THE UNIVERSITY OF CHICAGO GENETIC AND GENOMIC APPROACHES TO INVESTIGATE THE ROLES OF CIS- REGULATORY ELEMENTS IN DEVELOPMENT AND DISEASE 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 COMMITTEE ON GENETICS, GENOMICS, AND SYSTEMS BIOLOGY BY LINDSEY ELIZABETH MONTEFIORI CHICAGO, ILLINOIS JUNE 2019 Copyright © 2019 by Lindsey Elizabeth Montefiori All rights reserved TABLE OF CONTENTS List of figures ........................................................................................................................ viii List of tables ............................................................................................................................. x List of supplemental files (available online) ........................................................................... xi Acknowledgements ................................................................................................................ xii Abstract of the dissertation ................................................................................................... xiv Chapter 1: Introduction .......................................................................................................... 1 1.1 Overview of the control of gene expression regulation by cis-regulatory elements .......... 1 1.2 Molecular characteristics of cis-regulatory elements ....................................................... 2 1.3 The curious case of non-coding sequence conservation .................................................... 3 1.4 Gene regulation in the context of the 3D genome ............................................................. 5 1.5 Principles of genome organization: from large-scale compartmentalization of chromosomes to fine-scale mapping of enhancer-promoter loops .......................................................... 6 1.6 Gene regulation and human disease ................................................................................. 10 1.7 Genetic variation and complex disease: The Genome-Wide Association Study (GWAS) ........................................................................................................................... 11 1.8 Common genetic variation and cardiovascular disease ................................................... 13 1.9 Overview of thesis research projects ............................................................................... 14 Chapter 2: Reducing mitochondrial reads in ATAC-seq using CRISPR/Cas9.................... 17 2.1 Abstract ............................................................................................................................ 17 iii 2.2 Introduction ...................................................................................................................... 18 2.3 Results .............................................................................................................................. 19 2.3.1 Development and implementation of anti-mt CRISPR treatment ................................ 19 2.3.2 Effect of removing detergent from the original ATAC-seq protocol ........................... 24 2.3.3 Variations on the anti-mt CRISPR treatment................................................................ 30 2.4 Discussion ........................................................................................................................ 31 2.5 Methods............................................................................................................................ 32 2.5.1 Human lymphoblastoid cell line growth and harvesting .............................................. 32 2.5.2 Preparation of ATAC-seq libraries ............................................................................... 33 2.5.3 Anti-mitochondrial CRISPR/Cas9 treatment ................................................................ 33 2.5.4 Peak calling ................................................................................................................... 35 2.5.5 Fraction of TSS and enhancers intersecting peaks ....................................................... 36 2.5.6 Mean fraction of common peaks and mean Pearson’s R2 of read counts ..................... 36 2.5.7 Statistical tests ............................................................................................................... 36 2.6 Appendix A: Supplemental Figures ................................................................................. 38 2.7 Appendix B: Supplemental Tables .................................................................................. 42 Chapter 3: Deletion of an ultraconserved element causes reduced body weight in mice ....................................................................................................................... 44 3.1 Abstract ............................................................................................................................ 44 3.2 Introduction ...................................................................................................................... 44 3.3 Results .............................................................................................................................. 47 3.3.1 Extreme sequence conservation and functional characterization of the Irx UCEs ....... 47 iv 3.3.2 Deletion of UCE5, but not UCE3, results in reduced body weight on a high fat diet .. 49 3.3.3 UCE3 and UCE5 are not required for Irx3 or Irx5 expression in the adult hypothalamus ................................................................................................................ 50 3.4 Discussion ........................................................................................................................ 52 3.5 Methods............................................................................................................................ 57 3.5.1 Generation of UCE deletion mice ................................................................................. 57 3.5.2 High fat diet and body weight measurements ............................................................... 58 3.5.3 Quantitative real-time PCR ........................................................................................... 58 3.5.4 RNA-seq ....................................................................................................................... 59 3.6 Appendix C: Supplemental Figures ................................................................................. 60 3.7 Appendix D: Supplemental Tables .................................................................................. 61 Chapter 4: A promoter interaction map for cardiovascular disease genetics ................ 69 4.1 Abstract ............................................................................................................................ 69 4.2 Introduction ...................................................................................................................... 69 4.3 Results .............................................................................................................................. 71 4.3.1 iPSC-derived cardiomyocytes provide an effective model to study the architecture of CVD genetics ................................................................................................. 71 4.3.2 Promoter-capture Hi-C identifies distal regulatory elements in iPSCs and CMs ......... 72 4.3.3 Promoter interactions are enriched for tissue-specific transcription factor motifs ....... 77 4.3.4 Long-range promoter interactions are enriched for active cis-regulatory elements and correspond to gene expression dynamics ............................................................................... 80 v 4.3.5 Dynamic changes in genomic compartmentalization involve a subset of cardiac-specific genes ...................................................................................................................................... 83 4.3.6 CM promoter interactions link GWAS SNPs to target genes ....................................... 86 4.3.7 Using gene expression as a metric for interpreting disease-relevance of newly identified target genes ............................................................................................................................ 89 4.3.8 CM promoter interactions are informative to cardiovascular associations that do not directly involve cardiomyocytes ............................................................................................ 92 4.4 Discussion ........................................................................................................................ 95 4.5 Methods.......................................................................................................................... 100 4.5.1 Tissue culture of iPSCs ............................................................................................... 100 4.5.2 Cardiomyocyte differentiation .................................................................................... 100 4.5.3 Promoter capture Hi-C ................................................................................................ 102 4.5.4 Interaction calling ....................................................................................................... 105 4.5.5 4C-style plots .............................................................................................................