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Cis-Regulatory Mechanisms of Acute Phase Gene Expression Cis-regulatory Mechanisms of Acute Phase Gene Expression by Minggao Liang A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy The Department of Molecular Genetics University of Toronto © Copyright by Minggao Liang 2021 Cis-regulatory Mechanisms of Acute Phase Gene Expression Minggao Liang Doctor of Philosophy The Department of Molecular Genetics University of Toronto 2021 Abstract The accurate control of when and where genes are expressed is central to our understanding of development and disease. Elucidating the mechanisms that control this complex, multifaceted process is a major endeavor at the forefront of genetics and genome biology. The acute phase response (APR) is an evolutionarily conserved systemic response to inflammation that triggers rapid gene expression changes in the liver and other tissues. Genes induced during the APR are regulated via unique mechanisms that facilitate their rapid expression upon stimulation while maintaining their silencing under basal conditions. To dissect the mechanisms controlling APR gene expression, I employ a combination of epigenetic profiling, comparative genomics, and chromatin conformation capture to characterize the cis-regulation of a representative APR gene, PLAU. I investigate how PLAU is dysregulated in Quebec platelet disorder, a bleeding disorder in which a single tandem-duplication of PLAU causes its ectopic >100-fold overexpression in platelets and megakaryocytes. I demonstrate that the silencing of PLAU is normally facilitated by local 3-dimensional (3D) chromatin architecture which isolates it from the activity of nearby tissue-specific enhancers, while disruption of this architecture in QPD results in ectopic activation of PLAU by a conserved megakaryocyte enhancer. Employing a similar multi-omics approach, I investigate genome-wide transcriptional and epigenetic changes during the APR in the context of 3D chromatin architecture in the mouse liver. I demonstrate that enhancer-gene pairs within the same chromatin loop are more likely to show concordant changes in gene expression than equidistant genes in different domains, together illustrating a paradigm in which 3D chromatin architecture coordinates both the activation and silencing of acute-phase gene expression. ii Acknowledgments First and foremost, I would like to express my deep and sincere gratitude to my supervisor, Dr. Michael Wilson, for the opportunity to embark on my PhD journey as a member of his lab. I am deeply grateful for the incredible enthusiasm, excitement, creativity, insightfulness, sincerity, and patience that you have shown me over the past 7 years. I cannot imagine having a better advisor, mentor, or role model in any such capacity. I would also like to sincerely thank my thesis committee members, Dr. Lincoln Stein and Dr. Mathieu Lupien, for generously offering their time, support, encouragement and insight in guiding me throughout the course of my degree. Thank you also to my examination committee members Dr. Houshen He, Dr. Jeff Wrana, and Dr. Nathalie Bérubé for providing their time and insights in the evaluation of my dissertation. Thank you to Dr. Catherine Hayward for the invitation to participate in the QPD project; to the members of the Hayward lab Dr. Subia Tasneem and Dr. Asim Soomro, whose work and collaborative contributions made it possible to bring the QPD project to completion; to Dr. Bing Ren and members of the Ren lab for teaching me the 4C methodology; and to the QPD families and other blood donors for their generous contributions, without which none of the research on PLAU and QPD would have been possible. Thank you to my parents for raising and educating me and guiding me to pursue my path in science. It is upon the shoulders of their continued love, care, support, prayers, and sacrifices that I stand where I am today. Thank you to the fellow members of the Wilson lab, past and present, who have accompanied me in this journey through thick and thin as colleagues, collaborators, mentors, and friends. I will fondly remember the sound advice and mentorship, the group efforts to make last-minute pushes to meet deadlines, the adventures to find grad-student affordable food, the stimulating scientific conversations in the lab late after hours, and the all-around good humor we shared as a team. Finally, I would like to share the following verse from scripture which has inspired, and continues to inspire, my love for science and discovery: “Great are the works of the Lord; they are pondered by all who delight in them.” - Psalms 111:2 The work presented in this dissertation was supported by funding from the Canadian Institutes for Health Research, the National Sciences and Engineering Research Council, and the Hospital for Sick Children Research and Training Center. iii Table of Contents Acknowledgements iii Table of Contents iv List of Figures and Tables viii List of Appendicies x Introduction 1 Current outlook and challenges on the study of cis-regulation 2 Cis-regulatory variation as drivers of phenotype and disease 3 Molecular mechanisms of cis-regulation 5 Figure 1: cis-regulation of eukaryotic gene expression. 6 Steps of RNAP II transcription 7 Cis-reglatory elements: enhancers and promoters 8 Enhancers as platforms for TF recruitment 13 Histone post-translational modifications 11 Figure 2: Commonly studied histone PTMs and associated chromatin states 13 Enhancer-gene targeting and spatial genome organization 13 Cis-regulatory silencing 16 Cis-regulatory genomics methods and resources 18 Experimental methodologies 18 Genomics data repositories 21 Integrative genomics approaches 22 The Acute Phase Response (APR) 25 Definition and function of the APR 25 Regulation of APR gene expression 27 Figure 3: cytokine regulation of APR gene expression 28 Rationale 30 Cell type-specific overexpression of PLAU in Quebec platelet disorder 32 Background 32 The Urokinase-type plasminogen activator PLAU 32 Figure 4. The PLAU promoter 34 PLAU dysregulation in the pathogenesis of Quebec Platelet Disorder 36 Introduction 38 Results 39 uPA protein and mRNA levels in QPD and control leukocytes: 39 Figure 5. uPA in QPD and control leukocytes. 40 Figure 6. PLAU transcript levels in QPD and control leukocytes. 42 Megakaryocyte and granulocyte transcripts for the region that is duplicated in QPD 42 iv Figure 7. Comparison of gene expression at the PLAU locus in granulocytes and CD34+ derived megakaryocytes obtained using RNA-seq. 44 Figure 9. Exon and splice junction usage for PLAU in QPD versus control megakaryocytes and granulocytes, evaluated using JunctionSeq. 46 Figure 10. C10orf55 protein levels in QPD and control cells. 47 Figure 11. Exon and splice junction usage for C10orf55 in megakaryocytes and granulocytes 48 Global expression differences in QPD Megakaryocytes 49 Figure 12. Global expression differences in QPD megakaryocytes. 50 Figure 13. Splice junction analysis of EGR1 in megakaryocytes and granulocytes. 51 Discussion 53 Materials and Methods 59 Ethics Approval 59 Subjects and sample collection 59 Cell isolation 59 Preparation of samples for analysis 60 Protein analyses 60 Quantitative reverse transcription polymerase chain reaction 61 RNA-seq analysis 62 Genotyping 63 Statistical analyses 63 Rewiring of enhancer-gene interactions drives PLAU overexpression in QPD 65 Introduction 76 Results 67 Figure 14: Overview of QPD and study design 68 QPD is associated with gain of H3K36me3 and allele-specific loss of H3K27me3 at the PLAU promoter 68 Figure 15: QPD results in a loss of repressive chromatin and repositions PLAU relative to a candidate megakaryocyte enhancer. 71 Figure 16: ddPCR analysis of rs1916341 allele ratios in H3K27me3 ChIP and Input libraries. 71 QPD results in duplication and repositioning of a conserved megakaryocyte enhancer 72 Figure 17: Ranking analysis of H3K27ac enrichment at ENHQPD. 74 Figure 18: Phylogenetic analysis of ENHQPD. 75 Figure 19: ENHQPD is a conserved hematopoietic enhancer that acts in synergy with the PLAU and VCL promoters 76 Figure 20: PLAU and VCL expression during normal megakaryopoiesis. 77 Figure 21: Luciferase reporter assay for ENHQPD in K562 and megakaryocyte cultures 79 The QPD duplication spans a CTCF-associated subTAD boundary 79 v Figure 22: The QPD duplication spans the subTAD boundary separating PLAU and ENHQPD. 81 Figure 23: Hi-C interactions reveal tissue-specific subTAD architecture at the PLAU locus. 81 QPD results in ectopic enhancer-gene interactions specific to the disease chromosome 82 Figure 24: QPD results in ectopic enhancer-gene interactions specific to the disease chromosome 84 Discussion 85 Figure 25: Model of enhancer hijacking in QPD. 85 Materials and Methods 87 Ethics 87 Subjects and sample collection 87 Megakaryocyte culture 87 ChIP-seq library preparation and sequencing 87 4C-seq library preparation and sequencing 88 Sanger sequencing of 4C samples 89 H3K27me3 ChIP droplet digital PCR 89 ChIP-seq data analysis 90 Differential ChIP enrichment analysis 91 4C-seq data analysis 91 H3K27ac ranking analysis 91 Zebrafish reporter assays 92 Luciferase reporter assays 92 Primer Sequences 93 Accessions for published datasets used in this Chapter 93 Data sharing statement 94 Gene regulatory networks and cis-regulation of acute phase gene expression 95 Rationale 95 Results 95 Defining the ‘extended’ hepatic APR 95 Figure 26: Defining the extended APR. 96 Extended APR gene regulatory networks and functions 97 Figure 27: Extended APR genes detected at 2h versus 24h are associated with
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