Roles of long non coding RNAs in human embryonic stem cell pluripotency and neural differentiation 1

ROLES OF LONG NON-CODING RNAS IN HUMAN EMBRYONIC STEM CELL PLURIPOTENCY AND NEURAL DIFFERENTIATION NG SHI YAN B Sc (Honors) National University of Singapore, 2008 A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2012 Acknowledgements I would like to express my deepest gratitude to the many people who made this thesis possible I thank my supervisor Dr Lawrence Stanton for his invaluable guidance, advice, support and his belief in me His mentorship encouraged independence, creativity, and allowed me the freedom to grow and develop I have learnt a lot during the past four years, which has been an extremely enriching and inspiring experience I would also like to thank my thesis advisory committee, Dr Wang Hongyan, Dr Paul Robson, and Dr Gerald Udolph, for their critical feedback along the way I am especially grateful to Dr Rory Johnson, who introduced me to the world of lncRNAs I am thankful for the insightful discussions we had, the custom lncRNA microarray which he designed, as well as his feedback and comments I also thank Gireesh Bogu, for the RNA-seq analyses Dr Irene Aksoy was most generous in providing me with a human iPS cell line I am also thankful for stimulating discussions with Dr Akshay Bhinge It is also a pleasure to thank everyone in the GIS Stem Cell groups for companionship, support and helpful discussions Most importantly, my family has given me incredible support and encouragement Their love and understanding helped me overcome the obstacles in the process My parents have provided me with an incredible environment to grow and nurture; my sister was amazingly supportive, understanding and a great companion I also thank my fiancé for his patience and companionship, who made long hours in the lab more pleasant   ii  Table of contents Acknowledgements ii Table of contents iii Abstract vii List of figures ix List of tables xiii Abbreviations xiv Chapter I: Introduction ………………………………………………………… 1.1 Transcriptional control of stem cell pluripotency 1.1.1 The transcription factors OCT4, NANOG and SOX2 constitute the human embryonic stem cell transcriptional core 1.1.2 An expanded transcriptional regulatory circuit maintaining pluripotency 1.1.3 Non-coding RNAs modulate pluripotency by regulating expression of the core transcription factors and/or downstream genes 1.2 Directed neural differentiation of hESCs 1.2.1 1.2.1.1 1.2.1.2 1.2.2 1.2.3 Neural induction – lessons from the embryo Inhibiting the TGF-β signaling pathway enhances neural induction Stromal co-culture induces neural conversion Regional specification of midbrain dopamine neurons Radial glia cells are neuronal progenitors in vivo 10 11 14 1.3 Long non-coding RNAs in biology 15 1.3.1 1.3.2 1.3.2.1 1.3.2.2 1.3.2.3 Long non-coding RNAs in pluripotency Long non-coding RNAs in neural development Nkx2.2AS Evf2 Malat1 16 19 19 20 20 1.4 Molecular mechanisms of long non-coding RNA function 21 1.4.1 LncRNAs behave as scaffolds that target protein complexes to specific genomic loci to regulate gene transcription 23 1.4.2 LncRNAs with enhancer-like functions 24 1.4.3 LncRNAs regulate gene expression by behaving as competing endogenous RNAs or promoting mRNA decay 24 1.4.4 Other molecular functions of lncRNAs 25 Chapter II: Aims and Objectives ……………………………………………… 26 2.1 Main goals of the thesis 26 2.2 Thesis outline 27 Chapter III: Materials and Methods …………………………………………… 28 3.1 Feeder-free culture of human pluripotent stem cells 28 3.2 Expansion and mitotic inactivation of MEF cells 28 3.3 Preparation of MEF-conditioned medium 29   iii  3.4 Culture of PA6 mouse skull bone marrow stromal cells 29 3.5 Differentiation of hESCs into neural progenitors and dopaminergic neurons 30 3.5.1 3.5.2 3.5.3 Co-culture of PA6 and hESCs for stromal-derived inducing activity (SDIA) Isolation and expansion of neural progenitor cells (NPCs) Differentiation of NPCs into dopaminergic (DA) neurons 30 30 31 3.6 VM) Culture of human fetal mesencephalon-derived neural stem cells (ReN32 3.6.1 3.6.2 Maintenance of ReN-VM cells Differentiation of ReN-VM cells 32 32 3.7 Small-interfering RNA (siRNA)-mediated gene silencing 33 3.7.1 3.7.2 Transfection of H1 hESCs with siRNAs Transfection of ReN-VM cells with siRNAs 33 33 3.8 3.9 Stably transfected hESC lines RNA extraction 34 35 3.9.1 3.9.2 Extraction of total RNA Extraction of nuclear and cytoplasmic RNA (RNA fractionation) 35 35 3.10 3.11 3.12 Reverse transcription of RNA to cDNA Quantitative real-time PCR (qPCR) Analysis of gene expression by microarray 36 36 39 3.12.1 3.12.2 3.12.3 3.12.4 RNA amplification for Illumina bead chips Illumina bead chip hybridization RNA amplification and hybridization on custom designed Agilent arrays Statistical analysis of microarray data 39 40 40 40 3.13 RNA-sequencing (RNA-seq) 41 3.13.1 RNA-seq library preparation 3.13.2 RNA-seq data analysis 41 42 3.14 3.15 3.16 3.17 43 44 45 46 Western blot Immunocytochemistry Fluorescence-activated cell sorting (FACS) Co-immunoprecipitation (co-IP) 3.17.1 co-IP with overexpression constructus 3.17.2 Endogenous co-IP 46 46 3.18 3.19 3.20 3.21 47 48 49 50 Chromatin immunoprecipitation (ChIP) RNA immunoprecipitation (RIP) Biotinylated RNA pulldown RNA fluorescence in situ hybridization Chapter IV: Neural Differentiation of Human Pluripotent Stem Cells ……… 52 4.1 Introduction 52 4.2 Results 53 4.2.1 A homogenous population of neural progenitors was derived from hESCs by the modified SDIA method 53 4.2.2 Human ESC-derived neural progenitors differentiated into functional dopamine neurons with high efficiency 60 4.3 Discussion 67 4.3.1 Stromal induction induced neuronal differentiation from pluripotent stem cells with high efficiency 67 4.2.3 High efficiency neuronal differentiation by chemically defined methods 67 4.4   Conclusion 68 iv  Chapter V: Identification of Long Non-coding RNAs Associated Pluripotency and Neural Differentiation ……………………………………… 5.1 Introduction 5.2 Results with 70 70 72 5.2.1 Microarray expression profiling identifies differentially expressed lncRNAs 72 5.2.2 Identification of lncRNAs associated with pluripotency (pluripotent lncRNAs) 74 5.2.3 Identification of lncRNAs associated with neural progenitors (NPC lncRNAs) 77 5.2.4 Identification of lncRNAs associated with neuronal differentiation (neuronal lncRNAs) 79 5.3 5.4 Discussion Conclusion 81 82 Chapter VI: Long Non-coding RNAs Regulate Human Embryonic Stem Cell Pluripotency ……………………………………………………………………… 83 6.1 Introduction 83 6.2 Results 84 6.2.1 6.2.2 6.2.3 6.2.4 Screening for possibly functional pluripotent lncRNAs Pluripotent lncRNAs are regulated by transcription factors Knockdown of lncRNAs result in hESC differentiation Pluripotent lncRNAs physically associate with SUZ12 and SOX2 84 91 94 98 6.3 Discussion 101 6.3.1 6.3.2 LncRNAs join the pluripotency alliance LncRNAs possibly function as a modular scaffold 101 101 6.4 Conclusion 104 Chapter VII: Long Non-coding RNAs are Indispensable in Neurogenesis 105 7.1 Introduction 105 7.2 Results 107 7.2.1 7.2.2 7.2.3 7.2.4 Screening for possibly functional neuronal lncRNAs Neuronal lncRNAs are required for neuronal differentiation Neuronal lncRNAs support neurogenesis by associating with nuclear proteins Cytoplasmic lncRNA_N2 affects microRNA expression 107 112 118 121 7.3 Discussion 122 7.3.1 7.3.2 Neuronal lncRNAs act via diverse mechanisms Neuronal lncRNAs form part of a repressive complex to silence glia genes 122 122 7.4 Conclusion 124 Chapter VIII: Brain lncRNA RMST Regulates Neurogenesis by Association with SOX2 ……………………………………………………………………………… 125 8.1 Introduction 125 8.2 Results 127 8.2.1 RMST is highly expressed in the human brain and upregulated during neurogenesis 127 8.2.2 RMST is developmentally regulated by transcription factor REST 129 8.2.3 RMST is indispensable for neurogenesis, but not required for maintenance of neuronal identity 132 8.2.4 Nuclear-retained RMST physically associates with RNA-binding protein hnRNPA2B1 and transcription factor SOX2 134 8.2.5 RMST and SOX2 co-regulate a common pool of genes 140 8.2.6 RMST does not regulate SOX2 expression 144 8.3 Discussion 145 8.3.1 RMST forms part of a complex that is required for neurogenesis 145   v  8.3.2 8.3.3 RMST may change the binding patterns of SOX2 to chromatin RMST may bind to proteins other than hnRNPA2B1 and SOX2 146 147 8.4 Conclusion 147 Chapter IX: Conclusion and Perspectives ……………………………………… 149 9.1 Overall conclusions 149 9.1.1 9.1.2 9.1.3 Identification of functional human lncRNAs 149 LncRNAs specific to hESCs maintain the pluripotent state 150 Neuronal lncRNAs support neurogenesis by associating with transcription factors 150 9.2 Limitations and future work 151 9.2.1 9.2.2 9.2.3 9.2.4 9.2.5 Discovery of novel lncRNAs Epigenetic regulation of pluripotency RMST modulation SOX2 activity SOX2 and lncRNA association Long non-coding RNAs or short peptides 151 151 152 152 153 9.3 Concluding remarks 153 References ………………………………………………………………………… 155 Appendices ……………………………………………………………………… 169 Appendix I: List of 152 genes downregulated upon RMST and SOX2 knockdown 169 Appendix II: List of 331 genes upregulated upon RMST and SOX2 knockdown 170   vi  Abstract Long non-coding RNAs (lncRNAs) are a recently discovered class of transcripts encoded within the human genome LncRNAs have been proposed to be key regulators of biological processes, including stem cell pluripotency and neurogenesis However, at present very little functional characterization of lncRNAs involved in differentiation has been carried out in human cells In this thesis, functional characterization of lncRNAs in human development is addressed using human embryonic stem cells (hESCs) as a paradigm for pluripotency and neuronal differentiation Human ESCs were robustly and efficiently differentiated into neurons, and expression of lncRNAs was profiled using a custom-designed microarray Some hESC-specific lncRNAs involved in pluripotency maintenance were identified, and shown to physically interact with SOX2, and PRC2 complex component, SUZ12 Using a similar approach, we identified lncRNAs required for neurogenesis Knockdown studies indicated that loss of any of these lncRNAs blocked neurogenesis, and immunoprecipitation studies revealed physical association with REST and SUZ12 In particular, a neuronal lncRNA, RMST, was found to be essential for neurogenesis Knockdown of RMST in human neural stem cells prevented neurogenesis RNA pulldown and RNA immunoprecipitation indicated that RMST physically associated with the RNA-binding protein hnRNPA2B1 and the transcription factor SOX2 Perturbation studies, followed by genome-wide transcriptional profiling indicated that RMST and SOX2 co-regulate a large pool of targets Interestingly, knockdown of RMST resulted in reduced SOX2 occupancy at its   vii  target gene promoters, suggesting that RMST may alter SOX2 binding to chromatin during neurogenesis Together, this study represents important evidence for an indispensable role of lncRNAs in human brain development   viii  List of figures Figure 1.1 OCT4, SOX2 and NANOG form the core transcription factors governing pluripotency of hESCs Figure 1.2 An extended ES transcriptional network and regulatory circuit Figure 1.3 Patterning of the neural tube generates unique domains for neuronal progenitors 13 Figure 1.4 The differentiation of pluripotent stem cells into neuroepithelial stem cells and radial glia 15 Figure 1.5 Correlation of expression profiles of lncRNAs with protein-coding gene markers during embryoid body (EB) differentiation 17 Figure 1.6 A model for lincRNA integration into the molecular circuitry of embryonic stem cells 18 Figure 1.7 Paradigms for how lncRNAs function at the molecular level Figure 4.1 Stromal co-culture of H1 hESCs resulted in neural differentiation 55 Figure 4.2 Differentiation of hESCs into a monolayer neural progenitor population 57 Figure 4.3 Neural progenitors derived from H1 hESCs (H1-NPCs) homogenously expressed neural stem cell and radial glia markers 59 Figure 4.4 Schematic representation of the differentiation of hESC-derived radial glia-like NPCs into midbrain dopaminergic (DA) neurons 61 Figure 4.5 Immunofluorescence characterization of H1-derived neurons indicating their midbrain dopaminergic identity 62 Figure 4.6 Quantitative PCR (qPCR) analysis confirming midbrain dopaminergic identity 63 Figure 4.7 Flow cytometry analysis showed that H1-NPCs differentiated into TH+ midbrain dopamine neurons with high efficiency 65 Figure 4.8 Dopamine enzyme-linked immunosorbent assay indicated that H1derived dopamine neurons were responsive to depolarization 66 Figure 5.1 Microarray expression profilinf identified diferentially expressed lncRNAs during neural differentiation of hESCs 74   22 ix  Figure 5.2 Identification of lncRNAs important in pluripotency, neural induction, and neuronal differentiation 75 Figure 6.1 Three lncRNAs were exclusively expressed in hESCs and iPSCs 87 Figure 6.2 Pluripotent lnRNAs are low abundance transcripts Figure 6.3 RNA-seq analysis of pluripotent lncRNAs in H1 hESCs, indicating transcriptional start and end sites 90 Figure 6.4 Schematic showing OCT4 and NANOG binding sites in the vicinity of the lncRNAs 92 Figure 6.5 Pluripotent lncRNAs are possibly regulated by OCT4 and NANOG 93 Figure 6.6 Pluripotent lncRNAs can be effectively targeted by siRNAs Figure 6.7 Knockdown of pluripotent lncRNAs resulted in hESC differentiation 95 Figure 6.8 Knockdown of pluripotent lncRNAs resulted in loss of OCT4 Figure 6.9 Microarray analysis indicated that knockdown of pluripotent lncRNAs caused hESC differentiation 97 Figure 6.10 Pluripotent lncRNAs are preferentially localized in the nucleus Figure 6.11 Nuclear pluripotency lncRNAs physically associated with PRC2 component SUZ12, and the pluripotent transcription factor SOX2 100 Figure 6.12 Proposed mechanism for role of lncRNA in hESC pluripotency Figure 6.13 In silico prediction of lncRNA-protein interactions supporting the proposed mechanism of lncRNAs functioning as a modular scaffold for chromatin modifiers and transcription factors 103 Figure 7.1 In situ hybridization images of lncRNA expression showing that lncRNAs were specifically localized to the specific brain regions 106 Figure 7.2 RNA-seq analysis indicating transcription start and end sites of neuronal lncRNAs 110 Figure 7.3 Tissue specificity of the neuronal lncRNAs RMST, lncRNA_N1, lncRNA_N2 and lncRNA_N3 111 Figure 7.4 Relative abundance of neuronal lncRNAs, compared to that of GAPDH mRNA levels 112   88 94 96 98 102 x  Figure 4.1: Stromal co-culture of H1 hESCs resulted in neural differentiation (A) Following co-culture with PA6 stromal cells for days, H1 cells showed 80.5% and 66.3% decrease in OCT4 and NANOG mRNA expression respectively (B) At the same time, qPCR indicated that there was approximately 6-fold increased expression of neuroectodermal markers PAX6 and MSI1, and a 12-fold increase in SOX1 expression (C) Changes in mRNA expression in (A) and (B) were also detected at the protein level, by Western blot   55  In order to enrich for the NPC population, the differentiation hESC colonies were separated from the stromal cells by collagenase treatment Exposure to collagenase lifted the differentiating hESC colonies while the stromal cells remained adherent To effectively remove all stromal cells, the cell suspension was incubated on a gelatin-coated dish at 37 °C for an hour The suspended cell clusters were then broken into small clumps of less than 50 cells by vigorous pipetting and cultured as neural aggregates or spheres in low attachment plates, in neural proliferation medium with N2 supplement, bFGF and EGF The spheres were purified every to days by removing cystic structures under a stereomicroscope, vigorous pipetting to break the spheres into smaller aggregates, and replating on low-attachment plates Within three weeks, morphologically homogenous spheres were obtained These spheres were then trypsinized into single cells and expanded as a monolayer NPC population A schematic representation of the differentiation procedure is shown in Figure 4.2A The monolayer NPCs were maintained for at least 15 passages, and were karyotypically normal (Figures 4.2B-C)   56  Figure 4.2: Differentiation of hESCs into a monolayer neural progenitor population (A) Schematic representation of the differentiation of hESCs into NPCs by the modified SDIA method Human ESCs grown in feeder-free conditions was induced to differentiate by co-culturing with stromal feeder cells, termed stromal induction or SDIA Nine days later, the differentiating cells were separated from the stromal feeders and grown as aggregates called “neural spheres” These spheres were purified by periodically breaking them into smaller spheres and removing cystic structures A homogenous culture was obtained after a week under the suspension culture condition To obtain a monolayer culture, the spheres were trypsinized into single cells and plated onto Matrigel-coated tissue culture plates The NPCs were cultured in the presence of N2 supplement, bFGF and EGF The entire differentiation procedure took 21 days (B) Brightfield micrograph of the NPCs at passage number (P5) Scale bars represent 100 µm Cells at this stage were still proliferative and there were enough metaphase cells at this stage for karyotyping (C) Karyotype analysis of radial glia-like NPCs at P5 indicates that they have a normal XY chromosome profile   57  The neural progenitor cells expanded from the spheres were characterized by immunofluorescence, flow cytometry, and qPCR analyses These NPCs did not organize into rosette structures, but expanded as flat elongated cells in a monolayer, expressing neural progenitor markers NESTIN, MSI1 (Figures 4.3A-A’) and radial glia markers VIMENTIN (VIM), GFAP, and BLBP (Figures 4.3B-C’), indicating that they resembled radial glia cells Flow cytometry analyses indicated that almost all the NPCs generated by this protocol expressed NESTIN and VIM (more than 99%), while the vast majority also expressed BLBP (86%) and GFAP (92%) (Figure 4.3D) The main advantage of this protocol was that a homogeneous population of radial glia-like neural progenitors expressing NES, VIM, BLBP and GFAP could be derived from undifferentiated hESCs These NPCs were expandable for at least 15 passages in the presence of mitogens bFGF and EGF to produce large numbers of cells for subsequent differentiation In addition, these radial glia-like cells were karyotypically normal and could be cryopreserved with high cell viability   58  Figure 4.3: Neural progenitors derived from H1 hESCs (H1-NPCs) homogenously expressed neural stem cell and radial glia markers (A, A’) MUSASHI-1 (MSI1) and NESTIN (NES), both neural stem cell markers, were coexpressed in almost all the neural progenitor cells (B to C’) The elongated H1-NPCs co-expressed radial glia markers Brain Lipid Binding Protein (BLBP), VIMENTIN (VIM) and Glia Fibrillary Acidic Protein (GFAP) The scale bar indicates 100 µm (D) Flow cytometry quantification of lineage markers in neural progenitors Values show the percentage of immunopositive cells for the indicated antibodies   59  4.2.2 Human ESC-derived neural progenitors differentiated into functional dopamine neurons with high efficiency The NPCs derived from H1 hESCs (H1-NPCs) were differentiated into dopamine (DA) neurons by subjecting them to DA differentiation medium, consisting of SHH, FGF8 and ascorbic acid, represented in Figure 4.4 At the end of the 14-day differentiation process, neurons immunopositive for both the mature neuron marker, MAP2, and the dopaminergic marker, tyrosine hydroxylase (TH), were abundant (Figures 4.5A-C), indicating that H1-NPCs were differentiated into DA neurons (H1DANs) Further characterization revealed that other DA neuron markers such as VMAT2, PITX3 and dopamine (DA) were also expressed (Figures 4.5D-F) To further characterize the subtype of DA neurons derived, the gene expression profile of the derived neurons was compared against those of the whole brain and H1-NPC samples The enrichment of mRNA expression of LMX1A, LMX1B, EN1, PITX3, MAP2 and TH confirmed that midbrain DA neurons were derived (Figure 4.6A), whereas the lack or decreased expression of GAD65, ISLET1, HB9, TPH1, SERT and DBH indicated that contaminating GABAergic, motor, serotonergic and noradrenergic neurons were absent (Figure 4.6B)   60  Figure 4.4: Schematic representation of the differentiation of hESC-derived radial glia-like NPCs into midbrain dopaminergic (DA) neurons NPCs were exposed to ventralizing factors SHH and FGF8 for seven days in N2B27 neuronal differentiation medium After which, the ventralized cells were treated with BDNF, GDNF, ascorbic acid (AA) and cAMP in N2B27 medium for another seven days, to allow for neuronal maturation At the end of the 14-day differentiation procedure, neurons with long processes were observed   61  Figure 4.5: Immunofluorescence characterization of H1-derived neurons indicating their midbrain dopaminergic identity (A) MAP2-expressing mature neurons were abundant at the end of the 14-day differentiation of H1-NPCs (B-C) These neurons also expressed TH, indicative of a dopaminergic neuronal population A panel of midbrain dopaminergic markers including (D) VMAT2, (E) PITX3 and (F) DA were also expressed by the H1-derived dopamine neurons The white scale bar indicates 100 µm   62  Figure 4.6: Quantitative PCR analysis confirming midbrain dopaminergic identity (A) qPCR measurement of mRNAs encoding midbrain markers in cDNA from total brain, H1-derived neural precursors (H1-NPC) and dopaminergic neurons (H1-DAN) The H1-DAN sample was enriched in all the midbrain markers tested (B) qPCR measurement of various neuronal subtype markers: GABAergic subtype (GAD65), motor neuron subtype (ISLET1 and HB9), serotonergic neurons (TPH1 and SERT) and noradrenergic neurons (DBH), indicating the lack of these nondopaminergic neurons The results were indicative of an enriched dopaminergic population * and ** indicates p-values of