© 2000 • 640 pp., illus., appendices, index
Paperback • ISBN  0-87969-635-4

In the genome era, the analysis of gene expression has become a critical requirement in many laboratories. But there has been no comprehensive source of strategic, conceptual, and technical information to guide this often complex task. Transcriptional Regulation in Eukaryotes answers that need. Written by two experienced investigators, Michael Carey and Stephen Smale at the UCLA School of Medicine, and based in part on the Gene Expression course taught at Cold Spring Harbor Laboratory, this book directly addresses all the concerns of a laboratory studying the regulation of a newly isolated gene and the biochemistry of a new transcription factor.

Chapter 1 reviews the state-of-the-art in the RNA polymerase II transcription field;
Chapters 2–10 are a systematic guide to the investigation of regulatory mechanisms for newly identified genes;
Chapters 11–15 discuss approaches for synthesizing recombinant transcription factors, analyzing their binding, and deciphering their mode of action.

Transcriptional Regulation in Eukaryotes reveals not only what to do but why, and how to plan properly for success. This approach is ideal for graduate students, physician-scientists, postdocs, and others entering the field, but is also valuable for established investigators undertaking in-depth studies in specific systems.

Transcriptional Regulation in Eukaryotes is a powerful textbook for advanced instruction in molecular biology:

• supplementing clearly written text with extensive illustrations
• putting methods in the context of underlying theory
• giving expert recommendations on experimental strategies
• encouraging creativity in investigative design
• explaining protocols for essential techniques step by step, with extensive advice on troubleshooting

This important and unique book is essential reading for anyone pursuing the analysis of gene expression in model systems or disease states.

  "The field of transcriptional analysis of eukaryotic genes is complicated but this book brings it both order and insight. It is a rare book because it includes both strategical and tactical analysis, carrying the reader from conceptual issues through to the nuts and bolts of experimentation. The book provides perspective to the newcomer and the latest tools to the expert. In today's world of global gene expression analysis by DNA chips, this tome reminds us how hard it is to understand the regulation of any one gene."
      —DAVID BALTIMORE, California Institute of Technology

  "Carey and Smale have undertaken the heroic task of assembling a compendium of Concepts, Strategies and Techniques for analyzing the regulation of transcription in Eukaryotes. This is a remarkably comprehensive and well organized ‘working lab manual’ that should be particularly useful to those new to the field of eukaryotic transcription. [It] contains many of the experimental techniques and strategies that have been developed and used by the transcription community throughout the past two decades of research but never before compiled into one, well organized and comprehensive handbook of transcription."
      —ROBERT TJIAN, Howard Hughes Medical Institute, U.C. Berkeley

  "In keeping with the fact that gene expression in eukaryotes is regulated primarily at the level of transcription, the factors and mechanisms that execute and regulate transcription (by RNA polymerase II) have proved to be remarkably complex and sometimes daunting to the outsider. In this comprehensive work, the authors have done an excellent job of summarizing current facts and concepts in the field, and in describing experimental strategies and methodologies for application to the many genes whose regulatory factors and mechanisms remain to be established. There is both breadth and depth, as the authors describe, and in some cases detail, a variety of approaches (from biochemical to molecular genetic and cellular) that should help the investigator decide the most appropriate strategy for the particular problem and laboratory expertise at hand. For concepts and technologies not detailed, the text is well referenced. This work will be an invaluable reference for the new investigator, but should also prove useful to specialized investigators already in the field. Overall, it is an up-to-date, much-needed, and highly recommended treatise on both conceptual and practical aspects of transcriptional regulation in eukaryotes."
      —ROBERT G. ROEDER, The Rockefeller University

Printed-page renditions of Chapters 9 and 13 are offered online, in the form of optimized (byteserved) PDF files. See "Online Features" above.
The first selected chapter online is a guide to various experimental approaches for examining the functional relevance of a protein-DNA interaction for transcriptional regulation. The second one discusses the theory of DNA recognition, how to identify a high-affinity recognition site for a DNA-binding protein, and how to study and model a DNA-protein interaction.
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Preface

Overview

Abbreviations and Acronyms

1.    A PRIMER ON TRANSCRIPTIONAL REGULATION IN MAMMALIAN CELLS

INTRODUCTION

A general model for regulation of a gene

Overview

I.   PROMOTERS AND THE GENERAL TRANSCRIPTION MACHINERY

Core promoter architecture

The general transcription machinery

TAF_IIs

The holoenzyme and mediators

II.   ACTIVATORS AND REPRESSORS

Regulatory promoters and enhancers

Transcriptional activators

Repressors and corepressors

III.   CHROMATIN AND GENE REGULATION

Chromatin

ATP-dependent remodeling complexes

Acetylation of chromatin

Histone deacetylation, transcriptional repression, and silencing

Locus control regions, insulators, and matrix attachment regions

IV.   THE ENHANCEOSOME

Combinatorial control, cooperativity, and synergy

The enhanceosome theory

The interferon-beta enhanceosome

Biochemical mechanism of activation

Perspective

2.    INITIAL STRATEGIC ISSUES

INTRODUCTION

CONCEPTS AND STRATEGIES

The initial steps in a gene regulation analysis

Consider the time commitment and resources needed to reach a defined goal

Evaluate the feasibility of the analysis

Initiate an analysis of transcriptional regulation

Summary

3.    MODES OF REGULATING mRNA ABUNDANCE

INTRODUCTION

CONCEPTS AND STRATEGIES

Transcription initiation versus mRNA stability

Transcription elongation

Differential pre-mRNA splicing, mRNA transport, and polyadenylation

TECHNIQUES

Protocol 3.1    Nuclear run-on assay

4.    TRANSCRIPTION INITIATION SITE MAPPING

INTRODUCTION

CONCEPTS AND STRATEGIES

Initial considerations

Primer extension

RNase protection

S1 nuclease analysis

Rapid amplification of cDNA ends

TECHNIQUES

Protocol 4.1    Primer extension assay

Protocol 4.2    RNase protection assay

Protocol 4.3    S1 Nuclease assay

5.    FUNCTIONAL ASSAYS FOR PROMOTER ANALYSIS

INTRODUCTION

CONCEPTS AND STRATEGIES

Choosing an assay: Advantages and disadvantages of each assay

Transient transfection assays

Stable transfection assays by chromosomal integration

TECHNIQUES

Common transfection methods for mammalian cells

Common reporter enzyme assays

6.    IDENTIFICATION AND ANALYSIS OF DISTANT CONTROL REGIONS

INTRODUCTION

CONCEPTS AND STRATEGIES

DNase I hypersensitivity

Identification of matrix attachment regions

Functional approaches for the identification of distant control regions

Functional assays for the characterization of distant control regions

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

INTRODUCTION

CONCEPTS AND STRATEGIES

Identification of control elements by comprehensive mutant analysis

Identification of control elements using in vivo or in vitro protein-DNA interaction methods

Identification of control elements by database analysis

Mutagenesis techniques

8.    IDENTIFICATION OF DNA-BINDING PROTEINS AND ISOLATION OF THEIR GENES

INTRODUCTION

CONCEPTS AND STRATEGIES FOR THE IDENTIFICATION OF DNA-BINDING PROTEINS

Database methods

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

CONCEPTS AND STRATEGIES FOR CLONING GENES ENCODING DNA-BINDING PROTEINS

Cloning by protein purification and peptide sequence analysis

Cloning by methods that do not require an initial protein-DNA interaction assay

9.    CONFIRMING THE FUNCTIONAL IMPORTANCE OF A PROTEIN-DNA INTERACTION
[Selected Chapter online * PDF file (637 KB) * ]

 

INTRODUCTION

CONCEPTS AND STRATEGIES

Abundance of a protein-DNA complex in vitro

Relative expression patterns of the DNA-binding protein and target gene

Correlation between nucleotides required for protein binding and those required for activity of the control element

trans-Activation of a reporter gene or endogenous gene by overexpression of the DNA-binding protein

Cooperative binding and synergistic function of proteins bound to adjacent control elements

Comparison of genomic and in vitro footprinting patterns

Relative affinity of a protein-DNA interaction

Gene disruption or antisense experiments

Dominant-negative mutants

In vitro transcription strategies

In vivo protein-DNA crosslinking

Altered specificity experiment

10.    IN VIVO ANALYSIS OF AN ENDOGENOUS CONTROL REGION

INTRODUCTION

CONCEPTS AND STRATEGIES

In vivo analysis of sequence-specific protein-DNA interactions

Nucleosome positioning and remodeling

DNA methylation

Subnuclear localization of a gene

TECHNIQUES

Protocol 10.1    MNase-Southern blot assay

Protocol 10.2    LM-PCR methods

11.    APPROACHES FOR THE SYNTHESIS OF RECOMBINANT TRANSCRIPTION FACTORS

INTRODUCTION

CONCEPTS AND STRATEGIES

Prokaryotic expression systems

Strategies for overcoming expression problems in E. coli

Synthesizing large regulatory proteins

Synthesizing small quantities of crude protein

Synthesis and purification of macromolecular complexes

Choosing an appropriate system

12.    IDENTIFYING AND CHARACTERIZING TRANSCRIPTION FACTOR DOMAINS

INTRODUCTION

CONCEPTS AND STRATEGIES: DEFINING DOMAINS

Basic mutagenesis principles

Domains of a gene activator

Separating DNA-binding and activation domains of an activator

Subdividing DNA recognition and oligomerization subdomains

CONCEPTS AND STRATEGIES: PROTEIN-PROTEIN INTERACTIONS

Interaction of activation domains with coactivators and general factors

Affinity chromatography

Altered specificity genetic systems

Structure-function analysis of the general transcriptional machinery

TECHNIQUES

Protocol 12.1    PCR-mediated site-directed mutagenesis

13.    THEORY, CHARACTERIZATION, AND MODELING OF DNA BINDING BY REGULATORY TRANSCRIPTION FACTORS
[Selected Chapter online * PDF file (880 KB) * ]

 

INTRODUCTION

CONCEPTS AND STRATEGIES

General theory of DNA-protein interactions

Analysis and modeling of DNA-protein interactions

Analysis of promoter-specific multicomponent nucleoprotein complexes

TECHNIQUES

Protocol 13.1    DNase I footprinting

Protocol 13.2    Hydroxyl-radical footprinting

Protocol 13.3    Phosphate ethylation interference assay

Protocol 13.4    Methylation interference assay

Protocol 13.5    Electrophoretic mobility shift assays

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

14.    CRUDE AND FRACTIONATED SYSTEMS FOR IN VITRO TRANSCRIPTION

INTRODUCTION

CONCEPTS AND STRATEGIES

Preparation of extracts

Transcription assays

Fractionated systems

TECHNIQUES

Preparation of nuclear and whole-cell extracts

In vitro transcription assays

Transcription factor purification

15.    APPROACHES FOR STUDYING TRANSCRIPTION COMPLEX ASSEMBLY

INTRODUCTION

CONCEPTS AND STRATEGIES

Formation of the basal preinitiation complex

Open complex formation, initiation, and promoter escape

Assembly of activated complexes at a promoter

TECHNIQUES

Protocol 15.1    Potassium permanganate probing of Pol II open complexes

Protocol 15.2    Magnesium-agarose EMSA of TFIID binding to DNA

APPENDICES

CAUTIONS

SUPPLIERS

TRADEMARKS

INDEX