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Gene Regulation in Microglia and Genetic Risk for Complex Brain Disorders

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Gene Regulation in Microglia and Genetic Risk for Complex Brain Disorders Gene regulation in microglia and genetic risk for complex brain disorders A thesis submitted for the degree of Doctor of Philosophy at Cardiff University School of Medicine Darren Cameron 2019 ii Statements and Declarations Statement 1 This thesis is being submitted in partial fulfilment of the requirements for the degree of PhD. Signed _________________________ Date _________________________ Statement 2 This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is it being submitted concurrently for any other degree or award (outside of any formal collaboration agreement between the University and a partner organisation) Signed _________________________ Date _________________________ Statement 3 I hereby give consent for my thesis, if accepted, to be available in the University’s Open Access repository (or, where approved, to be available in the University's library and for inter-library loan), and for the title and summary to be made available to outside organisations, subject to the expiry of a University-approved bar on access if applicable. Signed _________________________ Date _________________________ Declaration This thesis is the result of my own independent work, except where otherwise stated, and the views expressed are my own. Other sources are acknowledged by explicit references. The thesis has not been edited by a third party beyond what is permitted by Cardiff University's Use of Third Party Editors by Research Degree Students Procedure. Signed _________________________ Date _________________________ Word Count: 34,385 iii Acknowledgments I would like to thank Nick, Matt, Manuela, Heath, Chris, Gareth, Derek, Katherine, Jo, Kate, Cath, Ann, Andrew, Ric, Antonio and the core team. In addition, I would like to thank the Medical Research Council for funding this work. iv ACRONYMS ADHD: Attention-deficit hyperactivity disorder ASD: Autism spectrum disorder ATAC-seq: Assay of transposase-accessible chromatin with sequencing BPD: Bipolar disorder ChIP-seq: Chromatin immunoprecipitation with sequencing CLOZUK: Genotype sample of patients with treatment resistant schizophrenia who have be prescribed clozapine C4AL: Complement component C4 long form CNS: Central nervous system CNV: Copy number variant DMSO: Dimethyl sulfoxide DNA: Deoxyribonucleic Acid EADI: European Alzheimer’s disease initiative EDTA: Ethylenediaminetetraacetic acid ENCODE: Encyclopaedia of DNA elements project EMSA: Electrophoresis mobility shift assay eQTL: Expression quantitative trait loci ETS: E twenty-six FACS: Fluorescence-activated cell sorting FSC-A: Forward scatter area FSC-H: Forward scatter height GARFIELD: GWAS analysis of regulatory or functional information enrichment with LD correction GERAD: Gene and environmental risk in Alzheimer’s disease consortium G-MCF: Granulocyte/macrophage colony stimulating factor GWAS: Genome-wide association study HDBR: Human developmental biology resource HM: Histone modification HOMER: Hypergeometric optimisation of motif enrichment IGAP: International genomics of Alzheimer’s disease project IGV: Integrative genomics viewer iPSC_MG: Immune pluripotent stem cell derived microglia iPSC_MΩpre: Immune pluripotent stem cell derived macrophage precursor cells LD: Linkage Disequilibrium v LOAD: Late onset Alzheimer’s disease M-CSF: Macrophage colony stimulating factor MDD: Major depressive disorder OCR: Open chromatin region PCA: Principal component analysis PCR: Polymerase chain reaction PCW: Post conception weeks PGC: Psychiatric Genomics Consortium PWM: Position weight matrix qPCR: Quantitative polymerase chain reaction REMC: United States Institute of Health Roadmap epigenetics consortium RNA: Ribonucleic acid SCZ: Schizophrenia SEB: Substrate equilibrium buffer sLDSC: Stratified linkage disequilibrium score regression SNP: Single nucleotide polymorphism SNV: Single nucleotide variant SPD: Schizophrenia patient derived SSC-A: Side scatter area SV40: Simian virus 40 large T antigen immortalised TBE: Tris-borate-Ethylenediaminetetraacetic acid TF: Transcription factor TSS: Transcription start site vlPFC: Ventrolateral prefrontal cortex vi Summary In recent years, genome wide association studies (GWAS) have established that common genetic variation plays an important role in complex brain disorders, such as autism spectrum disorder, schizophrenia and Alzheimer’s disease. However, as the vast majority of GWAS risk loci are located in poorly characterised non-coding regions, interpretation of GWAS data is difficult. As very few associations can be attributed to effects on protein-coding sequence, the majority of common risk loci for complex brain disorders are believed to operate through effects on gene regulation. In order to elucidate genetic risk mechanisms for complex brain disorders, it is important to establish in which cell types these risk loci are operating. Microglia are the primary immune cell of the central nervous system and are important regulators of brain function throughout life. As such, there has been growing interest in the potential role of microglia in brain disorders. One means of interpreting the function of the non-coding genome in a cell of interest is to measure the cell’s chromatin landscape. For example, the assay for transposase accessible chromatin with sequencing (ATAC-seq) maps regions of ‘open’ chromatin, which have the potential to regulate gene expression through binding of transcription factors. In this thesis, I use ATAC-seq to map open chromatin in adult ex vivo microglia, 2nd trimester foetal microglia, and in vitro human cell models of microglia. I then integrate this with complex brain disorder GWAS data to investigate whether gene regulatory processes in human microglia contribute to genetic risk for complex brain disorders. I find evidence that risk variants for late onset Alzheimer’s disease, autism spectrum disorder, bipolar disorder, major depressive disorder and schizophrenia are enriched within regulatory regions utilised by foetal and / or adult microglia, suggesting a primary role for this cell type in these conditions. Using the electrophoretic mobility shift assay, I further show that two single nucleotide polymorphisms associated with Alzheimer’s disease, and within regions of open chromatin in adult microglia, alter binding of protein from microglial nuclei. vii Table of Contents 1 GENERAL INTRODUCTION .......................................................................................... 1 1.1 DEFINITION OF BRAIN DISORDERS .............................................................................. 1 1.2 GENETICS OF COMPLEX BRAIN DISORDERS ................................................................. 4 1.2.1 Early molecular genetic studies of brain disorders ............................................ 5 1.2.2 Rare genetic variation ........................................................................................ 6 1.2.3 Genome-wide association studies ..................................................................... 7 1.2.4 Genome wide association studies of brain disorders ......................................... 8 1.2.5 Pathway analysis ............................................................................................... 9 1.2.6 Limitations of genome wide association studies .............................................. 10 1.3 THE REGULATION OF GENE EXPRESSION .................................................................. 11 1.3.1 Mapping the human epigenome ...................................................................... 11 1.3.2 Chromatin and chromatin accessibility ............................................................ 12 1.3.3 Regulatory elements ........................................................................................ 14 1.3.4 Transcription factors ........................................................................................ 15 1.3.5 Chromatin modifications .................................................................................. 17 1.3.6 Integration of genetic and epigenomic data ..................................................... 18 1.4 MICROGLIA ............................................................................................................. 19 1.4.1 Microglia ontogeny and turnover ...................................................................... 19 1.4.2 Microglia’s immunological functional repertoire ............................................... 21 1.4.3 Microglia’s brain architectural functional repertoire .......................................... 22 1.4.4 Microglia and brain disorders ........................................................................... 24 1.5 AIMS ...................................................................................................................... 26 2 THE OPEN CHROMATIN LANDSCAPE AND COMMON VARIANT DISEASE HERITABILITY IN HUMAN EX VIVO MICROGLIA .............................................................. 27 2.1 INTRODUCTION ....................................................................................................... 27 2.1.1 Aims ................................................................................................................. 28 2.2 METHODS .............................................................................................................. 28 2.2.1 Accessing publicly available datasets .............................................................. 28 2.2.2 Sequencing, QC and bed
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