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Methods in Molecular Biology 1443 Iain J. McEwan Editor The Nuclear Receptor Superfamily Methods and Protocols Second Edition M M B ETHODS IN OLECULAR IOLOGY Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB , UK For further volumes: http://www.springer.com/series/7651 The Nuclear Receptor Superfamily Methods and Protocols Second Edition Edited by Iain J. McEwan School of Medicine, Medical Sciences and Nutrition, Institute of Medical Sciences, University of Aberdeen, Aberdeen, Scotland, UK Editor Iain J . M cEwan School of Medicine Medical Sciences and Nutrition Institute of Medical Sciences University of Aberdeen Aberdeen, Scotland, UK ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-3722-6 ISBN 978-1-4939-3724-0 (eBook) DOI 10.1007/978-1-4939-3724-0 Library of Congress Control Number: 2016933673 © Springer Science+Business Media New York 2 016 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. 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Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC New York Prefa ce It is now 30 years since the fi rst steroid receptor cDNAs were cloned, a development that led to the concept of a superfamily of ligand-activated transcription factors: the nuclear receptors. Nuclear receptors share a common architecture at the protein level, but a remarkable diversity is observed in terms of natural ligands and xenobiotics that bind to and regulate receptor function. Natural ligands for nuclear receptors are generally lipophilic in nature and include steroid hormones, bile acids, fatty acids, thyroid hormones, certain vitamins, and prostaglandins. A signifi cant proportion of the family members have been described as orphans , as the natural ligand, if it exists, remains to be identifi ed. Nuclear receptors act prin- cipally to directly control patterns of gene expression and play vital roles during development and in the regulation of metabolic and reproductive functions in the adult organism. Since the original cloning experiments, considerable progress has been made in our understanding of the structure, mechanisms of action, and role in disease of this important family of pro- teins. This volume of M ethods in Molecular Biology follows on from an earlier edition (Volume 505) and aims to describe a complementary range of molecular, cell biological, and in vivo protocols used to investigate the structure–function of nuclear receptors, together with experimental approaches that may lead to new drugs to selectively target nuclear receptor- associated diseases. This volume will be of great benefi t and use to those starting out in the nuclear receptor research fi eld (life sciences graduate students and postdoctoral fellows) as well as to more established researchers who wish to apply different methods to a particular receptor/research problem. The volume will also be of use to medical students and clinicians undertaking research in this ever-growing fi eld of study. Aberdeen, UK Iain J. M cEwan v Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x PART I INTRODUCTION 1 The Nuclear Receptor Superfamily at Thirty . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Iain J. McEwan PART II NUCLEAR RECEPTOR AGONISTS AND ANTAGONISTS 2 Lipid Homeostasis and Ligands for Liver X Receptors: Identification and Characterization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Jean-Marc A. L obaccaro , Claude B eaudoin , B agora Bayala , Silvère Baron , and Amalia T rousson 3 U se of Differential Scanning Fluorimetry to Identify Nuclear Receptor Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 Kara A . D eSantis and Jeffrey L . R einking 4 D rug-Discovery Pipeline for Novel Inhibitors of the Androgen Receptor . . . . 3 1 Kush Dalal , R avi M unuganti , H élène Morin , N ada L allous , Paul S. Rennie , and Artem C herkasov PART III NUCLEAR RECEPTOR SIGNALLING 5 Use of BRET to Study Protein–Protein Interactions In Vitro and In Vivo. . . . 5 7 Shalini Dimri , S oumya B asu , and Abhijit De 6 S tudying Nuclear Receptor Complexes in the Cellular Environment . . . . . . . . 7 9 Fred S chaufele 7 Posttranslational Modifications of Steroid Receptors: Phosphorylation . . . . . . 1 05 Dagmara M cGuinness and Iain J. McEwan 8 M apping Protein–DNA Interactions Using ChIP-exo and Illumina-Based Sequencing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Stefan J. Barfeld and Ian G . M ills 9 M ethods to Identify Chromatin-Bound Protein Complexes: From Genome-Wide to Locus-Specific Approaches. . . . . . . . . . . . . . . . . . . . . 1 39 Charles E . M assie 10 Measuring the Expression of microRNAs Regulated by Androgens . . . . . . . . . 1 51 Mauro Scaravilli , Kati K ivinummi , T apio Visakorpi , and L eena Latonen 11 Methods for Identifying and Quantifying mRNA Expression of Androgen Receptor Splicing Variants in Prostate Cancer. . . . . . . . . . . . . . . 165 Yingming L i and S cott M. D ehm vii viii Contents PART IV MODEL SYSTEMS 12 Harvesting Human Prostate Tissue Material and Culturing Primary Prostate Epithelial Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 Fiona M. Frame , Davide P ellacani , Anne T. Collins , and Norman J. M aitland 13 I n Vivo Imaging of Nuclear Receptor Transcriptional Activity. . . . . . . . . . . . . 2 03 D. Alwyn Dart and Charlotte L . B evan 14 D evelopment and Characterization of Cell-Specific Androgen Receptor Knockout Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 19 Laura O’Hara and Lee B . S mith Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 49 Contributors STEFAN J. B ARFELD • Prostate Cancer Research Group, Centre for Molecular Medicine Norway (NCMM), N ordic EMBL Partnership University of Oslo and Oslo University Hospital , O slo, N orway SILVÈRE B ARON • Université Clermont Auvergne, Université Blaise Pascal, Génétique Reproduction et Développement , Clermont-Ferrand, F rance; CNRS, UMR 6293, GReD, Aubière, France; INSERM, UMR 1103, GReD, Aubière, France; Centre de Recherche en Nutrition Humaine d’Auvergne, Clermont-Ferrand, France SOUMYA B ASU • Molecular Functional Imaging Lab, ACTREC, Tata Memorial Centre , Navi Mumbai, I ndia BAGORA BAYALA • Université Clermont Auvergne, U niversité Blaise Pascal, Génétique Reproduction et Développement , Clermont-Ferrand, F rance; CNRS, UMR 6293, GReD, Aubière, France; INSERM, UMR 1103, GReD, Aubière, France; Centre de Recherche en Nutrition Humaine d’Auvergne, Clermont-Ferrand, France; University of Koudougou, Burkina, Faso CLAUDE B EAUDOIN • Université Clermont Auvergne, Université Blaise Pascal, Génétique Reproduction et Développement , Clermont-Ferrand, France; CNRS, UMR 6293, GReD, Aubière, France; INSERM, UMR 1103, GReD, Aubière, France; Centre de Recherche en Nutrition Humaine d’Auvergne, Clermont-Ferrand, France CHARLOTTE L. B EVAN • Androgen Signalling Laboratory, Department of Surgery and Cancer , I mperial College London , London , U K ARTEM C HERKASOV • Vancouver Prostate Centre, University of British Columbia , Vancouver, B C , C anada ANNE T. C OLLINS • YCR Cancer Research Unit, Department of Biology, U niversity of York , H eslington, North Yorkshire, UK KUSH D ALAL • Vancouver Prostate Centre, U niversity of British Columbia , Vancouver, B C, Canada D. A LWYN DART • Androgen Signalling Laboratory, Department of Surgery & Cancer, Imperial College London, London, UK; The Cardiff China Medical Research Collaborative, C ardiff University School of Medicine , C ardiff, U K ABHIJIT DE • Molecular Functional Imaging Lab, ACTREC, T ata Memorial Centre , N avi Mumbai, I ndia SCOTT M. DEHM • Masonic Cancer Center and Department of Laboratory Medicine and Pathology, U niversity of Minnesota , T win Cities, M N , U SA KARA A. DESANTIS • State University of New York at New Paltz , N ew Paltz, N Y , U SA SHALINI D IMRI • Molecular Functional Imaging Lab, ACTREC, T ata Memorial Centre , Navi Mumbai, I ndia FIONA M. F RAME • YCR Cancer Research Unit, Department of Biology, U niversity of York , Heslington, N orth Yorkshire, U K KATI KIVINUMMI • Prostate Cancer Research Center, Institute of Biosciences and Medical Technology – BioMediTech and Fimlab Laboratories, U niversity of Tampere and Tampere University Hospital , Tampere, Finland ix