Functional characterization of the new 8q21 Asthma risk locus Cristina M T Vicente B.Sc, M.Sc A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2017 Faculty of Medicine Abstract Genome wide association studies (GWAS) provide a powerful tool to identify genetic variants associated with asthma risk. However, the target genes for many allergy risk variants discovered to date are unknown. In a recent GWAS, Ferreira et al. identified a new association between asthma risk and common variants located on chromosome 8q21. The overarching aim of this thesis was to elucidate the biological mechanisms underlying this association. Specifically, the goals of this study were to identify the gene(s) underlying the observed association and to study their contribution to asthma pathophysiology. Using genetic data from the 1000 Genomes Project, we first identified 118 variants in linkage disequilibrium (LD; r2>0.6) with the sentinel allergy risk SNP (rs7009110) on chromosome 8q21. Of these, 35 were found to overlap one of four Putative Regulatory Elements (PREs) identified in this region in a lymphoblastoid cell line (LCL), based on epigenetic marks measured by the ENCODE project. Results from analysis of gene expression data generated for LCLs (n=373) by the Geuvadis consortium indicated that rs7009110 is associated with the expression of only one nearby gene: PAG1 - located 732 kb away. PAG1 encodes a transmembrane adaptor protein localized to lipid rafts, which is highly expressed in immune cells. Results from chromosome conformation capture (3C) experiments showed that PREs in the region of association physically interacted with the promoter of PAG1. Furthermore, results from luciferase reporter assays demonstrated that one of these PREs (PRE 3) acted as a transcriptional enhancer on PAG1 exclusively when it carried the rs2370615:C asthma predisposing allele. This variant, which is in complete LD with rs7009110, was found to disrupt the binding of the Foxo3a transcription factor to PRE3. As such, rs2370615 represents a putative functional variant underlying the association between rs7009110 and asthma. In addition to PAG1, other genes in the 8q21 region could be targets of asthma risk variants, despite the lack of evidence for an association between rs7009110 and gene expression. The closest gene is ZBTB10 (75 kb away), a repressor of the specificity proteins (Sp1, Sp3 and SP4), which are transcription factors known to regulate several immune-related genes. Therefore, based on distance to rs7009110 and its known function, we prioritised ZBTB10 to investigate if it could represent an additional target gene of 8q21 asthma risk variants. Results from 3C assays showed that the same PREs that interacted with the PAG1 promoter also interacted with the ZBTB10 promoter. Results from luciferase ii assays for ZBTB10 were inconclusive: PRE 2 enhanced the activity of the ZBTB10 promoter, and this effect was inhibited by an asthma risk allele but these differences were not statistically significant. Together, these results indicate that ZBTB10 is also likely to be a target of 8q21 asthma risk variants. To study the contribution of PAG1 and ZBTB10 to asthma pathophysiology, we performed in vivo experiments using wild type (WT), Pag1-/- and Zbtb10het mice, which included challenging mice with an allergen or virus, both able to induce airway inflammation. Results from these experiments indicate that Pag1 expression might regulate granulocyte, lymphocyte and/or airway epithelial cell function. On the other hand, the expression of Zbtb10 was critical for embryonic development and could play a role in immune cell development as well as activation during the allergen sensitization phase. In conclusion we have identified PAG1 and ZBTB10 as target genes of 8q21 asthma risk variants and showed that both genes play important roles in the immune system and development of airway inflammation. iii Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis. iv Publications during candidature Peer-reviewed papers: Vicente CT, Edwards SL, Hillman KM, Kaufmann S, Mitchell H, Bain L, Glubb DM, Lee JS, French JD, Ferreira MAR. (2015) “Long-range modulation of PAG1 expression by 8q21 allergy risk variants”. American Journal of Human Genetics 97: 1-8 Ferreira MA, Jansen R, Willemsen G, Penninx B, Bain LM, Vicente CT, Revez JA, Matheson MC, Hui J, Tung JY, Baltic S, Le Souëf P, Montgomery GW, Martin NG, Robertson CF, James A, Thompson PJ, Boomsma DI, Hopper JL, Hinds DA, Werder RB, Phipps S; Australian Asthma Genetics Consortium Collaborators. (2017) “Gene-based analysis of regulatory variants identifies 4 putative novel asthma risk genes related to nucleotide synthesis and signalling”. Journal of Allergy and Clinical Immunology 139(4):1148-1157. Conference abstracts: Vicente C, Edwards S, Hillman K, Kaufmann S, Mitchell H, Bain L, Glubb D, Lee J, French J, Ferreira M. “Long-range modulation of PAG1 expression by 8q21 allergy risk variants”. Annals of Translational Medicine 2015;3(S2):AB013 v Publications included in this thesis Vicente CT, Edwards SL, Hillman KM, Kaufmann S, Mitchell H, Bain L, Glubb DM, Lee JS, French JD, Ferreira MAR. (2015) “Long-range modulation of PAG1 expression by 8q21 allergy risk variants”. American Journal of Human Genetics 97: 1-8 – incorporated as Chapter 2. Contributor Statement of contribution Concept and design: 10% Cristina T. Vicente Analysis and interpretation: 30% Drafting and production: 30% Concept and design: 25% Stacey L. Edwards Analysis and interpretation: 15% Drafting and production: 10% Concept and design: 0% Kristine M. Hillman Analysis and interpretation: 10% Drafting and production: 0% Concept and design: 0% Susanne Kaufmann Analysis and interpretation: 5% Drafting and production: 0% Concept and design: 0% Hayley Mitchell Analysis and interpretation: 3% Drafting and production: 0% Concept and design: 0% Lisa Bain Analysis and interpretation: 3% Drafting and production: 0% Concept and design: 0% Dylan M. Glubb Analysis and interpretation: 3% Drafting and production: 0% Concept and design: 0% Jason S. Lee Analysis and interpretation: 6% Drafting and production: 0% Concept and design: 25% Juliet D. French Analysis and interpretation: 10% Drafting and production: 10% Concept and design: 40% Manuel A.R. Ferreira Analysis and interpretation: 15% Drafting and production: 50% vi Contributions by others to the thesis Overall Thesis The project was conceived and designed with the help of my main supervisor Dr. Manuel Ferreira and co-supervisors Dr. Juliet French and A/Prof. Simon Phipps, who also provided feedback on the interpretation and presentation of the results. Chapter 1: Introduction Review article submitted for publication together with Joana Revez and Dr. Manuel Ferreira. This publication is currently under review. Chapter 2: Identification of PAG1 and ZBTB10 as target genes of 8q21 asthma-risk variants Individual contributions as stated in the “Publication included in this thesis” section. Routine reagents used to perform experiments were prepared by several laboratories, depending on the technique. Contributing laboratories include the following from QIMR Berghofer: Asthma Genetics (Dr. Manuel Ferreira); Functional Genetics (Dr. Juliet French); Functional Cancer Genomics (Dr. Stacey Edwards); and Control of Gene Expression (Professor Frank Gannon). Chapter 3: Mechanisms of regulation of PAG1 and ZBTB10 expression Western blot and hypoxia experiments were done in collaboration with Dr. Jason Lee from the Control of Gene Expression laboratory. The correlation analyses were done by Dr. Manuel Ferreira. Chapter 4: PAG1 and ZBTB10 expression in human and mouse tissues This study makes use of data generated by the Blueprint Consortium. A full list of the investigators who contributed to the generation of the data is available from www.blueprint- epigenome.eu. Funding for the project was provided by the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no 282510 – BLUEPRINT. The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. The data used for the analyses described in this manuscript were obtained from the GTEx Portal on September 2017. Dr. Vivian Zhang assisted with mouse tissue collection, lung section slide preparation and staining. Dr. Jason Lynch performed mediastinal lymph node collection from experimental vii animals. Ms Rhiannon Werder collected airway epithelial cells from experimental animals.