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The Pennsylvania State University The Graduate School Department of Neural and Behavioral Sciences EPIGENETIC ANALYSIS OF IMMUNE ASSOCIATED SIGNALING MOLECULES DURING MOUSE RETINA DEVELOPMENT A Thesis in Anatomy by Chen Yang © 2013 Chen Yang Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science May 2013 The thesis of Chen Yang was reviewed and approved* by the following: Samuel Shao-Min Zhang Assistant Professor of Neural and Behavioral Sciences Thesis Advisor Colin J. Barnstable Department Head of Neural and Behavioral Sciences Professor of Neural and Behavioral Sciences Patricia J. McLaughlin Professor of Neural and Behavioral Sciences Director of Graduate Program in Anatomy *Signatures are on file in the Graduate School. ii ABSTRACT The retina is an immune-privileged organ. Many autoimmune diseases, such as AMD, glaucoma, and diabetic retinopathy, are caused by excessive inflammatory responses targeting self-tissue. The physiological functions of extracellular and intracellular signaling molecules of immune responses have been well characterized. The epigenetic aspects of these molecules in the retina, however, have not been well elucidated. In this study, we examined the expression of selected immune-related genes, and their transcriptional accessibility via epigenetic mapping, cluster analysis, and RT-PCR. Among these genes, interleukin receptor related genes and intracellular signaling molecules exhibit higher transcriptional accessibility. Epigenetic mapping of the toll-like receptor (TLR) family revealed that 3 out of 13 TLRs exhibit H3K4me2 accumulation during retina development, suggesting that TLR2, TLR3, and TLR9 are the only TLR members expressed in the retina. Most of the NF-κB signaling molecules exhibited transcriptional accessibility, implying their essential roles in inflammatory regulation during retina maturation. We have also identified two isoforms each of two NF-κB negative feedback regulator genes, Tnfaip3/A20 and Pcbp2, as well as another NF-κB negative feedback regulator gene, Trafd1, that are differentially expressed in mouse retina and spleen in response to LPS treatment. iii TABLE OF CONTENTS Page Number LIST OF FIGURES viii LIST OF TABLES ix LIST OF ABBREVIATIONS x ACKNOWLEDGEMENTS xvi CHAPTER 1. INTRODUCTION 1.1 Developing retina is a great model to study central nerve system 2 1.1.1 Gross Structures of retina 2 1.1.2 Histological structures of retina 2 1.1.3 Development of retina 5 1.2 Inflammation/immune response signaling is associated with retinal pathogenesis 6 1.2.1 Diabetic retinopathy 6 1.2.2 Age-related macular degeneration (AMD) 7 1.2.3 Glaucoma 9 1.3 STAT3 is an important molecule for retinal development and retinal stress responses 10 1.3.1 STAT expression in retina 10 iv 1.3.2 STAT3 is a signaling mediator for photoreceptor development 12 1.3.3 Cross-talk of PKC and STAT3 signaling in retina differentiation 13 1.3.4 STAT3 mediates stress responses in retina 14 1.4 NF-κB, the master control for inflammatory and immune responses 16 1.4.1 Basics of NF-κB 16 1.4.2 NF-κB mediated signaling 16 1.4.3 Regulation of NF-κB signaling 18 1.5 Negative feedback for NF-κB signaling 20 1.5.1 Tnfaip3/A20 is a specific ubiquitinases for NF-κB signaling 20 1.5.2 Tnfaip3/A20 is a universal inhibitor for NF-κB signaling by varies of activators 21 1.5.3 Loss of NF-κB negative feedback and human diseases 22 1.6 Summary 24 CHAPTER 2. OBJECTIVES 2.1 Overall hypothesis 27 2.2 Specific Aim 1: Epigenetic analysis on extracellular signaling of inflammatory/immune response genes during retina development 27 2.3 Specific Aim 2: Epigenetic analysis on intracellular signaling of inflammatory/immune response genes during retina development 28 2.4 Specific Aim 3: Examination of NF-κB negative feedback signaling in v retina 28 CHAPTER 3. METHODS AND MATERIALS 3.1 Animals 30 3.2 Tissue collection 30 3.2.1 Retina evisceration 30 3.2.2 Spleen isolation 30 3.3 Drug treatment 31 3.4 Retina explants culture 31 3.5 RNA extraction and purification 31 3.6 Spectrophotometry 32 3.7 RNA reverse transcription 32 3.8 Primer design and synthesis 33 3.9 Genomic-PCR / RT-PCR 34 3.10 ChIP-Seq database and data collection 35 3.11 Cluster analysis 35 3.12 Image J and densitometry analysis 36 3.13 Statistical analysis 36 CHAPTER 4. RESULTS 4.1 Epigenetic analysis on extracellular signaling of inflammatory/immune vi response genes during retina development 38 4.1.1 Introduction 38 4.1.2 Collection of genes 38 4.1.3 Epigenetic analysis 38 4.1.3.1 Cluster analysis of interleukin ligands and their receptors 38 4.1.3.2 Cluster analysis of complements 42 4.1.3.3 Epigenetic mapping of complements 44 4.1.3.4 Epigenetic mapping of Toll-like receptors 45 4.2 Epigenetic analysis on intracellular signaling of inflammatory/immune response genes during retina development 46 4.2.1 Introduction 46 4.2.2 Collection of genes 47 4.2.3 Epigenetic analysis 47 4.2.3.1 Cluster analysis of PI3K signaling 47 4.2.3.2 Cluster analysis of STAT signaling 48 4.2.3.3 Cluster analysis of NF-κB signaling 50 4.3 Examination of NF-κB negative feedback signaling in retina 51 4.3.1 Introduction 51 4.3.2 Collection of genes 51 4.3.3 Epigenetic mapping of NF-κB negative feedback genes 51 4.3.3.1 Epigenetic mapping of NF-κB signaling genes 52 vii 4.3.3.2 Epigenetic mapping of NF-κB negative feedback genes 55 4.3.4 Gene expression of NF-κB negative feedback genes in retina 56 4.3.4.1 Tnfaip3/A20 expression during retina development 56 4.3.4.2 Tissue specific Tnfaip3/A20 responses to LPS 57 4.3.4.3 Differential response of other NF-κB negative feedback genes 61 CHAPTER 5. DISCUSSION AND CONCLUSION 5.1 Interleukin receptors compared to interleukin themselves have more transcriptional accessibilities in retina 66 5.2 A number of complements are transcriptionally accessible at the alternative transcription start site in the retina 68 5.3 The toll-like receptor family has limited transcriptional accessibilities in the retina 69 5.4 Most of genes in NF-κB signaling pathway are transcriptionally accessible in the retina 71 5.5 Constitutive expression of the genes related to NF-κB negative feedback in mature retina 73 5.6 Conclusion 74 REFERENCES 75 viii LIST OF FIGURES Figure 1 PCR Scheme 34 Figure 2 Cluster analysis and Tree-view for interleukins and interleukin receptors 39 Figure 3 Bar chart for interleukin and interleukin receptor comparison 40 Figure 4 Cluster analysis and Tree-view for complements 41 Figure 5 Epigenetic mapping of complements 42 Figure 6 Epigenetic mapping of Toll-like receptors 43 Figure 7 Cluster analysis and Tree-view of PI3K-AKT signaling molecules 46 Figure 8 Cluster analysis and Tree-view of JAK-STAT signaling molecules 47 Figure 9 Cluster analysis and Tree-view of NF-κB signaling molecules 50 Figure 10 Epigenetic mapping of NF-κB signaling genes part 1 52 Figure 11 Epigenetic mapping of NF-κB signaling genes part 2 53 Figure 12 Epigenetic mapping of NF-κB negative feedback genes 54 Figure 13 Tnfaip3/A20 expression during developmental stages 55 Figure 14 Tnfaip3/A20 primer design 56 Figure 15 Tnfaip3/A20 PCR results with/without LPS treatment 57 Figure 16 Statistical analysis for Tnfaip3 expression in the retina and spleen 58 Figure 17 Pcbp2 and Trafd1 PCR results with/ without LPS treatment 59 Figure 18 Statistical analyses for Pcbp2 and Trafd1 expression in the retina and spleen 61 ix LIST OF TABLES Table 1 Primer information 34 x LIST OF ABBREVIATIONS A20 another name for tnfaip3 AMD age-related macular degeneration ATP adenosine triphosphate ATSS alternative transcription start site AXK axokine BCR B cell receptor β beta bFGF basic fibroblast growth factor bZIP basic Leucine Zipper Domain °C degrees Centigrade CFH complement factor H CNV choroidal neovascularisation CRP C-reactive protein CD cluster of differentiation CD Crohn’s disease cIAP cellular inhibitor of apoptosis CNTF ciliary neurotrophic factor CO2 carbon dioxide Cre cyclization recombination xi Crx cone-rod homeobox protein CYLD cylindromatosi d day DC dendritic cell diH2O dionized water DNA deoxyribonucleic acid DUB deubiquitylating enzyme et al and others E/ED embryonic day EGF epidermal growth factor EGFRs epidermal growth factor receptors ELM external limiting membrane ERK extracellular signal-regulated kinase FCS fetal calf serum flox flanked by loxP g gram GCL ganglion cell layer GCAP guanylatecyclase-activating protein GFAP glial fibrillary acidic protein gp130 glycoprotein 130 h hour xii IACUC Institutional Animal Care and Use Committee IBD inflammatory bowel disease IFN interferon IGF insulin-like growth factor IKK IκB kinase IκB inhibitor of kappa B ILM inner limiting membrane IL interleukin INL inner nuclear layer IOP intraocular pressure IPL inner plexiform layer IRBP Interphotoreceptor retinoid-binding protein JAK janus kinases κ kappa LIF leukemia inhibitory factor LPS lipopolysaccharide LUBAC E3 ligase linear ubiquitin chain assembly complex Lys lysine MCP monocyte chemotactic protein MDP muramyl dipeptide min(s) minute(s) xiii ml milliliter M molar MALT mucosa-associated tissue MAPK mitogen-activated protein kinases NEMO NF-kappa-B essential modulator NFL nerve fiber layer NPG normal pressure glaucoma OPL outer plexiform layer OSM oncostatin M Otx2 orthodenticlehomeobox 2 RGCs retinal ganglion cells RIP receptor-interacting protein RPE retinal pigment epithelium μ mu MEF mouse embryonic fibroblast MDP muramyl dipeptide μg microgram μm micrometer μl microliter NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells xiv