THE REGULATION OF URIDINE 5’DIPHOSPHO- GLUCURONOSYLTRANSFERASE 1A (UGT1A) GENE FAMILY MEMBERS BY VITAMIN D RECEPTOR Khanyile Merritt Dube BSc (Hons) Biomedical Sciences Research conducted within The School of Biomedical Science Faculty of Life and Health Sciences University of Ulster A thesis submitted for the degree of Doctor of Philosophy October 2020 I confirm that the word count of this thesis is less than 100,000 words 1 Contents DECLARATION 13 ACKNOWLEDGEMENTS 14 SUMMARY 15 ABBREVIATIONS 17 1: Chapter 1: Introduction 1.1.1 Vitamin D and its biosynthesis 25 1.1.2 Hepatic and Renal Metabolism of Vitamin D 25 1.1.3.Homeostatic control of Vitamin D 26 1.2 Actions of Vitamin D 28 1.2.1 Regulation of Mineral Homeostasis (Classical Role of Vitamin D) 1.3 Clinical Recommendation of Vitamin D 29 1.4 Genomic Mechanisms of Vitamin D 31 1.4.1 Introduction to Nuclear Receptors 31 1.4.2 Vitamin D Receptor 35 1.4.3 Genome-wide studies of VDR/RXR binding in various cell lines 35 1.5 Non-classical roles of Vitamin D 38 1.5.1 Vitamin D and Immunology 38 1.5.2 Vitamin D and Cardiovascular Diseases 38 1.5.3 Vitamin D and Neurodegenerative Disorders 39 1.5.4 Vitamin D and Malignancies 39 1.5.5 Vitamin D and Metabolism 40 1.6. UDP-Glucuronosyltransferases (UGTs) 42 2 1.6.1 UGT1A Tissue Distribution 45 1.6.2 Transcriptional Regulation of UGT1A gene family members 47 1.6.3 UGT1A Clinical Relevance 50 1.7 NRF2: The master regulator of oxidative stress 53 1.7.1 NRF2 Discovery and Function 54 1.7.2 NRF2 activation and repression 57 1.7.3 NRF2 and Target genes 58 1.7.4 NRF2 in Health and Disease 59 1.8 Vitamin D – ‘A custodian for phenotypic stability’ 60 1.9 Research Aims and Objectives 64 2 Chapter 2: Material and Methods 2.1 Reagents 67 2.2 Cell Culture 67 2.2.1 LS180 cells 67 2.2.2 Human Embryonic Kidney cells (HEK293 cells) 68 2.2.3 LNCaP cells 68 2.2.4 Sub-culture routine 68 2.2.5 Cell Storage 68 2.2.6 Thawing cells 69 2.2.7 Cell Density 69 2.2.8 Dosage with Ligands 70 2.3 Polymerase Chain Reaction (PCR) 71 2.3.1 RNA Extraction 71 2.3.2 Ethanol Precipitation 72 3 2.3.3 cDNA Synthesis 72 2.3.4 Primer Design and Preparation 73 2.3.5 Endpoint PCR 75 2.3.6 Gel Electrophoresis 76 2.3.7 Real Time PCR 76 2.4 DNA Extraction 78 2.4.1 DNA Amplification 79 2.4.2 Gel Excision and DNA purification 80 2.5 Western Blot Analysis 81 2.5.1 Extraction Protein from Whole Cell Lysates 81 2.5.2 Extraction of Protein from Microsomal fractions 81 2.5.3 Quantification of Protein 82 2.5.4 Blotting 82 2.5.5 Probing with antibody 83 2.6 Glucuronidation Activity Assay 85 2.6.1 Preparation of cell homogenates 85 2.6.2 Glucuronidation Assay using UGT-glo 85 2.6.3 UGT Activity Detection 86 2.7 Reporter Activity Assay 86 2.7.1 Plasmids 86 2.7.2 Transformation of plasmids into Library Efficiency® DH5α E.coli (Invitrogen) 88 2.7.3 Transfection Methodology 89 2.7.3.1 Lipofectamine Transfection 89 4 2.7.3.2 Calcium Phosphate Transfection 89 2.7.4 Reporter Assay Reading 90 2.8 Site-directed Mutagenesis 90 2.9 Sequencing 92 2.10 Cell Viability Assay 94 2.11 Growth Inhibition Assay 94 2.12 In Silico Analysis 94 2.12.1 In Silico Screening for putative response elements 94 2.13 CloneJet PCR cloning 95 2.13.1 Ligation Formula 95 2.13.2 Analysis of recombinant clones 96 2.14 Clustered Regulatory Interspaced short palindromic repeats interference (CRISPRi) engineering 97 2.14.1 Single guide RNA design 97 2.14.2 Ribonucleoprotein (RNP) complex formation and Lipofection 98 3 Chapter 3 Induction of UGT1A gene family members by Vitamin D Receptor 3.1 Introduction 102 3.2 Results 107 3.2.1 Vitamin D regulates the expression of UGT1A gene family members 107 3.3 VDR is an autonomous regulator of the UGT1A gene 112 3.4 Direct repeat 3 (DR3) motif within the UGT1A1 proximal gene promoter is a functional Vitamin D response element (VDRE). 116 5 3.5 Vitamin D enhances UGT1A protein expression in LS180 cells 122 3.6 Vitamin D increases glucuronidation activity in LS180 cells 126 3.7 Discussion 128 3.8 Summary of key findings 135 4 Chapter 4: Examining the cross-talk between Vitamin D Receptor (VDR) and nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathways 4.1 Introduction 137 4.2 Results 139 4.2.1 Cell Viability Assay 139 4.2.2 NRF2 and VDR ligands induce ARE-signaling in LS180 cells 140 4.2.3 ARE-signaling in Vitamin D pre-treated LS180 cells 143 4.2.4 ARE minimal promoter activity is modulated by NRF2 and VDR ligands 145 4.2.5 The effects of NRF2 and VDR ligands co-treatment in LS180 cells 146 4.2.6 Establishing co-dependency of VDR and NRF2 signaling 149 4.2.7 Effects of VDR and NRF2 ligands in combinatorial Treatment 151 4.2.8 VDRE minimal promoter activity is suppressed by sulphorafane (SFN) 154 4.2.9 Vitamin D pre-treatment enhances NRF2-mediated 6 UGT1A1 activity 156 4.2.10 Modulation of detoxification products in pre-treated LS180 cells 159 4.2.11 Anti-tumoural actions of VDR and NRF2 signaling Pathways 158 4.3 VDR and NRF2 signaling interplay in LNCaP cells 168 4.3.1 NRF2 and VDR interplay in LNCaP cells 168 4.3.2 VDR and NRF2 ligands inhibit LNCaP and LS180 cell growth 173 4.4 Discussion 174 4.5 Summary of key findings 184 5 Chapter 5: Characterization of UGT1A loci 5.1 Introduction 186 5.2 Results 190 5.2.1 The UGT1A locus is regulated by novel VDR/RXR binding 190 5.2.2 Characterization of the UGT1A gene using CRISPRi 196 5.3 Discussion 201 5.4 Summary of key findings 205 6 Chapter 6: General Discussion 6.1 Discussion 207 6.2 Clinical Relevance 212 6.3 Limitations of this study 216 7 6.4 Future Experiments 217 6.5 Summary of key findings 219 6.6 Concluding Remarks 220 7 References 221 8 Appendix 263 List of Figures 1: Chapter 1 1.1Chemical Structure and Vitamin D Biosynthesis 27 1.2 Structural organization of VDR 33 1.3 Gene transcription activity 37 1.4 A representation of cellular detoxification 41 1.5 The endoplasmic reticulum (ER) glucuronidation pathway 43 1.6 The Genomic Organization of the UGT1A loci on chromosome 2q37 45 1.7 Differential expression of UGT1A and UGT2B genes in normal tissue 46 1.8 The phenobarbital responsive enhancer module nucleotide sequence 50 1.9 NRF2 structural domain 56 1.10 The domain structure of small Maf proteins 57 1.11 Examples of NRF2 target genes 59 1.12 A proposed model of Vitamin D and NRF2 co-operative effects 63 2 Chapter 2 2.1 The pGL4.37[luc2P/ARE/Hygro] Vector 87 2.2 The pGL3-Basic Luciferase Reporter Vector 88 2.3 UGT1A1 promoter sequence 91 2.4 PJET1.2/blunt is a blunt- end-cloning vector 97 8 2.5 CRISPRi transcriptional silencing model 100 3 Chapter 3 3.1 Gene expression in LS180 cells 107 3.2 UGT1A expression profile in LS180 cells 109 3.3 mRNA expression of VDR target genes in LS180 cells 111 3.4 Time-dependent mRNA expression of UGT1A genes by VDR and PXR ligands. 112 3.5 The UGT1A1 phenobarbital responsive enhancer module 114 3.6 VDR regulates UGT1A1 promoter activity 115 3.6 Identification of a functional VDRE within the UGT1A1 promoter region. 3.7 UGT1A sequence 118 3.8 VDR and PXR share the same DR3-type binding motif within the UGT1A1 promoter region. 120 3.10 Defining the DR3-type motif specificity within the UGT1A1 promoter region 3.11 Western Blot analysis showing UGT1A protein expression in LS180 cells 124 3.12 UGT1A protein expression in LS180 cells by VDR and PXR ligands 125 3.13 Nuclear receptor expression in various cancer cell lines 125 3.14 VDR ligands enhance glucuronidation activity in LS180 cells 127 4 Chapter 4 4.1 Cell Viability of NRF2 ligands in LS180 cells 140 9 4.2 Titration curve of ARE activation in LS180 and HEK293 cells. 142 4.3 ARE activation by NRF2 ligands following pre-treatment in LS180 cells 144 4.4 ARE minimal promoter activity is mediated by NRF2 and VDR ligands. 146 4.5 VDR and NRF2 ligands co-treatment in LS180 cells. 148 4.6 VDR and NRF2 co-dependency in UGT1A1 induction. 150 4.7 Effects of VDR and NRF2 target genes in combinatorial treatments 4.8 VDRE signalling in LS180 cells. 154 4.9 UGT1A1 induction in LS180 cells pre-treated with 1,25D 156 4.10 Effects of VDR and NRF2 target genes in combinatorial treatments 4.11 Effects of VDR and NRF2 target genes in combinatorial treatments 4.12 ARE signalling in LNCaP cells 167 4.13 NRF2 target gene regulation in LNCaP cells pre-treated with 1,25D 170 4.14 VDR target gene regulation in LNCaP cells pre-treated with 1,25D 4.15 Growth inhibition assay in LNCaP and LS180 cells 174 5 Chapter 5 5.1 CRISPRi –based approach model 189 5.2 RSAT output of putative VDREs within UGT1A4 promoter region 5.3 UGT1A loci is regulated by VDR/RXR binding motifs 195 5.4UGT1A plasmid construction by sub-cloning 196 5.5 RT-PCR product of amplified of the 1532by DNA fragment of the UGT1A locus 196 10 5.6 Lipofection procedure for CRISPRi technique.