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Epigenetic Trajectories to Childhood Asthma Epigenetic Trajectories to Childhood Asthma Item Type text; Electronic Dissertation Authors DeVries, Avery Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction, presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 04/10/2021 09:24:24 Link to Item http://hdl.handle.net/10150/630233 1 EPIGENETIC TRAJECTORIES TO CHILDHOOD ASTHMA by Avery DeVries __________________________ Copyright © Avery DeVries 2018 A Dissertation Submitted to the Faculty of the DEPARTMENT OF CELLULAR AND MOLECULAR MEDICINE In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 2018 2 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of the requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that an accurate acknowledgement of the source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: Avery DeVries 4 ACKNOWLEDGEMENTS I would like to thank my dissertation mentor, Donata Vercelli, for endless support throughout this journey and for always encouraging and challenging me to be a better scientist. And to my committee members, thank you for your support, critiques and comments, all of which have been invaluable. 5 DEDICATION For my loving parents, my children, and my amazing husband, David. 6 TABLE OF CONTENTS LIST OF FIGURES ……………………………………………………………. 8 LIST OF TABLES …………………………………………………………..... 9 LIST OF ABBREVIATIONS …………………………………………... 10 ABSTRACT ………………………………………………………………..…. 11 CHAPTER 1: INTRODUCTION …………………………………………... 13 1.1 – THE ASTHMA PUZZLE …………………………………... 13 1.1.1 – ASTHMA EPIDEMIOLOGY …………………………... 14 1.1.1.1. Asthma as a disease of childhood ……….….. 14 1.1.1.2. Risk factors for asthma …………………... 15 1.1.1.3. Molecular phenotypes (endotypes) of asthma ………..….………….…...……….…….………….……. 16 1.1.2 – ASTHMA AS A COMPLEX DISEASE …….…..… 17 1.1.2.1 – Asthma genetics …………………….…..… 17 1.1.2.2 – Asthma and the environment ……...…… 18 1.1.2.3 – Development and natural history ….…..…… 19 1.2 – EPIGENETICS …….…………..…….…………………..……… 21 1.2.1 – DNA METHYLATION ………………………..…. 21 1.2.2 – DNA METHYLATION PROFILING TECHNIQUES ………………………..…………………………………………. 23 1.3. – ASTHMA EPIGENETICS ………………………………..…. 26 1.3.1 – EPIGENOME-WIDE STUDIES …………..………. 26 1.3.1.1 – Asthma-associated epigenetic mechanisms in blood immune cells ………………………………..…. 28 1.3.1.2 – Asthma-associated epigenetic mechanisms in the airways ………………………………………..…. 32 1.3.1.3 – Conclusions ………………………………..…. 36 1.4 – DISSERTATION OUTLINE ………………………………..…. 39 CHAPTER 2: EPIGENOME-WIDE ANALYSIS LINKS SMAD3 METHYLATION AT BIRTH TO ASTHMA IN CHILDREN OF ASTHMATIC MOTHERS ……………………………………………………..……………. 42 2.1 – INTRODUCTION ………………………..…………………. 42 2.2 – METHODS …………..………………………………………. 45 2.3 – RESULTS ………………..…………………………………. 52 2.3.1 – Cord blood cells from IIS children harbor asthma- associated DMRs …………………………………………... 52 2.3.2 – Asthma-associated DMRs cluster in regulatory and pro- inflammatory gene networks ………….…….…………………. 55 2.3.3 – The SMAD3 promoter is significantly hypermethylated in asthmatic children of asthmatic mothers ………..…………. 57 2.3.4 – The association between SMAD3 promoter hypermethylation and childhood asthma replicates in two independent birth cohorts ………………..…………………. 60 2.3.5 – Maternal asthma modifies the relation between neonatal SMAD3 methylation and IL-1 producing capacity ………..…. 62 7 2.4 – DISCUSSION ……………………………..……………………. 65 CHAPTER 3: NEONATAL DNA METHYLATION PROFILES AND THE MATERNAL PRENATAL IMMUNE MILIEU …………………..………. 69 3.1 – INTRODUCTION …………………………..………………. 69 3.1.1 – Association between maternal immune profiles during pregnancy and risk for childhood asthma ………..…………. 70 3.2 – METHODS ………………………………..…………………. 72 3.3 – RESULTS ……………………………………..……………. 76 3.3.1 – Profiles of neonatal DNA methylation are associated with the maternal prenatal immune profile selectively in neonates born to non-asthmatic mothers …………..………………………. 76 3.3.2 – Neonatal CpG sites associated with the maternal IFN/IL- 13 ratio cluster in the TGFB1 pathway …………..………. 81 3.4 – DISCUSSION ……..……………………………..…...….....…... 86 CHAPTER 4: LEVERAGING TRANSCRIPTOMICS AND NETWORK APPROACHES TO EXPLORE THE ASTHMA-PROTECTIVE EFFECTS OF FARM EXPOSURE …………………………………………………………... 89 4.1 – INTRODUCTION ………………………………………..…. 89 4.1.1 – Intranasal treatment with allergen and Amish dust extract induces T cells in the lung ……………………….………….. 90 4.1.2 – T cells in the lungs of mice treated with Amish dust extract express V4 …………..………………………………. 91 4.1.3 – Network approaches to characterize the lung and 17 T cell transcriptomes in associated with asthma protection …... 92 4.2 – METHODS …………………………………………………... 94 4.3 – RESULTS …………………………………………………. 100 4.3.1 – Network analysis of transcriptome profiles in unfractionated lung cells …………………………………. 100 4.3.2 – Characterization of the transcriptome of isolated T cells .….….…………….……………………………………. 103 4.3.3 – Determining the relationships between modules associated with protection and the 17 T cell gene signature …………. 111 4.4 – DISCUSSION …………………………………………………. 118 CHAPTER 5: CONCLUSIONS AND FUTURE DIRECTIONS …………. 120 5.1 – CONCLUSIONS …………………………………………. 120 5.2 – FUTURE DIRECTIONS …………………………………. 125 APPENDIX: SUPPLEMENTARY MATERIALS FOR CHAPTER 2 …. 133 A.1 – METHODS …………………………………………………. 133 A.2 – SUPPLEMENTARY FIGURES …………………………. 137 A.3 – SUPPLEMENTARY TABLES …………………………. 145 REFERENCES …………………………………………………………. 239 8 LIST OF FIGURES Figure 1.1. Literature on epigenetics and human asthma and allergic diseases, 2007-2017 …………………………………………………………………... 27 Figure 2.1. Overview of study design …………………………………... 47 Figure 2.2. Molecular interaction network of asthma-associated DMRs …... 54 Figure 2.3. Effects of maternal asthma on the association between neonatal SMAD3 methylation and childhood asthma in IIS, MAAS and COAST …... 59 Figure 2.4. Effects of maternal asthma on the relation between neonatal SMAD3 methylation and IL-1 protein production …………………………………... 63 Figure 3.1. Samples selected for pilot DNA methylation study …………... 74 Figure 3.2. Number of CpG sites associated with the maternal prenatal IFN/IL- 13 ratio at various levels of statistical significance ……………………….….. 78 Figure 3.3. Chromosomal location of neonatal CpG sites associated with the maternal prenatal IFN/IL-13 ratio in children of non-asthmatic mothers …... 79 Figure 3.4. Network of genes harboring differential neonatal methylation associated with the maternal prenatal IFN/IL-13 ratio in children of non- asthmatic mothers …………………………………………………………... 84 Figure 3.5. Relationships between neonatal DNA methylation in genes in the aryl hydrocarbon receptor and retinoic receptor pathways, the maternal prenatal IFN/IL-13 ratio, and childhood asthma in children of non-asthmatic mothers …………………………………………………………………………………... 85 Figure 4.1. Flow cytometry analysis of lung immune cells from a murine model of asthma …………………………………………………………….…..… 93 Figure 4.2. Experimental models and samples included in RNA-sequencing analysis ……………………………………………………………….….. 99 Figure 4.3. Module identification by WGCNA ………………...……….. 101 Figure 5.1. Working model: the relationship between TGF- signaling and potential trajectories to asthma …………………………………………. 124 Figure E1. Workflow of DNA methylation profiling …………………. 137 Figure E2. Correlation of bisulfite sequencing and DNA methylation microarray results …………………………………………………………………………. 138 Figure E3. Distribution of asthma-associated DMRs by chromosome and genomic location …………………………………………………………. 139 Figure E4. Regulatory potential of the SMAD3 DMR …………………. 140 Figure E5. Association between SMAD3 DNA methylation at birth and childhood asthma in the IIS cohort …………………………………………………. 141 Figure E6. SMAD3 CpG7 methylation in IIS and MAAS …………………. 142 Figure E7. Estimation of CBMC composition in the COAST cohort …. 143 Figure E8. Effects of maternal asthma on the association between neonatal SMAD3 methylation and childhood asthma in COAST after adjusting for CBMC composition …………………………………………………………………. 144 9 LIST OF TABLES Table 1.1. Replicated asthma associations from GWAS ....………….….…. 20 Table 1.2. Epigenome-wide studies of asthma ………………..…………. 38 Table 2.1. Upstream regulators of genes containing asthma-associated DMRs ……....…………………………………………………………………………... 56 Table 2.2. LPS-stimulated CBMC-production of IL-1 in IIS children with or without a history of maternal asthma …………………………….…………….. 64 Table 3.1. Genomic distribution of neonatal CpG sites associated with the maternal prenatal IFN/IL-13 ratio in children of non-asthmatic mothers …... 80 Table 3.2. Top 5 upstream regulators of genes containing maternal IFN/IL-13 ratio-associated CpGs in children of non-asthmatic mothers ……….…….…… 83 Table 3.3. Top 5 canonical pathways enriched for genes containing
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