Chromatin and Chromatin Remodeling Enzymes Part C

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Functional Analyses of Chromatin Modifications in Yeast......Page 9
Nucleosome Structure......Page 10
Using S. cerevisiae to Study Histone Function......Page 12
Yeast Histone Mutants: What We Have Learned about Histone Function From Mutational Analysis......Page 16
Histone H3......Page 21
Histone H4......Page 29
Histone H2A......Page 32
Histone H2B......Page 37
Histone Deacetylases......Page 38
Many Histone Mutations Cause Transcriptional Silencing Defects......Page 40
Protocola......Page 42
Experimental Strategies When the Human Gene Shares a Yeast Homolog......Page 43
Testing Complementation When the Yeast Homolog is Essential......Page 51
Using Functional Conservation to Explore the Cellular Function of a Protein......Page 52
Experimental Strategies When the Human Gene Lacks a Yeast Homolog......Page 53
The p53 Paradigm: How to Gain Insightful Information through Ectopic Expression in Yeast......Page 55
Genetic Resources......Page 57
Cell Biological and Biochemical Resources......Page 59
Conclusions......Page 60
Acknowledgments......Page 61
Genetic Analysis of Chromatin Remodeling Using Budding Yeast as a Model......Page 62
Genetic Selection for PHO5 Uninducible Mutants......Page 63
Screening the Collection of 4847 Viable Haploid Yeast Deletion Strains for Those That Exhibit Impaired PHO5 Expression......Page 66
Acknowledgments......Page 67
Introduction to Trx-G and Pc-G Genes......Page 68
The Sex Combs Reduced Homeotic Gene and the "Extra Sex Combs" Phenotype......Page 69
The Abdominal-B Homeotic Gene of the BXC......Page 72
The Deformed (Dfd) Homeotic Gene of the ANTC......Page 74
The Antennapedia (Antp) and Ultrabithorax (Ubx) Homeotic Genes......Page 75
The ETP Group of Homeotic Gene Regulators......Page 76
Genetic and Cytological Analysis of Drosophila Chromatin-Remodeling Factors......Page 78
Generation and Characterization of Dominant-Negative Transgenes......Page 80
Preparation of Protein Extracts From Small Quantities of Drosophila Embryos......Page 81
Eye-based Screens for Identifying Genes that Interact with Chromatin-Remodeling Factors In vivo......Page 82
Antibody Staining of Polytene Chromosomes......Page 87
Staining Polytene Chromosomes with Two Antibodies......Page 89
Using Fab Fragments to Stain Polytene Chromosomes with Two Primary Antibodies Raised in Rabbits......Page 90
Analysis of Chromosome Defects Resulting from the Loss of Chromatin-Remodeling Factor Function......Page 91
Acknowledgments......Page 93
Genetic Analysis of H1 Linker Histone Subtypes and Their Functions in Mice......Page 94
Production of Mice Lacking a Single H1 Subtype......Page 96
Methods for ES Cell Transfection and Selection......Page 99
Preparation of ES Cell DNA for Screening Clones......Page 100
Method for Preparation of Total Histones......Page 102
Compensation Among Linker Histone Subtypes in Single H1 Null Mice......Page 104
Deletion of Specific H1 Linker Histone Subtypes Affects Expression of Transgenes in Mice......Page 106
Generation of Compound H1 Knockout Mice......Page 108
Future Prospects......Page 112
Histone Modifications......Page 117
Mass Spectrometry......Page 118
Over Expression of Drosophila Histones in Bacteria......Page 120
Preparation of Bulk Histones from Drosophila Embryos......Page 121
Mass Spectrometry......Page 122
Trypsin Digestion......Page 123
Target Preparation......Page 127
Sample Preparation for Nano-ESI......Page 128
In Vitro Modification of Histones to Generate Specifically Modified Histones......Page 132
Expression of GST-GCN5......Page 134
In Vitro Modification of Peptides......Page 135
Acknowledgments......Page 136
Histone Modification Patterns During Gene Activation......Page 137
Measurement of Peptide Concentration Using Ellman’s Reagent......Page 140
Preparation of Enzymes for Testing Activity In Vitro......Page 141
HAT and HK Complex Purification......Page 142
HAT and HK Assays......Page 143
Effect of Phosphorylation on HAT Activity......Page 144
Effect of Acetylation on HK Activity......Page 146
Design and Purification of Single and Dual Modification Antibodies......Page 147
Testing Antibodies for Specificity......Page 150
Examining Global and Local Histone Modification Patterns In Vivo......Page 152
Bulk Histone Purification from Yeast......Page 153
ChIP Analysis Using Quantitative PCR in Real Time......Page 155
RT-PCR of RNA Extracted from Cells......Page 157
Acknowledgments......Page 160
Purification of Native Yeast HAT Complexes......Page 161
Assembly of Recombinant HAT Complexes......Page 165
Liquid Assay......Page 166
Gel Fluorography......Page 167
Activity Gel Assay......Page 168
Mapping Acetylation Sites......Page 169
Purification of Native Human HAT Complexes......Page 170
Generation of Cell Lines......Page 171
HAT Complex Purification......Page 173
Acknowledgments......Page 174
Histone Deacetylases: Purification of the Enzymes, Substrates, and Assay Conditions......Page 175
Generation of Recombinant Baculoviruses and Expression and Purification of HDAC2......Page 177
Purification of HDACs from Mammalian Cells......Page 178
Immunoaffinity Purification of an Endogenous HDAC3 Complex from HeLa Cells......Page 179
Expression of Epitope-Tagged HDAC5 Using Recombinant Adenoviruses and Purification of an HDAC5 Complex with Immunoaffinity Chro......Page 181
Protocols Related to Histone Deacetylase Assay......Page 183
Labeling of H4 Peptide with [3H] for Histone Deacetylase Assays......Page 184
Histone Deacetylase Assay......Page 185
Preparation and Electrophoresis of TAU Gels......Page 186
Acknowledgments......Page 187
Measurement of Mammalian Histone Deacetylase Activity......Page 188
Mammalian Cell Culture Systems......Page 190
Immunoprecipitation of HDACs from Mammalian Cell Culture Systems......Page 193
In vitro Translated Protein......Page 196
Purification of Enzymatically Active Class III HDACs......Page 197
Mammalian Cell Culture Systems......Page 198
Expression in Escherichia coli......Page 200
Enzymatic Substrates......Page 201
Procedure......Page 203
Acknowledgments......Page 204
Analysis of Histone Phosphorylation: Coupling Intracellular Signaling to Chromatin Remodeling......Page 205
Mitosis......Page 207
Activation of Gene Expression......Page 208
Programmed Cell Death......Page 209
Chromatin Immunoprecipitation......Page 210
In Gel Histone Kinase Assay......Page 212
In Vitro Kinase Assay......Page 215
GST-Pulldown Assays on Cellular Extracts......Page 216
Immunofluorescence......Page 217
Concluding Remarks......Page 220
Preparation of Histone Substrates and Assay for HMTase Activities......Page 221
Sucrose Gradient Purification of Oligonucleosomes......Page 222
Purification of Histone Octamers......Page 223
Purification of Histone Methyltransferases from Cultured HeLa Cells......Page 224
Distribution of HMTase Activities in NE and NP Fractions from HeLa Cells......Page 225
Purification of Histone H4-R3 Methyltransferase PRMT1......Page 227
Purification of Histone H4-K20 Methyltransferase SET8......Page 228
Purification of the Histone H3-K4 Methyltransferase SET7/9......Page 230
Purification of the Histone H3-K27 Methyltransferase EED-EZH2 Complex......Page 232
Acknowledgments......Page 234
Global Proteomic Analysis of S. cerevisiae (GPS) to Identify Proteins Required for Histone Modifications......Page 235
Reagents......Page 237
GPS Analysis in Identification of the Components of COMPASS and Rad6 Complex Involvement in Histone Methylation......Page 239
Potential of GPS for Defining Pathways of Posttranslational Modifications......Page 241
Acknowledgments......Page 242
Purification of Sir2 Proteins from Yeast......Page 243
Tagging Sir2p for Affinity Purification......Page 246
Method......Page 248
Mating Assays......Page 249
Deacetylase Assays......Page 250
Solutions......Page 251
Method......Page 252
Solutions......Page 253
Method......Page 254
Solutions......Page 255
Solutions......Page 256
Solutions......Page 257
Calmodulin Affinity Chromatography......Page 258
Method......Page 259
Materials......Page 260
Materials......Page 261
Solutions......Page 262
Method......Page 263
Purification and Biochemical Properties of the Drosophila TAC1 Complex......Page 264
TAC1 Complex Purification......Page 265
Preparation of Nuclear Extract......Page 266
TAC1 Fractionation Procedure by Conventional Chromatography......Page 267
Size Fractionation of TAC1......Page 270
Immunoaffinity Purification of TAC1......Page 271
Acetylation of Histones by TAC1......Page 272
Depletion Experiments......Page 273
Solutions......Page 274
Acknowledgments......Page 275
Isolation and Characterization of CHRASCH, a Polycomb-Containing Silencing Complex......Page 276
Stable Cell Lines......Page 278
Cell Staining......Page 280
Protein Purification......Page 281
Preparation of Acetylated Histone......Page 283
DNA Binding Assays......Page 285
In Vivo Studies......Page 286
Imaginal Disc Staining......Page 287
Polytene Staining......Page 289
Acknowledgments......Page 291
Biochemical Analysis of Mammalian Polycomb Group Protein Complexes and the Identification of Genetic Elements that Block Polyc......Page 292
Immunoprecipitation of Polycomb Group Proteins......Page 294
Coimmunoprecipitation Experiments......Page 296
Methods to Isolate Antirepressor Elements......Page 297
Construction of a Library of Genomic DNA Fragments......Page 298
Construction of Host Strains......Page 301
Library Screening, Transfection, and Selection Scheme......Page 302
The Strategy......Page 307
Purification with Cognate Antibodies Against Components of SWI/SNF......Page 308
Use Existing Antibodies to Purify Epitope-Tagged SWI/SNF......Page 310
Immobilizing the Antibodies......Page 312
Silver-Staining Analysis......Page 317
Coomassie-Blue Staining and Mass Spectrometry Analysis......Page 318
A Chromatin-Remodeling Assay......Page 319
Detecting Physiological Binding Targets of Mammalian SWI/SNFs Using Chromatin Immunoprecipitation (ChIP) Assay......Page 321
Acknowledgments......Page 324
Preparation of RSC......Page 325
Assay of RSC Activity......Page 327
DNA Translocation and Nucleosome Remodeling Assays by the RSC Chromatin Remodeling Complex......Page 332
Purification of TAP-RSC......Page 333
IgG-Sepharose Affinity Chromatography......Page 334
Remodelers Couple DNA-Stimulated ATPase Activity to DNA Translocation......Page 335
ATPase Assay......Page 336
Triple-Helix Displacement Assay......Page 337
Formation of the Triple-Helix Substrate......Page 338
Reconstitution of Yeast Nucleosomes Using Nap1......Page 340
Inclusion Body Purification......Page 341
Purification of the Histone Subunits......Page 342
Reconstitution of the Histone Octamer......Page 343
Purification of Nap1......Page 344
Reconstitution of Nucleosomes Using Nap1......Page 346
Purification of the Nucleosomes......Page 347
Nucleosome Binding Assay......Page 348
Endonuclease Access Assays......Page 349
DNAse I Sensitivity Assay......Page 350
Acknowledgments......Page 353
A Nucleosome Sliding Assay for Chromatin Remodeling Factors......Page 354
Generating the DNA Fragment......Page 355
Histone Sources......Page 356
Reference Remodelers: Expression of ISWI and ACF via Baculovirus Vectors......Page 357
Nucleosome Assembly and Purification......Page 358
Nucleosome Sliding Assay......Page 361
Concluding Remarks......Page 362
Acknowledgments......Page 363
Acknowledgments......Page 364
Expression and Purification of Recombinant Core Histones......Page 366
DNA Preparation......Page 367
Nucleosome Reconstitution......Page 368
Native Gel Electrophoresis......Page 369
Mapping Nucleosome Positions......Page 370
Expression and Purification of Recombinant NURF......Page 371
Nucleosome Sliding Assay......Page 373
Methods for Preparation and Assays for Xenopus ISWI Complexes......Page 375
Egg Extract Preparation......Page 376
Extract Fractionation......Page 377
ATPase Assay......Page 379
Nucleosome Mobility Assay......Page 380
Nucleosome Spacing Assay......Page 382
Nucleosome Spacing in Xenopus Oocyte Extract......Page 383
Salt Dialysis......Page 385
Functional Analysis of ISWI Complexes in Mammalian Cells......Page 387
Selection and Preparation of siRNA Oligonucleotides......Page 389
Procedure......Page 390
Preparation of Whole Cell Extracts for Western Analysis......Page 391
Fixation of Cells for Flow Cytometry......Page 392
Analysis of Subnuclear Localization of SNF2H in Combination with PCNA or BrdU Staining......Page 394
Procedure......Page 395
Analysis of Replication of Pericentric Heterochromatin in ACF1-Depleted NIH 3T3 Cells......Page 397
Acknowledgments......Page 400
Preparation and Assays for Mammalian ISWI Complexes......Page 401
Reagents......Page 402
Purification of WCRF/hACF Complex......Page 403
Protocol......Page 404
Evaluation of WCRF by ATPase Assay (Gel Electrophoresis Assay)......Page 405
Purification of the SNF2H-Cohesin Complex......Page 406
Protocol......Page 407
Human NURF......Page 408
Protocol for Preparing SNF2L Associated Complexes......Page 409
Preparation and Analysis of the INO80 Complex......Page 414
Principle......Page 416
Hardware......Page 417
Epitope-Tagging of INO80......Page 418
Making Yeast Whole Cell Extract......Page 419
Purification of INO80 Complex by FLAG-Immunoaffinity Chromatography......Page 421
In Vitro Assays for IN080 Functions......Page 422
Acknowledgments......Page 424
Assay of Z-DNA Induction by Chromatin Remodeling Factors......Page 426
Overexpression and Purification of ZaaFOK......Page 428
Analysis of ZaaFOK Activity In Vitro......Page 429
Detection of Z-DNA Structure in the Episomal CSF1 Promoter Construct in SW-13 Cells......Page 430
Detection of Z-DNA Structure in the Endogenous CSF1 Promoter in SW-13 Cells......Page 433
Immuno-Depletion and Purification Strategies to Study Chromatin-Remodeling Factors In Vitro......Page 435
General Buffers......Page 436
Generation of Antigens for Immunization and Affinity Matrices......Page 437
Protein Expression in E. Coli......Page 438
Preparation of Antigen from Pellet Fraction......Page 439
Purification of GST Fusion Proteins......Page 440
Purification of Soluble His-Tagged Fusion Proteins......Page 441
Preparation of Peptides Conjugated to Keyhole Limpet Hemocyanin......Page 442
Generation of Affinity Matrices Using Soluble Antigen......Page 443
Generation of Affinity Matrices Using Insoluble Protein......Page 444
Generation of Affinity Matrix by Direct Coupling of Purified GST-Fusion Proteins......Page 445
Generation of Affinity Matrices by Coupling Peptides......Page 446
Affinity Purification of Polyclonal Antibodies......Page 447
Protocol......Page 448
Immunodepletion of Transcription and Chromatin Remodeling Extracts......Page 449
Immunopurification of Protein Complexes from Crude Extracts or Partially Purified Fractions......Page 450
TCA Precipitation......Page 452
Illustrative Results Using Immunodepletion and Immunopurification Techniques......Page 453
Acknowledgments......Page 456
Transcription Through the Nucleosome by mRNA-Producing RNA Polymerases......Page 457
Materials and Methods......Page 459
Design and Purification of the DNA Fragments Containing the T7A1 Promoter and the Nucleosome Positioning Sequence......Page 460
Transcription Buffers......Page 461
Reconstitution and Analysis of Mononucleosomes for Transcription......Page 462
Transcription and Analysis of RNA......Page 465
Transcription in Solid Phase and Analysis of Released Templates......Page 466
Reconstitution and Purification of Hexasomes from Purified Histones......Page 470
Ligation of the 204 bp Nucleosomes or Hexasomes to the T7A1 Promoter Fragment for Use as Mobility Controls in Native P......Page 471
Acknowledgments......Page 472
Reconstitution and Transcriptional Analysis of Chromatin In Vitro......Page 473
Preparation of Recombinant and Native Histones......Page 474
Chromatin Reconstitution In Vitro......Page 476
Chromatin Assembly......Page 477
Structural Analysis of Assembled Chromatin......Page 478
Micrococcal Nuclease Digestion Assay......Page 479
Histone Modification Assay with Assembled Chromatin......Page 481
In Vitro Transcription Assay with Assembled Chromatin......Page 483
Transcription Reaction......Page 485
Techniques Used to Study Transcription on Chromatin Templates......Page 488
ATP-Dependent Chromatin Remodeling Complexes......Page 489
In Vitro Transcription Systems......Page 491
Native FACT and RSF......Page 492
Recombinant FACT......Page 495
Recombinant RSF......Page 497
Recombinant Histones......Page 498
Synthetic Histones Containing Site-Specific Modifications......Page 500
Chromatin Assembly......Page 505
Partial Micrococcal Nuclease Digestion and Southern Blotting......Page 507
Chromatin Purification......Page 509
Chromatin Transcription Derived from Nuclear Extract......Page 510
Reconstituted Transcription......Page 511
Concluding Remarks and Future Directions......Page 512
Acknowledgments......Page 513
Analysis of DNA Repair on Nucleosome Templates......Page 514
Sequence Selection......Page 516
Synthetic DNA-Based Approach......Page 517
Fragment Design......Page 518
Procedure......Page 519
DNA Damage: Effect on Nucleosome Formation......Page 520
Footprinting......Page 521
Acknowledgments......Page 522