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Characterizing Thyroid Hormone Mediated Action on Gene

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Characterizing Thyroid Hormone Mediated Action on Gene Characterizing thyroid hormone mediated action on gene expression in mice: mechanistic insight into thyroid hormone response elements, thyroid hormone receptor-binding sites, and microRNAs by Martin A. Paquette A thesis submitted to the Faculty of Graduate and Postdoctoral Affairs in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology Carleton University Ottawa, Ontario ©2013 Martin A. Paquette ABSTRACT Thyroid hormone (TH) exerts its effects by binding to the TH receptor (TR), which binds to TH response elements (TREs) to regulate target gene expression. Disruption of TH action can have detrimental health effects. The precise molecular mechanisms involved in TH mediated gene expression remain unclear. The overall objectives of this thesis were to: i) characterize global gene and microRNA (miRNA) expression in early response to TH perturbation in mouse liver; ii) identify TREs and TR-binding sites found throughout the mouse genome; and iii) compare TRE half-site organizations and their ability to drive gene expression. Transcriptional profiling of mRNA liver samples from TH disrupted mice enabled the identification of genes that were under direct TH-regulation. TREs in the promoter region of Tor1a, Hectd3, Slc25a45 and 2310003H01Rik were validated in vitro, adding four genes to the battery of only 13 known TRE- containing mouse genes. Hepatic miRNAs were also found to be significantly altered following perturbations in TH levels. In vitro analyses confirmed TH regulation of miR-206. Moreover, Mup1 and Gpd2 were confirmed to be targeted by miR-206 in response to TH, demonstrating that miRNAs can act as master regulators of the TH response pathway. ChIP-chip analysis identified TR-DNA interactions in juvenile mouse liver revealing only a few TR-binding sites consistent between all analyzed samples, suggesting that relatively few genes are under direct TH/TR control. Reporter assays confirmed the presence of TREs in the promoter regions of Ddx54 and Thrsp, thus validating two additional functional mouse TREs. Finally, we investigated the relative ability of liganded homodimers of TR and retinoid X receptor (RXR), and the heterodimer TR/RXR, to regulate gene expression for three TRE half-site organizations. We found that there were fundamental differences between TRE configurations that affect nuclear receptor interactions with the response element and the ability to specifically bind their ii ligands. These studies provide mechanistic insight into TREs, TR-binding sites and TH action. Collectively this thesis increases our understanding of how TH operates to control genome function and provides a basis to develop appropriate testing strategies for environmental chemicals that may disrupt TH-associated genes expression by interaction with TR. iii ACKNOWLEDGEMENTS I would like to express my deep and sincere gratitude to my advisor Dr. Carole Yauk – thank you for your guidance and support throughout this project. Thank you to the committee members; Dr. Iain Lambert, Dr. Mike Wade, Dr. Vance Trudeau and Dr. Michel Dumontier for helpful discussions and input. Special thanks to Dr. Ella Atlas for guidance with the cell culture work, Dr. Hongyan Dong for performing the animal work, Rémi Gagné for performing bioinformatics analyses and to Andrew Williams for helping with statistical analyses. Thank you to the many wonderful people that I have met at Health Canada’s Environmental Health Center that have supported and helped me in different ways, notably, Alexandra Long, Jaime Nead, Meghan Murphy, Julie Cox, Sarah Labib, Julie Bourdon, Amal Malik, Julie Buick and Andrea Rowan-Carroll. Finally, a big thank you to my family, friends and loved ones for the support and motivation provided throughout the past five years. iv TABLE OF CONTENTS ABSTRACT .............................................................................................................................. II ACKNOWLEDGEMENTS .................................................................................................... IV TABLE OF CONTENTS ........................................................................................................ V LIST OF TABLES ................................................................................................................... IX LIST OF FIGURES ................................................................................................................. X LIST OF ABBREVIATIONS ................................................................................................. XII STATEMENET OF CONTRIBUTION ................................................................................ XV CHAPTER 1: INTRODUCTION ........................................................................................... 1 1.1 Thyroid Function and Regulation .................................................................................... 1 1.1.1 Thyroid Hormones ............................................................................................................ 1 1.1.2 Thyroid Hormone Regulation .......................................................................................... 2 1.1.3 Thyroid Hormone Receptors and Thyroid Hormone Response Elements......................... 4 1.1.4 Alternate Regulatory Mechanisms Mediated by Thyroid Hormones ................................ 6 1.2 Characterizing Thyroid Hormone Action in the Liver .................................................. 8 1.2.1 Characterizing Thyroid Hormone-mediated Gene Expression ......................................... 9 1.2.2 Characterizing Thyroid Receptor Binding Sites ............................................................... 11 1.2.3 Characterizing Thyroid Hormone Response Elements ..................................................... 12 1.3 Disrupting Thyroid Hormone Levels ............................................................................... 14 1.4 Screening Methods for Thyroid Hormone Disruptors ................................................... 17 1.5 Thesis Overview ................................................................................................................. 19 1.5.1 Rationale ........................................................................................................................... 19 1.5.2 Specific Objectives and Hypotheses .................................................................................. 20 CHAPTER 2: THYROID HORMONE-REGULATED GENE EXPRESSION IN JUVENILE MOUSE LIVER: IDENTIFICATION OF THYROID RESPONSE ELEMENTS USING MICROARRAY PROFILING AND IN SILICO ANALYSES....... 22 2.1 Abstract ............................................................................................................................... 22 2.2 Background ........................................................................................................................ 23 2.3 Methods ............................................................................................................................... 25 v 2.3.1 Animals and Exposures ..................................................................................................... 25 2.3.2 Tissue Collection, RNA Extraction and Purification ........................................................ 26 2.3.3 Microarray Hybridization ................................................................................................. 27 2.3.4 Experimental Design and Statistical Analysis of Microarray Data .................................. 28 2.3.5 Bioinformatics ................................................................................................................... 29 2.3.6 Gene Ontology Analysis and Principal Component Analysis ........................................... 31 2.3.7 Real-Time Quantitative PCR Analysis .............................................................................. 31 2.3.8 ChIP-PCR ......................................................................................................................... 32 2.3.9 Electrophoretic Mobility Shift Assays ............................................................................... 33 2.4 Results ................................................................................................................................. 34 2.4.1 Validation of the MMI/sodium Perchlorate Mouse Model ............................................... 34 2.4.2 Genes Significantly Altered by TH Perturbations ............................................................. 36 2.4.3 Microarray Data Validation and Hypothyroid Replacement Analysis ............................. 40 2.4.4 Bioinformatics Analysis of Direct TH-Regulated Genes .................................................. 43 2.4.5 Electrophoretic Mobility Shift Assays ............................................................................... 48 2.5 Discussion............................................................................................................................ 50 CHAPTER 3: THYROID HORMONE MAY REGULATE MRNA ABUNDANCE IN LIVER BY ACTING ON MICRORNAS .............................................................................. 60 3.1 Abstract ............................................................................................................................... 60 3.2 Background ........................................................................................................................ 61 3.3 Methods ..............................................................................................................................
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