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Exploring the Roles of KAT2A and KAT2B in Keratinocyte Biology

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Exploring the Roles of KAT2A and KAT2B in Keratinocyte Biology Exploring the Roles of KAT2A and KAT2B in Keratinocyte Biology A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health. School of Medical Sciences 2019 Benjamin W. Walters 2 LIST OF CONTENTS LIST OF FIGURES ...................................................................................................................... 7 LIST OF TABLES ...................................................................................................................... 11 LIST OF ABBREVIATIONS ...................................................................................................... 12 ABSTRACT ............................................................................................................................... 15 DECLARATION ........................................................................................................................ 16 COPYRIGHT STATEMENT ...................................................................................................... 16 ACKNOWLEDGMENTS ........................................................................................................... 17 CHAPTER 1 - INTRODUCTION 1.1 Human Skin Anatomy and Physiology................................................................................... 19 1.1.1 The Subcutaneous Tissue ........................................................................................ 19 1.1.2 The Dermis ............................................................................................................ 19 1.1.3 The Epidermal-Dermal Junction ............................................................................... 20 1.1.4 The Epidermis ........................................................................................................ 20 1.1.4.1 The Basal Layer ...................................................................................................... 20 1.1.4.2 The Spinous Layer .................................................................................................. 21 1.1.4.3 The Granular Layer ................................................................................................. 22 1.1.4.4 The Stratum Corneum ............................................................................................. 22 1.2 In Vitro Systems of Epidermal Homeostasis .......................................................................... 23 1.2.1 Propagation of Primary Keratinocytes Using a Feeder Layer ........................................ 23 1.2.2 Propagation of Keratinocytes in Serum-free Media without a Feeder Layer ................... 24 1.2.3 Generation of Immortalised Keratinocytes ................................................................. 24 1.2.4 Clonal Types of Cultured Keratinocytes .................................................................... 25 1.2.5 Differentiation of Keratinocytes in Conventional Cultures ........................................... 25 1.3 Molecular Control of Epidermal Differentiation..................................................................... 26 1.3.1 Maintenance of Keratinocyte Stemness ..................................................................... 26 1.3.2 Regulation of the Basal to Spinous Cell Switch .......................................................... 29 1.3.3 Molecular Drivers of Late Epidermal Differentiation .................................................. 29 1.4 Chromatin: Modifiers, Modifications, and Gene Regulation ................................................... 33 3 1.4.1 Nucleosome Structure and Function .......................................................................... 33 1.4.2 Histone Modifications Regulate Gene Expression....................................................... 34 1.4.3 Histone Acetylation: A Mark of Transcriptional Activation ......................................... 35 1.4.4 Histone Acetyltransferases: Classification, Structure and Function ............................... 36 1.4.4.1 The MYST Family .................................................................................................. 36 1.4.4.2 The p300/CBP Family ............................................................................................. 38 1.4.4.3 The GNAT Family .................................................................................................. 40 1.4.5 STAGA and Related Complexes in Humans .............................................................. 43 1.5 KAT2A and KAT2B in Stemness and Differentiation ............................................................ 47 1.5.1 KAT2A and KAT2B Developmental Expression Patterns ........................................... 47 1.5.2 Developmental Roles of KAT2A and KAT2B............................................................ 47 1.5.3 KAT2A and KAT2B in the Regulation of the Cell Cycle ............................................. 48 1.5.4 KAT2A as a Regulator of Stemness .......................................................................... 49 1.5.5 KAT2A as a Regulator of Cellular Differentiation ...................................................... 50 1.5.6 KAT2B as a Regulator of Cellular Differentiation ...................................................... 51 1.5.7 Concluding Remarks ............................................................................................... 52 1.6 Hypothesis and Aims ............................................................................................................. 53 CHAPTER 2 - MATERIALS AND METHODS 2.1 Cell Culture ........................................................................................................................... 54 2.1.1 Conditions for Maintaining Cell Cultures .................................................................. 54 2.1.2 Differentiation of Keratinocyte Cultures .................................................................... 55 2.1.3 Short Hairpin RNA (shRNA) Expression Constructs................................................... 56 2.1.4 Generation of KAT2A Expression Constructs ............................................................ 57 2.1.5 Lentiviral Production and Transduction ..................................................................... 59 2.2 Proliferation Assays .............................................................................................................. 60 2.2.1 Trypan Blue Cell Counting Assay ............................................................................ 60 2.2.2 MTT Cell Proliferation Assay .................................................................................. 60 2.2.3 Cell Confluency Assay ............................................................................................ 60 2.3 Gene Expression Analysis ..................................................................................................... 61 4 2.3.1 RNA Extraction ..................................................................................................... 61 2.3.2 cDNA Synthesis (Reverse Transcription) .................................................................. 61 2.3.3 Quantitative-PCR ................................................................................................... 62 2.3.4 RNA-seq Library Preparation and Resulting Sequencing Data Analysis ........................ 65 2.3.5 Polysome Profiling ................................................................................................. 65 2.3.6 RNAscope® In Situ Hybridization ............................................................................. 66 2.4 Analysis of Relative Levels and Subcellular Localisation of Proteins ..................................... 67 2.4.1 Whole Cell Protein Extraction .................................................................................. 67 2.4.2 Subcellular Fractionation for Protein Analysis ........................................................... 68 2.4.3 SDS PAGE and Western Blotting ............................................................................. 68 2.4.4 Immunofluorescence-labelling in Cultured Cells and Tissues ....................................... 71 CHAPTER 3 - DYNAMICS IN GLOBAL HISTONE H3 MODIFICATIONS AND A SWITCH IN KAT2A AND KAT2B EXPRESSION ARE HALLMARKS OF DIFFERENTIATING KERATINOCYTES 3.1 Introduction ........................................................................................................................... 73 3.1 Calcium-induced differentiation of keratinocyte monolayers mirrors in vivo epidermal homeostasis................................................................................................................................. 74 3.2 Global levels of key histone acetylation modifications are reduced upon differentiation of immortalised keratinocytes. ......................................................................................................... 82 3.3 KAT2A and KAT2B are Inversely Expressed in Differentiating Keratinocytes In Vitro ........... 86 3.4 KAT2A and KAT2B are Inversely Expressed in Differentiating Keratinocytes In Vitro ......... 89 3.5 KAT2A and KAT2B are Inversely Expressed in the Adult Human Epidermis .......................... 91 3.6 KAT2A and KAT2B are Inversely Expressed in the Developing Mouse Epidermis .................. 94 CHAPTER 4 - KAT2A FUNCTIONS IN UNDIFFERENTIATED KERATINOCYTES TO MAINTAIN STEMNESS FEATURES WHILST KAT2B PRIMARILY FUNCTIONS IN CALCIUM TREATED CELLS TO DRIVE DIFFERENTIATION
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