transcriptional regulation in eukaryotes

Genetic links between gene activation and chromatin, 27SWI/SNF complexes, 27 Mechanisms and targeting, 29 Mammalian acetylases, 32 TAFs and chromatin remodeling, 32 Histone deacetylation

Transcriptional Regulation

in Eukaryotes

Concepts, Strategies, and Techniques

COLD SPRING HARBOR LABORATORY PRESS

Michael Carey

Stephen T Smale

Cover illustration: The cover schematically illustrates the structure of the RNA polymerase II transcription

complex emerging from a black box It is a composite illustration of the TFIIA-TBP-TATA (Geiger et al Science 272: 830–836 [1996]; Tan et al Nature 381: 127–151 [1996]), and TFIIB-TBP-TATA (Nikolov et al Nature 377:

119–128 [1995]) crystal structures rendered by Michael Haykinson (UCLA) using the Molecular Graphics structure modeling computer program Insight II.

Library of Congress Cataloging-in-Publication Data

Carey, Michael (Michael F.)

Transcriptional regulation in eukaryotes: concepts, strategies, and techniques/Michael

Carey, Stephen T Smale.

p cm.

Includes bibliographical references and index.

ISBN 0-87969-537-4 (cloth) ISBN 0-87969-635-4 (pbk.)

1 Genetic transcription Regulation 2 Transcription factors 3 Genetic

transcription Regulation Research Methodology I Smale T II Title.

Procedures for the humane treatment of animals must be observed at all times Check with the local animal facility for guidelines.

Certain experimental procedures in this manual may be the subject of national or local legislation or agency restrictions Users of this manual are responsible for obtaining the relevant permissions, certificates, or licens-

es in these cases Neither the authors of this manual nor Cold Spring Harbor Laboratory assumes any sibility for failure of a user to do so.

respon-The polymerase chain reaction process is covered by certain patent and proprietary rights Users of this ual are responsible for obtaining any licenses necessary to practice PCR or to commercialize the results of such use COLD SPRING HARBOR LABORATORY MAKES NO REPRESENTATION THAT USE OF THE INFOR- MATION IN THIS MANUAL WILL NOT INFRINGE ANY PATENT OR OTHER PROPRIETARY RIGHT Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients,

man-is granted by Cold Spring Harbor Laboratory Press, provided that the appropriate fee man-is paid directly to the Copyright Clearance Center (CCC) Write or call CCC at 222 Rosewood Drive, Danvers, MA 01923 (978-750- 8400) for information about fees and regulations Prior to photocopying items for educational classroom use, contact CCC at the above address Additional information on CCC can be obtained at CCC Online at http://www.copyright.com/

All Cold Spring Harbor Laboratory Press publications may be ordered directly from Cold Spring Harbor Laboratory Press, 500 Sunnyside Boulevard, Woodbury, New York 11797-2924 Phone: 1-800-843-4388 in Continental U.S and Canada All other locations: (516) 422-4100 FAX: (516) 422-4097 E-mail: cshpress@cshl.org For a complete catalog of all Cold Spring Harbor Laboratory Press publications, visit our World Wide Web Site http://www.cshlpress.com/

Developmental Editor: Judy Cuddihy

Assistant Developmental Editor: Birgit Woelker

Project Coordinator: Maryliz M Dickerson

Production Editor: Patricia Barker

Desktop Editors: Danny deBruin, Susan Schaefer Interior Book Design: Denise Weiss

Cover Design: Tony Urgo Cover art rendered by Michael Haykinson

Transcriptional Regulation in Eukaryotes

Concepts, Strategies, and Techniques

All rights reserved

© 2000 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

Printed in the United States of America

Preface, xvii Overview, xix Abbreviations and Acronyms, xxv

Activating a gene, 3 Inactivating a gene, 5

CONCEPTS AND STRATEGIES: I PROMOTERS AND THE GENERAL

Basal transcription complex assembly, 11 Conformational changes during transcription complex assembly, 11

Discovery of the Pol II holoenzyme, 14 Composition of the yeast holoenzyme, 15 Mammalian holoenzymes, 16

Modular activators, 20 DNA-binding domains, 21 Activation domains, 21 Structural aspects of activation domains, 22

General mechanisms, 23 Sequence-specific repressors, 24

CONCEPTS AND STRATEGIES: III CHROMATIN AND

Genetic links between gene activation and chromatin, 27

SWI/SNF complexes, 27 Mechanisms and targeting, 29

Mammalian acetylases, 32 TAFs and chromatin remodeling, 32

Histone deacetylation, transcriptional repression, and silencing 32

Repression and deacetylases, 33 Linking deacetylation and ATP-remodeling machines, 33 Methylation and repression, 34

Transcriptional silencing, 35

Locus control regions, insulators, and matrix attachment regions 35

Locus control regions, 35 Boundary elements, 37 MARs, 38

Consider the time commitment and resources needed

Two general strategies that provide preliminary albeit superficial insight into transcriptional regulation mechanisms, 54

An example of a rigorous, yet incomplete gene regulation analysis:

The immunoglobulin µheavy-chain gene, 55 Defining the project goals, 57

Appropriate source of cells for functional studies, 57 Source of cells for protein extract preparation, 59 Success in developing an appropriate functional assay, 59

Beginning with the promoter or distant control regions, 61 Initiating an analysis of a promoter, 62

Initiating an analysis of distant control regions, 62

vi ■ Contents

3 MODES OF REGULATING mRNA ABUNDANCE 65

Basic mRNA degradation pathways, 67 Regulation of mRNA stability and degradation, 68 Interrelationship between mRNA stability and transcription initiation, 70 Confirming that the rate of transcription initiation

contributes to gene regulation, 71 Nuclear run-on transcription assay (Box 3.1), 72 Measuring mRNA stabilities, 73

Recommended approach for demonstrating regulation of transcription initiation or mRNA stability, 77

Basic mechanism of elongation, 78 Regulation of transcription elongation in prokaryotes, 79 Regulation of transcription elongation in eukaryotes, 80 Strategies for distinguishing between regulation of elongation and regulation of initiation, 82

Recommended approach for demonstrating regulation

of transcription initiation or elongation, 83 Extending an analysis of elongation regulation, 84

Differential pre-mRNA splicing, mRNA transport, and polyadenylation 85

Basic principles, 85 Identifying regulation of pre-mRNA splicing, transport, and polyadenylation, 86

Primer annealing and reverse transcription, 104 Analysis of example data, 104

Contents ■ vii

RNase protection 105

Advantages and disadvantages, 105 Probe preparation, 105

Method (Box 4.2), 106 Probe annealing and RNase digestion, 108 Analysis of example data, 108

Advantages and disadvantages, 109 Probe preparation, 109

Method (Box 4.3), 109 Analysis of example data, 111

Advantages and disadvantages, 112 Data analysis, 112

Method (Box 4.4), 112 Effect of introns on the interpretation of start-site mapping results (Box 4.5), 114

Choosing an assay: Advantages and disadvantages of each assay 141

Transient transfection assay, 142 Stable transfection assay by integration into host chromosome, 144 Stable transfection of episomally maintained plasmids, 145

In vitro transcription assay, 145 Transgenic assays, 146

Homologous recombination assay, 147

Cells, 148 Transfection procedures (Box 5.1), 148 Reporter genes, vectors, and assays (Boxes 5.2, 5.3, 5.4), 150 Plasmid construction, 155

Initial transfection experiments, 157 Assessing appropriate promoter regulation (Boxes 5.5, 5.6), 159

General strategies, 160 Cells and transfection procedures, 162

viii ■ Contents

Reporter genes and assays, 165 Drug-resistance genes and vectors, 165 Plasmid construction, 168

Drug selection, 169 Controls and interpretation of results, 171

Protocol 5.1 Calcium phosphate transfection of 3T3 fibroblasts 174Protocol 5.2 DEAE-dextran transfection of lymphocyte cell lines 176Protocol 5.3 Transfection by electroporation of RAW264.7 macrophages 178

Protocol 5.5 Chloramphenicol acetyltransferase assay 183

Basic principles of the nuclear matrix and of MARs and SARs, 200 Advantages and disadvantages of using MARs to identify

distant control regions, 200 Methods for identifying MARs (Box 6.2), 201

Functional approaches for the identification of distant control regions 201

Basic advantages and disadvantages of functional approaches, 201 Functional approach beginning with a large genomic DNA fragment, 203 Functional approach beginning with smaller fragments directing

expression of a reporter gene, 204

Functional assays for the characterization of distant control regions 205

Transient transfection assays, 205 Stable transfection assays, 206 Demonstration of LCR activity, 208 Demonstration of silencer activity, 209 Demonstration of insulator activity, 209

Contents ■ ix

7 IDENTIFYING cis-ACTING DNA ELEMENTS WITHIN A CONTROL REGION 213

Identification of control elements by comprehensive mutant analysis 215

Rationale for a comprehensive analysis, 215 The Ig µgene example, 216

Disadvantages of using mutagenesis to identify control elements, 219 Strategies for a comprehensive analysis, 220

Methodology for mutating a control region, 235

Identification of control elements using in vivo or in vitro

Advantages and disadvantages, 235

Advantages and disadvantages, 237

CONCEPTS AND STRATEGIES FOR THE IDENTIFICATION OF

Development of a protein-DNA interaction assay for crude cell lysates 253

Standard methods for detecting protein–DNA interactions, 253 Electrophoretic mobility shift assay (Box 8.1), 257

DNase I footprinting, 268

CONCEPTS AND STRATEGIES FOR CLONING GENES ENCODING

Cloning by protein purification and peptide sequence analysis (Box 8.2) 276

Amount of starting material, 276 Conventional chromatography steps, 277 DNA affinity chromatography, 277 Identification of the relevant band following SDS-PAGE (Box 8.3), 278 Amino acid sequence analysis and gene cloning, 279

Confirmation that the gene isolated encodes the DNA-binding activity

of interest, 282

Cloning by methods that do not require an initial

9 CONFIRMING THE FUNCTIONAL IMPORTANCE OF A

Relative expression patterns of the DNA-binding protein

Correlation between nucleotides required for protein binding

trans-Activation of a reporter gene or endogenous gene

Cooperative binding and synergistic function of

In vivo analysis of sequence-specific protein-DNA interactions 321

DNase I and DMS genomic footprinting (Box 10.1), 321

In vivo protein–DNA crosslinking/immunoprecipitation, 326

Model systems, 326 Low-resolution analysis of nucleosome positioning by the MNase-Southern blot method (Box 10.2), 328

High-resolution analysis of nucleosome positioning by an MNase-LM-PCR method and DNase I genomic footprinting (Box 10.3), 329

In vivo methods for analyzing nucleosome remodeling (Box 10.4), 332

Restriction enzyme accessibility to monitor nucleosome remodeling, 347 DMS genomic footprinting, 347

Yeast systems (Box 11.3), 377 Baculovirus system (Box 11.4), 379 Vaccinia virus (Box 11.5), 382 Retroviral expression systems (Box 11.6), 384

Specialized inducible expression systems (Box 11.7), 386

In vitro transcription/translation systems (Box 11.8), 388 Mammalian expression vectors (Box 11.9), 389

Separating DNA-binding and activation domains of an activator 403

General considerations, 403 DNA binding, 404

Activation (Box 12.1), 406 Limitations of the domain swap, 406

Subdividing DNA recognition and oligomerization

Interaction of activation domains with coactivators and general factors 410

Principles, 413 Caveats of the affinity approach, 415

Structure–function analysis of the general transcriptional machinery 420

xii ■ Contents

13 THEORY, CHARACTERIZATION, AND MODELING OF DNA BINDING BY

Theory of DNA recognition, 436 Chemical basis of the interactions, 437 The role of the α-helix in DNA recognition, 437 Major and minor groove specificity, 439

Monomers and dimers; energetic and regulatory considerations, 441 Dissociation constant analysis (Box 13.1), 444

K d determination, 447

Identification of a high-affinity DNA recognition site, 448 Basic theory, 449

General methods (Boxes 13.2 and 13.3), 449 Minor groove/DNA backbone probes (Box 13.4), 454 Major groove probes, 458

Modeling DNA–protein interactions, 459

Analysis of promoter-specific multicomponent nucleoprotein complexes 463

DNA binding cooperativity, 465 DNA looping and bending, 466 Mechanisms of DNA bending, 468 Approaches for studying bending, 469

Protocol 13.6 Preparation of 32 P-end-labeled DNA fragments 497

Cell choice, 507 Extract preparation method, 508

General considerations (Box 14.1), 510 Choice of template, 514

Chromatin systems, 516 Optimization of conditions, 519

Contents ■ xiii

Fractionated systems (Box 14.2) 519

Holoenzyme, 520 Mediator subcomplexes, 521 Partially fractionated systems, 521 Factor-depleted systems, 525 Highly fractionated systems, 526

Protocol 14.1 The Dignam and Roeder nuclear extract 528Protocol 14.2 Preparation of nuclear extracts from rat liver 532Protocol 14.3 Preparation of whole-cell extract 536

Protocol 14.4 In vitro transcription using HeLa cell extracts and

Protocol 14.5 G-less cassette in vitro transcription using

Protocol 14.6 Preparation of a crude fractionated system 551

Protocol 14.7 Purification of recombinant TFIIB from E coli 556Protocol 14.8 Purification of recombinant TFIIA 560Protocol 14.9 Affinity purification of RNA Pol II 562Protocol 14.10 Purification of epitope-tagged TFIID 567

Kinetic studies, 582 Sarkosyl probing, 582 Template commitment experiment, 584 DNase I footprinting and EMSA studies of transcription complex assembly, 584

Photocrosslinking, 586 Structure–function analyses of the general machinery, 589

ATP-analogs and an energy-dependent step, 589 Permanganate probing, 590

Premelted templates, 590 The transition to elongation, 591

The immobilized template approach, 594 Gel filtration, 596

Permanganate probing to study activation, 596

xiv ■ Contents

EMSA and DNase I footprinting analyses of the TFIID–TFIIA complex, 599 Assembly and analysis of TFIID subcomplexes, 600

Future directions, 601

Protocol 15.1 Potassium permanganate probing of Pol II open complexes 603

Protocol 15.2 Magnesium-agarose EMSA of TFIID binding to DNA 607

Preface

Since the advent of recombinant DNA technology three decades ago, thousands of otic genes have been isolated The differential expression of these genes is critical for bothnormal cellular processes and abnormal processes associated with disease To understandthese processes, a growing number of investigators from diverse fields of biology havebegun to study the molecular mechanisms regulating gene transcription Furthermore, thegenome projects under way throughout the world have led to the identification of the

eukary-entire gene complements of Saccharomyces cerevisiae, Caenorhabditis elegans, and

numer-ous archaeal and eubacterial organisms Within the next few years, the approximately100,000 genes within the human genome will have been identified After this goal is real-ized, the need to dissect mammalian transcriptional control regions and regulatory mech-anisms rigorously will increase dramatically

Despite the global interest in elucidating mechanisms of transcriptional regulation, acomprehensive source of strategic, conceptual, and technical information has not been avail-able for those entering the field for the first time Although protocols for numerous tech-niques have been published, the strategic decisions necessary to carry out a step-by-stepanalysis have not been outlined This deficiency became apparent to us while we were serv-ing as instructors for the Eukaryotic Gene Expression course held each summer at ColdSpring Harbor Laboratory This laboratory course was designed for physician-scientistsinterested in understanding the regulation of a specific disease-related gene, Ph.D scientiststrained in other fields who became interested in the regulatory mechanisms for a geneinvolved in a particular biological process, and graduate students or postdoctoral fellows whowere initiating transcriptional regulation projects This book is targeted toward this samediverse group of scientists who have developed an interest in transcriptional regulation

In writing this book, we have focused on issues that the average investigator faces whenundertaking a transcriptional regulation analysis, and we have outlined recommendedstrategies for completing the analysis One risk of describing a prescribed step-by-stepapproach is that it may suppress creativity and may not be applicable to all regulatory sce-narios To the contrary, our hope is that our recommendations will enhance creativity byallowing it to evolve from an informed perspective

We thank the many participants in the Eukaryotic Gene Expression Course from 1994through 1998 for providing the inspiration and motivation for this book We also acknowl-edge our colleagues at UCLA, the members of our laboratories, and our co-instructors forthe Eukaryotic Gene Expression course, including Marc Learned, Ken Burtis, Grace Gill,David Gilmour, and Jim Goodrich, for many valuable discussions We are deeply indebted

to a number of colleagues for specific contributions and reading of sections, includingDoug Black, Mike Haykinson, Leila Hebshi, Reid Johnson, Ranjan Sen, and Amy

Weinmann We are particularly grateful to our editor Judy Cuddihy and the book’s ers, Grace Gill, Bill Tansey, and Steve Hahn, whose generous contribution of time and ideasmade the undertaking intellectually rewarding and personally enjoyable The book wasgreatly improved by the work of Birgit Woelker and Maryliz Dickerson at Cold SpringHarbor Laboratory Press, as well as Jan Argentine, Pat Barker, and Denise Weiss Finally, weacknowledge Cold Spring Harbor Laboratory Press Director John Inglis, whose encour-agement was essential for the completion of this novel project.

review-M.C and S.T.S.

xviii ■ Preface

The goal of this book is to provide a detailed description of the approaches to be employedand issues to be considered when undertaking a molecular analysis of the transcriptionalregulatory mechanisms for a newly isolated gene, or a biochemical analysis of a new tran-scription factor Our emphasis is on mammalian transcription, which is complicated bythe combinatorial nature of regulation and the lack of facile genetics We refer periodical-

ly to studies in yeast, Drosophila, and other organisms where more tractable genetic

approaches have led to a detailed understanding of particular mechanistic issues The ics covered in the book extend from the determination of whether a gene is in fact regu-lated at the level of transcription initiation to advanced strategies for characterizing thebiochemical mechanism underlying its combinatorial regulation by activators Althoughnumerous specialized and detailed techniques are included, the unique characteristics ofthis book are its strategic and conceptual emphasis on analysis of individual genes and thetranscription factors that regulate them

top-Chapter 1 reviews the current state of the RNA polymerase II transcription field Thischapter provides an investigator entering the field with a comprehensive introduction intoareas of active research and the types of regulatory strategies that will be confronted Wehave defined the general properties of known regulatory regions (i.e., enhancers, promot-ers, silencers), components of the transcriptional machinery (mediator components andthe general transcription factors), activators, and repressors Select review articles and on-line information sources are included for the novice interested in additional details on thevarious topics Emphasis is placed on the role of macromolecular complexes in regulation.Chapters 2–9 were conceived as a step-by-step guide for an investigator who wants to pur-sue the regulatory mechanisms for a new gene that has been identified Chapter 2 presentsgeneral strategic issues to consider before the analysis is initiated First and foremost is a dis-cussion of the goals of the analysis This topic was included because it has become apparentthat many investigators enter the transcription field with unrealistic expectations.Presumably, these expectations arise because a preliminary analysis of a control region, usingbasic reporter assays and electrophoretic mobility shift assays, is relatively straightforward Tothe contrary, a substantial amount of effort is usually required to make meaningful progresstoward an understanding of a gene’s regulatory mechanisms Chapter 2 also contains a dis-cussion of the feasibility of achieving the goals The feasibility is largely dependent on theavailability of particular tools, including appropriate cell lines for functional and biochemi-cal studies, and an appropriate functional assay The chapter concludes with a discussion ofwhether to begin the analysis by studying the promoter or, alternatively, distant controlregions, with a brief description of the initial steps required for each starting point In thisbook, the phrase “distant control regions” is used in reference to any control region that isdistinct from the promoter, such as enhancers, locus control regions, and silencers

One issue that will become apparent in Chapter 2 and in all subsequent chapters is thatspecific protocols are not included for many of the methods described Instead, references

xix

are given to standard methods manuals, in particular Sambrook et al (1989) and Ausubel

et al (1994) The intention was to avoid duplication of the valuable information provided

in pre-existing manuals and to instead focus on strategic advice Although the book couldhave been written without any protocols, since they all can be found in the literature, wechose to include selected protocols for three reasons First, some of the protocols were cho-sen because we felt that the reader would benefit from a detailed explanation of the spe-cific steps and history of the methodology, information generally not found in other man-uals Second, in some instances we felt it necessary to provide the reader with a sense of themechanics of a technique while reading the book Finally, several protocols were includedbecause of their special nature (e.g., permanganate footprinting, TFIID binding studies)and the fact that no general source exists for such methods

Chapter 3 continues the step-by-step guide by describing how to determine the mode

of regulation for a new gene At the outset, this chapter emphasizes the fact that the lation of a biochemical activity does not necessarily mean that the gene encoding the pro-tein is subject to regulation Alternative possibilities are the regulation of protein synthesis

regu-or degradation, regu-or posttranslational regulation of the biochemical activity itself.Furthermore, if the gene is found to be regulated, it is not necessarily regulated at the level

of transcription initiation Rather, it may be regulated at the level of transcription tion, mRNA stability, pre-mRNA splicing, polyadenylation, or mRNA transport Becauseregulation at the level of transcription initiation is most difficult to distinguish from regu-lation of mRNA stability and transcription elongation, the basic principles of these lattermodes of regulation are discussed Furthermore, strategies for distinguishing between thevarious modes of regulation are presented, along with a detailed protocol for one impor-tant technique, the nuclear run-on

elonga-As stated above, one critical decision discussed in Chapter 2 is whether to begin ananalysis of transcriptional regulation by studying the promoter or, alternatively, the distantcontrol regions for the gene If the investigator opts to study the promoter, the approach-

es detailed in Chapters 4 and 5 should be followed if the gene is found to be regulated atthe level of transcription initiation Chapter 4 describes methods for determining the loca-tion of the transcription start site, an essential first step in every promoter analysis Fourmethods for start-site mapping are described, including the primer extension, RNase pro-tection, S1 nuclease, and RACE methods The advantages and limitations of each methodare discussed, and detailed protocols are included for the first three

Chapter 5 considers the development of a functional assay for a promoter; in otherwords, the development of an assay that can be used to identify, by mutagenesis (see Chapter7), the individual control elements required for promoter activity Transient and stabletransfection assays are discussed in detail, including an overview of transfection procedures,reporter genes, vectors, and assays, and the initial design and interpretation of experiments.Alternative functional assays, including in vitro transcription and transgenic assays, are alsobriefly mentioned, along with their advantages and disadvantages Chapter 5 is the first ofseveral chapters where the text becomes strongly focused toward a discussion of transcrip-tional activation, with very little discussion of transcriptional repression The intention wasnot to minimize the importance of repression mechanisms for transcriptional regulation;however, a discussion of each point from the perspective of both activation and repressionwould have been unmanageable In most cases, it therefore is left to the reader to determinehow the principles discussed can be applied to a repression analysis

If an investigator chooses to pursue distant control regions instead of, or in addition to,the promoter, Chapters 5 and 6 are designed to follow Chapter 3 Chapter 5, as described

xx ■ Overview

above, contains basic information regarding the design of functional assays This tion is applicable to both promoters and distant regions Chapter 6 describes approaches foridentifying distal control regions, including the recommended starting point of performingDNase I hypersensitivity experiments Chapter 6 also describes special strategies not dis-cussed in Chapter 5 for developing functional assays to analyze distant control regions.After a functional assay is developed for a promoter (Chapters 4 and 5) or distant con-trol region (Chapters 5 and 6), the next step is to dissect the individual DNA elements con-stituting the region These procedures, which usually involve a systematic mutant analysis,are described in Chapter 7 This chapter stresses the benefits of a mutant analysis, but alsodescribes other strategies that may lead to the identification of important DNA elementswithin a control region.

informa-After the DNA elements are identified, the proteins that bind to them must be fied and their genes cloned These procedures are described in Chapter 8, beginning withthe development of EMSA and DNase I footprinting assays for use with crude nuclearextracts These assays are discussed in greater detail in Chapter 13 from the perspective of

identi-an identi-analysis of a pure recombinidenti-ant protein An attempt was made to minimize the tion of information between these two chapters However, to maintain the logical progres-sion of the book, some redundancy was unavoidable Various strategies that can be used toclone the gene encoding a DNA-binding protein are then described, including proteinpurification, the yeast one-hybrid screen, in vitro expression library screen, mammalianexpression cloning, degenerate PCR, and database approaches

duplica-Chapter 9 completes the step-by-step outline of the characterization of a new gene byfocusing on a crucial issue: After a factor that binds an important DNA element in vitro isidentified, how can one determine whether that factor is indeed responsible for the func-tion of the control element in vivo? Although no experiment is available that can provideconclusive evidence that the protein is functionally relevant, twelve experimental strategiesare described that can be used to test the hypothesis As with all science, the strength of thehypothesis will correspond to the number of rigorous tests to which it has been subjected.The analysis of a control region, using the strategies described in Chapters 2–9, relies

on the use of artificial assays, such as transfection assays and in vitro DNA-binding assays

To complement these approaches, it can be helpful to study the properties of the nous control region within its natural environment Chapter 10 describes experimentalstrategies for such a characterization, beginning with genomic footprinting and in vivocrosslinking/immunoprecipitation strategies for visualizing specific protein–DNA interac-tions at the endogenous locus Chromatin structure is also known to be an important con-tributor to gene regulation and is best studied in the context of the endogenous locus.Therefore, strategies are included for determining nucleosome positioning and remodel-ing Strategies for analyzing DNA methylation status and subnuclear localization of a geneare also briefly discussed

endoge-From a biochemical point of view, an understanding of the mechanism of gene tion involves recreating regulated transcription in vitro and delineating the precise pro-tein–protein and protein–DNA interactions involved in the process Chapters 11–15describe approaches for recreating and studying gene regulation in vitro using purified andreconstituted biochemical systems

regula-The initial starting point in a biochemical analysis of any regulatory protein is to thesize the protein and its derivatives in recombinant form Chapter 11 provides a list ofapproaches for expressing proteins, and guides the investigator through the strategic andtechnical decisions encountered in choosing an appropriate system for diverse applica-

syn-Overview ■ xxi

tions The chapter outlines the fundamentals of using E coli to generate small regulatory

molecules (e.g., DNA-binding domains of activators and repressors) and baculovirus andretroviral systems to generate multi-protein complexes

Typically, as an investigator proceeds through different stages of an analysis, it becomesimperative to delineate the protein domains engaged in interactions with other regulatoryproteins and with the transcriptional machinery This information is essential for com-pleting a biochemical analysis of mechanism The approach employed to gain such insights

is termed “structure–function” analysis This is not a trivial task, and the approach anddecision-making are often based on the particular type of regulatory protein being stud-ied Chapter 12 discusses structure–function analysis from several perspectives.Approaches for studying protein interactions are described briefly to permit the investiga-tor to design specific assays for analyzing the relevant domains Simple deletion analysis isdiscussed as a means to delineate how different regions of a regulatory protein contribute

to different aspects of DNA binding and transcriptional regulation This discussion serves

as a springboard to more advanced approaches, including domain swapping, a ward means to ascribe precise functions to portions of proteins Most importantly, how-ever, a molecular understanding of transcription is often derived from knowledge of thespecific amino acid residues mediating the relevant contacts Particular emphasis is placed

straightfor-on guiding the investigator through different cstraightfor-onceptual approaches to generating directed mutants, how such mutants are modeled, and case studies in which mutagenesis

site-is compared with the results of crystal structures Finally, the chapter dsite-iscusses the excitingand emerging concept that structural information can be employed to generate novel

“altered specificity” genetic systems for analyzing transcriptional mechanisms

DNA recognition by combinations of proteins is the major contributor to the cell anddevelopmental specificity of a transcriptional response The mechanisms employed byproteins to bind a promoter or enhancer, both alone and cooperatively with other proteins,are key areas of study in the transcription field As new transcription factors are identifiedfrom the genome project, even more focus will be placed on understanding DNA-bindingcooperativity and combinatorial interactions Chapter 13 describes the fundamentals ofequilibrium binding It introduces the concepts of DNA recognition, describes the chem-istry of DNA–protein interactions to the novice, and finally, discusses how chemical andnuclease probes can be employed to generate detailed models for DNA binding.Furthermore, the chapter outlines case studies where models derived from chemical prob-ing are compared with the results of crystal structures of DNA–protein co-complexes.Finally, but most importantly, the chapter provides a basic introduction to the concept andstudy of nucleoprotein complexes called enhanceosomes, an emerging area of research thatunderlies the combinatorial action of transcription factors

Ultimately, the investigator may wish to understand the detailed biochemical stepsaffected by activators This goal involves two undertakings: First, development of a robust

in vitro transcription system that recreates the regulatory phenomenon in vitro and, ond, design of mechanistic experiments with highly specialized reagents including purifiedtranscription factors and chromatin templates Chapter 14 guides the investigator throughthe logistical decisions and reagents necessary to design the appropriate reporter templatesand to develop active transcription systems The chapter discusses how in vitro transcrip-tion reactions are measured and optimized, including G-less cassettes and primer exten-sion, while expanding on the nuances of in vitro systems presented originally in Chapter

sec-8 Descriptions of the available methods for generating reconstituted systems with crude

or pure general factors and Pol II and the development of systems for analyzing chromatintemplates are also presented

xxii ■ Overview

Once activators are shown to stimulate transcription in vitro, the investigator may wish

to further pursue the biochemical mechanism of activated transcription using purifiedtranscription reagents This is a rapidly evolving area in terms of both new concepts andspecialized reagents Chapter 15 presents a historical overview of how different methodswere originally applied for understanding basal and activated transcription The chapterthen outlines numerous strategies employed to study specific steps in activated transcrip-tion using crude and pure reagents These include approaches for analyzing transcriptioncomplex assembly including sarkosyl sensitivity, the immobilized template approach, per-manganate probing, and others The emphasis is on assay development and data interpre-tation The chapter also attempts to provide an up-to-date tabulation of sources for spe-cialized reagents including systems for expressing and purifying recombinant transcriptionfactors and multi-component complexes such as the human holoenzyme, chromatinremodeling machines, human mediator, and TFIID

REFERENCES

Ausubel F.M., Brent R.E., Kingston E., Moore D.D., Seidman J.G., Smith J.A., and Struhl K 1994

Current protocols in molecular biology John Wiley and Sons, New York.

Sambrook J., Fritsch E.F., and Maniatis T 1989 Molecular cloning: A laboratory manual Cold Spring

Harbor Laboratory Press, Cold Spring Harbor, New York

Overview ■ xxiii

Abbreviations and Acronyms

In addition to standard abbreviations for metric measurements (e.g., ml) and chemical bols (e.g., HCl), the abbreviations and acronyms below are used throughout this manual

sym-A, adenine

AcPNV, Autographa californica polyhedrosis virus

AdMLP, adenovirus major late promoter AMV, avian myeloblastosis virus

AR, androgen receptor ARC, activator-recruited co-factor ARS, autonomous replication sequence AOX1, alcohol oxidase

ARE, AU-rich response element ATP, adenosine triphosphate att site, attachment site

BAC, bacterial artificial chromosome BEAF, boundary element-associated factor bHLH, basic helix-loop-helix

BrdU, bromodeoxyuridine BRE, TFIIB recognition element BSA, bovine serum albumin bZIP, basic leucine zipper

C, cytosine CAP, catabolite activator protein CAT, chloramphenicol acetyltransferase CBP, CREB-binding protein

cDNA, complementary DNA C/EBP, CCAAT enhancer-binding protein CHD, chromodomain SWI/SNF-like helicase/ATPase domain and DNA-binding domain CITE, cap-independent translational enhancers

CMV, cytomegalovirus CREB, cAMP receptor element binding protein cRNA, complementary RNA

cs, cold sensitive CTD, carboxy-terminal domain CTP, cytosine triphosphate

DAN, deadenylating nuclease dATP, deoxyadenosine triphosphate dCTP, deoxycytidine triphosphate DEPC, diethyl pyrocarbonate dGTP, deoxyguanosine triphosphate DHFR, dihydrofolate reductase DMP, dimethyl pimelidate dihydrochloride DMS, dimethyl sulfate

DMSO, dimethyl sulfoxide DPE, downstream core promoter element

DR, direct repeat DRB, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole

ES, embryonic stem (cells) EST, expressed sequence tag ETL, early-to-late promoter

ExoIII, exonuclease III

FACS, fluorescence-activated cell sorting FISH, fluorescence in situ hybridization FBS, fetal bovine serum

β-gal,β-galactosidase

G, guanine GFP, green fluorescent protein GTFs, general transcription factors

HEBS, HEPES-buffered saline

HEPES, N-2-hydroxyethylpiperazine-N´-2-ethanesulfonic acid

HisD, histidinol dehydrogenase HIV, human immunodeficiency virus HIV-1, human immunodeficiency virus type 1

HKLM, heat-killed Listeria monocytogenes

HLH, helix-loop-helix HMBA, hexamethylene bisacetamide

xxvi ■ Abbreviations and Acronyms

HMG, high mobility group

HMK, heart muscle kinase

hpi, hours post induction

HPLC, high-performance liquid chromatography

HS, hypersensitive

hsp70, heat shock protein 70

HSTF, heat shock transcription factor

HSV, herpes simplex virus

HSV-1, herpes simplex virus type 1

HSV-TK, herpes simplex virus thymidine kinase

IPTG, isopropyl-β-D-thiogalactoside

IRE, iron-responsive element

IRP, iron-regulating protein

ISWI, imitation SWI

LCR, locus control region

LIS, lithium diiodosalicylate

LM-PCR, ligation-mediated PCR

LPS, lipopolysaccharide

LTR, long terminal repeat

M, molar

MAR, matrix attachment region

MBP, maltose binding protein

MEL, mouse erythroleukemia (cells)

MMLV, Moloney murine leukemia virus

MMTV, mouse mammary tumor virus

MNase, micrococcal nuclease

moi, multiplicity of infection

MOPS, 3-(N-morpholino) propanesulfonic acid

MPE, methidium propyl EDTA

mRNA, messenger RNA

MTX, methotrexate

NAT, negative activator of transcription

NER, nucleotide excision repair

neo, aminoglycoside phosphotransferase

NHP, nonhistone proteins

Ni-NTA, nickel-nitriloacetic acid

NMR, nuclear magnetic resonance

NP-40, Nonidet P-40

Abbreviations and Acronyms ■ xxvii

NTP(s), nucleotide triphosphate(s) NURF, nucleosome remodeling factor

O L, leftward operator

O R, rightward operator

OH-radical, hydroxyl-radical ONPG, O-nitrophenyl-β-D-galactopyranoside

OP-Cu, Cu-phenanthroline ORC, origin recognition complex ori, origin of replication

P R, promoter in rightward direction

P RM, promoter for repressor maintenance

PAGE, polyacrylamide gel electrophoresis PAN, poly(A) nuclease

PBS, phosphate-buffered saline

pc, positive control PCR, polymerase chain reaction PCV, packed cell volume PEG, polyethylene glycol PEI, polyethylenimine PIC, preinitiation complex

PIPES, piperazine-N,N´-bis(2-ethanesulfonic acid)

PMSF, phenylmethylsulfonyl fluoride PNK, polynucleotide kinase

PPAR-γ, peroxisome proliferator-activated receptor-γ

RACE, rapid amplification of cDNA ends RAR, retinoic acid receptor

Rb, retinoblastoma rbs, ribosome binding site RDA, representative difference analysis RNA, ribonucleic acid

RNAP, RNA polymerase RRE, Rev-responsive element RSV, Rous sarcoma virus

RT, reverse transcriptase RT-PCR, reverse transcription polymerase chain reaction

SAAB, selected and amplified binding site analysis SAGA, SPT-ADA-GCN acetyltransferase

SAR, scaffold-associated region SCAP, SREBP cleavage-activating protein SDS, sodium dodecyl sulfate

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis SEAP, secreted alkaline phosphatase

SMCC, SRB MED co-activator complex

sn, small nuclear SPR, surface plasmon resonance

xxviii ■ Abbreviations and Acronyms

SRB, suppressor of RNA polymerase B

SREBP-1, sterol response element binding protein

SSC, standard saline citrate

Su(Hw), suppressor of hairy wing

Tac, Trp-Lac (promoter)

TE, Tris/EDTA buffer

TES, N-Tris[hydroxymethyl]methyl-2-amino ethane sulfonic acid

Tet, tetracycline

TetR, Tet repressor

TFII, transcription factor for Pol II

TICS, TAFII- and initiator-dependent cofactors

tRNA, transfer RNA

TRRD, transcription regulatory region database

TSA, trichostatin A

U, uracil

UAS, upstream activating sequence

UAS G, galactose upstream activating sequence

USA, upstream stimulatory activity

UTL, untranslated leader

UTP, uridine triphosphate

VAF, virus-inducible transcription activator complex

VHL, von Hippel-Lindau

VSV, vesicular stomatitis virus

WCE, whole cell extract

Xis, excision protein

YAC, yeast artificial chromosome

Abbreviations and Acronyms ■ xxix

CONCEPTS AND STRATEGIES: I PROMOTERS AND THE GENERAL

Basal transcription complex assembly, 11 Conformational changes during transcription complex assembly, 11

Discovery of the Pol II holoenzyme, 14 Composition of the yeast holoenzyme, 15 Mammalian holoenzymes, 16

Modular activators, 20 DNA-binding domains, 21 Activation domains, 21 Structural aspects of activation domains, 22

General mechanisms, 23 Sequence-specific repressors, 24

CONCEPTS AND STRATEGIES: III CHROMATIN AND GENE

Structure and organization, 25 Binding of transcription factors to chromatin, 26 Genetic links between gene activation and chromatin, 27

SWI/SNF complexes, 27 Mechanisms and targeting, 29

Acetylation of chromatin 31

Mammalian acetylases, 32 TAFs and chromatin remodeling, 32

Histone deacetylation, transcriptional repression, and silencing 32

Repression and deacetylases, 33 Linking deacetylation and ATP-remodeling machines, 33 Methylation and repression, 34

Transcriptional silencing, 35

Locus control regions, insulators, and matrix attachment regions 35

Locus control regions, 35 Boundary elements, 37 MARs, 38

Combinatorial control, cooperativity, and synergy 38

INTRODUCTION

One of the central goals of the gene expression field is understanding how a mammalianorganism regulates transcription of approximately 30,000–40,000 genes in the proper spa-tial and temporal patterns Knowledge of how transcription factors function during this

“differential” gene expression can be applied to fundamental issues in the fields of biologyand medicine To decipher these mechanisms, we need to understand the numerousprocesses influencing transcription and develop technical and strategic approaches foraddressing them This chapter provides an introduction to basic aspects of RNA poly-merase II transcription The goal is to prepare the novice for the issues raised in subsequentchapters and to provide a general overview of the field as of this writing However, this field

is evolving rapidly and the reader is encouraged to consult recent reviews in the literature

Current Opinion in Cell Biology and Current Opinion in Genetics and Development publish

such reviews in the June and April issues, respectively Some of the topics are quiteadvanced, although we have cited numerous review articles to allow the novice to exploreunfamiliar areas Almost all of the topics are covered in subsequent chapters and may helpclarify concepts discussed briefly in this chapter

A General Model for Regulation of a Gene

In eukaryotes, DNA is assembled into chromatin, which maintains genes in an inactivestate by restricting access to RNA polymerase and its accessory factors Chromatin is com-posed of histones, which form a structure called a nucleosome Histones can be modifiedposttranslationally to decrease the ability of the nucleosome to inhibit transcription factorbinding Nucleosomes themselves are assembled into higher-order structures with differ-ent properties depending on the regulatory context During the process of development,

2 ■ Chapter 1

genes are turned on and off in a pre-programmed fashion, a process that eventually erates cell specificity This developmental program is orchestrated by transcription factors,which bind to specific DNA sites near genes they control A single transcription factor isnot dedicated to each regulatory event Instead, a mechanism called combinatorial control

gen-is employed In combinatorial control, different combinations of ubiquitous and specific regulatory proteins are used to turn genes on and off in different regulatory con-texts (Britten and Davidson 1969) The ability of an organism to employ small numbers ofregulatory proteins to elicit a larger number of regulatory decisions is based on the princi-ples of cooperativity and synergy, issues we discuss later in the chapter

cell-type-Activating a Gene

To provide a framework for the issues involved in transcription regulation, consider amodel for how a typical gene is turned on (Fig 1.1) and then off again In a typical gene,

a DNA sequence called the core promoter is located immediately adjacent to and upstream

of the gene The core promoter binds RNA polymerase II (Pol II) and its accessory factors(“the general transcription machinery”) and directs the Pol II to begin transcribing at thecorrect start site In vivo, in the absence of regulatory proteins, the core promoter is gener-ally inactive and fails to interact with the general machinery A caveat is that some core pro-

moters such as the heat-shock promoter in Drosophila and the Cyc-1 promoter in yeast

appear to contain partial complements of general factors (i.e., RNA Pol and TATA binding protein [TBP], respectively) when inactive, but these factors are insufficient fortranscription in the absence of regulatory proteins Immediately upstream of the core pro-moter is a regulatory promoter, and farther away either upstream or downstream areenhancer sequences (Fig 1.1A) Regulatory promoters and enhancers bind proteins calledactivators, which “turn on” or activate transcription of the gene Activation generallyoccurs by recruitment of the general machinery to the core promoter via interactionsbetween the activator bound to promoter DNA and the general machinery in solution.Some activators are ubiquitously expressed, whereas others are restricted to certain celltypes, regulating genes necessary for a particular cell’s function

box-To activate a gene, the chromatin encompassing that gene and its control regions must

be altered or “remodeled” to permit transcription There are different levels of tion needed at different levels and stages of the transcription process Higher-order chro-matin structures comprising networks of attached nucleosomes must be decondensed,specific nucleosomes over gene-specific enhancers and promoters must be made accessible

modifica-to cell-specific activamodifica-tors, and, finally, nucleosomes within the gene itself must be eled to permit passage of the transcribing RNA polymerases (Fig 1.1B) There are differ-ent types of enzymes involved in chromatin remodeling and these must be directed, per-haps by a limited set of activators or other sequence-specific DNA-binding proteins, to the

remod-“target” genes These enzymes fall into two broad classes: ATP-dependent remodelingenzymes and histone acetyltransferases (or simply histone acetylases) Once they bind near

a gene, these enzymes remodel the chromatin so that other activators and the generalmachinery can bind The mechanisms of remodeling are unclear, but they involve changes

in the structure of chromatin and in modification of histones that somehow increaseaccessibility to transcription factors Remodeling achieved at a local level affects only thechromatin close to a gene In some instances, however, a single gene or locus of relatedgenes might spread over 100 kb or more In these cases, genes might be under control ofnot simply specific enhancers and regulatory promoters but also of locus control regions

A Primer on Transcriptional Regulation in Mammalian Cells ■ 3

(LCRs), which remodel chromatin and control global access of activators over an

extend-ed region Once enhancers are accessible, they can stimulate transcription of a gene.However, because enhancers are known to activate transcription when they are positionedlarge distances from a gene, they could inadvertently activate other nearby genes in theabsence of appropriate regulation To focus the action of the enhancer or LCR on theappropriate gene or set of genes, the gene and its regulatory regions are thought to beassembled into a domain Domain formation appears to involve boundary elements andmatrix attachment regions (MARs) Boundary or insulator elements are thought to flankboth sides of an individual gene or gene locus These elements bind proteins that preventthe enhancer from communicating with genes on the opposite side of the insulator MARsalso flank some active genes and tether them as loops to the nuclear periphery or matrixalthough a gene function for MARs has not been established

The current view is that once the enhancer and promoter are accessible they bind tocombinations of activators Binding of activators is generally cooperative, where one pro-tein binds weakly, but multiple activators engage in protein–protein interactions thatincrease each of their affinities for the regulatory region The nucleoprotein structurescomprising these combinatorial arrays of activators are called enhanceosomes (Fig 1.1B).The enhanceosome interacts with the general transcription machinery and recruits it to acore promoter to form “the preinitiation complex.” The enhanceosome, the generalmachinery, and the core promoter form a complicated network of protein–protein and

4 ■ Chapter 1

B.

A.

Matrix attachment region (MAR)

Boundary element

–4000

Regulatory promoter

Boundary element Core

promoter –500 –40 +50

Chromatin Remodeling

USA CBP

IIB IIA TBP

IIH TAFs

Pol II

Enhancer Locus control region

General machinery

Mediator

FIGURE 1.1 (A) Model of typical gene and components involved in gene activation and

inactiva-tion (B) Acitvation of a gene and assembly of the Pol II preinitiation complex (Redrawn, with

per-mission, from Carey 1998 [copyright Cell Press].)

protein–DNA interactions that dictate the frequency of transcription initiation The actions between the enhanceosome and components of the general machinery are rarelydirect but are bridged or linked by proteins called coactivators It is important to note thatthe term “coactivator” has several definitions depending on the regulatory context: In somecases, coactivators are part of the general machinery and in other cases they are not Theterm will be defined on a case-by-case basis in this chapter.

inter-Inactivating a Gene

In many instances, genes are activated transiently and then later turned off In these cases,the hypothetical sequence of events would include inactivation of the preinitiation com-plex and establishment of a repressive chromatin environment over the gene and its regu-latory regions Establishment of an inactive chromatin environment involves ATP-depen-dent remodeling and histone deacetylases However, higher-order interactions with thenuclear periphery may also occur to form domains of inactive “heterochromatin.” Themechanisms for inactivating a gene vary, but generally they involve the binding ofsequence-specific repressors to silencer elements Genes are often methylated to maintainthe inactive state Methylation also leads to recruitment of histone deacetylases

Although the sequence of events described above provides a framework for gene vation and inactivation, the regulatory strategies employed vary considerably We attempt

acti-in the followacti-ing sections to evaluate different aspects of this simple model and to alert thereader to alternate regulatory strategies

Overview

In Section I, we summarize the basic mechanics of the transcription process, including anoverview of core promoter structure and the composition of the general machinery The gen-eral machinery consists of general transcription factors, or GTFs, and Pol II, which are nec-essary for the catalytic process of transcription However, the machinery also comprises coac-tivators and corepressors, which allow activators and repressors to communicate with theGTFs and chromatin In Section II, we discuss regulatory DNA sequences, includingenhancers and silencers, and regulatory proteins, including activators and repressors InSection III, we consider the structure of chromatin and the enzymes involved in “remodel-ing” it There is an emphasis on the roles of remodeling enzymes in establishing the activeand repressed states of genes Finally, we end with Section IV, entitled “the enhanceosome,”where we discuss the concepts of enhancer complexes and the basis for combinatorial con-trol Note that some of the topics are covered in greater detail in the ensuing chapters and wehave abbreviated our description of these here to prevent unnecessary redundancy

CONCEPTS AND STRATEGIES: I PROMOTERS AND THE GENERAL

TRANSCRIPTION MACHINERY

A typical core promoter encompasses DNA sequences between approximately –40 and +50relative to a transcription start site (Smale 1994) Core promoter DNA elements (1) bind toand control assembly of the preinitiation complex containing Pol II, the general transcrip-tion factor, and coactivators; (2) position the transcription start site and control the direc-tionality of transcription; and (3) respond to nearby or distal activators and repressors in acell In most cases, the core promoter elements do not play a direct role in regulated tran-scription The core promoter alone is generally inactive in vivo, but in vitro it can bind to

A Primer on Transcriptional Regulation in Mammalian Cells ■ 5

TABLE 1.1 Composition of coactivator/mediator complexes

Factor gene(s) (kD) Essential Characteristics gene(s) (kD)

19 ( β )

affects start site selection; zinc ribbon

elongation; functional interaction with TFIIB

DNA interactions

catalytic activities; functions in promoter melting and clearance;

zinc-binding domain

dependent DNA helicase (3´-5´); DNA-dependent ATPase; ATPase/helicase required for both transcription and NER

A Primer on T

ATPase; ATPase/helicase required for NER but not ERCC2 transcription

not a subunit of kinase/cyclin subcomplex

Data reprinted, with permission, from Orphanides et al 1996 (Copyright 1996 Cold Spring Harbor Laboratory Press); Hampsey 1998 (Copyright 1998 American Society for Microbiology); Myer and Young 1998 (Copyright 1998 American Society for Biochemistry and Molecular Biology); Björklund et al 1999 (Copyright 1999 Cell Press); and Coulombe and Burton 1999 (copy- right 1999 American Society for Microbiology).

8 ■ Chapter 1

the general machinery and support low or “basal” levels of transcription The amount ofbasal transcription is dictated by the DNA sequences in the core promoter Activators great-

ly stimulate transcription levels, and the effect is called activated transcription

The preinitiation complex that binds the core promoter comprises two classes of factors(see Table 1.1): (1) the GTFs including Pol II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH(Orphanides et al 1996); and (2) coactivators and corepressors that mediate response toregulatory signals (see Hampsey 1998; Myer and Young 1998) In mammalian cells, coacti-vator complexes are heterogeneous and occasionally can be purified as discrete entities or aspart of a larger Pol II holoenzyme, a point on which we elaborate below This sectiondescribes the properties of core promoters and the general transcription machinery

Core Promoter Architecture

TFIID is the only general transcription factor capable of binding core promoter DNA bothindependently and specifically TFIID is a multisubunit protein containing TBP and 10 ormore TBP-associated factors or TAFIIs (described in more detail below) The TFIID DNase

I footprint extends from –40 to +50 and encompasses most of the DNA constituting thecore promoter TFIID does not contact all of the bases in the footprint, and other generalfactors bind in a sequence-specific fashion within these “open” regions The other generalfactors bind DNA weakly on their own, however, and it is the network of cooperative pro-tein–protein interactions with TFIID and each other that allows them to form stable, spe-cific DNA interactions The role of cooperativity in assembly of transcription complexes is

a recurring theme in gene regulation, one that we return to throughout this book Theamount of basal transcription and the ability to respond to activators are likely related tothe affinities of the GTFs and TFIID for the core promoter (discussed in Lehman et al.1998) A typical core promoter contains the following DNA sequence elements (Fig 1.2):

1 The TATA motif This sequence element, with the consensus TATAAA, was originally

dis-covered by David Hogness and is called the Hogness box in the older literature It is

locat-ed 25–30 bp upstream of the transcription start site The TATA box is capable of pendently directing a low level of transcription by Pol II on naked DNA templates in vitro

inde-or transfected DNA templates in vivo The TATA box is sufficient finde-or directing activatedtranscription when an activator protein binds to a nearby regulatory element The TBPsubunit (Hernandez 1993; Burley and Roeder 1996) of TFIID (Table 1.1) makes directcontact with the TATA motif The binding of TFIID to the TATA box nucleates the bind-ing of the remaining general transcription factors, currently thought to be present in theform of a multifactor “holoenzyme,” an issue we discuss below (for review, see Myer andYoung 1998) We discuss TBP-binding mechanisms in Chapters 13 and 15

–38

Initiator TFIIB recognition

element

Downstream core promoter element TATA motif

IIE, IIH, Pol II:

FIGURE 1.2 Sequence elements in a typical core promoter.

A Primer on Transcriptional Regulation in Mammalian Cells ■ 9

2 The initiator element A second type of core promoter element that appears to be

func-tionally analogous to the TATA box is the initiator (Inr; Smale 1994) Although thiselement carries out the same functions as TATA by directing the formation of a preini-tiation complex, determining the location of the start site, and mediating the action ofupstream activator proteins, it directly overlaps the transcription start site Function-

al Inr activity depends on a loose consensus sequence of approximately PyPyA+1NT/APyPy The basal Inr appears to be recognized by two independent proteins: a TAFIIand Pol II (for review, see Smale 1997) The TAFIIthat binds the Inr has not been firm-

ly established but may be TAFII250, with binding further stabilized by TAFII150(Kaufmann et al 1998) TFIID binding to the Inr appears to be influenced by bothTFIIA and cofactors called TICs (TAFII- and initiator-dependent cofactors) (Emami et

al 1997; Martinez et al 1998) A plausible model for the initiation of transcriptionfrom a TATA-less promoter containing an Inr is as follows: The TFIID complex rec-ognizes the Inr, possibly with the assistance of TFIIA and TIC-1 At some promoters,this recognition event directs the TBP subunit of TFIID to associate with the –30region of the promoter in a TATA sequence-independent manner, although at somepromoters TBP binding may be unnecessary Following the stable binding of TFIID tothe core promoter, the remaining steps leading to formation of a functional preinitia-tion complex and transcription initiation proceed by a similar mechanism and require

a similar set of general transcription factors as TATA-containing promoters The cific interactions between Pol II and the Inr may become important at later steps inpreinitiation complex formation

spe-3 The downstream core promoter element The downstream core promoter element (DPE) is a 7-nucleotide sequence first identified in Drosophila The DPE bears the

consensus sequence RGA/TCGTG and is centered approximately 30 bp downstream of

the initiation site It is found in many, but not all, Drosophila promoters and most

like-ly many mammalian promoters (Burke and Kadonaga 1996) In Drosophila, where the

DPE element has been studied in the greatest detail, the DPE is found in TATA-lesspromoters and acts in conjunction with the Inr element to direct specific initiation oftranscription Crosslinking and DNA-binding studies suggest that the DPE recognizes

Drosophila TAFII60 and perhaps TAFII40 directly (Burke and Kadonaga 1997)

4 The TFIIB recognition element The TFIIB recognition element (BRE) was discovered

by Ebright and colleagues (Lagrange et al 1998), who recognized the potential for cific DNA binding by TFIIB based on the position of TFIIB relative to the majorgroove in the crystal structure of the TBP–TFIIB–TATA ternary complex (for review,see Burley and Roeder 1996) Binding-site-selection experiments (discussed inChapter 13) revealed that TFIIB bound specifically to a sequence with the consensus

spe-G/CG/CG/ACGCC located from –32 to –38, just upstream of the TATA box The BRE isfound in a substantial number of eukaryotic promoters

It is likely that other general transcription factors display a limited degree of cific recognition TFIIA, TFIIF, and Pol II all interact with the major groove as revealed in thecrystal structures of TFIIA–TBP–TATA and in photocrosslinking experiments with Pol II andTFIIF (Kim et al 1997; Robert et al 1998) Additional TAFIIs may also bind DNA specifically,based on photocrosslinking data (Oelgeschlager et al 1996) Further research is needed tounderstand the significance of the interactions occurring on the core promoter However, asdiscussed above, preliminary indications are that the ability of the core promoter to respond

sequence-spe-to activasequence-spe-tors and direct high levels of transcription is dependent on cooperative binding ofmultiple general factors