Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Winter 12-15-2019 Elucidating Enhancer Function in Epidermal Development and Filaggrin Loss-of-Function Variants in African American Atopic Dermatitis Mary Elizabeth Mathyer Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Genetics Commons, and the Molecular Biology Commons Recommended Citation Mathyer, Mary Elizabeth, "Elucidating Enhancer Function in Epidermal Development and Filaggrin Loss-of- Function Variants in African American Atopic Dermatitis" (2019). Arts & Sciences Electronic Theses and Dissertations. 2008. https://openscholarship.wustl.edu/art_sci_etds/2008 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology and Biomedical Sciences Molecular Genetics and Genomics Dissertation Examination Committee: Cristina de Guzman Strong, Chair Shiming Chen John Edwards Jacqueline Payton Celia Santi Elucidating Enhancer Function in Epidermal Development and Filaggrin Loss-of-Function Variants in African American Atopic Dermatitis by Mary Elizabeth Mathyer A dissertation presented to the The Graduate School of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy December 2019 St. Louis, Missouri © 2019, Mary Elizabeth Mathyer Table of Contents List of Figures .................................................................................................................................. v List of Tables .................................................................................................................................. vi Acknowledgments ........................................................................................................................ vii Abstract of the Dissertation ............................................................................................................. x Chapter 1: Introduction .................................................................................................................... 1 1.1 Overview ......................................................................................................................... 2 1.2 Skin Barrier Structure and Function ................................................................................ 3 1.3 The Epidermal Differentiation Complex ......................................................................... 4 1.3.1 Discovery of the EDC ............................................................................................ 6 1.3.2 Involucrin ............................................................................................................... 6 1.3.3 Filaggrin ................................................................................................................. 7 1.4 Regulation of EDC Gene Expression .............................................................................. 9 1.4.1 Epigenetic Modifications of the EDC ................................................................. 10 DNA Methylation ................................................................................................... 11 Histone Methylation ............................................................................................... 12 Histone Acetylation ................................................................................................ 14 1.4.2 Nonrandom Nuclear Chromatin Localization and Accessibility ......................... 14 1.4.3 Enhancer-Centric Regulation of the EDC ........................................................... 17 Elucidating Enhancers ............................................................................................ 17 CNE 923 ................................................................................................................. 19 1.4.5 Chromosome Conformation Capture Further Elucidates Dynamic Chromatin Remodeling ......................................................................................................... 20 1.4.6 Beyond Enhancer Predictions .............................................................................. 22 1.5 Atopic Dermatitis .......................................................................................................... 23 1.5.1 Epidemiology ....................................................................................................... 23 1.5.1 FLG in AD ........................................................................................................... 24 1.6 Scope of Thesis .............................................................................................................. 25 1.7 References ..................................................................................................................... 26 Chapter 2: The 923 enhancer is required for cis-regulation of the proximal target gene involucrin in keratinocytes ............................................................................................................ 37 2.1 Abstract .......................................................................................................................... 38 2.2 Introduction ................................................................................................................... 38 2.3 Results ........................................................................................................................... 41 2.3.1 Generation and Phenotypic Characterization of 923 KO Mice ........................... 41 2.3.2 Deletion of the 923 Enhancer in Mice Results in Decreased Expression of the Proximal Gene Involucrin in the Skin ............................................................... 44 ii 2.3.3 923 Enhancer Regulates Ivl Target Gene Expression in Cis ............................... 46 2.3.4 Deletion of 923 Enhancer Affects the Chromatin Landscape in the EDC .......... 47 2.4 Discussion ...................................................................................................................... 49 2.5 Materials and Methods .................................................................................................. 54 2.5.1 Mice Genotyping ................................................................................................. 54 2.5.2 Histology ............................................................................................................. 54 2.5.3 Dye Penetration Barrier Assays ........................................................................... 55 2.5.4 Cornified Envelope Preparations ......................................................................... 55 2.5.5 Trans-epidermal Water Loss Assay ..................................................................... 55 2.5.6 Generation of 923 enhancer knockout alleles in mice by CRISPR/Cas9 genome editing ................................................................................................... 55 2.5.7 RNA-seq .............................................................................................................. 56 2.5.8 Real-time qPCR ................................................................................................... 58 2.5.9 Allele-specific gene expression ........................................................................... 58 2.5.10 ATAC-Seq ......................................................................................................... 59 2.5.11 Western Blots .................................................................................................... 60 2.6 References ..................................................................................................................... 61 2.7 Supplemental Material ................................................................................................... 63 Chapter 3: Tiled array-based sequencing identified enrichment of loss-of-function variants in the highly homologous filaggrin gene in African American children with severe atopic dermatitis ............................................................................................ 72 3.1 Abstract .......................................................................................................................... 73 3.2 Introduction ................................................................................................................... 74 3.3 Results ........................................................................................................................... 76 3.4 Discussion ...................................................................................................................... 79 3.5 Materials and Methods .................................................................................................. 79 3.5.1 Patients ................................................................................................................. 79 3.5.2 FLG array-based sequencing ............................................................................... 80 3.6 References ....................................................................................................................