REGULATION OF BOVINE β-DEFENSIN EXPRESSION THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF DUBLIN FOR THE DEGREE OF DOCTOR OF PHILOSOPHY 2017 ANNE BARRY-REIDY SCHOOL OF BIOCHEMISTRY & IMMUNOLOGY TRINITY COLLEGE DUBLIN SUPERVISORS: PROF. CLIONA O’FARRELLY & DR. KIERAN MEADE TABLE OF CONTENTS DECLARATION ................................................................................................................................. vii ACKNOWLEDGEMENTS ................................................................................................................... viii ABBREVIATIONS ................................................................................................................................ix LIST OF FIGURES............................................................................................................................. xiii LIST OF TABLES .............................................................................................................................. xvii ABSTRACT ........................................................................................................................................xix Chapter 1 Introduction ........................................................................................................ 1 1.1 Antimicrobial/Host-defence peptides ..................................................................... 1 1.2 Defensins................................................................................................................. 1 1.3 β-defensins .............................................................................................................. 4 1.3.1 Antimicrobial properties .................................................................................. 6 1.3.2 Immunomodulatory properties......................................................................... 9 1.3.3 β-defensin 3 .................................................................................................... 12 1.3.4 β-defensins in reproduction ........................................................................... 14 1.4 Regulation of β-defensins ..................................................................................... 16 1.4.1 Regulation of syntenic cluster D β-defensins ................................................ 16 1.4.2 Regulation of syntenic cluster B β-defensins................................................. 21 1.4.3 The non-coding genome and regulation of β-defensins ................................. 22 1.5 A potential role for bovine β-defensins in fertility ............................................... 24 1.5.1 Fertility in Irish beef and dairy herds ............................................................. 24 1.5.2 β-defensins in bull fertility and uterine disease ............................................. 26 1.6 Aims and objectives .............................................................................................. 28 Chapter 2 Materials and Methods .................................................................................... 29 2.1 Bioinformatics analysis ......................................................................................... 29 2.1.1 Generation of β-defensin ortholog promoter sequence sets. .......................... 29 ii 2.1.2 Transcription factor binding site over-representation analysis- JASPAR, oPOSSUM3............................................................................................................. 31 2.1.3 Detection of conserved motifs- DREME and TOMTOM (MEME suite) ..... 35 2.1.4 Prediction of conserved non-coding sequences- MULAN multi-species alignment................................................................................................................. 36 2.1.5 Modelling of transcription factor binding site features within conserved non- coding regions- CLARE ......................................................................................... 37 2.1.6 Prediction of transcription factors binding conserved motifs in intergenic evolutionarily-conserved regions ............................................................................ 38 2.2 Collection of female reproductive tract tissue samples ........................................ 39 2.3 List of reagents, materials and equipment used .................................................... 40 2.4 Primary endometrial stromal cell culture .............................................................. 43 2.4.1 Generation of primary endometrial stromal cell cultures .............................. 43 2.4.2 Quantification of bovine herpesvirus DNA in endometrial tissue samples ... 44 2.4.3 Immunofluorescent staining of endometrial cell cytoskeletal proteins ......... 44 2.4.4 Detection of leukocyte contamination of primary endometrial cell cultures via PTPRC polymerase chain reaction (PCR) .............................................................. 45 2.4.5 Histological processing of endometrial samples............................................ 45 2.4.6 Stimulation of cells ........................................................................................ 46 2.5 Quantitative PCR (PCR) ....................................................................................... 48 2.5.1 RNA extraction .............................................................................................. 48 2.5.2 cDNA synthesis ............................................................................................. 49 2.5.3 Quantitative PCR primer design .................................................................... 49 2.5.4 Quantitative PCR ........................................................................................... 50 2.5.5 Quantitative PCR assay optimisation............................................................. 51 2.5.6 qPCR data processing .................................................................................... 52 2.5.7 Statistical Analysis ......................................................................................... 53 iii Chapter 3 In-silico prediction of transcriptional regulation mechanisms of β-defensin genes ..................................................................................................................................... 54 3.1 Introduction ........................................................................................................... 54 3.1.1 Computational analysis of gene regulation .................................................... 54 3.1.2 Transcriptional regulation of β-defensins ...................................................... 57 3.2 Hypothesis and specific aims ................................................................................ 61 3.3 Results ................................................................................................................... 62 3.3.1 TFBS over-representation analysis of syntenic cluster B β-defensin promoters ................................................................................................................................. 62 3.3.2 DREME detection of conserved DNA motifs in syntenic cluster B β-defensin promoters ................................................................................................................ 85 3.3.3 Multi-species alignment of syntenic cluster B β-defensin gene region and detection of evolutionarily-conserved regions ........................................................ 87 3.3.4 TFBS modelling in the DEFB127-DEFB126 intergenic ECR ...................... 93 3.3.5 TFBS modelling in the DEFB116-DEFB117 intergenic ECR ...................... 95 3.3.6 TFBS modelling in the DEFB123-DEFB124 intergenic ECR ...................... 97 3.3.7 Prediction of TFs binding conserved motifs within ECRs ............................ 98 3.3.8 oPOSSUM3 TFBS over-representation analysis of identified ECRs .......... 101 3.3.9 oPOSSUM3 TFBS over-representation analysis of syntenic cluster D genes DEFB103 and DEFB106 ...................................................................................... 104 3.3.10 DREME detection of conserved DNA motifs in syntenic cluster D β-defensin promoters .............................................................................................................. 107 3.4 Discussion ........................................................................................................... 108 Chapter 4 Proinflammatory cytokines regulate β-defensin expression in primary endometrial stromal cells ................................................................................................. 116 4.1 Introduction ......................................................................................................... 116 4.1.1 Inflammation within the cow reproductive tract .......................................... 118 4.1.2 The structure of the endometrium ................................................................ 123 iv 4.2 Hypothesis and specific aims .............................................................................. 126 4.3 Results ................................................................................................................. 127 4.3.1 Several chromosome 27 β-defensin genes are expressed in the female reproductive tract .................................................................................................. 127 4.3.2 Validation of the predicted gene DEFB106 is expressed in the female reproductive tract .................................................................................................