1 A PRIMER ON TRANSCRIPTIONAL REGULATION IN MAMMALIAN CELLS 1
INTRODUCTION 2
A general model for regulation of a gene 2
Activating a gene 3
Inactivating a gene 5
Overview 5
CONCEPTS AND STRATEGIES：Ⅰ．PROMOTERS AND THE GENERALTRANSCRIPTION MACHINERY 5
Core promoter architecture 8
The general transcription machinery 10
Basal transcription complex assembly 11
Conformational changes during transcription complex assembly 11
TAFⅡs 12
Discovery ofthePol Ⅱ holoenzyme 14
The holoenzyme and mediators 14
Composition of the yeast holoenzyme 15
Mammalian holoenzymes 16
ContentsPreface 17
CONCEPTS AND STRATEGIES：Ⅱ．ACTIVATORS AND REPRESSORS 18
Regulatory promoters and enhancers 18
Transcriptional activators 20
Modular activators 20
Overviewx 21
DNA-binding domains 21
Activation domains 21
Structural aspects of activation domains 22
Repressors and corepressors 23
General mechanisms 23
Sequence-specific repressors 24
Abbreviations and Acronyms 25
CONCEPTS AND STRATEGIES：Ⅲ．CHROMATIN ANDGENE REGULATION 25
Chromatin 25
Structure and organization 25
Binding of transcription factors to chromatin 26
Genetic links between gene actwation and chromatin 27
ATP-dependent remodeling complexes 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 deacetfylases 33
Linking deacetylation and ATP-remodeling machines 33
Methylation and repression 34
Locus control regions,insulators,and matrix attachment regions 35
Locus control regions 35
Transcriptional silencing 35
Boundary elements 37
MARs 38
CONCEPTS AND STRATEGIES：Ⅳ．THE ENHANCEOSOME 38
Combinatorial control,cooperativity,and synergy 38
The enhanceosome theory 39
The interferon-β enhanceosome 40
Biochemical mechanism of activation 41
Perspective 42
2 INITIAL STRATEGIC ISSUES 51
The initial steps in a gene regulation analysis 52
CONCEPTS AND STRATEGIES 52
INTRODUCTION 52
Consider the time commitment and resources needed to reach a defined goal 54
Two general strategies that provide preliminary albeit superficial insight into transcriptional regulation mechanisms 54
An example ofa rigorous,yet incomplete gene regulation analysis：The immunoglobulin μ heavy-chain gene 55
Defining the project goals 57
Evaluate the feasibility of the analysis 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
Initiate an analysis of transcriptional regulation 61
Beginning with the promoter or distant control regions 61
Summary 62
Initiating an analysis of a promoter 62
Initiating an analysis of distant control regions 62
3 MODES OF REGULATING mRNA ABUNDANCE 65
INTRODUCTION 66
CONCEPTS AND STRATEGIES 66
Transcription initiation versus mRNA stability 66
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
Transcription elongation 78
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
Protocol 3.1 Nuclear run-on assay 87
TECHNIQUES 87
4 TRANSCRIPTION INITIATION SITE MAPPING 97
INTRODUCTION 98
CONCEPTS AND STRATEGIES 99
Initial considerations 99
Reagents needed before proceeding 99
Information provided by the DNA sequence 99
Primer extension 102
Advantages and disadvantages 102
Design of oligonucleotide primers 102
Method(Box 4.1) 103
Analysis of example data 104
Primer annealing and reverse transcription 104
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
S1 nuclease analysis 109
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
Rapid amplification of cDNA ends 112
Effect of introns on the interpretation of start-site mapping results(Box 4.5) 114
TECHNIQUES 116
Protocol 4.1 Primer extension assay 116
Protocol 4.2 RNase protection assay 124
Protocol 4.3 S1 nuclease assay 130
5 FUNCTIONAL ASSAYS FOR PROMOTER ANALYSIS 137
I NTRODUCTION 138
Choosing an assay：Advantages and disadvantages of each assay 141
CONCEPTS AND STRATEGIES 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
Transient transfection assays 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
Stable transfection assays by chromosomal integration 160
General strategies 160
Cells and transfection procedures 162
Reporter genes and assays 165
Drug-resistance genes and vectors 165
Plasmid construction 168
Drug selection 169
Controls and interpretation of results 171
Common transfection methods for mammalian cells 172
TECHNIQUES 172
Protocol 5.1 Calcium phosphate transfection of 3T3 fibroblasts 174
Protocol 5.2 DEAE-dextran transfection of lymphocyte cell lines 176
Protocol 5.3 Transfection by electroporation of RAW264.7 macrophages 178
Common reporter enzyme assays 180
Protocol 5.4 Luciferase assay 181
Protocol 5.5 Chloramphenicol acetyltransferase assay 183
Protocol 5.6 β-Galactosidase assay 186
6 IDENTIFICATION AND ANALYSIS OF DISTANT CONTROL REGIONS 193
INTRODUCTION 194
DNase I hypersensitivity 195
Basic principles of DNase I sensitivity and hypersensitivity 195
CONCEPTS AND STRATEGIES 195
Advantages and disadvantages of using DNase I hypersensitivity to identify control regions 197
DNaseI hypersensitivity assay(Box 6.1) 198
Data interpretation 200
Identification of matrix attachment regions 200
Basic principles of the nuclear matrix and of MARs and SARs 200
Advantages and disadvantages of usingMARs 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
Functionalapproach beginningwith a large genomic DNA fragment 203
Functional approach beginning with smaller fragments directing expression of a reportergene 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
7 IDENTIFYING cis-ACTING DNA ELEMENTS WITHIN A CONTROL REGION 213
INTRODUCTION 214
CONCEPTS AND STRATEGIES 215
Identification of control elements by comprehensive mutant analysis 215
Rationale for a comprehensive anialysis 215
The Ig μ gene example 216
Disadvantages of using mutagenesis to identify control elemen 219
Strategies for a comprehensive analysis 220
Methodology for mutating a control region 235
Identification of control elements using in vivo or in vitro protein-DNA interaction methods 235
Advantages and disadvantages 235
Identification of control elements by database analysis 237
Advantages and disadvantages 237
Mutagenesis techniques(Boxes 7.1-7.6) 238
8 IDENTIFICATION OF DNA-BINDING PROTEINS AND ISOLATION OF THEIR GENES 249
INTRODUCTION 250
Database methods 252
CONCEPTS AND STRATEGIES FOR THE IDENTIFICATION OF DNA-BINDING PROTEINS 252
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 DNA-BINDING PROTEINS 272
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 protein-DNA interaction assay 283
One-hybrid screen 283
In vitro expression library screening with DNA or antibody probes 285
Mammalian expression cloning methods 287
Genome database methods and degenerate PCR 288
9 CONFIRMING THE FUNCTIONAL IMPORTANCE OF A PROTEIN-DNA INTERACTION 291
INTRODUCTION 292
CONCEPTS AND STRATEGIES 294
Abundance of a protein-DNA complex in vitro 294
Relative expression patterns of the DNA-binding protein and target gene 295
Correlation between nucleotides required for protein binding and those required for activity of the control element 296
trans-Activation of a reporter gene or endogenous gene by overexpression of the DNA-binding protein 297
Cooperative binding and synergistic function of proteins bound to adjacent control elements 299
Comparison of genomic and in vitro footprinting patterns 301
Relative affinity of a protein-DNA interaction 302
Gene disruption or antisense experiments 304
Dominant-negative mutants 305
In vitro transcription strategies 308
In vivo protein-DNA crosslinking 310
Altered specificity experiments 313
10 IN VIVO ANALYSIS OF AN ENDOGENOUS CONTROL REGION 319
INTRODUCTION 320
DNase I and DMS genomic footprinting(Box 10.1) 321
In vivo analysis of sequence-specific protein-DNA interactions 321
CONCEPTS AND STRATEGIES 321
In vivo protein-DNA crosslinking/immunoprecipitation 326
Nucleosome positioning and remodeling 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 footprin ting(Box 103) 329
In vivo methods for analyzing nucleosome remodeling(Box 10.4) 332
DNA methylation 335
Subnuclear localization of a gene 337
TECHNIQUES 338
Protocol 10.1 MNase-Southern blot assay 338
Restriction enzyme accessibility to monitor nucleosome remodeling 347
DMS genomicfootprinting 347
Protocol 10.2 LM-PCR methods 347
MNase mapping of nucleosome positioning 347
DNase genomic footprinting 347
11 APPROACHES FOR THE SYNTHESIS OF RECOMBINANT TRANSCRIPTION FACTORS 365
INTRODUCTION 366
CONCEPTS AND STRATEGIES 367
Prokaryotic expression systems(Boxes 11.1 and 11.2) 367
Strategies for overcoming expression problems in E.coli 374
Synthesizing large regulatory proteins 377
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
Synthesizing small quantities of crude protein 385
Specialized inducible expression systems(Box 11.7) 386
In vitro transcription/translation systems(Box 11.8) 388
Mammalian expression vectors(Box 11.9) 389
Synthesis and purification of macromolecular complexes 390
Choosing an appropriate system 391
12 IDENTIFYING AND CHARACTERIZING TRANSCRIPTION FACTOR DOMAINS 399
CONCEPTS AND STRATEGIES：DEFINING DOMAINS 400
Basic mutagenesis principles 400
INTRODUCTION 400
Domains of a gene activator 402
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 subdomains(Box 12.2) 409
CONCEPTS AND STRATEGIES：PROTEIN-PROTEIN INTERACTIONS 410
Interaction of activation domains with coactivators and general factors 410
Affinity chromatography 413
Principles 413
Caveats of the affinityapproach 415
Altered specificity genetic systems 416
Structure-function analysis of the general transcriptional machinery 420
TECHNIQUES 422
Protocol 12.1 PCR-mediated site-directed mutagenesis 422
13 THEORY,CHARACTE RIZATION,AND MODELING OF DNA BINDING BY REGULATORY TRANSCRIPTION FACTORS 433
INTRODUCTION 434
CONCEPTS AND STRATEGIES 436
General theory and examples of DNA-protein interactions 436
Theory of DNA recognition 436
Chemical basis of the interactions 437
The role of the α-helix in DNA recognition 437
Major and minorgroove specificity 439
Monomers and dimers；energetic and regulatory considerations 441
Dissociation constant analysis(Box 13.1) 444
Kd determination 447
Analysis and modeling of DNA-protein interactions 448
Identification of a high-affinity DNA recognition site 448
Basic theory 449
General methods(Boxes 13.2 and 133) 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
TECHNIQUES 472
Protocol 13.1 DNase I footprinting 472
Protocol 13.2 Hydroxyl-radical footprinting 482
Protocol 13.3 Phosphate ethylation interference assay 485
Protocol 13.4 Methylation interference assay 488
Protocol 13.5 Electrophoretic mobility shift assays 493
Protocol 13.6 Preparation of 32P-end-labeled DNA fragments 497
14 CRUDE AND FRACTIONATED SYSTEMS FOR IN VITRO TRANSCRIPTION 505
INTRODUCTION 506
CONCEPTS AND STRATEGIES 507
Preparation of extracts 507
Cell choice 507
Extract preparation method 508
Transcription assays 510
General considerations(Box 14.1) 510
Choice of template 514
Chromatin systems 516
Optimization of conditions 519
Fractionated systems(Box 14.2) 519
Holoenzyme 520
Partially fractionated systems 521
Mediator subcomplexes 521
Factor-depleted systems 525
Highly fractionated systems 526
TECHNIQUES 526
Preparation of auclear and whole-cell extracts 526
Protocol 14.1 The Dignam and Roeder nuclear extract 528
Protocol 14.2 Preparation of nuclear extracts from rat liver 532
Protocol 14.3 Preparation of whole-cell extract 536
In vitro transcription assays 539
Protocol 14.4 In vitro transcription using HeLa cell extracts and primer extension 539
Protocol 14.5 G-less cassette in vitro transcription using HeLa cell nuclear extracts 545
Transcription factor purification 549
Protocol 14.6 Preparation of a crude fractionated system 551
Protocol 14.7 Purification of recombinant TFIIB from E.coli 556
Protocol 14.8 Purification of recombinant TFIIA 560
Protocol 14.9 Affinity purification of RNA Pol Ⅱ 562
Protocol 14.10 Purification of epitope-tagged TFIID 567
15 APPROACHES FOR STUDYING TRANSCRIPTION COMPLEX ASSEMBLY 579
INTRODUCTION 580
CONCEPTS AND STRATEGIES 582
Formation of the basal preinitiation complex 582
Kinetic studies 582
Sarkosyl probing 582
DNase Ifootprinting and EMSA studies oftranscription complex assembly 584
Template commitment experiment 584
Photocrosslinking 586
Structure-function analyses of the general machinery 589
Open complex formation,initiation,and promoter escape 589
ATP-analogs and an energy-dependent step 589
Permanganate probing 590
Premelted templates 590
The transition to elongation 591
Assembly of activated complexes at a promoter 594
The immobilized template approach 594
Permanganate probing to study activation 596
Gel filtration 596
EMSA and DNase I footprinting analyses of the TFIID-TFIIA complex 599
Assembly and analysis of TFIID subcomplexes 600
Future directions 601
TECHNIQUES 603
Protocol 15.1 Potassium permanganate probing of Pol Ⅱ open complexes 603
Protocol 15.2 Magnesium-agarose EMSA of TFIID binding to DNA 607
APPENDICES 617
Ⅰ．CAUTIONS 617
Ⅱ．SUPPLIERS 623
Ⅲ．TRADEMARKS 625
INDEX 627