Table Of ContentM E T H O D S I N M O L E C U L A R B I O L O G YTM
John M. Walker, Series Editor
188.Epithelial Cell Culture Protocols, edited by Clare Wise, 2002 158.Gene Knockoout Protocols, edited by Martin J. Tymms and
187.PCR Mutation Detection Protocols, edited by Bimal D. M. Ismail Kola, 2001
Theophilus and Ralph Rapley, 2002 157.Mycotoxin Protocols, edited by Mary W. Trucksess and Albert
186.Oxidative Stress and Antioxidant Protocols, edited by E. Pohland, 2001
Donald Armstrong, 2002 156.Antigen Processing and Presentation Protocols, edited by
185.Embryonic Stem Cells: Methods and Protocols, edited by Joyce C. Solheim, 2001
Kursad Turksen, 2002 155.Adipose Tissue Protocols, edited by Gérard Ailhaud, 2000
184.Biostatistical Methods, edited by Stephen W. Looney, 2002 154.Connexin Methods and Protocols, edited by Roberto Bruzzone
183.Green Fluorescent Protein: Applications and Protocols, edited and Christian Giaume, 2001
byBarry W. Hicks, 2002 153.Neuropeptide Y Protocols, edited by Ambikaipakan
182.In Vitro Mutagenesis Protocols, 2nd ed., edited by Jeff Balasubramaniam, 2000
Braman, 2002 152.DNA Repair Protocols: Prokaryotic Systems, edited by Patrick
181.Genomic Imprinting: Methods and Protocols, edited by Vaughan, 2000
Andrew Ward, 2002 151. Matrix Metalloproteinase Protocols, edited by Ian M. Clark, 2001
180.Transgenesis Techniques, 2nd ed.: Principles and Protocols, 150.Complement Methods and Protocols, edited by B. Paul Mor-
edited by Alan R. Clarke, 2002 gan, 2000
179.Gene Probes: Principles and Protocols, edited by Marilena 149.The ELISA Guidebook,edited by John R. Crowther, 2001
Aquino de Muro and Ralph Rapley, 2002 148.DNA–Protein Interactions: Principles and Protocols (2nd
178.`Antibody Phage Display: Methods and Protocols, edited by ed.), edited by Tom Moss, 2001
Philippa M. O’Brien and Robert Aitken, 2001 147.Affinity Chromatography: Methods and Protocols, edited by
177.Two-Hybrid Systems: Methods and Protocols, edited by Paul Pascal Bailon, George K. Ehrlich, Wen-Jian Fung, and
N. MacDonald, 2001 Wolfgang Berthold, 2000
176.Steroid Receptor Methods: Protocols and Assays, edited by 146.Mass Spectrometry of Proteins and Peptides, edited by John
Benjamin A. Lieberman, 2001 R. Chapman, 2000
175.Genomics Protocols, edited by Michael P. Starkey and 145.Bacterial Toxins: Methods and Protocols, edited by Otto Holst,
Ramnath Elaswarapu, 2001 2000
174.Epstein-Barr Virus Protocols, edited by Joanna B. Wilson 144.Calpain Methods and Protocols, edited by John S. Elce, 2000
and Gerhard H. W. May, 2001 143.Protein Structure Prediction: Methods and Protocols,
173.Calcium-Binding Protein Protocols, Volume 2: Methods and edited by David Webster, 2000
Techniques,edited by Hans J. Vogel, 2001 142.Transforming Growth Factor-Beta Protocols, edited by
172.Calcium-Binding Protein Protocols, Volume 1: Reviews and Philip H. Howe, 2000
Case Histories, edited by Hans J. Vogel, 2001 141.Plant Hormone Protocols, edited by Gregory A. Tucker and
171.Proteoglycan Protocols, edited by Renato V. Iozzo, 2001 Jeremy A. Roberts, 2000
170.DNA Arrays: Methods and Protocols, edited by Jang B. 140.Chaperonin Protocols, edited by Christine Schneider,2000
Rampal, 2001 139.Extracellular Matrix Protocols, edited by Charles Streuli and
169.Neurotrophin Protocols, edited by Robert A. Rush, 2001 Michael Grant,2000
168.Protein Structure, Stability, and Folding, edited by Kenneth 138.Chemokine Protocols, edited by Amanda E. I. Proudfoot, Timo-
P. Murphy, 2001 thy N. C. Wells, and Christine Power, 2000
167.DNA Sequencing Protocols, Second Edition, edited by Colin 137.Developmental Biology Protocols, Volume III, edited by
A. Graham and Alison J. M. Hill, 2001 Rocky S. Tuan and Cecilia W. Lo, 2000
166.Immunotoxin Methods and Protocols, edited by Walter A. 136.Developmental Biology Protocols, Volume II, edited by
Hall, 2001 Rocky S. Tuan and Cecilia W. Lo, 2000
165.SV40 Protocols, edited by Leda Raptis, 2001 135.Developmental Biology Protocols, Volume I, edited by Rocky
164.Kinesin Protocols, edited by Isabelle Vernos, 2001 S. Tuan and Cecilia W. Lo, 2000
163.Capillary Electrophoresis of Nucleic Acids, Volume 2: 134.T Cell Protocols: Development and Activation, edited by Kelly
Practical Applications of Capillary Electrophoresis, edited by P. Kearse, 2000
Keith R. Mitchelson and Jing Cheng, 2001 133.Gene Targeting Protocols, edited by Eric B. Kmiec, 2000
162.Capillary Electrophoresis of Nucleic Acids, Volume 1: 132.Bioinformatics Methods and Protocols, edited by Stephen
Introduction to the Capillary Electrophoresis of Nucleic Acids, Misener and Stephen A. Krawetz, 2000
edited by Keith R. Mitchelson and Jing Cheng, 2001 131.Flavoprotein Protocols, edited by S. K. Chapman and G. A.
161.Cytoskeleton Methods and Protocols, edited by Ray H. Gavin, 2001 Reid, 1999
160.Nuclease Methods and Protocols, edited by Catherine H. 130.Transcription Factor Protocols, edited by Martin J. Tymms,
Schein, 2001 2000
159.Amino Acid Analysis Protocols, edited by Catherine Cooper, 129.Integrin Protocols, edited by Anthony Howlett, 1999
Nicole Packer, and Keith Williams, 2001 128.NMDA Protocols, edited by Min Li, 1999
M E T H O D S I N M O L E C U L A R B I O L O G YTM
Steroid Receptor
Methods
Protocols and Assays
Edited by
Benjamin A. Lieberman
Blueprint Technologies, Englewood, CO
Humana Press Totowa, New Jersey
© 2001 Humana Press Inc.
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Cover Illustration: Fig. 4B from Chapter 7, “Physical Structure of Nuclear Receptor–DNAComplexes” by
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Library of Congress Cataloging in Publication Data
Steroid receptor methods : protocols and assays / edited by Benjamin A. Lieberman.
p. cm. -- (Methods in molecular biology ; 176)
Includes bibliographical references and index.
ISBN 0-89603-754-1 (alk. paper)
1. Steroid hormones--Receptors--Laboratory manuals. I. Lieberman, Benjamin A. II.
Series.
QP572.S7 .S756 2001
612.4'05--dc21
2001024304
Preface
This volume of the Methods in Molecular Biology series is entirely devoted
to the study of steroid receptor biology. Steroid hormone receptors represent a
powerful system for the study of both the most fundamental molecular mecha-
nisms of gene regulation and control and the gross physiological responses of
organisms to steroid hormones. Research in this field has brought forth advances
in the treatment of cancer, endocrine disorders, and reproductive biology, and
allowed elucidation of the fundamental biological mechanisms of gene expres-
sion. In Steroid Receptor Methods: Protocols and Assays, the reader will find a
collection of methods and protocols submitted by many fine steroid receptor
researchers from throughout the world. These authors have been instructed to
create a highly informative cross-section of the latest research techniques avail-
able. The resulting work is timely, useful, and approachable for both the expe-
rienced researcher and the novice to the field. Because the steroid receptor family
is represented by a wonderfully diverse, yet strongly interrelated set of steroid
receptor proteins, Steroid Receptor Methods contains protocols for the produc-
tion and purification of a variety of receptor forms, including the progesterone,
glucocorticoid, and androgen receptors. These procedures provide the raw mate-
rial needed to conduct sophisticated biochemical analysis of receptor properties.
Other techniques presented allow the reader to perform biochemical experiments
on DNA binding characteristics, hormone binding assays, and protocols using
combinatorial chemistry for drug discovery. Because steroid receptor effective-
ness is influenced by a variety of cellular proteins, there are included in this
volume a series of novel protocols utilizing the latest advances in immunochem-
istry, yeast two-hybrid screening, fluorescence, and other biochemical and
cellular techniques to detect and detail these interactions. These techniques
include both in vitro and in vivo approaches to provide the widest possible
selection of tools to the modern biological researcher. Finally, in recognition of
the growing importance of bioinformatics in biological research, several chapters
have been included to guide and assist the modern research biologist in harness-
ing this increasingly valuable resource. These chapters locate and make acces-
sible to the researcher the diverse computational tools currently available via the
Internet. Taken together these chapters provide both novice and experienced
researchers alike a set of invaluable tools to advance and extend their research.
Ben Lieberman, PhD
v
Contents
Preface .............................................................................................................v
Contributors.....................................................................................................xi
PART I. BIOINFORMATICS....................................................................................1
1 Bioinformatics of Nuclear Receptors
Mark Danielsen.......................................................................................3
2 Phylogenetic Inference and Parsimony Analysis
Llewellyn D. Densmore III...................................................................23
PART II. PURIFICATION PROTOCOLS.....................................................................37
3 Expression and Purification of Recombinant Human Progesterone
Receptor in Baculovirus and Bacterial Systems
Vida Senkus Melvin and Dean P. Edwards.......................................39
4 High-Yield Purification of Functionally Active Glucocorticoid Receptor
Terace M. Fletcher, Barbour S. Warren, Christopher T. Baumann,
and Gordon L. Hager.......................................................................55
5 Production and Purification of Histidine-Tagged Dihydrotestosterone-
Bound Full-Length Human Androgen Receptor
Mingmin Liao and Elizabeth M. Wilson.............................................67
6 Large Scale Production of Nuclear Receptor Ligand-Binding Domains
Li-Zhi Mi and Fraydoon Rastinejad...................................................81
7 Physical Structure of Nuclear Receptor–DNA Complexes
Scott A. Chasse and Fraydoon Rastinejad......................................91
8 Isolation of Steroid-Regulated Genes from the Uterus by mRNA
Differential Display
Sushma Kumar, Maarit Angervo, Milan K. Bagchi,
and Indrani C. Bagchi...................................................................105
9 Identification of Nuclear Hormone Receptor Homologs by Screening
Libraries with Highly Degenerate Oligonucleotide Probes
Bruce Blumberg.................................................................................119
PART III. STEROID HORMONE-BINDING ASSAYS...................................................131
10 Use of [99mTc] Technetium-Labeled Steroids as Probes for
Steroid Hormone Receptors
Frank Wüst..........................................................................................133
vii
viii Contents
11 Steroid Hormone Metabolites and Hormone Binding Assays
Rosemary Bland and Martin Hewison.............................................145
12 Vitamin D Analog Screening
3
Sami Väisänen, Sanna Ryhänen, and Pekka H. Mäenpää............163
PART IV. PROTEIN INTERACTION ASSAYS ...........................................................177
13 Application of Green Fluorescent Protein to the Study
of Dynamic Protein–Protein Interactions and Subcellular
Trafficking of Steroid Receptors
Steven K. Nordeen, Paul R. Housley, Yihong Wan,
and Richard N. Day.......................................................................179
14 Knockout Mice and Steroid Receptor Research
Per Flodby, Stephan Teglund, and Jan-Åke Gustafsson.............201
15 Yeast Two-Hybrid Screening for Proteins that Interact with Nuclear
Hormone Receptors
Bertrand Le Douarin, David M. Heery, Claudine Gaudon,
Elmar vom Baur, and Régine Losson.........................................227
16 Isolation of a p300/CBP Cointegrator-Associated Protein
Coactivator Complex
Rabindra N. Bhattacharjee, Caroline Underhill,
and Joseph Torchia......................................................................249
17 Nonradioactive Photoaffinity Labeling of Steroid Receptors
Using Western Blot Detection System
Simon J. Evans and Frank L. Moore...............................................261
18 Analysis of Steroid Hormone-Induced Histone Acetylation
by Chromatin Immunoprecipitation Assay
James R. Lambert and Steven K. Nordeen....................................273
19 Analyzing the Contributions of Chromatin Structure
in Nuclear Hormone Receptor Activated Transcription In Vivo
Christy J. Fryer and Trevor K. Archer.............................................283
20 Cotransfection Assays and Steroid Receptor Biology
Shimin Zhang and Mark Danielsen.................................................297
21 Estrogen Receptor mRNA In Situ Hybridization Using Microprobe
System
Hironobu Sasano, Sachiko Matsuzaki, and Takashi Suzuki........317
PART V. CANCER RESEARCH AND DRUG DISCOVERY...........................................327
22 Solid Tumor Cancer Markers and Applications to Steroid Hormone
Research
Marcia V. Fournier, Katherine J. Martin, and Arthur B. Pardee.....329
Contents ix
23 Assessing Modulation of Estrogenic Activity of Environmental
and Pharmaceutical Compounds Using MCF-7 Focus Assay
Kathleen F. Arcaro and John F. Gierthy.........................................341
24 Combinatorial Chemistry in Steroid Receptor Drug Discovery
John A. Flygare, Daniel P. Sutherlin, and S. David Brown..........353
25 Identification of Nuclear Receptor Interacting Proteins Using Yeast
Two-Hybrid Technology: Applications to Drug Discovery
Sunil Nagpal, Corine R. Ghosn,
and Roshantha A.S. Chandraratna.............................................359
Index............................................................................................................377
Contributors
MAARIT ANGERVO• Population Council and The Rockefeller University,
New York, NY
KATHLEEN F. ARCARO •Department of Environmental Health
and Toxicology, School of Public Health, University at Albany,
Rensselaer, NY
TREVOR K. ARCHER• The Chromatin and Gene Expression Section,
Laboratory of Reproductive and Developmental Toxicology, National
Institute of Environmental Health Sciences, National Institutes of Health,
Research Triangle Park, NC
INDRANI C. BAGCHI •Department of Veterinary Biosciences, University of
Illinois at Urbana-Champaign, Urbana, IL
MILAN K. BAGCHI•Department of Molecular and Integrative Physiology,
University of Illinois at Urbana-Champaign, Urbana, IL
CHRISTOPHER T. BAUMANN• Laboratory of Receptor Biology and Gene
Expression, NCI, NIH, Bethesda, MD
RABINDRA N. BHATTACHARJEE • London Regional Cancer Centre, London,
Ontario, Canada
ROSEMARY BLAND • Molecular Physiology, Leicester/Warwick Medical
School, Department of Biological Sciences, University of Warwick,
Coventry, UK
BRUCE BLUMBERG • Department of Developmental and Cell Biology,
University of California-Irvine, Irvine, CA
S. DAVID BROWN •Exelixis, South San Francisco, CA
ROSHANTHA A. S. CHANDRARATNA • Retinoid Research, Departments of
Biology and Chemistry, Allergan Inc., Irvine, CA
SCOTT A. CHASSE• Department of Pharmacology, University of Virginia
Health Sciences Center, Charlottesville, VA
MARK DANIELSEN • Department of Biochemistry and Molecular Biology,
Georgetown University, Washington, DC
RICHARD N. DAY •Departments of Medicine and Cell Biology, National
Science Foundation Center for Biological Timing, University of Virginia
Health Sciences Center, Charlottesville, VA
xi
xii Contributors
LLEWELLYN D. DENSMORE III • Department of Biological Sciences, Texas
Tech University, Lubbock, TX
DEAN P. EDWARDS • Department of Pathology, University of Colorado Health
Sciences Center, Denver, CO
SIMON J. EVANS •Mental Health Research Institute, University of Michigan,
Ann Arbor, MI
TERACE M. FLETCHER •Laboratory of Receptor Biology and Gene Expression,
NCI, NIH, Bethesda, MD
PER FLODBY•Department of Medical Nutrition, Karolinska Institutet, NOVUM,
Huddinge, Sweden
JOHN A. FLYGARE •Genentech, South San Francisco, CA
MARCIA V. FOURNIER •Adult Oncology Division, Dana-Farber Cancer
Institute, Boston, MA
CHRISTY J. FRYER •Departments of Obstetrics and Gynaecology, Oncology
and Biochemistry, University of Western Ontario
CLAUDINE GAUDON •Institut de Génétique et de Biologie Moléculaire et
Cellulaire, CNRS/INSERM/ULP/Collège de France, Strasbourg, France
CORINE R. GHOSN •Retinoid Research, Department of Biology, Allergan Inc.,
Irvine, CA
JOHN F. GIERTHY •Wadsworth Center for Laboratories and Research, New
York State Department of Health, Albany, NY
JAN-ÅKE GUSTAFSSON • Department of Medical Nutrition, Karolinska
Institutet, NOVUM, Huddinge, Sweden
GORDON L. HAGER• Laboratory of Receptor Biology and Gene Expression,
NCI, NIH, Bethesda, MD
DAVID M. HEERY • Department of Biochemistry, University of Leicester,
Leicester, UK
MARTIN HEWISON •Department of Medicine, University of Birmingham,
Birmingham, UK
PAUL R. HOUSLEY •Department of Pharmacology and Physiology,
University of South Carolina School of Medicine, Columbia, SC
SUSHMA KUMAR• Department of Obstetrics/Gynecology, Nassau University,
East Meadow, NY
JAMES R. LAMBERT• Department of Pathology, University of Colorado
Health Sciences Center, Denver, CO
BERTRAND LE DOUARIN •Institut de Génétique et de Biologie Moléculaire et
Cellulaire, CNRS/INSERM/ULP/Collège de France, Strasbourg, France
MINGMIN LIAO•Loeb Health Research Institute, University of Ottawa,
Ottawa, Ontario, Canada
