真核生物转录调控 概念，策略和方法 英文版PDF电子书下载

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