outliers are shown as black dots. Images were collected from at least three biological replicates and ≥100 nuclei were quantified for each sample. Statistical differences determined by one-way ANOVA (p < 0.05) are indicated by different letters.

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Figure 3.15

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Figure 3.15: SOX2 increases the stability of KLF4 protein in ES cells maintained in LIF/2i.

A) Total transcript levels of Sox2 and Klf4 were quantified relative to Gapdh and F1 levels, in three biological replicates of F1, ΔSCR129/cast and Sox2-t2A-GFP transfected ΔSCR129/cast. Error bars represent standard deviation. Statistical differences for each transcript were determined by one way ANOVA (P<0.05) and displayed as upper case letters for Sox2 and lower case letters for Klf4.

B) Immunoblots for SOX2 and KLF4 in F1, ΔSCR129/cast and Sox2-t2A-GFP transfected ΔSCR129/cast cultured with LIF/2i. GAPDH levels indicate equal sample loading.

C) Quantification of KLF4 and SOX2 protein intensity levels relative to GAPDH and F1 levels, in three biological replicates of F1, ΔSCR129/cast and Sox2-t2A-GFP transfected ΔSCR129/cast. Error bars represent standard deviation. Statistical differences for each protein were determined by one way ANOVA (P<0.05) and displayed as upper case letters for SOX2 and lower case letters for KLF4.

D) Immunoblot for KLF4 and GAPDH in untransfected and Sox2-2A-GFP transfected ΔSCR129/cast cultured with LIF/2i and sampled at 0, 2, 4, 6, 8 and 12 hr after CHX treatment. GAPDH levels were used as the CHX chase assay control and displayed the expected protein half-life (t½ >30 hr).

E) The percent remaining KLF4 and GAPDH protein at specific time point (0, 2, 4, 6, 8, and 12 hr) was calculated from the intensity of CHX treated immunoblots, measured in three biological replicates. Half-life was calculated for each time series replicate by best fit to exponential decay. Error bars represent standard deviation. Statistical differences determined by t-test (p< 0.001) are indicated as ***.

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Figure 3.16

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Figure 3.16: SOX2 stabilizes KLF4 protein by stabilizing its association with transcriptional complexes. A) KLF4 immunoprecipitation (IP) from F1 ES, untransfected and Sox2-t2A-GFP transfected ΔSCR129/cast was probed with anti-SOX2 and anti-KLF4 (PD, pull down). GAPDH levels reveal equal loading of the input.

B) Proximity ligation amplification (PLA) displays the interaction between KLF4/SOX2 in untransfected and Sox2-2A-GFP transfected ΔSCR129/cast. Images shown are maximum-intensity projections. Merged images display DAPI in blue and PLA in red. Scale bar = 10 μm.

C) Box-and-whisker plots indicate the number of KLF4/SOX2 PLA foci per nucleus. Boxes indicate interquartile range of intensity values and whiskers indicate the 10th and 90th percentiles; outliers are shown as black dots. Images were collected from at least three biological replicates and ≥100 nuclei were quantified for each sample. Statistical differences determined by t test (p < 0.001) are indicated as ***.

D) Box-and-whisker plots indicate the total intensity value of each KLF4/SOX2 PLA focus. Boxes indicate interquartile range of intensity values and whiskers indicate the 10th and 90th percentiles; outliers are shown as black dots. Images were collected from at least three biological replicates and ≥100 nuclei were quantified for each sample.

E) PLA displays the interaction between KLF4 and RNAPII, OCT4 or NANOG in untransfected and Sox2-2A-GFP transfected ΔSCR129/cast. Images shown are maximum-intensity projections. Merged images display DAPI in blue and PLA in red. Scale bar = 10 μm.

F) Box-and-whisker plots indicate the number PLA foci per nucleus between KLF4 and RNAPII, OCT4 or NANOG. Boxes indicate interquartile range of intensity values and whiskers indicate the 10th and 90th percentiles; outliers are shown as black dots. Images were collected from at least three biological replicates and ≥100 nuclei were quantified for each sample.

G) Box-and-whisker plots indicate the total intensity value of each PLA focus. Boxes indicate interquartile range of intensity values and whiskers indicate the 10th and 90th percentiles; outliers are shown as black dots. Images were collected from at least three biological replicates and ≥100 nuclei were quantified for each sample. Statistical differences determined by t test (p < 0.001) are indicated as ***.

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3.4 Discussion

The naïve pluripotent state of ES cells is known to be regulated by pluripotency associated transcription factors which act in a complex interconnected network, by binding their own regulatory elements, as well as the regulatory elements of other genes throughout the genome to regulate transcription. Klf4 transcriptional regulation was studied in detail by Xie et al., 2017 who identified downstream enhancer regions bound by OCT4, SOX2, STAT3, KLF4 and ESRRB which are required to maintain Klf4 transcript and protein levels in mouse ES cells maintained in LIF/serum. By contrast, I found that for ES cells maintained in LIF/2i the same enhancers had little role in maintaining KLF4 protein levels as the high stability of the KLF4 protein in LIF/2i conditions buffered against the greatly reduced levels of Klf4 transcription in the absence of the enhancers. Removal of MEK inhibition greatly disrupted KLF4 stability accounting for the dramatic differences between the requirements for Klf4 enhancers in LIF/serum compared to LIF/2i conditions. Furthermore, I found that KLF4 protein is stabilized both by domains within the protein that anchor KLF4 in the nucleus and by interaction with RNAPII and the other pluripotency transcription factors. These findings detail a new way in which the function of the core pluripotency regulatory circuitry is integrated at a post-translational level by controlling KLF4 protein levels.

Nuclear proteins generally contain a NLS, which is required to localize them to the nucleus. As expected, KLF4 requires an intact NLS for nuclear import as NLS mutant KLF4 protein is cytoplasmic (Dhaliwal et al., 2018). In addition, nuclear localization increases the stability of KLF4 protein as the NLS mutant displayed greatly reduced stability compared to the wild type protein. Other mutations which disrupted KLF4 nuclear anchoring also disrupted KLF4 protein stability in LIF/2i conditions. My data reveal that deletion of the KLF4 DNA binding zinc finger domains or mutation of the K275 sumoylation site, involved in transactivation, cause a partial disruption to KLF4 nuclear anchoring. Interestingly, the stability of the protein was completely abolished in these mutants suggesting that even a small disruption to nuclear anchoring can greatly affect KLF4 protein stability. In COS-1 cells, mutation of mouse KLF4 at K275 did not affect protein stability (Du et al., 2010). Moreover, in mutated human KLF4 at an equivalent site, K269, transfected in HEK293 cells showed no effect on localization and stability further suggesting KLF4 stability is context dependent (Tahmasebi et al., 2013). The transactivation function of

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KLF4 has been shown to be regulated in a SUMO1-dependent manner; mutation of K275 in mouse KLF4 impairs the ability of KLF4 to transactivate target promoters by disrupting the SUMO- interacting motif in KLF4 (Du et al., 2010). I found that this mutation also reduced recruitment of KLF4 to RNAPII-S5P rich nuclear compartments which would explain reduced transactivation and reduced nuclear anchoring of the K275 mutant protein. However, sumoylation of human KLF4 at K269 has been reported to induce adipocyte differentiation and inhibit induced pluripotency in MEFs during reprogramming (Tahmasebi et al., 2013). My data showed that a compound mutant containing both K275 sumoylation site and S132 ERK phosphorylation site mutations, was not able to maintain pluripotency in the absence of LIF/2i whereas the single S132 mutant was, suggesting that K275 site is important for pluripotency maintenance in ES cells. Together both studies indicate that sumoylation of KLF4 is not required for pluripotency induction but is important in maintenance of pluripotency. Sumoylation of KLF4 has been reported to occur through the action of an E2 enzyme, Ubc9 (ubiquitin conjugating enzyme 9) also named as ubiquitin conjugating enzyme E2I [UBE2I] (Tahmasebi et al., 2014). Downregulation of Ubc9 inhibited induced pluripotency and stimulated apoptosis in ES cells but not in MEFs suggesting that Ubc9 is essential for ES cell survival and pluripotency maintenance (Tahmasebi et al., 2014). I propose that Ubc9 will be involved in KLF4 K275 sumoylation in ES cells to maintain their pluripotency but during pluripotency induction in MEFs, Ubc9 might be involved in some other unknown mechanisms.

ERK activation induced KLF4 nuclear export is a critical first step in pluripotency exit which relies on KLF4 phosphorylation at S132 and intact NES allowing for interaction with XPO1 (Dhaliwal et al., 2018). In addition, we determined that mono-ubiquitination of KLF4 at K249 is required for KLF4 nuclear export and pluripotency exit. All three of S132, K249 and the KLF4 NES are required for the disruption to KLF4 stability that occurs as ES cells exit the pluripotent state preventing KLF4 phosphorylation, ubiquitination or interaction with XPO1 blocks pluripotency exit in the absence of LIF/2i. By contrast, even in the presence of LIF/2i, KLF4 stability can be disrupted by interfering with KLF4 function as a transcription factor by deletion of the DNA binding zinc finger domains or mutation of the K275 sumoylation site, involved in transactivation. The compound mutants (S132AZNF, S132AK275R), however, maintain KLF4 stability but not

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KLF4 transcription factor function or interaction with RNAPII-S5P and therefore do not block ES cell differentiation. All the KLF4 mutant and their phenotypes are summarized in Figure 3.17.

Figure 3.17: Summary of KLF4 mutations and their effects

KLF4 site Modification Effect

NES1, 2 Leucine(L) to Glutamine (Q) Prevent nuclear export of for NES1 in 97-107 amino KLF4, KLF4 is stable, acids increased interaction with RNAPII-S5P Alanine to Glycine for NES2 in 117-127 amino acids

Serine 132 (S132) required Serine modified to Alanine Inhibit phosphorylation by for phosphorylation by ERK2 ERK, inhibit nuclear export KLF4(S132A)-GFP of KLF4, increases KLF4 stability, increases interaction with RNAPII-S5P and prevent differentiation.

Lysine 249 (K249) required Lysine modified to Arginine Inhibit ubiquitination of for ubiquitination and KLF4, inhibit nuclear export KLF4(K249R)-GFP degradation of KLF4 and degradation of KLF4 at 24 hr, increases KLF4 stability and prevent differentiation.

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Lysine 266 (K275) required Lysine modified to Arginine Makes KLF4 unstable, for sumoylation and localization of KLF4 is both KLF4(K275R)-GFP transactivation of KLF4 nuclear and cytoplasmic after mutation, reduced interaction with RNAPII-S5P, differentiates normally.

S132 and K275 compound KLF4(S132A, K275R)-GFP Inhibit nuclear export, mutant increases KLF4 stability, could not prevent differentiation.

NLS Arginine1 to Ser; Arginine 2 Makes KLF4 unstable, to Ser; Arginine 3 to Glycine localization of KLF4 is in 382-396 amino acids. cytoplasmic after mutation, no interaction with RNAPII- S5P.

Zinc fingers Deletion using restriction Makes KLF4 unstable, enzyme approach localization of KLF4 is both nuclear and cytoplasmic after KLF4(ΔZNF)-GFP mutation, reduced interaction with RNAPII-S5P, differentiates normally.

S132 and ZNF compound KLF4(S132A, ΔZNF)-GFP Inhibit nuclear export, increases KLF4 stability, could not prevent differentiation.

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Naïve pluripotent ES cells cultured in LIF/2i are thought to maintain a more balanced and stable state through higher and more uniform expression of the major pluripotency transcription factors in the cell population (Sim et al., 2017; Tosolini and Jouneau, 2016). Protein interactome studies determined that these core pluripotency transcription factors bind each other and form nuclear complexes in order to maintain the pluripotent state (Gao et al., 2013; Gao et al., 2012; Morey et al., 2015). Removal of LIF/2i from the media disrupts the balanced state by disrupting transcription factor/RNAPII-S5P complexes (Dhaliwal et al., 2018). Disruption in these complexes might begin with loss of nuclear p-STAT3 due to the absence of LIF (Niwa et al., 1998), loss of nuclear KLF4 due to phosphorylation by ERK2 (Dhaliwal et al., 2018) and reduced levels of NANOG protein that occurs during the first 24 hr of differentiation (Zhang et al., 2010). In this study we found that KLF4 protein stability is maintained through interaction of KLF4 with p-STAT3, NANOG, and SOX2 in RNAPII-S5P rich nuclear complexes (Figure 3.18). Interestingly, the stability of other pluripotency factors are not affected in the same way by these complexes as their stability does not change during pluripotency exit. Mutations to the KLF4 protein that increase KLF4 stability, without affecting KLF4 transcription factor function, could be deployed in a reprogramming context to improve the efficiency of reprogramming protocols by prolonging KLF4 protein function in reprogramming cells.

In addition to the role of KLF4 protein in pluripotency maintenance and reprogramming, KLF4 protein level modulation is also involved in both the oncogenic and tumor suppressor roles of KLF4 in adult tissues. Increased stability of the KLF4 protein, mediated by the ubiquitin- proteosomal pathway, is linked to an oncogenic role in breast cancer (Hu et al., 2012) and decreased stability is linked to its tumor suppressor role in colorectal cancer (Gamper et al., 2012). KLF4 stability modulation by interaction with the pluripotency transcription factors may also be important in tumorigenesis as co-expression of these factors is associated with variety of cancers such as breast cancer, squamous cell carcinoma, gastrointestinal cancers progression (Almozyan et al., 2017; Gwak et al., 2017; Hu et al., 2012; Lu et al., 2014; Piva et al., 2014; Soheili et al., 2017; Palla et al, 2015; as reviewed in Muller et al, 2016 ).

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Figure 3.18

Figure 3.18: The summary figure showing transcription factor status and complex after 24 hr differentiated ES cell

The nuclear KLF4 is stabilized by its association with other transcription factors including OCT4, SOX2, NANOG and STAT3-P dimer and other protein in RNAPII-S5P transcriptional complexes. The removal of LIF/2i induces differentiation in naïve ES cells maintained in their ground state by LIF/2i. After 24 hr of differentiation, due to loss of associating partners including phosphorylate STAT3 dimer and NANOG from the active transcriptional complexes, KLF4 becomes more unstable in transcriptional complexes made at 24 hr which becomes accessible to E3 ligases (unknown) in order to monoubiquitinate it and export via exportins.

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3.5 Materials and Methods 3.5.1 Embryonic stem Cell culture

The mouse ES cell line E14TG2a (E14) was obtained from the ATCC (CRL-1821). F1 (M. musculus129 × M. castaneus) ES cells were obtained from Barbara Panning (Mlynarczyk-Evans et al., 2006). All cells were maintained in feeder free conditions on 0.1% gelatin in DMEM supplemented with 15% (v/v) fetal bovine serum (FBS), 0.1mM non-essential amino acids, 1mM sodium pyruvate, 2mM GlutaMAX, 0.1mM 2-mercaptoethanol, 1,000U/mL LIF, 3μM CHIR99021 (GSK3β inhibitor, Biovision) and 1μM PD0325901 (MEK inhibitor, Invivogen); referred to as LIF/2i medium. The differentiation medium contained the same components with the exception of LIF and the two inhibitors.

For protein half-life analysis cells were treated with 10μg/ml cyclohexamide (Sigma Aldrich) for 2, 4 6, 8 and 12 hr, collected as cell pellets, and lysed in RIPA buffer containing protease inhibitor complete EDTA free (Roche) and phosphatase inhibitor cocktail (Millipore) to generate cell lysates for further analysis by western blotting. The percent remaining protein at specific time point (0, 2, 4, 6, 8, and 12 hr) was calculated from the intensity of CHX treated immunoblots using Image lab 5.0 (Bio-Rad), measured in three biological replicates. Half-life was calculated for each time series replicate by best fit to exponential decay. All the half life analysis are displayed in a tabular form in Appendix 2.Treatment with 10µM MG132 proteasome inhibitor (Sigma Aldrich) was used to indicate the role of proteosomal degradation in KLF4 function.

3.5.2 Cellular fractionation, co-immunoprecipitation and western blotting

Nuclear and cytoplasmic fractions were generated according to the protocol described in (Dhaliwal and Mitchell, 2016). Protein was extracted from cell fractions using RIPA buffer containing protease inhibitor complete EDTA free (Roche) and phosphatase inhibitor cocktail (Millipore) and quantified using bicinchoninic acid (Thermo Fisher Scientific). Protein samples were analyzed by SDS-PAGE (Bis-Tris, 5% stacking, 10% resolving). Blots were probed with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (Table S1). Blots were quantified by relative intensity using background correction from adjacent regions. At least three biological replicates were analyzed for each experiment.

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For co-immunoprecipitation of protein, fractions or total cell lysates in RIPA were incubated overnight with the appropriate antibody and then incubated overnight with a 50:50 mixture of protein A and protein G Dynabeads (Thermo Fisher Scientific). Beads were washed three times with non-denaturing lysis buffer (20 mM Tris-HCl, 137 mM NaCl, 10% glycerol, 1% NP-40, 2 mM EDTA, 1 mM PMSF, and proteinase inhibitors), twice with PBS, eluted in SDS-PAGE loading buffer, and analyzed by SDS-PAGE. Antibodies used are listed in table 3.1.

Table 3.1: Antibody list

Name Company Catalog # Experiment rabbit anti-KLF4 Abcam AB129473 PLA (1:2000), WB (1:1000), IP mouse anti-KLF4 Santa Cruz sc-393462 PLA (1:2000), IPWB (1:1000) rabbit anti-NANOG Cosmo Bio RCAB0002P-F PLA (1:1000), WB (1:1000) rabbit anti-NANOG Abcam AB80892 PLA (1:2000), WB (1:1000) mouse anti-NANOG BD Biosciences 560259 PLA (1:2000),WB (1:1000) mouse anti-SOX2 R&D Systems MAB2018 PLA (1:1000), WB (1:1000) mouse anti-OCT3/4 Santa Cruz sc-5279 PLA (1:1000), WB (1:1000) mouse anti-RNAPII-PS5 Abcam AB5408 PLA (1:2000), WB (1:1000)

145 rabbit anti-RNAPII-PS5 Abcam AB5131 PLA (1:2000), WB (1:1000) mouse anti-RNAPII core Millipore Sigma CBL221 PLA (1:2000), WB (ARNA3) (1:1000) mouse anti-XPO1 (CRM1) Santa Cruz sc-74454 PLA (1:2000), IP-WB (1:1000) mouse anti-p(Tyr705)- Santa Cruz sc-8059 WB (1:500) STAT3 mouse anti-STAT3 Santa Cruz sc-8019 PLA (1:1000), WB (1:1000) mouse anti-GFP Origene TA150041 PLA (1:500), IP chicken anti-GFP Abcam AB13970 WB (1:1000) rabbit anti-CYPA Abcam AB131334 WB (1:1000) rabbit anti-UBF1 Santa Cruz sc-13125 WB (1:1000) mouse anti-GAPDH Santa Cruz sc-365062 WB (1:1000) goat anti-rabbit-HRP Bio-Rad 170-6515 WB (1:2000) goat anti-mouse-HRP Bio-Rad 170-6516 WB (1:2000)

Rabbit anti ubiquitin Abcam AB7780 WB (1:500)

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3.5.3 Proximity Ligation Amplification (PLA)

PLA was conducted using Duolink (Sigma-Aldrich) following the manufacturer's instructions. Images were collected using a Leica TCS SP8 and a 63× magnification objective lens. The number of PLA foci per nucleus was quantified using Imaris 7.1 by manual 3D masking of nuclei in ESC colonies defined by the DAPI signal. The two antibodies against two different epitopes in same protein (RNAPII core (ARNA3) and RNAPII-S5P) were used as positive control whereas two antibodies against proteins in two different compartments (nuclear RNAPII and nucleolar UBF1) were used as negative control for PLA. Also, no antibody and single antibody controls were performed. All PLA experiments were carried out on at least three biological replicates. The total intensity per focus data was obtained from Imaris 7.1.

3.5.4 CRISPR/Cas9 deletion

Cas9-mediated deletions were carried out as previously described (Moorthy and Mitchell, 2016; Zhou et al., 2014). Cas9 targeting guides flanking Klf4 enhancer regions were selected (Table 3.2). Only gRNAs predicted to have no off-target binding in the F1 mouse genome were chosen. Guide RNA plasmids were assembled using the protocol described by Mali et al.2013 (Mali et al., 2013). Briefly, two partially complementary 61-bp oligos were annealed and extended using Phusion polymerase (New England Biolabs). The resulting 100-bp fragment was assembled into an AflII- linearized gRNA empty vector (Addgene, ID#41824) using the Gibson assembly protocol (New England Biolabs). The sequence of the resulting guide plasmid was confirmed by sequencing.

F1 ES cells were transfected with 5 µg each of 5′ gRNA, 3′ gRNA, and pCas9_GFP (Addgene, ID#44719)(Ding et al., 2013) plasmids using the Neon Transfection System (Life Technologies). Forty-eight hours post-transfection, GFP-positive cells were collected and sorted on a BD FACSAria. Ten to twenty thousand GFP positive cells were seeded on 10-cm gelatinized culture plates and grown for 5–6 d until large individual colonies formed. Colonies were picked and propagated for genotyping and gene expression analysis as previously described (Moorthy and Mitchell, 2016; Zhou et al., 2014). All deletions were confirmed by sequence analysis using

147 primers 5′ and 3′ from the gRNA target sites; SNPs within the amplified product confirmed the genotype of the deleted allele.

Table 3.2: List of CRISPR/Cas9 guide sequences

Name Sequence

5’Δ1 Forward TTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCCAAGAGCGT TCGTGCCCCG

5’Δ1 Reverse GACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACCGGGGCACGAA CGCTCTTGGC

3’Δ1 Forward TTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGAGCACAGACG GATTGAGTGA

3’Δ1 Reverse GACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACTCACTCAATCCG TCTGTGCTC

5’Δ2 Forward TTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGCAGATGAATT GACACGACGT

5’Δ2 Reverse GACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACACGTCGTGTCAA TTCATCTGC

3’Δ2 Forward TTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGACTAGGGGCT CACGCGTGGT

3’Δ2 Reverse GACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACACCACGCGTGA GCCCCTAGTC

3.5.5 Real-Time qPCR

Total RNA was purified as per manufacturer’s protocol using RNAeasy (Qiagen). Following a DNaseI (turbo DNaseI from Invitrogen) digestion to remove DNA, total RNA was reverse transcribed with random primers using the High Capacity cDNA Synthesis Kit (Applied Biosystems). Gene expression was monitored by qPCR using genomic DNA to generate standard curves. Gapdh expression was used to normalize expression values. At least three biological replicates were analyzed for each experiment. Primers used are listed in Table 3.3.

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Table 3.3: List of expression primers

Gene Forward primer Reverse primer Amplicon size

Klf4_cast CCCTCGTGGGAAGACAaTG CACTACCGCAAACACACAGG 192bp

Klf4_129 CTCGTGGGAAGACAgTGTGA ACAGGCGAGAAACCTTACCA 266bp

Klf4 GAAGACGAGGATGAAGCTGAC TGGACCTAGACTTTATCCTTTCC 94bp

Nanog TCCCAAACAAAAGCTCTCAAG ATCTGCTGGAGGCTGAGGTA 165bp

Sox2 ACGCCTTCATGGTATGGTC CGGACAAAAGTTTCCACTC 114bp

Oct4 ATGAGGCTACAGGGACACCTT GTGAAGTGGGGGCTTCCATA 100bp

Gapdh GCACCAGCATCCCTAGACC CTTCTTGTGCAGTGCCAGGTG 109bp

3.5.6 Expression of KLF4 Mutants

A mouse KLF4-GFP vector (RG206691) obtained from Origene and KLF4(S132A)-GFP mutant published in Dhaliwal et al. 2018 were subjected to site-directed mutagenesis (SDM, QuikChange Lightning, Agilent Technologies) to introduce additional mutations. Primers for SDM are indicated in Table 3.4. All the sites used to mutate are conserved among different species and shown as multiple sequence alignment by Clustal W in Appendix 1. A MluI/AleI restriction digestion deleted zinc fingers from the Klf4 sequence cloned in the pUC 19 vector. Klf4 in pUC19 without Zinc fingers (3881bp) was ligated by blunt end ligation. After sequence confirmation, the zinc finger deleted Klf4 fragment was inserted into a Kpn1/Not1 digested KLF4-GFP vector. Sequence-confirmed plasmids were transfected by electroporation into E14 ES cells and selected with 400 μg/mL G418. The cells were sorted by fluorescence-activated cell sorting, and individual clones selected and maintained in 50 μg/mL G418 to obtain KLF4-GFP-positive clones.

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Table 3.4: List of primers for Site directed mutagenesis

Primer name Sequence

K249R TCGGTCATCAGTGTTAGCAGAGGAAGC forward

K249R GCTTCCTCTGCTAACACTGATGACCGA reverse

K275R GCATGTGCCCCAAGATTAGGCAAGAGGCGGTC forward

K275R GACCGCCTCTTGCCTAATCTTGGGGCACATGC reverse

S132A CCACCTCGGCGTCAGCTTCATCCTCGTCTGCCCCAGCGAGCAGCGGCCCTGCC forward

S132A reverse GGCAGGGCCGCTGCTCGCTGGGGCAGACGAGGATGAAGCTGACGCCGAGGTGG

NLS forward CGGGGCCACGACCCGCTTCCGCTCTTTGGCTTGG

NLS reverse CCAAGCCAAAGAGCGGAAGCGGGTCGTGGCCCCG

NES1 forward AAAGGATAAAGTCTAGGTCCTGTTGGTCGTTGAACTCCTCGGTC

NES1 reverse GACCGAGGAGTTCAACGACCAACAGGACCTAGACTTTATCCTTT

3.5.7 Transient transfections

CMV-Sox2-T2A-GFP was generated by amplifying the human SOX2 sequence from a donor construct (HsCD00079917, Harvard Institute of Proteomics)(Zuo et al., 2007) using NheI and XbaI overhang primers and Phusion High-Fidelity Polymerase (NEB). A T2a-GFP backbone was subcloned from pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-GFP (Addgene plasmid #71237, a

150 gift from Charles Gersbach)(Thakore et al., 2015) and inserted into hCas9 (Addgene plasmid #41815, a gift from George Church)(Mali et al., 2013) using AgeI/XbaI digestion and T4 DNA ligase (Thermo Fisher Scientific). Next, dCas9-KRAB was excised out using NheI/XbaI to create a lineralized CMV-T2a-GFP construct. The SOX2 PCR product was purified and inserted into CMV-T2a-GFP using the In-Fusion HD cloning kit (Takara Bio USA) and transformed into Stellar Competent Cells (Takara Bio USA). Bacterial colonies were PCR-screened, and positive inserts were sequence confirmed. CMV-Sox2-T2A-GFP was extracted with an Endotoxin-free Plasmid Midiprep Kit (Geneaid™ Midi Plasmid Kit Endotoxin Free).

The SOX2 compromised SCR129/Cast deleted cells were transfected with CMV-Sox2-T2A-GFP and 24 hr differentiated ES cells were transfected with Nanog-2A-GFP using neon electroporation transfection system as per manufacturer’s instruction manual (Thermo Fisher Scientific). Nanog- 2A-GFP was a gift from Rudolf Jaenisch (Addgene plasmid # 59994)(Faddah et al., 2013).

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Chapter 4 Global Discussion and Future Directions

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4.1 Summary and discussion

Mammalian development relies on the crucial transient cellular property of “Pluripotency” which holds great potential for therapeutic use in regenerative cellular therapies. In order to completely harness this potential, a full understanding of naïve pluripotency maintenance, exit from naïve pluripotency and differentiation is required. Several studies have collaboratively identified important molecular mechanisms underlying pluripotency maintenance and differentiation to specific cell types; however, what has been less well studied is how pluripotency regulatory control is disrupted as cells exit the naïve pluripotent state. Pluripotency is a finely balanced state modulated by interconnected networks of multiple regulatory layers. These regulatory layers involve multiple complex networks at transcriptional levels further guided by post transcriptional and post translational regulatory mechanisms under the influence of epigenetic and extracellular signaling pathways. This complexity makes it difficult to understand the key mechanisms that allow cells to shift to a differentiation program; however, our extensive understanding of transcriptional regulatory control in pluripotency provides an important opportunity to investigate the dynamic mechanisms that allow cells to shift phenotypes and the mechanisms that can prevent these shifts. Mouse ES cells provide a robust in vitro system to investigate the molecular regulation of pluripotency while still allowing for in vivo investigation of these mechanisms and the impact that perturbation has on later development.

At a transcriptional level, the pluripotent state of ES cells is known to be regulated by an extensively interconnected network of a core set of pluripotency associated transcription factors which bind cooperatively throughout the genome and regulate genes required for pluripotency (Loh et al., 2006; Chen et al., 2008). These transcription factors also bind the promoters and regulatory enhancers of their own genes as a larger transcriptional regulatory complex indicating they support a pluripotency auto regulatory feedback loop (Kim et al., 2008). KLF4 is a part of this core network of pluripotency transcription factors and controlled at a transcriptional level by the LIF signaling pathway (Chen et al., 2008; Chen et al., 2012; Zhou et al., 2014; Jiang et al., 2008). As removal of LIF is a key signal that initiates differentiation and Klf4 is the first of the pluripotency associated transcription factors affected by differentiation induction it is an important factor to consider in determining the molecular events that destabilize the pluripotency transcriptional regulatory network (Zhang et al., 2010). I, therefore, hypothesized that KLF4

153 followed by NANOG would be the first proteins of this network to be removed from nuclear transcription complexes to initiate exit from the naïve pluripotent state. I initially investigated the dynamic spatial and temporal behavior of pluripotency associated transcription factors (OCT4, SOX2, NANOG and KLF4) in pluripotent ES cells and during the first 24 hr of differentiation. After observing nuclear export of KLF4, I focused on investigating the gene and protein regulatory mechanisms that modulate the function of KLF4 in the naïve pluripotent state and how these mechanisms are altered as ES cells exit pluripotency.

Based on my findings I generated a model of early ES cell differentiation detailing the mechanisms underlying naïve pluripotency exit. Mouse ES cells cultured in LIF/2i conditions are considered the closest representation of naïve pluripotent preimplantation ICM cells. The naïve pluripotent state of mouse ES cells cultured with LIF/2i is maintained by an interconnected network of core pluripotency associated transcription factors under the influence of extracellular signaling cues (as reviewed in Nichols et al., 2008). Presence of LIF/ STAT3 signaling and inhibition of FGF/ERK and GSK3 signaling in LIF/2i culture conditions promotes self-renewal by upregulating expression of pluripotency associated transcription factors (Ying et al., 2008, as reviewed in Nichols et al., 2008). Moreover, these cells also express high levels of naïve pluripotency markers such as Rex1, NrOb1, Fgf and Klf4 (as reviewed in Nichols et al., 2008; Ginis et al.,2004). KLF4 protein levels are maintained both by transcription and through maintaining high KLF4 protein stability. This high stability is maintained by the nuclear anchoring provided by KLF4 zinc finger domains and the transactivation domain mediated by sumoylation at K275 (Du et al., 2010) in naïve ES culture conditions. Removal of LIF/2i from the culture media disturbs the balanced extracellular and intracellular environment by allowing for ERK activation within 6-12 hr. Active ERK phosphorylates KLF4 at Ser132 which removes KLF4 from active nuclear complexes. Phosphorylated KLF4 associates with XPO1 and is exported from the nucleus through nuclear pore complexes. Exported KLF4 accumulates in cytoplasm where it is degraded by the proteosomal pathway. At the same time removal of LIF prevents STAT3 phosphorylation which causes STAT3 to leave the active transcriptional complexes required to maintain the expression of naïve pluripotency genes. Loss of p-STAT3 and nuclear export of KLF4 disrupts these transcriptional complexes causing significant downregulation in Klf4 transcription as early as 6 hr, followed by Nanog transcript and protein downregulation. KLF4 export is required for initiating mouse ES cell exit from the naïve pluripotent state as inhibiting this process blocks differentiation. 154

24 hr after LIF/2i removal Klf4 transcription is greatly reduced and newly synthesized KLF4 protein is localized in the nucleus. As KLF4 is stabilized by interaction with other pluripotency transcription factors the loss of NANOG protein at this time further disrupts nuclear transcriptional complexes leading to a dramatic loss of KLF4 protein stability. This destabilization of KLF4 requires nuclear monoubiquitination at K246 which is required for export to the cytoplasm, polyubiquitination and degradation by the proteosomal pathway. This change in active nuclear complexes after 24 hr of differentiation causes downregulation of some pluripotency associated genes while silencing others. Also, at this time the change in transcription factor expression causes upregulation of differentiation associated genes (Figure 4.1). Although Klf4 null mice survive embryogenesis, I found that inhibiting the mechanisms that downregulate KLF4 protein, KLF4 nuclear export or monoubiquitination and degradation, impairs differentiation of ES cells suggesting that persistent high levels of KLF4 protein due to failed downregulation could result in phenotypic consequences during early development. In support of this idea I found that KLF4 interacts with XPO1 during blastocyst development and preventing this interaction interferes with KLF4 downregulation.

Klf4 null mice survive embryogenesis but die postnatally indicating that Klf4 is dispensable for early embryogenesis (Jiang et al., 2008, Segre et al., 1999). As Klf4 is not required for early development I hypothesized that deletion of the Klf4 gene would generate a Klf4 null ES cell line that could allow me to study the phenotype of Klf4 null ES cells. I used CRISPR/Cas9 to delete the Klf4 gene in mouse ES cells and I isolated six clones with a homozygous deletion of the Klf4 gene. Interestingly, none of these Klf4 deleted clones would expand in culture beyond two passages suggesting that although Klf4 is dispensable for pluripotency in vivo it may be required for long term self-renewal of mouse ES cells maintained in culture. This highlights one of the major differences between pluripotency in vitro and in vivo. In vivo pluripotency is a transient cellular state required through only a few cell divisions before the cells differentiate. Clonal expansion of naïve pluripotent cells in vitro, however, requires self-renewal through many cell divisions to maintain the clonal line. Both extrinsic signaling stimulation and intrinsic transcriptional networks converge on Klfs to maintain and expand pluripotent stem cells (Hall et al., 2009). Klf4 and Klf5 transcription is induced upon addition of LIF in 2i culture indicating them as direct targets of LIF; Klf4 is upregulated directly by STAT3 and Klf5 is also stimulated by LIF in a STAT3-dependent manner but involving an additional factor (Hall et al., 2009). Another Klf factor namely Klf2 does 155 not respond to the addition of LIF, instead it is directly regulated by Oct4 (Hall et al., 2009). Klf2 overexpression in serum-free culture containing BMP4 can maintain the self-renewal program in the absence of LIF (Hall et al., 2009). Klf4 is directly downstream of STAT3 and therefore a key mediator of the positive effects of LIF stimulation on mouse ES cell self-renewal (Niwa et al., 2009). Mouse ES cells maintained in LIF/2i acquire highly clonal naïve state expressing high levels of naïve pluripotency markers including Klf4 whereas when derived in FGF and Activin A they acquire a less clonal primed state of EpiSC which had very low Klf4 expression (Ginis et al., 2004; as reviewed in Nichols et al., 2008; Ying et al., 2008). Taken together these data suggested a need for Klf4 to promote a clonal population might depend on the culture conditions in which the cells were maintained.

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Figure 4.1

Figure 4.1: The naïve pluripotency exit model for ES cells

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Sox2 is required for pluripotency maintenance both in vitro and in vivo (Avilion et al. 2003; Maruyama et al., 2005; Masui et al., 2007). As I determined that KLF4 protein was stabilized by interaction with SOX2, and expression of the stable nuclear KLF4(S132A)-GFP mutant delayed differentiation of mouse ES cells and maintained the expression of the pluripotency transcription factors I hypothesized that stable KLF4 might replace the need for Sox2 in pluripotency maintenance. To investigate this, I attempted to delete the Sox2 gene using CRISPR/Cas9 in cells expressing the stable KLF4(S132A)-GFP mutant. Although I identified twelve heterozygous deletions of the Sox2 gene, I was unable to expand any of three homozygous deleted clones beyond the first passage. This suggested that Sox2 is indeed required to maintain the pluripotent state in ES cells regardless of KLF4 stability. SOX2 regulates a unique set of target gene(s) including nuclear receptor subfamily 5 group A member 2 (Nr5a2) along with other OCT/SOX enhancer- dependent genes and Oct3/4 expression needed to maintain pluripotency (Takahashi et al., 2006; Masui et al., 2007). Mice homozygous for disruptions in Nr5a2 die around embryonic day 7.5 (Stergiopoulos et al., 2016). Moreover, three of the Yamanaka reprogramming factors Oct3/4, c- Myc and Klf4 induced cells morphologically similar to ES cells expressing Nanog, Fgf4 and Fbxo15, but these cells were not pluripotent as they could not generate mouse chimeras (Masui et al., 2007). Additionally, Sox2-null ES cells rescued by Oct3/4 overexpression could not generate adult chimeric mice suggesting that Sox2 is required for maintaining a true pluripotent state needed to produce complete and normally fertile organisms (Masui et al., 2007).

Although, OCT4/SOX2 binding at the Klf4 enhancer has been shown to be critical for Klf4 transcription (Xie et al., 2018), I did not observe any change in Klf4 transcript levels by stable downregulation of SOX2 in ΔSCR129/Cast cells, or any increase in Klf4 transcription after transfection of a Sox2 expression vector. One explanation for these observations is that the small amount of SOX2 protein present in ΔSCR129/Cast cells is enough to maintain binding at the Klf4 enhancer and maintain Klf4 transcription in LIF/2i conditions. In contrast, this small amount of SOX2 protein is not enough to maintain the stability of the KLF4 protein. Reduced SOX2 protein level disrupts the active nuclear transcriptional complexes freeing the KLF4 protein from them. In chapter 3, I showed that the fate of constitutively cytoplasmic and unstable nuclear localization mutant of KLF4(NLS)-GFP is to be degraded by the proteosomal pathway regardless of the culture conditions. Moreover, in chapter 3 I showed that unstable endogenous KLF4 and WT KLF4-GFP

158 protein in cells differentiated for 24 hr is exported to the cytoplasm by XPO1 after monoubiquitinaton at K249 and the degraded by the proteosomal pathway. This observation also suggested that the unbound fraction of nuclear KLF4 might be tagged for export and proteosomal degradation by monoubiquitination. Similar mechanism might also be responsible for the degradation of the free pool of KLF4 protein in SOX2 compromised ΔSCR129/Cast cells even in LIF/2i conditions.

Modulation of KLF4 protein stability is not only involved in mechanisms such as regulation of pluripotency and reprogramming but also in both oncogenesis and tumor suppression. KLF4 appears to function as an oncogene or tumor suppressor depending on the tissue type. Increased levels of the KLF4 protein promote tumorigenesis in skin and breast, but act as a tumor suppressor in colon (as reviewed in Rowland et al., 2006). The molecular mechanisms underlying this ambiguity are not very well understood. Moreover, nuclear localization of KLF4 was found to be a prognostic factor for an aggressive phenotype in breast cancers (Rowland et al., 2005). The tumor suppressor role of KLF4 has been shown to be via induction of p21-dependent cell cycle arrest, whereas by inhibiting B-cell lymphoma 2-associated X protein (Bax)/p53-tumorsupressor complex in the presence of pro-oncogenic signals, such as oncogenic RAS, KLF4 acts as a context dependent oncogene (Rowland et al., 2005; as reviewed in Müller et al, 2016). KLF4 protein with high stability has an oncogenic role in breast cancer (Hu et al., 2012) and reduced KLF4 stability is oncogenic in colorectal cancer (Gamper et al, 2012). The pluripotency transcription factors are co-expressed in different types of cancers such as breast cancer, squamous cell carcinoma, and gastrointestinal cancers (as reviewed in Müller et al, 2016). Reprogramming with the Yamanaka factors has been shown to generate a “cancer-poised” but not yet “cancer-committed” state (Ohnishi et al., 2014). Furthermore, three factors OCT4, KLF4 and SOX2 caused persistent dysplasia (Ohnishi et al., 2014). However, the presence of Yamanaka factors in various cancers supports the hypothesis that functional regulation of cancer cells appears to be very similar to the pluripotency status maintained by transcription factors of the pluripotency network (as reviewed in Müller et al, 2016). Based on my findings I propose that KLF4 protein stability modulation by interaction with the pluripotency transcription factors might be an important regulatory switch that determines the outcome of KLF4 signaling, thus affecting its role as an oncogene or tumor suppressor.

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4.2 Future Directions

The results described in my thesis have several areas inviting further investigation. In my investigation of KLF4 protein stability, I chose a biochemical assay using cyclohexamide (CHX) to inhibit protein synthesis and assess protein half-life by decay following CHX treatment. This is a straight forward way to assess half-life of a protein but there are some drawbacks to this method. Treating cells with a chemical to inhibit global protein synthesis could affect other metabolic activities and following cells for longer time points is difficult due to toxicity of the CHX compound. My results showed that the KLF4 protein is very stable in ES cells with a half-life longer than 24 hrs; however, as the CHX treatment was only carried out for 12 hr it is difficult to know the absolute stability of KLF4. Using another method such as pulse chase labeling with heavy stable isotope of nitrogen (15N) referred to metabolic labelling similar to Oda et al., 1999; McClatchy et al., 2007; Savas et al., 2012 and Toyama et al, 2013 would allow for longer investigation of labeled protein and a more accurate determination of KLF4 stability. In addition, I tested my hypothesis that KLF4 is stabilized by interaction with other transcription factors by using transient transfections of Sox2-t2A-GFP and Nanog-2A-GFP. This does not allow me to investigate how rapidly these proteins can function as their transcription and translation is required before their effect can be observed. It would be interesting to look at how dynamic these interactions are by using proteins that can be controlled at the level of nuclear localization. One way to evaluate that would be stably transfected Nanog and Sox2 constructs fused to an estrogen receptor (ER) ligand-binding domain; nuclear localization of these proteins can be controlled with drug treatment such as fulvestrant (David et al., 2012). Protein expressed from these constructs would remain in the cytoplasm until induced, allowing better temporal resolution in the experiments. Moreover, I observed KLF4 stability was altered through involvement in active transcriptional complexes and by association with pluripotency associated transcription factors and RNAPII-S5P. What remains unknown is whether it is the direct protein-protein interaction of KLF4 with pSTAT3, NANOG, and SOX2 that increases KLF4 stability or whether it is co- association to a larger complex containing these transcription factors and a currently unknown KLF4 partner protein that is responsible for KLF4 stability. Investigating the role of candidate KLF4 interaction domains contained in pSTAT3, NANOG and SOX2 and the role of these in KLF4 stability could clarify this point.

160

I chose to investigate KLF4 protein stability 24 hr after LIF/2i removal as at this time point KLF4 was again nuclear indicating there was no difference in localization of the KLF4 protein compared to undifferentiated ES cells. In addition, experiments I describe in Chapter 2 indicated that KLF4 protein that was nuclear at 24 hr was predominantly newly synthesized protein. The ERK activation burst that I observed at 6 hr was not apparent at 24 hr suggesting no involvement of the ERK pathway in mediating KLF4 protein at that time point. I found that KLF4 protein becomes unstable after removal of LIF/2i for 24 hr and monoubiquitination of nuclear KLF4 caused its export and degradation. It would be interesting to investigate the stability and monoubiquitination of KLF4 before 24 hr to determine how early these mechanisms start to be involved in the regulation of KLF4 function. I predict the stability of KLF4 would be low at 6 and 12 hr when KLF4 is predominantly cytoplasmic as mutation of the KLF4 NLS affected protein stability. I also observed reduced nuclear pSTAT3 as early as 6 hr and this would likely also cause reduced KLF4 stability.

It would be interesting to investigate naïve human ES cells to determine whether similar mechanisms regulating KLF4 function are important in pluripotency exit for human cells. If this is the case it would indicate that my findings could be important in designing differentiation and reprogramming approaches for human therapeutic purposes. In the context of development, it would be interesting to determine whether or not KLF4(S132A) mutant cells would differentiate appropriately in vivo. This would reveal whether or not nuclear export of KLF4 is critical in early development for exit from the pluripotent state. Based on my findings I predict that persistent nuclear KLF4 would be a dominant mutant phenotype that interferes with differentiation of the ICM in vivo resulting in incomplete embryogenesis.

Better understanding of regulatory mechanisms underlying pluripotency maintenance and exit would be useful to design improved efficiency of reprogramming mechanisms. Improved reprogramming efficiency is needed to generate clinical grade induced pluripotent stem cells which is a basic requirement for regenerative medicine procedures. As my data has shown some important sites in KLF4 protein that makes it more stable. Also the stability of KLF4 is known to maintain pluripotent state and its loss is necessary for pluripotency exit. Therefore, I predict designing KLF4 transgene with modifications such as S132A and K249R would improve reprogramming efficiency.

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In summary my work has revealed a new way in which the pluripotency transcription factors are interconnected to maintain the pluripotent state. Not only are these transcription factors required in a coordinated manner to maintain transcription of pluripotency genes they are also connected by in protein complexes that have a role in modulating protein stability and function. Also pluripotency associated transcription factors are co-expressed in a variety of cancers, similar regulation might occur in cancer cells. Designing strategies to disrupt these transcriptional complexes might be important in developing cancer treatments.

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Appendix 1

Figure A1.1: The schematic of mouse KLF4 and mutated/deleted sites.

(CTC & CTG CAA or CAG for Leucine[L] to Glutamine[Q])NES site1 mutation.

(GCC GGC for Alanine to Glycine)NES site2 mutation.

(TCC GCC for Serine to Alanine)Ser-132 phosphorylation site mutation.

(Lysine to arginine) K249 ubiquitination mutation

(Lysine to arginine) K275 sumoylation mutation

(Arginine1 to Ser; Arginine 2 to Ser; Arginine 3 to Glycine)NLS mutation.

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Figure A1.2: KLF4 orthologue sequences in different primate and non-primates.

>tr|F6VVB3|F6VVB3_MACMU Krueppel-like factor 4 OS=Macaca mulatta OX=9544 GN=KLF4 PE=2 SV=2

MRQPPGESDMAVSDALLPSFSTFASGPAGREKTLRQAGAPNNRWREELSHMKRLPPVLPGRPYDLAAATVATDLESG VAGAACGGSNLAPLPRRETEEFNDLLDLDFILSNSLTHPPESVAATVSSSASASSSSSPSSSGPASAPSTCSFTYPI RAGSDPGVAPGGTGGGLLYGRESAPPPTAPFNLADINDVSPSGGFVAELLRPELDPVYIPPQQPPPPGGGLMGKFVL KASLSAPGSEYGSPSVISVSKGSPDGSHPVVVAPYNGGPPRTCPKIKQEAVSSCTHLGAGPPLSNGHRPAAHDFPLG RQLPSRTTPTLGLEEVLSSRDCHPALPLPPGFHPHPGPNYPSFLPDQMQPQVPPLHYQELMPPGSCMPEEPKPKRGR RSWPRKRTATHTCDYAGCGKTYTKSSHLKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFQCQKCD RAFSRSDHLALHMKRHF

>tr|H2QXN2|H2QXN2_PANTR KLF4 isoform 3 OS=Pan troglodytes OX=9598 GN=KLF4 PE=2 SV=1

MRQPPGESDMAVSDALLPSFSTFASGPAGREKTLRQAGAPNNRWREELSHMKRLPPVLPGRPYDLAAATVATDLESG GAGAACGGSNLAPLPRRETEEFNDLLDLDFILSNSLTHPPESVAATVSSSASASSSSSPSSSGPASAPSTCSFTYPI RAGNDPGVAPGGTGGGLLYGRESAPPPTAPFNLADINDVSPSGGFVAELLRPELDPVYIPPQQPQPPGGGLMGKFVL KASLSAPGSEYGSPSVISVSKGSPDGSHPVVVAPYNGGPPRTCPKIKQEAVSSCTHLGAGPPLSNGHRPAAHDFPLG RQLPSRTTPTLGLEEVLSSRDCHPALPLPPGFHPHPGPNYPSFLPDQMQPQVPPLHYQELMPPGSCMPEEPKPKRGR RSWPRKRTATHTCDYAGCGKTYTKSSHLKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFQCQKCD RAFSRSDHLALHMKRHF

>tr|Q52JJ4|Q52JJ4_PIG Kruppel-like factor 4 isoform b OS=Sus scrofa OX=9823 GN=KLF4 PE=2 SV=1

MAVSDALLPSFSTFASGPAGREKTLRPAGAPNNRWREELSHMKQRLPPVLPGRPYDLAAATVATDLESGGVGAACGS SNPALLPRRETEEFNDLLDLDFILSNSLSHQESVAATVSSSASASSSSSPSSSGPASAPSTCSFSYPIRAGGDPGVA PGSTGGSLLYGRESAPPPTAPFNLADINDVSPSGGFVAELLRPELDPVYIPPQQSQPPGGGLMGKFVLKASLSAPGS EYGSPSVISVSKGSPDGSHPVVVAPYSGGPPRMCPKIKQEAVSSCTVGRPLEAHLGTGPPLSNGHRPPAHDFPLGRQ LPSRTTPTLGAEELLSSRDCHPALPLPPGFHPHHGPNYPPFLPDQLQPQVPPLHYQGQSRGIVVGAGEPCICRPSGA HGMVLTPPSSPLELMPPGSCMPEEPKPKRGRRSWPRKRTATHTCDYAGCGKTYTKSSHLKAHLRTHTGEKPYHCDWD GCGWKFARSDELTRHYRKHTGHRPFQCQKCDRAFSRSDHLALHMKRHF

>tr|Q923V7|Q923V7_RAT Gut-enriched kruppel-like factor OS=Rattus norvegicus OX=10116 GN=Klf4 PE=1 SV=1

MRQPPGESDMAVSDALLPSFSTFASGPAGREKTLRPAGAPTNRWREELSHMKRLPPLPGRPYDLAATVATDLESGGA GAACSSNNPALPRRETEEFNDLLDLDFILSNSLSHQESVAATVTTSASASSSSSPASSGPASAPSTCSFSYPIRAGG DPGVAAGNTGGGLLYSRESAPPPTAPFNLADINDVSPSGGFVAELLRPELDPVYIPPQQPQPPGGGLMGKFVLKASL STPGSEYTSPSVISVSKGSPDGSHPVVVAPYSGGPPRMCPKIKQEAVPSCTVSRSLEAHLSAGPQLSNGHRPNTHDF PLGRQLPTRTTPTLSPEELLNSRDCHPGLPLPPGFHPHPGPSYPPFLPDQMQSQVPSLHYQELMPPGSCLPEEPKPK RGRRSWPRKRTATHTCDYAGCGKTYTKSSHLKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFQCQ KCDRAFSRSDHLALHMKRHF

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>tr|Q7SZU5|Q7SZU5_XENLA Biklf-A protein OS=Xenopus laevis OX=8355 GN=klf17 PE=2 SV=1

MSVAFSTLQPVEDKQLGMVSVMAMEELRPLADMSYTMIEHCIKKEEDDLGKFVDLDFILAHTSSSQNGGTPVGHGGA YPLPETPESCSTTYDSDGSYTASQKYGGGSFAGSPHHSYVAELLTPDVPCIEVSPDFGLKAGMHSRKYTELRVSAMD TPGHLPSEHLQHLSHRIKKERPEQSCMLGAGSPTSPDCISPIMEQKAGIMQMHGQLLPNPAYSQHRLSPPVPTEDMP PNDCHMIPDMHCLSLMSMAQHYPMASPYQTHFTSQPNVQFHGQFGVYRDPMKVHPSMHGMIVTPPSSPLLEYYPAMA ATDDCKPKRGRRSWAKKRTATHSCEYPGCGKTYTKSSHLKAHMRTHTGEKPYHCNWEGCGWKFARSDELTRHFRKHT GHRPFQCHLCERAFSRSDHLALHMKRHM

>tr|Q90XE8|Q90XE8_DANRE Kruppel-like factor 4 OS=Danio rerio OX=7955 GN=klf17 PE=2 SV=1

MALADAMLPSINTFSNNHILDEKQSEIVRDWKVDIAKSNPRAGDVRPLIEVEFSIVESPPLAKDEDDLSKFLDLEFI LSNTVTSDNDNTSAPPPAYSLPESPESCSTVYDSDGCHPTPNAYCGTNFNSRPGHSLVAELFTPDMNYQGEYNLKGH LDRLEYTELRALNTRNQQHLTNSNNAGYKIKTENQEQSCMMVNDYMGHYYAQEPQRVMQHQTYGHHVQDVPRDILGR KDCILTEMNTQHHIDISHQQQFINNAHFPPQYAQHQQYHGHFNMFSEPLRANHPAMSGVMLTPPSSPLLGFLSPEDS KPKRGRRSWARKRTATHSCEFPGCGKTYTKSSHLKAHMRTHTGEKPYHCSWEGCGWKFARSDELTRHYRKHTGHRPF QCHLCERAFSRSDHLALHMKRHM

>sp|Q60793|KLF4_MOUSE Krueppel-like factor 4 OS=Mus musculus OX=10090 GN=Klf4 PE=1 SV=3

MRQPPGESDMAVSDALLPSFSTFASGPAGREKTLRPAGAPTNRWREELSHMKRLPPLPGRPYDLAATVATDLESGGA GAACSSNNPALLARRETEEFNDLLDLDFILSNSLTHQESVAATVTTSASASSSSSPASSGPASAPSTCSFSYPIRAG GDPGVAASNTGGGLLYSRESAPPPTAPFNLADINDVSPSGGFVAELLRPELDPVYIPPQQPQPPGGGLMGKFVLKAS LTTPGSEYSSPSVISVSKGSPDGSHPVVVAPYSGGPPRMCPKIKQEAVPSCTVSRSLEAHLSAGPQLSNGHRPNTHD FPLGRQLPTRTTPTLSPEELLNSRDCHPGLPLPPGFHPHPGPNYPPFLPDQMQSQVPSLHYQELMPPGSCLPEEPKP KRGRRSWPRKRTATHTCDYAGCGKTYTKSSHLKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFQC QKCDRAFSRSDHLALHMKRHF

>sp|O43474|KLF4_HUMAN Krueppel-like factor 4 OS=Homo sapiens OX=9606 GN=KLF4 PE=1 SV=3

MRQPPGESDMAVSDALLPSFSTFASGPAGREKTLRQAGAPNNRWREELSHMKRLPPVLPGRPYDLAAATVATDLESG GAGAACGGSNLAPLPRRETEEFNDLLDLDFILSNSLTHPPESVAATVSSSASASSSSSPSSSGPASAPSTCSFTYPI RAGNDPGVAPGGTGGGLLYGRESAPPPTAPFNLADINDVSPSGGFVAELLRPELDPVYIPPQQPQPPGGGLMGKFVL KASLSAPGSEYGSPSVISVSKGSPDGSHPVVVAPYNGGPPRTCPKIKQEAVSSCTHLGAGPPLSNGHRPAAHDFPLG RQLPSRTTPTLGLEEVLSSRDCHPALPLPPGFHPHPGPNYPSFLPDQMQPQVPPLHYQGQSRGFVARAGEPCVCWPH FGTHGMMLTPPSSPLELMPPGSCMPEEPKPKRGRRSWPRKRTATHTCDYAGCGKTYTKSSHLKAHLRTHTGEKPYHC DWDGCGWKFARSDELTRHYRKHTGHRPFQCQKCDRAFSRSDHLALHMKRHF

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Figure A1.3: Mouse KLF4 homologue, KLF2 and KLF5 sequences

>sp|Q60843|KLF2_MOUSE Krueppel-like factor 2 OS=Mus musculus OX=10090 GN=Klf2 PE=1 SV=2

MALSEPILPSFATFASPCERGLQERWPRNEPEAGGTDEDLNNVLDFILSMGLDGLGAENPPEPPPQPPPPAFYYPEP GAPPPYSIPAASLGTELLRPDLDPPQGPALHGRFLLAPPGRLVKAEPPEVDGGGYGCAPGLAHGPRGLKLEGAPGAT GACMRGPAGRPPPPPDTPPLSPDGPLRIPASGPRNPFPPPFGPGPSFGGPGPALHYGPPAPGAFGLFEDAAAAAAAL GLAPPATRGLLTPPSSPLELLEAKPKRGRRSWPRKRAATHTCSYTNCGKTYTKSSHLKAHLRTHTGEKPYHCNWEGC GWKFARSDELTRHYRKHTGHRPFQCHLCDRAFSRSDHLALHMKRHM

>sp|Q60793|KLF4_MOUSE Krueppel-like factor 4 OS=Mus musculus OX=10090 GN=Klf4 PE=1 SV=3

>sp|Q9Z0Z7|KLF5_MOUSE Krueppel-like factor 5 OS=Mus musculus OX=10090 GN=Klf5 PE=1 SV=2

MPTRVLTMSARLGPLPQPPAAQDEPVFAQLKPVLGAANPARDAALFSGDDLKHAHHHPPAPPPAAGPRLPSEELVQT RCEMEKYLTPQLPPVPIISEHKKYRRDSASVVDQFFTDTEGIPYSINMNVFLPDITHLRTGLYKSQRPCVTQIKTEP VTIFSHQSESTAPPPPPAPTQALPEFTSIFSSHQTTAPPQEVNNIFIKQELPIPDLHLSVPSQQGHLYQLLNTPDLD MPSSTNQTAVMDTLNVSMAGLNPHPSAVPQTSMKQFQGMPPCTYTMPSQFLPQQATYFPPSPPSSEPGSPDRQAEML QNLTPPPSYAATIASKLAIHNPNLPATLPVNSPTLPPVRYNRRSNPDLEKRRIHFCDYNGCTKVYTKSSHLKAHLRT HTGEKPYKCTWEGCDWRFARSDELTRHYRKHTGAKPFQCMVCQRSFSRSDHLALHMKRHQN

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Figure A1.4: Conservation of NES sites among primate and non-primate species.

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Figure A1.5: Conservation of S132, K249, and K275 sites among primate and non-primate species.

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Figure A1.6: Conservation of S132, K249, and K275 sites among other KLFs expressed in mammalian ES cells (KLF2, KLF5)

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Appendix 2

Table A2.1: Half life summary table

Protein Condition Half life

KLF4 +LIF/2i 24.23±0.15

KLF4 24 hr -LIF/2i 1.46±2.34

OCT4 +LIF/2i 4.06±0.15

OCT4 24 hr -LIF/2i 4.02±0.23

SOX2 +LIF/2i 2.56±1.12

SOX2 24 hr -LIF/2i 3.03±3.10

NANOG +LIF/2i 3.20±0.15

NANOG 24 hr -LIF/2i 3.57±0.57

KLF4 in Δ2129 +LIF/2i 23.29±0.43

KLF4 in Δ2129 24 hr -LIF/2i 1.53±0.01

KLF4 in Δ2129/CAST +LIF/2i 22.32±1.10

KLF4 in Δ2129/CAST 24 hr -LIF/2i 1.56±0.03

KLF4 in ΔSCR +LIF/2i 1.54±0.04

KLF4 in ΔSCR 24 hr -LIF/2i 1.31±0.20

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KLF4 24 hr -LIF 6.18±0.34

KLF4 24 hr -MEKi 3.16±0.03

KLF4 24 hr –GSK3i 22±0.13

KLF4(WT)-GFP 24 hr -LIF 7.08±2.45

KLF4(WT)-GFP 24 hr -MEKi 3.40±2.01

KLF4(WT)-GFP 24 hr –GSK3i 22.10±1.10

KLF4(WT)-GFP +LIF/2i 25.57±2.23

KLF4(WT)-GFP 24 hr -LIF/2i 1.54±0.04

KLF4(S132A)-GFP +LIF/2i 53.12±0.70

KLF4(S132A)-GFP 24 hr -LIF/2i 51.40±1.39

KLF4(NES1)-GFP +LIF/2i 56.08±3.35

KLF4(NES1)-GFP 24 hr -LIF/2i 54.21±3.25

KLF4(K249R)-GFP +LIF/2i 53±0.70

KLF4(K249R)-GFP 24 hr -LIF/2i 53±4.61

KLF4(K275R)-GFP +LIF/2i 3±0.11

KLF4(K275R)-GFP 24 hr -LIF/2i 3±0.09

KLF4(NLS)-GFP +LIF/2i 1.27±0.18

KLF4(NLS)-GFP 24 hr -LIF/2i 1.13±0.13

KLF4(ΔZNF)-GFP +LIF/2i 3±0.10

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KLF4(ΔZNF)-GFP 24 hr -LIF/2i 3±0.11

KLF4(S132A, K275R)-GFP +LIF/2i 54±3.17

KLF4(S132A, K275R)-GFP 24 hr -LIF/2i 54±3.2

KLF4(S132A, ΔZNF)-GFP +LIF/2i 56±3.35

KLF4(S132A, ΔZNF)-GFP 24 hr -LIF/2i 54±3.25

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