THE UNIVERSITY OF CHICAGO GENETICALLY ENCODED MECHANOTRANSDUCTION RESPONSE IN ENDOTHELIAL CELLS MEDIATES HUMAN CARDIOVASCULAR TRAITS A DISSERTATION SUBMITTED TO THE FACULTY OF THE DIVISION OF THE BIOLOGICAL SCIENCES AND THE PRITZKER SCHOOL OF MEDICINE IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PATHOLOGY BY MATTHEW DANIEL KRAUSE CHICAGO, ILLINOIS MARCH 2020 Copyright © 2020 by Matthew Daniel Krause All rights reserved ii TABLE OF CONTENTS List of Figures………………………………………………………….……...…………………vii List of Tables………………………………………………………………….………...………. xi Acknowledgements…………………………………………………………...………………….xii Abstract of the dissertation……………………………………………………………………...xiv Chapter 1: Introduction…………………………………………………………………………1 1.1 Atherosclerosis in Coronary Artery Disease and Ischemic Stroke……………………………1 1.2 Endothelial Mechanotransduction in Atherosclerosis…………………………….….……....2 1.3 Mechanosensitive Transcription Factors in Endothelium………………….…………………4 1.4 Genetics in Atherosclerosis……………………………………………………………………7 1.5 Enhancer regulation of gene expression in endothelial cells………………………………...13 1.6 Aims of this dissertation to understand genetic mechanisms governing flow-sensitive genes and enhancers in human endothelial cells………………………………………………………..17 CHAPTER 2: Genetic Variant at Coronary Artery Disease and Ischemic Stroke Locus 1p32.2 Regulates Endothelial Responses to Hemodynamics………………………………...18 2.1 Abstract………………………………………………………………………………………18 2.2 Introduction………………………………………..........……………………………………19 2.3 Results………………………………………......……………………………………………21 2.3.1 Bayesian refinement and conditional and joint multiple-SNP analyses predict that rs17114036 and rs2184104 are possible causal SNPs located in CAD/IS locus 1p32.2….... ….21 2.3.2 CAD/IS-associated SNP rs17114036 is located in an enhancer element in HAECs…....…22 2.3.3 CRISPR-based approaches identified rs17114036-containing region as a cis-regulatory element for endothelial PLPP3 expression……………………….……….……………………24 2.3.4 Unidirectional flow increases the enhancer activity in vascular endothelium……….…….25 2.3.5 CAD/IS-protective allele C at rs17114036 confers a higher enhancer activity……...…….26 2.3.6 CAD/IS-protective allele C at rs17114036 promotes flow-induced, KLF2-mediated enhancer activity………………………......…………………………….………………………28 2.4 Discussion……………………………………………………………………………………29 iii 2.5 Materials and methods……………………………………………………………………….33 2.5.1 Cell culture…………………………………………………………………………………33 2.5.2 H3K27ac and H3K4me2 chromatin immuno-precipitation with whole genome sequencing (ChIP-seq) ………………………………………………………………………………….……34 2.5.3 Chromatin accessibility quantitative trait locus (caQTL) mapping and allelic imbalance...34 2.5.4 RNA-seq…………………………………………………………………………………...36 2.5.5 Normalization of high-throughput sequencing data……………….……………………...36 2.5.6 Dual luciferase assay……………………………………………………………………….37 2.5.7 CRISPR Cas9-mediated deletion of enhancer in teloHAECs……………………………...37 2.5.8 CRISPR interference (CRISPRi).…............................................................................….…39 2.5.9 Leukocyte adhesion assay………………………………………………………………….39 2.5.10 Measurement of transendothelial electrical resistance………...…………………………40 2.5.11 Chromatin Immunoprecipitation PCR……………………………………………………40 2.5.12 mRNA quantitative real-time PCR……………………………………………………….41 2.5.13 Reagents and antibodies…………....................……………….............…………………41 2.5.14 Application of athero-relevant flows………..........………………………………………42 CHAPTER 3: Genome-Wide Identification of Endothelial Mechanosensitive transcriptome and cis-Regulatory Elements......................................................................................................80 3.1 Abstract....................................................................................................................................80 3.2 Introduction..............................................................................................................................80 3.3 Results......................................................................................................................................82 3.3.1 RNA-sequencing identifies 1,432 unique differentially expressed genes under flow..........82 3.3.2 ATAC-sequencing identifies 2,473 unique differentially accessible chromatin regions regulated by athero-relevant flows.................................................................................................83 3.3.3 Nucleosome-free clusters in endothelial cells.......................................................................84 3.3.4 ERG, KLF, JUN, and NFB transcription factors bind many flow-sensitive regions in endothelial genome........................................................................................................................85 3.3.5 GWAS loci in flow-sensitive cis-regulatory elements..........................................................85 3.3.6 Promoter capture Hi-C in endothelial cells...........................................................................86 iv 3.4 Discussion................................................................................................................................87 3.4.1 2% of chromatin accessible regions are differentially induced by flow...............................87 3.4.2 Majority of flow-sensitive genes are indirect transcriptional targets....................................88 3.4.3 Nearly all nucleosome-free clusters contacts at least one gene promoter............................89 3.4.4 One in three nucleosome-free clusters co-localizes with GWAS SNPs...............................90 3.5 Materials and Methods.............................................................................................................91 3.5.1 Tissue culture of human aortic endothelial cells...................................................................91 3.5.2 RNA-seq for mechanosensitive genes..................................................................................91 3.5.3 ATAC-seq for mechanosensitive chromatin accessibility....................................................92 3.5.4 Quantification and heatmap visualization.............................................................................92 3.5.5 Nucleosome-free cluster analysis.........................................................................................92 3.5.6 Motif analysis........................................................................................................................93 3.5.7 Gene ontology analysis.........................................................................................................93 3.5.8 Promoter-capture Hi-C and enhancer-promoter interaction analysis...................................93 3.5.9 Genomic annotation of ATAC peaks....................................................................................94 3.5.10 Transcription factor binding analysis..................................................................................94 3.5.11 GWAS analysis...................................................................................................................94 CHAPTER 4: Summary and Conclusions...............................................................................135 4.1 Endothelial PLPP3 expression in humans is regulated by human genetic variation and is relevant to CAD...........................................................................................................................135 4.2 A causal nucleotide(s) in the CAD loci 1p32.2.....................................................................138 4.3 Additional cis-regulatory mechanisms associated with CAD locus 1p32.2..........................139 4.4 Allelic Distribution and Selection Signals at rs17114036.....................................................140 4.5 Additional causal SNPs in the CAD locus 1p32.2.................................................................141 4.6 Increasing gene-editing efficiency in primary human endothelial cells................................142 4.7 Additional GWAS SNPs located in mechanosensitive cis-regulatory elements...................142 4.8 Flow-sensitive transcription factors in the regulation of mechano-sensitive cis-regulatory elements.......................................................................................................................................143 v 4.9 Necessity of patho-physiological flow in endothelial biology studies..................................144 4.10 Future studies to identify additional cis-regulatory elements at the interface of genetic predisposition and endothelial mechano-sensing mechanisms....................................................145 Works Cited……………………….….......................................................…………………….146 vi LIST OF FIGURES Figure 2.1: Fine mapping strategies at 1p32.2 locus.....................................................................43 Figure 2.2: Genome browser image of chromatin state at PLPP3 locus with SNPs within high LD of rs17114036................................................................................................................................44 Figure 2.3: Chromatin and histone modifications cross cell lines.................................................45 Figure 2.4: Genome browser image of chromatin state at PLPP3 locus.......................................46 Figure 2.5: Electroporation of plasmids results in high and stable transfection efficiency in teloHAEC.......................................................................................................................................47