TRANSCRIPTIONAL REGULATION IN EUKARYOTES
CONTENTS A PRIMER ON TRANSCRIPTIONAL REGULATION IN MAMMALIAN CELLS INTRODUCTION A GENERAL MODEL FOR REGULATION OF A GENE ACTIVATING A GENE, INACTIVATING A GENE, OVERVIEW CONCEPTS AND STRATEGIES: I PROMOTERS AND THE GENERAL TRANSCRIPTION MACHINERY CORE PROMOTER ARCHITECTURE THE GENERAL TRANSCRIPTION MACHINERY BASAL TRANSCRIPTION COMPLEX ASSEMBLY, CONFORMATIONAL CHANGES DURING TRANSCRIPTION COMPLEX ASSEMBLY, TAFIIS THE HOLOENZYME AND MEDIATORS DISCOVERY OF THE POL II HOLOENZYME, COMPOSITION OF THE YEAST HOLOENZYME, MAMMALIAN HOLOENZYMES, CONCEPTS AND STRATEGIES: II ACTIVATORS AND REPRESSORS REGULATORY PROMOTERS AND ENHANCERS TRANSCRIPTIONAL ACTIVATORS MODULAR ACTIVATORS, DNA-BINDING DOMAINS, ACTIVATION DOMAINS, STRUCTURAL ASPECTS OF ACTIVATION DOMAINS, REPRESSORS AND COREPRESSORS GENERAL MECHANISMS, SEQUENCE-SPECIFIC REPRESSORS, CONCEPTS AND STRATEGIES: III CHROMATIN AND GENE REGULATION CHROMATIN STRUCTURE AND ORGANIZATION, BINDING OF TRANSCRIPTION FACTORS TO CHROMATIN, GENETIC LINKS BETWEEN GENE ACTIVATION AND CHROMATIN, ATP-DEPENDENT REMODELING COMPLEXES SWI/SNF COMPLEXES, MECHANISMS AND TARGETING, ACETYLATION OF CHROMATIN MAMMALIAN ACETYLASES, TAFS AND CHROMATIN REMODELING, HISTONE DEACETYLATION, TRANSCRIPTIONAL REPRESSION, AND SILENCING REPRESSION AND DEACETYLASES, LINKING DEACETYLATION AND ATP-REMODELING MACHINES, METHYLATION AND REPRESSION, TRANSCRIPTIONAL SILENCING, LOCUS CONTROL REGIONS, INSULATORS, AND MATRIX ATTACHMENT REGIONS LOCUS CONTROL REGIONS, BOUNDARY ELEMENTS, MARS, CONCEPTS AND STRATEGIES: IV THE ENHANCEOSOME COMBINATORIAL CONTROL, COOPERATIVITY, AND SYNERGY THE ENHANCEOSOME THEORY THE INTERFERON-? ENHANCEOSOME BIOCHEMICAL MECHANISM OF ACTIVATION PERSPECTIVE INITIAL STRATEGIC ISSUES CONCEPTS AND STRATEGIES THE INITIAL STEPS IN A GENE REGULATION ANALYSIS CONSIDER THE TIME COMMITMENT AND RESOURCES NEEDED TO REACH A DEFINED GOAL TWO GENERAL STRATEGIES THAT PROVIDE PRELIMINARY ALBEIT SUPERFICIAL INSIGHT INTO TRANSCRIPTIONAL REGULATION MECHANISMS, AN EXAMPLE OF A RIGOROUS, YET INCOMPLETE GENE REGULATION ANALYSIS: THE IMMUNOGLOBULIN µ HEAVY-CHAIN GENE, DEFINING THE PROJECT GOALS, EVALUATE THE FEASIBILITY OF THE ANALYSIS APPROPRIATE SOURCE OF CELLS FOR FUNCTIONAL STUDIES, SOURCE OF CELLS FOR PROTEIN EXTRACT PREPARATION, SUCCESS IN DEVELOPING AN APPROPRIATE FUNCTIONAL ASSAY, INITIATE AN ANALYSIS OF TRANSCRIPTIONAL REGULATION BEGINNING WITH THE PROMOTER OR DISTANT CONTROL REGIONS, INITIATING AN ANALYSIS OF A PROMOTER, INITIATING AN ANALYSIS OF DISTANT CONTROL REGIONS, SUMMARY MODES OF REGULATING MRNA ABUNDANCE TRANSCRIPTION INITIATION VERSUS MRNA STABILITY BASIC MRNA DEGRADATION PATHWAYS, REGULATION OF MRNA STABILITY AND DEGRADATION, INTERRELATIONSHIP BETWEEN MRNA STABILITY AND TRANSCRIPTION INITIATION, CONFIRMING THAT THE RATE OF TRANSCRIPTION INITIATION CONTRIBUTES TO GENE REGULATION, NUCLEAR RUN-ON TRANSCRIPTION ASSAY (BOX 3 1), MEASURING MRNA STABILITIES, RECOMMENDED APPROACH FOR DEMONSTRATING REGULATION OF TRANSCRIPTION INITIATION OR MRNA STABILITY, TRANSCRIPTION ELONGATION BASIC MECHANISM OF ELONGATION, REGULATION OF TRANSCRIPTION ELONGATION IN PROKARYOTES, REGULATION OF TRANSCRIPTION ELONGATION IN EUKARYOTES, STRATEGIES FOR DISTINGUISHING BETWEEN REGULATION OF ELONGATION AND REGULATION OF INITIATION, RECOMMENDED APPROACH FOR DEMONSTRATING REGULATION OF TRANSCRIPTION INITIATION OR ELONGATION, EXTENDING AN ANALYSIS OF ELONGATION REGULATION, DIFFERENTIAL PRE-MRNA SPLICING, MRNA TRANSPORT, AND POLYADENYLATION BASIC PRINCIPLES, IDENTIFYING REGULATION OF PRE-MRNA SPLICING, TRANSPORT, AND POLYADENYLATION, TECHNIQUES PROTOCOL 3 1 NUCLEAR RUN-ON ASSAY TRANSCRIPTION INITIATION SITE MAPPING INITIAL CONSIDERATIONS REAGENTS NEEDED BEFORE PROCEEDING, INFORMATION PROVIDED BY THE DNA SEQUENCE, PRIMER EXTENSION ADVANTAGES AND DISADVANTAGES, DESIGN OF OLIGONUCLEOTIDE PRIMERS, METHOD (BOX 4 1), PRIMER ANNEALING AND REVERSE TRANSCRIPTION, ANALYSIS OF EXAMPLE DATA, RNASE PROTECTION PROBE PREPARATION, METHOD (BOX 4 2), PROBE ANNEALING AND RNASE DIGESTION, S1 NUCLEASE ANALYSIS METHOD (BOX 4 3), RAPID AMPLIFICATION OF CDNA ENDS DATA ANALYSIS, METHOD (BOX 4 4), EFFECT OF INTRONS ON THE INTERPRETATION OF START-SITE MAPPING RESULTS (BOX 4 5), PROTOCOL 4 1 PRIMER EXTENSION ASSAY PROTOCOL 4 2 RNASE PROTECTION ASSAY PROTOCOL 4 3 S1 NUCLEASE ASSAY FUNCTIONAL ASSAYS FOR PROMOTER ANALYSIS CHOOSING AN ASSAY: ADVANTAGES AND DISADVANTAGES OF EACH ASSAY TRANSIENT TRANSFECTION ASSAY, STABLE TRANSFECTION ASSAY BY INTEGRATION INTO HOST CHROMOSOME, STABLE TRANSFECTION OF EPISOMALLY MAINTAINED PLASMIDS, IN VITRO TRANSCRIPTION ASSAY, TRANSGENIC ASSAYS, HOMOLOGOUS RECOMBINATION ASSAY, TRANSIENT TRANSFECTION ASSAYS CELLS, TRANSFECTION PROCEDURES (BOX 5 1), REPORTER GENES, VECTORS, AND ASSAYS (BOXES 5 2, 5 3, 5 4), PLASMID CONSTRUCTION, INITIAL TRANSFECTION EXPERIMENTS, ASSESSING APPROPRIATE PROMOTER REGULATION (BOXES 5 5, 5 6), STABLE TRANSFECTION ASSAYS BY CHROMOSOMAL INTEGRATION GENERAL STRATEGIES, CELLS AND TRANSFECTION PROCEDURES, REPORTER GENES AND ASSAYS, DRUG-RESISTANCE GENES AND VECTORS, DRUG SELECTION, CONTROLS AND INTERPRETATION OF RESULTS, COMMON TRANSFECTION METHODS FOR MAMMALIAN CELLS PROTOCOL 5 1 CALCIUM PHOSPHATE TRANSFECTION OF 3T3 FIBROBLASTS PROTOCOL 5 2 DEAE-DEXTRAN TRANSFECTION OF LYMPHOCYTE CELL LINES PROTOCOL 5 3 TRANSFECTION BY ELECTROPORATION OF RAW264 7 MACROPHAGES COMMON REPORTER ENZYME ASSAYS PROTOCOL 5 4 LUCIFERASE ASSAY PROTOCOL 5 5 CHLORAMPHENICOL ACETYLTRANSFERASE ASSAY PROTOCOL 5 6 ?-GALACTOSIDASE ASSAY IDENTIFICATION AND ANALYSIS OF DISTANT CONTROL REGIONS DNASE I HYPERSENSITIVITY BASIC PRINCIPLES OF DNASE I SENSITIVITY AND HYPERSENSITIVITY, ADVANTAGES AND DISADVANTAGES OF USING DNASE I HYPERSENSITIVITY TO IDENTIFY CONTROL REGIONS, DNASE I HYPERSENSITIVITY ASSAY (BOX 6 1), DATA INTERPRETATION, IDENTIFICATION OF MATRIX ATTACHMENT REGIONS BASIC PRINCIPLES OF THE NUCLEAR MATRIX AND OF MARS AND SARS, ADVANTAGES AND DISADVANTAGES OF USING MARS TO IDENTIFY DISTANT CONTROL REGIONS, METHODS FOR IDENTIFYING MARS (BOX 6 2), FUNCTIONAL APPROACHES FOR THE IDENTIFICATION OF DISTANT CONTROL REGIONS BASIC ADVANTAGES AND DISADVANTAGES OF FUNCTIONAL APPROACHES, FUNCTIONAL APPROACH BEGINNING WITH A LARGE GENOMIC DNA FRAGMENT, FUNCTIONAL APPROACH BEGINNING WITH SMALLER FRAGMENTS DIRECTING EXPRESSION OF A REPORTER GENE, FUNCTIONAL ASSAYS FOR THE CHARACTERIZATION OF DISTANT CONTROL REGIONS TRANSIENT TRANSFECTION ASSAYS, STABLE TRANSFECTION ASSAYS, DEMONSTRATION OF LCR ACTIVITY, DEMONSTRATION OF SILENCER ACTIVITY, DEMONSTRATION OF INSULATOR ACTIVITY, IDENTIFYING CIS-ACTING DNA ELEMENTS WITHIN A CONTROL REGION IDENTIFICATION OF CONTROL ELEMENTS BY COMPREHENSIVE MUTANT ANALYSIS RATIONALE FOR A COMPREHENSIVE ANALYSIS, THE IG µ GENE EXAMPLE, DISADVANTAGES OF USING MUTAGENESIS TO IDENTIFY CONTROL ELEMENTS, STRATEGIES FOR A COMPREHENSIVE ANALYSIS, METHODOLOGY FOR MUTATING A CONTROL REGION, IDENTIFICATION OF CONTROL ELEMENTS USING IN VIVO OR IN VITRO PROTEIN–DNA INTERACTION METHODS IDENTIFICATION OF CONTROL ELEMENTS BY DATABASE ANALYSIS MUTAGENESIS TECHNIQUES (BOXES 7 1–7 6) IDENTIFICATION OF DNA-BINDING PROTEINS AND ISOLATION OF THEIR GENES CONCEPTS AND STRATEGIES FOR THE IDENTIFICATION OF DNA-BINDING PROTEINS DATABASE METHODS DEVELOPMENT OF A PROTEIN-DNA INTERACTION ASSAY FOR CRUDE CELL LYSATES STANDARD METHODS FOR DETECTING PROTEIN–DNA INTERACTIONS, ELECTROPHORETIC MOBILITY SHIFT ASSAY (BOX 8 1), DNASE I FOOTPRINTING, CONCEPTS AND STRATEGIES FOR CLONING GENES ENCODING DNA-BINDING PROTEINS CLONING BY PROTEIN PURIFICATION AND PEPTIDE SEQUENCE ANALYSIS (BOX 8 2) AMOUNT OF STARTING MATERIAL, CONVENTIONAL CHROMATOGRAPHY STEPS, DNA AFFINITY CHROMATOGRAPHY, IDENTIFICATION OF THE RELEVANT BAND FOLLOWING SDS-PAGE (BOX 8 3), AMINO ACID SEQUENCE ANALYSIS AND GENE CLONING, CONFIRMATION THAT THE GENE ISOLATED ENCODES THE DNA-BINDING ACTIVITY OF INTEREST, CLONING BY METHODS THAT DO NOT REQUIRE AN INITIAL PROTEIN–DNA INTERACTION ASSAY ONE-HYBRID SCREEN, IN VITRO EXPRESSION LIBRARY SCREENING WITH DNA OR ANTIBODY PROBES, MAMMALIAN EXPRESSION CLONING METHODS, GENOME DATABASE METHODS AND DEGENERATE PCR, CONFIRMING THE FUNCTIONAL IMPORTANCE OF A PROTEIN–DNA INTERACTION 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 EXPERIMENTS IN VIVO ANALYSIS OF AN ENDOGENOUS CONTROL REGION IN VIVO ANALYSIS OF SEQUENCE-SPECIFIC PROTEIN-DNA INTERACTIONS DNASE I AND DMS GENOMIC FOOTPRINTING (BOX 10 1), IN VIVO PROTEIN–DNA CROSSLINKING/IMMUNOPRECIPITATION, NUCLEOSOME POSITIONING AND REMODELING MODEL SYSTEMS, LOW-RESOLUTION ANALYSIS OF NUCLEOSOME POSITIONING BY THE MNASE-SOUTHERN BLOT METHOD (BOX 10 2), HIGH-RESOLUTION ANALYSIS OF NUCLEOSOME POSITIONING BY AN MNASE-LM-PCR METHOD AND DNASE I GENOMIC FOOTPRINTING (BOX 10 3), IN VIVO METHODS FOR ANALYZING NUCLEOSOME REMODELING (BOX 10 4), DNA METHYLATION SUBNUCLEAR LOCALIZATION OF A GENE PROTOCOL 10 1 MNASE-SOUTHERN BLOT ASSAY PROTOCOL 10 2 LM-PCR METHODS DNASE GENOMIC FOOTPRINTING, MNASE MAPPING OF NUCLEOSOME POSITIONING, RESTRICTION ENZYME ACCESSIBILITY TO MONITOR NUCLEOSOME REMODELING, DMS GENOMIC FOOTPRINTING, APPROACHES FOR THE SYNTHESIS OF RECOMBINANT TRANSCRIPTION FACTORS PROKARYOTIC EXPRESSION SYSTEMS (BOXES 11 1 AND 11 2) STRATEGIES FOR OVERCOMING EXPRESSION PROBLEMS IN E COLI SYNTHESIZING LARGE REGULATORY PROTEINS YEAST SYSTEMS (BOX 11 3), BACULOVIRUS SYSTEM (BOX 11 4), VACCINIA VIRUS (BOX 11 5), RETROVIRAL EXPRESSION SYSTEMS (BOX 11 6), SYNTHESIZING SMALL QUANTITIES OF CRUDE PROTEIN SPECIALIZED INDUCIBLE EXPRESSION SYSTEMS (BOX 11 7), IN VITRO TRANSCRIPTION/TRANSLATION SYSTEMS (BOX 11 8), MAMMALIAN EXPRESSION VECTORS (BOX 11 9), SYNTHESIS AND PURIFICATION OF MACROMOLECULAR COMPLEXES CHOOSING AN APPROPRIATE SYSTEM IDENTIFYING AND CHARACTERIZING TRANSCRIPTION FACTOR DOMAINS CONCEPTS AND STRATEGIES: DEFINING DOMAINS BASIC MUTAGENESIS PRINCIPLES DOMAINS OF A GENE ACTIVATOR SEPARATING DNA-BINDING AND ACTIVATION DOMAINS OF AN ACTIVATOR GENERAL CONSIDERATIONS, DNA BINDING, ACTIVATION (BOX 12 1), LIMITATIONS OF THE DOMAIN SWAP, SUBDIVIDING DNA RECOGNITION AND OLIGOMERIZATION SUBDOMAINS (BOX 12 2) CONCEPTS AND STRATEGIES: PROTEIN–PROTEIN INTERACTIONS INTERACTION OF ACTIVATION DOMAINS WITH COACTIVATORS AND GENERAL FACTORS AFFINITY CHROMATOGRAPHY PRINCIPLES, CAVEATS OF THE AFFINITY APPROACH, ALTERED SPECIFICITY GENETIC SYSTEMS STRUCTURE–FUNCTION ANALYSIS OF THE GENERAL TRANSCRIPTIONAL MACHINERY PROTOCOL 12 1 PCR-MEDIATED SITE-DIRECTED MUTAGENESIS THEORY, CHARACTERIZATION, AND MODELING OF DNA BINDING BY REGULATORY TRANSCRIPTION FACTORS GENERAL THEORY AND EXAMPLES OF DNA–PROTEIN INTERACTIONS THEORY OF DNA RECOGNITION, CHEMICAL BASIS OF THE INTERACTIONS, THE ROLE OF THE ?-HELIX IN DNA RECOGNITION, MAJOR AND MINOR GROOVE SPECIFICITY, MONOMERS AND DIMERS; ENERGETIC AND REGULATORY CONSIDERATIONS, DISSOCIATION CONSTANT ANALYSIS (BOX 13 1), KD DETERMINATION, ANALYSIS AND MODELING OF DNA–PROTEIN INTERACTIONS IDENTIFICATION OF A HIGH-AFFINITY DNA RECOGNITION SITE, BASIC THEORY, GENERAL METHODS (BOXES 13 2 AND 13 3), MINOR GROOVE/DNA BACKBONE PROBES (BOX 13 4), MAJOR GROOVE PROBES, MODELING DNA–PROTEIN INTERACTIONS, ANALYSIS OF PROMOTER-SPECIFIC MULTICOMPONENT NUCLEOPROTEIN COMPLEXES DNA BINDING COOPERATIVITY, DNA LOOPING AND BENDING, MECHANISMS OF DNA BENDING, APPROACHES FOR STUDYING BENDING, 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 CRUDE AND FRACTIONATED SYSTEMS FOR IN VITRO TRANSCRIPTION PREPARATION OF EXTRACTS CELL CHOICE, EXTRACT PREPARATION METHOD, TRANSCRIPTION ASSAYS GENERAL CONSIDERATIONS (BOX 14 1), CHOICE OF TEMPLATE, CHROMATIN SYSTEMS, OPTIMIZATION OF CONDITIONS, FRACTIONATED SYSTEMS (BOX 14 2) HOLOENZYME, MEDIATOR SUBCOMPLEXES, PARTIALLY FRACTIONATED SYSTEMS, FACTOR-DEPLETED SYSTEMS, HIGHLY FRACTIONATED SYSTEMS, PREPARATION OF NUCLEAR AND WHOLE-CELL EXTRACTS PROTOCOL 14 1 THE DIGNAM AND ROEDER NUCLEAR EXTRACT PROTOCOL 14 2 PREPARATION OF NUCLEAR EXTRACTS FROM RAT LIVER PROTOCOL 14 3 PREPARATION OF WHOLE-CELL EXTRACT IN VITRO TRANSCRIPTION ASSAYS PROTOCOL 14 4 IN VITRO TRANSCRIPTION USING HELA CELL EXTRACTS AND PRIMER EXTENSION PROTOCOL 14 5 G-LESS CASSETTE IN VITRO TRANSCRIPTION USING HELA CELL NUCLEAR EXTRACTS TRANSCRIPTION FACTOR PURIFICATION PROTOCOL 14 6 PREPARATION OF A CRUDE FRACTIONATED SYSTEM PROTOCOL 14 7 PURIFICATION OF RECOMBINANT TFIIB FROM E COLI PROTOCOL 14 8 PURIFICATION OF RECOMBINANT TFIIA PROTOCOL 14 9 AFFINITY PURIFICATION OF RNA POL II PROTOCOL 14 10 PURIFICATION OF EPITOPE-TAGGED TFIID APPROACHES FOR STUDYING TRANSCRIPTION COMPLEX ASSEMBLY FORMATION OF THE BASAL PREINITIATION COMPLEX KINETIC STUDIES, SARKOSYL PROBING, TEMPLATE COMMITMENT EXPERIMENT, DNASE I FOOTPRINTING AND EMSA STUDIES OF TRANSCRIPTION COMPLEX ASSEMBLY, PHOTOCROSSLINKING, STRUCTURE–FUNCTION ANALYSES OF THE GENERAL MACHINERY, OPEN COMPLEX FORMATION, INITIATION, AND PROMOTER ESCAPE ATP-ANALOGS AND AN ENERGY-DEPENDENT STEP, PERMANGANATE PROBING, PREMELTED TEMPLATES, THE TRANSITION TO ELONGATION, ASSEMBLY OF ACTIVATED COMPLEXES AT A PROMOTER THE IMMOBILIZED TEMPLATE APPROACH, GEL FILTRATION, PERMANGANATE PROBING TO STUDY ACTIVATION, EMSA AND DNASE I FOOTPRINTING ANALYSES OF THE TFIID–TFIIA COMPLEX, ASSEMBLY AND ANALYSIS OF TFIID SUBCOMPLEXES, FUTURE DIRECTIONS, PROTOCOL 15 1 POTASSIUM PERMANGANATE PROBING OF POL II OPEN COMPLEXES PROTOCOL 15 2 MAGNESIUM-AGAROSE EMSA OF TFIID BINDING TO DNA