Transcriptional Regulation in Eukaryotes

Автор(ы):Carey Michael
06.10.2007
Год изд.:2000
Описание: The goal of this book is to provide a detailed description of the approaches to be employed and issues to be considered when undertaking a molecular analysis of the transcriptional regulatory mechanisms for a newly isolated gene, or a biochemical analysis of a new transcription factor. Our emphasis is on mammalian transcription, which is complicated by the combinatorial nature of regulation and the lack of facile genetics. We refer periodically to studies in yeast, Drosophila, and other organisms where more tractable genetic approaches have led to a detailed understanding of particular mechanistic issues. The topics covered in the book extend from the determination of whether a gene is in fact regulated at the level of transcription initiation to advanced strategies for characterizing the biochemical mechanism underlying its combinatorial regulation by activators. Although numerous specialized and detailed techniques are included, the unique characteristics of this book are its strategic and conceptual emphasis on analysis of individual genes and the transcription factors that regulate them.
Оглавление:
Transcriptional Regulation in Eukaryotes — обложка книги.
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 GENERAL TRANSCRIPTION MACHINERY [5]
    Core promoter architecture [8]
    The general transcription machinery [10]
      Basal transcription complex assembly [11]
      Conformational changes during transcription complex assembly [11]
    TAFns [12]
    The holoenzyme and mediators [14]
      Discovery of the Pol II holoenzyme [14]
      Composition of the yeast holoenzyme [15]
      Mammalian holoenzymes [16]
  CONCEPTS AND STRATEGIES: II. ACTIVATORS AND REPRESSORS [18]
    Regulatory promoters and enhancers [18]
    Transcriptional activators [20]
      Modular activators [20]
      DNA-binding domains [21]
      Activation domains [21]
      Structural aspects of activation domains [22]
    Repressers and corepressors [23]
      General mechanisms [23]
      Sequence-specific repressers [24]
  CONCEPTS AND STRATEGIES: III. CHROMATIN AND GENE REGULATION [25]
    Chromatin [25]
      Structure and organization [25]
      Binding of transcription factors to chromatin [26]
      Genetic links between gene activation 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 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]
  CONCEPTS AND STRATEGIES: IV. THE ENHANCEOSOME [38]
    Combinatorial control, cooperativity, and synergy [38]
    The enhanceosome theory [39]
    The interferon-(?) enhanceosome [40]
    Biochemical mechanism of activation [41]
    Perspective [42]
2 INITIAL STRATEGIC ISSUES [51]
  INTRODUCTION [52]
  CONCEPTS AND STRATEGIES [52]
    The initial steps in a gene regulation analysis [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 of a rigorous, yet incomplete gene regulation analysis: The immunoglobulin ц 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]
      Initiating an analysis of a promoter [62]
      Initiating an analysis of distant control regions [62]
    Summary [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 ofpre-mRNA splicing, transport, and polyadenylation [86]
  TECHNIQUES [87]
    Protocol 3.1 Nuclear run-on assay [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]
      Primer annealing and reverse transcription [104]
      Analysis of example data [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]
    SI nuclease analysis [109]
      Advantages and disadvantages [109]
      Probe preparation [109]
      Method (Box 4.3) [109]
      Analysis of example data [111]
    Rapid amplification of cDNA ends [112]
      Advantages and disadvantages [112]
      Data analysis [112]
      Method (Box 4.4) [112]
      Effect ofintrons 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 SI nuclease assay [130]
5 FUNCTIONAL ASSAYS FOR PROMOTER ANALYSIS [137]
  INTRODUCTION [138]
  CONCEPTS AND STRATEGIES [141]
    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 ofepisomally 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]
  TECHNIQUES [172]
    Common transfection methods for mammalian cells [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 (3-Galactosidase assay [186]
6 IDENTIFICATION AND ANALYSIS OF DISTANT CONTROL REGIONS [193]
  INTRODUCTION [194]
  CONCEPTS AND STRATEGIES [195]
    DNase I hypersensitivity [195]
      Basic principles of DNase I sensitivity and hypersensitivity [195]
      Advantages and disadvantages of using DNase I hypersensitivity to identify control regions [197]
      DNase I hypersensitivity assay (Box 6.1) [198]
      Data interpretation [200]
    Identification of matrix attachment regions [200]
      Basic principles of the nuclear matrix and ofMARs 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 ofLCR activity [208]
      Demonstration of silencer activity [209]
      Demonstration of insulator activity [209]
7 IDENTIFYING cis-ACTING DMA 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 analysis [215]
      The Ig (?) gene 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 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]
  CONCEPTS AND STRATEGIES FOR THE IDENTIFICATION OF DNA-BINDING PROTEINS [252]
    Database methods [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]
      Ammo add 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]
  CONCEPTS AND STRATEGIES [321]
    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]
    Nucleosome positioning and remodeling [326]
      Model systems [326]
      Low-resolution analysis of nudeosome positioning by the MNase-Southern blot method (Box 10.2) [328]
      High-resolution analysis of nudeosome positioning by an MNase-LM-PCR method and DNase I genomic footprinting (Box 10.3) [329]
      In vivo methods for analyzing nudeosome remodeling (Box 10.4) [332]
    DNA methylation [335]
    Subnuclear localization of a gene [337]
  TECHNIQUES [338]
    Protocol 10.1 MNase-Southern blot assay [338]
    Protocol 10.2 LM-PCR methods [347]
      DNase genomic footprinting [347]
      MNase mapping of nudeosome positioning [347]
      Restriction enzyme accessibility to monitor nucleosome remodeling [347]
      DMS 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]
  INTRODUCTION [400]
  CONCEPTS AND STRATEGIES: DEFINING DOMAINS [400]
    Basic mutagenesis principles [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 affinity approach [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, CHARACTERIZATION, 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 (?)-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(?) 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 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]
      DATA 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 (?)P-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]
      Mediator subcomplexes [521]
      Partially fractionated systems [521]
      Factor-depleted systems [525]
      Highly fractionated systems [526]
  TECHNIQUES [526]
    Preparation of nuclear 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]
      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]
    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]
      Gel filtration [596]
      Permanganate probing to study activation [596]
      EMSA and DNase Ifootprinting 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]
Формат: djvu
Размер:8221394 байт
Язык:ENG
Рейтинг: 168 Рейтинг
Открыть: