Regulation of UDP- glucuronosyltransferase 1A genes in the intestine by Siti Nurul Mubarokah Thesis Submitted to Flinders University for the degree of Doctor of Philosophy College of Medicine and Public Health 18 October 2018 TABLE OF CONTENTS List of Figures ................................................................................................................................ vi List of Tables ................................................................................................................................. x Summary ................................................................................................................................ xi Declaration ............................................................................................................................... xiii Acknowledgements ....................................................................................................................... xiv Publication ............................................................................................................................... xvi Conference Proceedings ............................................................................................................... xvi Scholarships and Awards in Support of This Thesis .................................................................... xviii Abbreviations ............................................................................................................................... xix CHAPTER 1. INTRODUCTION ....................................................................................................... 1 1.1 Glucuronidation and human UGTs ......................................................................................... 1 1.1.1 Glucuronidation .......................................................................................................... 1 1.1.2 Human UDP-Glucuronosyltransferase (UGT) enzymes .............................................. 3 1.1.3 The UGT gene super family ........................................................................................ 5 1.2 Expression of UGT genes: general aspects ......................................................................... 10 1.2.1 Tissue specific distribution of human UGTs .............................................................. 10 1.2.2 Constitutive and inducible regulation of UGTs .......................................................... 12 1.3 Intestinal UGT enzymes ....................................................................................................... 15 1.3.1 Intestinal UGTs in phenolic compound glucuronidation ............................................ 15 1.3.2 Intestinal microbiota and UGT activities .................................................................... 18 1.3.3 Expression of intestinal UGTs .................................................................................. 19 1.4 Intestinal development and homeostasis ............................................................................. 20 1.4.1 Intestinal development is critically regulated by homeobox factors including the master regulator CDX2 ............................................................................................. 20 1.4.2 The intestine is maintained by continual epithelial turnover that is controlled by developmental regulators. ........................................................................................ 22 1.4.3 Constitutive regulation of intestinal UGTs by CDX2 and its partners ......................... 27 1.5 In vitro models for intestinal UGT studies ............................................................................. 31 1.5.1 Caco-2 as a model for enterocytes ........................................................................... 31 1.5.2 The intestinal organoid model ................................................................................... 32 1.5.3 ‘Humanised’ UGT transgenic mouse models ............................................................ 34 1.6 UGT1A8 is the prototypical intestinal-specific UGT gene ..................................................... 36 i 1.6.1 UGT1A8 expression, activities and substrates .......................................................... 36 1.6.2 UGT1A8 polymorphisms in drug metabolism and cancer risk ................................... 38 1.6.3 UGT1A8 gene regulation .......................................................................................... 40 1.7 Project overview and experimental aims .............................................................................. 44 CHAPTER 2. MATERIALS AND GENERAL METHODS ............................................................... 46 2.1 Materials .............................................................................................................................. 46 2.1.1 Chemicals, reagents and buffers .............................................................................. 46 2.1.2 Mammalian cell lines ................................................................................................ 46 2.1.3 Expression vectors and reporters ............................................................................. 46 2.1.4 Oligonucleotides ....................................................................................................... 47 2.2 Methods ............................................................................................................................... 47 2.2.1 Mammalian cell culture, maintenance and stocks ..................................................... 47 2.2.2 Bacterial culture and stocks ...................................................................................... 47 2.2.3 Cryopreservation of cells (freezing stocks) ............................................................... 47 2.2.4 Polymerase chain reaction (PCR) amplification ........................................................ 48 2.2.5 Total RNA extraction ................................................................................................ 49 2.2.6 cDNA synthesis ........................................................................................................ 50 2.2.7 Quantification of nucleic acid (DNA or RNA) ............................................................. 50 2.2.8 Molecular cloning using restriction enzymes ............................................................. 51 2.2.9 Competent cell preparation ....................................................................................... 51 2.2.10 Plasmid DNA extraction ............................................................................................ 52 2.2.11 Agarose gel electrophoresis ..................................................................................... 52 2.2.12 DNA Sequencing ...................................................................................................... 53 2.2.13 Plasmid-DNA transfection......................................................................................... 53 2.2.14 Luciferase reporter assays ....................................................................................... 54 2.2.15 Chromatin Immunoprecipitation (ChIP) assays ......................................................... 55 2.2.16 Electrophoretic mobility shift assay (EMSA) ............................................................. 56 2.2.17 Western blotting ....................................................................................................... 57 2.2.18 Cell imaging (EVOS® FL) ......................................................................................... 58 2.2.19 Statistics ................................................................................................................... 58 CHAPTER 3. REGULATION OF INTESTINAL UGTs BY A COMBINATORIAL MECHANISM OF CDX2 AND HNF4 ................................................................................................ 59 3.1 Introduction .......................................................................................................................... 59 3.2 Methods ............................................................................................................................... 63 3.2.1 Plasmids ................................................................................................................... 63 ii 3.2.2 Mutagenesis of binding sites in UGT1A8, -1A9 and -1A10 promoter constructs ....... 63 3.2.3 Transfections of CDX2 and HNF4 expression vectors ............................................ 64 3.2.4 Chromatin Immunoprecipitation (ChIP) assays in Caco-2 cells ................................. 64 3.2.5 Non-radioactive EMSA assays in Caco-2 cells ......................................................... 64 3.2.6 CDX2 and HNF4α siRNA design and transfection .................................................... 64 3.2.7 Transcriptomic data profiling of Colon Adenocarcinoma (COAD) from The Cancer Genome Atlas (TCGA) ............................................................................................. 65 3.2.8 Oligonucleotides ....................................................................................................... 65 3.3 Results and Discussion ........................................................................................................ 67 3.3.1 CDX2 synergistically interacts with HNF4 to further induce UGT1A8 promoter activity .....................................................................................................................