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The Role of Mek/Erk Signalling Inhibition and Krüppel‑Like Factor 2 in Mouse Ground State Pluripotency

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The Role of Mek/Erk Signalling Inhibition and Krüppel‑Like Factor 2 in Mouse Ground State Pluripotency This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. The role of Mek/Erk signalling inhibition and Krüppel‑like factor 2 in mouse ground state pluripotency Yeo, Jia Chi 2015 Yeo, J. C. (2015). The role of Mek/Erk signalling inhibition and Krüppel‑like factor 2 in mouse ground state pluripotency. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/62942 https://doi.org/10.32657/10356/62942 Downloaded on 10 Oct 2021 22:21:26 SGT The Role of Mek/Erk Signalling Inhibition and Krüppel-like Factor 2 in Mouse Ground State Pluripotency YEO JIA CHI B.Sc (Hons) School of Biological Sciences 2015 The Role of Mek/Erk Signalling Inhibition and Krüppel-like Factor 2 in Mouse Ground State Pluripotency YEO JIA CHI B.Sc (Hons) School of Biological Sciences A thesis submitted to the Nanyang Technological University in partial fulfillment of the requirement for the degree of Doctor of Philosophy 2015 Acknowledgements This thesis would not have been possible for the numerous people who have extended their guidance and help throughout these 4 years. Among these, I would firstly like to thank my PhD supervisors Dr Ng Huck Hui, as well as Dr Su I-Hsin, for their invaluable teaching, advice and support in allowing me to explore and cultivate my scientific interests. I also like to extend my appreciation to Dr Jiang Jianming and Qi Lihua who have been instrumental in getting me up to speed on this project on ground state pluripotency when I first started my PhD. My laboratory colleagues at the Genome Institute of Singapore also played a big role in helping me throughout my research. These people include Dr Winston Chan, Dr Ng Jia Hui, Dr Lu Xinyi, Dr Daniel Yim, Dr Liang Hongqing, Dr Jonathan Goke, Dr Chuti Laowtammathron, Tan Zi Ying, Kevin Gonzales, Friedrich Sachs, Bobby Tan, Lim Yee Siang, and Yaw Lai Ping. My research collaborators have also played critical roles in my research with their kind assistance with various experiments. My thesis may not have been completed if it were not for their help. I am grateful to Dr Thomas Lufkin and Dr Petra Kraus for generating the Klf2-LacZ transgenic mice, as well as for their help in teaching me the various mouse experimental procedures. I also thank Dr Andrew Tan, as well Kelvin Chong for their help in performing the Proximity Ligation Assay (PLA), which helped linked my data coherently. I would also like to thank my thesis advisory committee members Dr Andrew Tan and Dr Newman Sze for their time and advice throughout my PhD. I would like to thank my parents and friends for their understanding and support for my pursuit in scientific research. I also thank Rachel Lim for walking with me through these 4 years, and I am greatly indebted to her. Lastly, I want to thank God for his strength and blessings in sustaining me. I also eagerly await the exciting things that God has in store for me. List of Publications Peer-Reviewed Articles 1. Yeo JC, Jiang J, Tan ZY, Yim GR, Ng JH, Göke J, Kraus P, Liang HQ, Gonzales KA, Chong HC, Tan CP, Lim YS, Tan NS, Lufkin T, Ng HH “Klf2 Is an Essential Factor that Sustains Ground State Pluripotency” Cell Stem Cell (2014) Jun 5; 14(6) : 864-72 2. Ng JH, Kumar V, Muratani M, Kraus P, Yeo JC, Yaw LP, Xue K, Lufkin T, Prabhakar S, Ng HH "In vitro epigenomic profiling of germ cells reveals germ cell molecular signatures." Dev Cell (2013) Feb 11 ; 24(3) : 324-33 3. Percharde M, Lavial F, Ng JH, Kumar V, Tomaz RA, Martin N, Yeo JC, Gil J, Prabhakar S, Ng HH, Parker MG, Azuara V "Ncoa3 functions as an essential Esrrb coactivator to sustain embryonic stem cell self-renewal and reprogramming." Genes Dev (2012) Oct 15; 26(20):2286-98 Review Articles 4. Yeo JC, Ng HH "The transcriptional regulation of pluripotency." Cell Res (2013) Jan ; 23(1) : 20-32 5. Yeo JC, Ng HH. “Transcriptomic analysis of pluripotent stem cells: insights into health and disease.” Genome Med. (2011) Oct 27; 3(10): 68. doi: 10.1186/gm284. i Table of Contents List of Figures vi List of Tables ix List of Abbreviations x Work performed by collaborators xiii Abstract xiv 1. Introduction 2 1.1 - Mouse embryonic stem cells (mESCs) 3 1.1.1 – Origins and applications of mESCs 3 1.1.2 – The transcriptional regulatory network of mESCs 4 1.1.3 – External signalling and the integration with the ESC transcriptional 11 regulatory core 1.2 - Mouse epiblast stem cell (EpiSCs) 14 1.2.1 – Origins and the pluripotential of EpiSCs 14 1.2.2 – The concept of “naïve” and “primed” pluripotency 16 1.2.3 – Interconversion between naïve and primed pluripotent states 18 1.3 – Ground state pluripotency in mESCs 20 1.3.1 – mESC culture via dual Fgf/Mek/Erk and Gsk3/Tcf3 pathway 20 inhibition 1.3.2 – Characteristics of 2i ground state cultured mESCs 22 1.3.3 – 2i ground state mESCs resemble E4.5 epiblast cells of the 25 pre-implantation embryo 1.4 – Dissecting the mechanism of ground state pluripotency 27 ii 1.4.1 – Gsk3-inhibition alleviated Tcf3 mediated transcriptional repression 27 of Esrrb 1.4.2 – The Klf transcription factors 28 1.4.3 – The role of Klf proteins in mESCs and their association with 32 ground state pluripotency 1.5 – Objectives of this study 35 2. Materials and Methods 37 2.1 – Cell culture 38 2.2 – Generation of Klf2-LacZ reporter mice 39 2.3 – Embryo collection and mESC derivation 41 2.4 – Proximity ligation assay (PLA) 41 2.5 – Cell cycle and apoptosis detection 42 2.6 – RNAi knockdown 42 2.7 – RNA extraction, reverse transcription and quantitative real-time PCR 43 2.8 – Microarray analysis 45 2.9 – Southern blot and PCR genotyping 46 2.10 – Transfection, Co-immunoprecipitation and Immunoblotting 48 2.11 – Generation of stable Klf2 expressing Klf2-null mESCs 49 2.12 – Recombinant GST-Klf2 expression and in vitro kinase assay 50 2.13 – Chromatin immunoprecipitation (ChIP) and ChIP-seq analysis 51 3. Results 53 3.1 – Klf2 is negatively regulated by Mek/Erk signalling 54 iii 3.1.1 – Gene expression changes of Klf2, Klf4 and Klf5 during ground 54 state pluripotency 3.1.2 – Protein level changes of Klf2, Klf4 and Klf5 during ground state 56 pluripotency 3.1.3 – Klf2 protein is negatively regulated by the Mek/Erk pathway 58 3.1.4 – Klf2 protein is proteasomally degraded in mESCs 61 3.2 – Klf2 directly interacts with, and is phosphorylated by, Erk2 63 3.2.1 – Bioinformatics analysis of Klf2 reveals potential Erk 63 phosphorylation motifs 3.2.2 – Klf2 is phosphorylated in mESCs 65 3.2.3 – Klf2 physically interacts with Erk2 protein 66 3.2.4 – Erk2 is able to phosphorylate Klf2 in vitro 69 3.3 – Klf2 is essential for mESCs under 2i conditions 70 3.3.1 – Generation of Klf2 knockout mice 70 3.3.2 – Derivation of Klf2-null mESCs using LIF+KSR 74 3.3.3 – Functional validation of Klf2-null mESCs 75 3.3.4 – Loss of Klf2 in mESCs results in cell death under 2i 77 3.3.5 – Klf2 overexpression can restore self-renewal in 2i-cultured 82 Klf2-null mESCs 3.3.6 – Klf2 overexpression confers independence from 83 Mek-inhibition during ground state pluripotency 3.3.7 – Klf2 is important for the establishment of mESCs under 2i 84 conditions 3.4 – Klf2-null mESCs exhibit aberrant PGC gene expression under 86 2i conditions iv 3.4.1 – Klf2-null mESCs retain a pluripotent signature despite 86 massive apoptosis under 2i conditions 3.4.2 – Klf2-null mESCs under 2i exhibit aberrant PGC gene expression 88 3.4.3 – Klf2 binds to the promoters of key PGC genes 90 4. Discussion 94 4.1 – Model of Klf regulation in LIF/Serum and 2i mESC conditions 95 4.2 – Varying potencies of Klf2, Klf4 and Klf5 in mESCs 97 4.3 – Post-translational modification of Klf2 by the Mek/Erk signalling 99 pathway 4.4 – The role of Klf2 in pre-implantation embryonic development 101 4.5 – The potential role of Klf2 in mESC metabolic regulation 102 4.6 – The association of KLF2 with naïve hESC pluripotency 107 4.7 – Future perspectives and conclusion 112 5. References 115 6. Appendix 129 A – List of the top 200 upregulated genes in Klf2-null mESCs 130 (relative to Klf2-WT) under LIF/Serum, 2i passage 1, 2, and 3 B – List of the top 200 downregulated genes in Klf2-null mESCs 138 (relative to Klf2-WT) under LIF/Serum, 2i passage 1, 2, and 3 C – Author reprints of articles by Yeo Jia Chi 146 v List of Figures Fig. 1 – The classical ESC Transcriptional Network 7 Fig. 2 – An alternative view: ESC transcription factors as lineage specifiers 10 Fig. 3 – Development of the mouse epiblast from pre-implantation to 15 post-implantation stage Fig. 4 – Molecular differences between mESCs and EpiSCs 17 Fig. 5 – The maintenance of mESC identity using chemical inhibitors 21 Fig. 6 – The molecular features of ground state pluripotent mESCs 24 Fig. 7– Phylogenetic tree of human KLFs 31 Fig. 8 – The transcriptional regulation of Klf2, Klf4 and Klf5 in mESCs 34 Fig. 9 – Gene targeting of Klf2 loci 40 Fig. 10 – Gene expression changes of key mESC genes under LIF/Serum or 2i 55 culture Fig. 11 – Western blot for key mESC factors in LIF/Serum or 2i (P3) conditions 56 Fig.
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