Chromatin Remodeling: Methods and Protocols

Cover......Page 1
Frontmatter......Page 4
Preface......Page 6
Contents......Page 8
Contributors......Page 10
1. Introduction......Page 14
2.1. Construct the Reporter Strain......Page 16
2.2. Prepare Histone Mutants for Integration......Page 17
2.4. Confirm Correct Integration......Page 18
3. Methods......Page 19
3.1. Construct the Reporter Strain......Page 20
3.2. Prepare Histone Mutants for Integration......Page 21
3.3. Integrate the Mutants into the Reporter Strain......Page 22
3.4. Confirm Correct Integration......Page 23
4. Notes......Page 25
References......Page 27
1. Introduction......Page 28
2.1. Media......Page 30
2.2. Reagents, Solutions, and Other Materials......Page 31
3.1. Cell Culture and Transcription Induction......Page 32
3.3. ChIP and Reversal of Cross-Linking......Page 34
3.4. PCR Analysis of Precipitated DNA......Page 36
4. Notes......Page 37
References......Page 40
1. Introduction......Page 42
2.1. Chromatin Immunoprecipitation......Page 44
2.3. Chromosome Conformation Capture (3C)......Page 45
3.1. ChIP (Results Are Shown in Fig.  1)......Page 47
3.2. DNase I Sensitivity by RT-qPCR (Results Are Shown in Fig.  2)......Page 51
3.3. Chromosome Conformation Capture (3C) (Results Are Shown in Fig.  3)......Page 53
4. Notes......Page 56
References......Page 58
1. Introduction......Page 60
2.1. Cell Growth and Harvest......Page 61
2.2. Micrococcal Nuclease Digest......Page 62
2.3. DNA Measurement......Page 63
3.1. Cell Culture and Harvest......Page 64
3.3. DNA Measurement......Page 65
3.4. Data Analysis......Page 66
4. Notes......Page 69
References......Page 74
1. Introduction......Page 76
2.3. Micrococcal Nuclease Digestion......Page 80
2.5. Quantitative Real-Time PCR Analysis of Isolated DNA......Page 81
3.1. Harvesting Cells for NuSA......Page 82
3.4. Protein Degradation and DNA Purification......Page 83
3.6. Data Analysis and Interpretation ( see Note 7)......Page 84
3.7. Chromatin Immunoprecipitation-NuSA Protocol ( see Note 9)......Page 85
4. Notes......Page 87
References......Page 99
1. Introduction......Page 102
2.1. Cell Culture......Page 103
2.4. In Vitro Redigestion and Purification of DNA......Page 105
3. Methods......Page 106
3.2. Isolation of Nuclei......Page 107
3.4. In Vitro Redigestion and Purification of DNA......Page 108
3.6. Reiterative Primer Extension......Page 109
3.7. Analysis of Polymerase Extension Products......Page 110
3.8. Concluding Remarks......Page 111
4. Notes......Page 113
References......Page 114
1. Introduction......Page 116
2.1.2. Recombinase Target Sites and Excision Reactions......Page 118
2.2. Generating Chromosomal Recombination Cassettes......Page 119
2.3. Generating a Recombination-Proficient Strain......Page 120
2.4.2. Reagents to Isolate DNA by Glass Beads Lysis......Page 121
3.2. Isolation of DNA for Analysis by Restriction Digestion......Page 122
4. Notes......Page 123
References......Page 125
1. Introduction......Page 128
2.2. Preparation of Whole Cell Extract......Page 129
2.3. Coupling Anti-FLAG M2 Antibody with Magnetic Beads......Page 130
3.2. Preparation of Whole Cell Extract......Page 131
3.4. Purification of TALO8 from Cell Extract......Page 132
4. Notes......Page 134
References......Page 136
1. Introduction......Page 138
2. Materials......Page 141
2.1. Nuclei Isolation......Page 143
2.3. Probing Chromatin......Page 144
2.5. Amplification and Cloning......Page 145
3.1. Nuclei Isolation......Page 146
3.2. Chromatin Reconstitution and Remodeling......Page 147
3.3. Probing Chromatin......Page 148
3.5. Amplification and Cloning ( see Note 10)......Page 149
3.6. Mapping Methylation in Sequences......Page 150
4. Notes......Page 151
References......Page 153
1. Introduction......Page 156
2.2. Affinity Purification......Page 158
3. Methods......Page 159
3.1. Cell Culture and Cryogenic Lysis......Page 160
3.2. Affinity Purification......Page 161
4. Notes......Page 163
References......Page 165
1. Introduction......Page 166
2.4. Software......Page 168
3.1.1. Set Up Time-Lapse Imaging......Page 169
3.1.2. Set Up the Intentional Photobleach......Page 172
3.1.4. Process the FRAP Data......Page 173
3.1.5. Compare FRAP Curves Under Different Biological Conditions......Page 174
3.2.2. Semi-quantitative Measurements......Page 175
3.3. Protocol for Quantitative FRAP......Page 177
3.3.2. Process and Fit the FRAP Data......Page 178
4. Notes......Page 183
References......Page 189
1.1. Mobility of Nuclear Proteins......Page 190
1.2. Fluorescence Correlation Spectroscopy and Temporal Image Correlation Spectroscopy in the Nucleus......Page 191
2.1. Sample Preparation for FCS and TICS......Page 195
2.2.2. TICS......Page 196
3.1.1. Calibration of the FCS Measurement Volume......Page 197
3.1.2. Acquisition of FCS Data on Living Cells......Page 198
3.2. Data Acquisition and Analysis for TICS......Page 200
3.2.2. Confirming/Refining the TICS Measurement Volume......Page 201
3.2.3. Acquisition of TICS Data in Live Cells......Page 202
3.3. Quantitative Analysis of FCS/TICS Data......Page 203
4. Notes......Page 205
References......Page 212
1. Introduction......Page 214
2.1.1. Isolation of Nuclei and Mononucleosome Preparation......Page 216
2.3.1. Isolation and Fragmentation of Chromatin......Page 217
3.1.1. Isolation of Nuclei......Page 219
3.1.2. Mononucleosome Preparation (Micrococcal Nuclease Digestion and Purification)......Page 220
3.2. Analysis of Nucleosome......Page 221
3.2.1. Analysis of Nucleosome Using QPCR......Page 223
3.2.2. Nucleosome Border Mapping with PAGE......Page 224
3.3. Deciphering Histone Modifications and Protein Interactions on Chromatin Using Chromatin Immunoprecipitation in Plants......Page 226
3.3.1. Isolation and Fragmentation of Chromatin......Page 228
3.3.2. Immunoprecipitation......Page 230
3.3.3. Collection of Complexes Bound to Antibody and DNA Purification......Page 231
3.3.4. Analysis of the Precipitated DNA Using QPCR......Page 232
4. Notes......Page 233
References......Page 235
1. Introduction......Page 238
2.2. Analysis of Histones......Page 240
3.1. Acid Extraction of Histones......Page 241
3.2. Analysis of Histones Using Acid Urea Gel Electrophoresis......Page 243
3.3. Fractionation of Histones by Reverse-Phase-HPLC......Page 245
4. Notes......Page 247
References......Page 248
1. Introduction......Page 250
2.2. Installing Methyl-Lysine Analog onto Histones......Page 252
2.3. Preparation of Radioactive-Labeled Nucleosomes for EMSA and Sliding......Page 253
2.5. Histone Deacetylase Assays......Page 254
3.1. Preparation of Recombinant Histone Octamers......Page 255
3.2. Installing Methyl-Lysine Analog to Histones......Page 256
3.3. Preparation of Radioactive Nucleosomes for EMSA and Sliding Assays......Page 257
3.4. A Large-Scale Preparation of Modified Nucleosomes......Page 259
3.5. Histone Deacetylase Assays Using Nucleosomal Templates......Page 263
4. Notes......Page 264
References......Page 266
1. Introduction......Page 268
2.1.1. Preparation of DNA......Page 269
2.2.3. Control of Histone Stoichiometry......Page 270
3.1.1. Preparation of DNA......Page 271
3.1.2. Radioactive End-Labeling of the HindIII-End......Page 272
3.2. Quality Controls of Nucleosomal Arrays......Page 273
3.2.1. Electrophoretic Mobility Shift Assays......Page 274
3.3.1. Chromatin Accessibility Assay......Page 275
3.3.2. Chromatin Footprint Assay Using Micrococcal Nuclease......Page 276
3.3.3. Nucleosome Repositioning Assay Using Primer Extension......Page 277
4. Notes......Page 279
References......Page 283
1. Introduction......Page 284
2.2. Chromatin Assembly by Salt Gradient Dialysis......Page 287
2.4. Chromatin Digestion with DNaseI, MNase, or Restriction Enzymes......Page 288
3.1. Preparation of Yeast WCE......Page 289
3.2.2. Dialysis Mini Chamber......Page 291
3.2.4. Salt Gradient Dialysis......Page 292
3.4. Chromatin Digestion with DNaseI, MNase, or Restriction Enzymes......Page 293
4. Notes......Page 294
References......Page 299
1. Introduction......Page 302
2.1. The HTLV-1 DNA Template......Page 304
2.2. Nucleosomal Array Reconstitution and Biophysical Characterization......Page 306
2.4. HTLV-1 Nucleosomal Array Folding and Oligomerization Assays......Page 307
2.6. In Vitro Transcription......Page 308
3. Methods......Page 310
3.1. Purification of the HTLV-1 DNA Template......Page 311
3.2. Reconstitution of HTLV-1 Nucleosomal Arrays and Determination of Nucleosome Saturation......Page 312
3.4. Chromatin Folding and Oligomerization Assays......Page 314
3.5. Preparation of Nuclear Extract from CEM Cells......Page 315
3.6. In Vitro Transcription......Page 317
4. Notes......Page 319
References......Page 321
1. Introduction......Page 324
2.2. For Nucleosome Assembly......Page 326
2.3. For Remodeling Reactions and Nucleosome Mapping......Page 327
3.1. Choice of Region to Analyze, and Preparation of Template and Probe DNAs......Page 329
3.2. Polynucleosome Assembly......Page 330
3.3. MNase Footprint/Restriction Enzyme Mapping, and Southern Blotting......Page 332
3.4. Mapping and Quantitation of Nucleosome Positions......Page 338
4. Notes......Page 344
References......Page 349
1. Introduction......Page 350
2.1. Site-Specific Labeling of Histones with Fluorescent Dyes......Page 352
2.3. Large-Scale PCR Preparation of Fluorescently Labeled DNA......Page 353
3.1. Site-Specific Labeling of Histones with Fluorescent Dyes......Page 354
3.2. Preparation of Histone Octamer from Purified Core Histones......Page 355
3.3. Large-Scale PCR Preparation of Fluorescently Labeled DNA......Page 357
3.4. Measurement of Remodeling End Points......Page 359
3.5. Measurement of Remodeling Kinetics......Page 360
4. Notes......Page 361
References......Page 362
1. Introduction......Page 364
2.1.1. DNA Preparation......Page 366
2.1.5. H2A–H2A Intranucleosome Cross-Linking......Page 367
2.3. Nucleosome Remodeling......Page 368
3.1.1. DNA Preparation......Page 369
3.1.2. Core Histone Protein Preparation......Page 370
3.1.4. Nucleosome Gradient Purification......Page 371
 Centrifugation Method......Page 372
 H2A–H2A Intranucleosome Cross-Linking......Page 373
3.1.6. Nucleosome Labeling and Purification......Page 374
3.2. Preparing H2A–H2A Cross-Linked Nucleosomes with DNA Containing a Unique Radioactive End Label......Page 376
3.2.1. DNA Fragment Preparation and Labeling......Page 377
3.2.2. Nucleosome Reconstitution......Page 378
3.3.1. Restriction Enzyme Assay......Page 379
3.3.2. Nucleosome Mobility Assay......Page 380
4. Notes......Page 381
References......Page 383
1. Introduction......Page 386
2.1. Preparation of Reduced Histone Tetramers......Page 388
3.1. Preparation of Reduced H3 K115C Tetramers......Page 389
3.2. Targeted Cross-Linking as a Probe of Tetramer Structure......Page 391
3.3. Targeted Cross-Linking as a Method of Tetramer Stabilisation......Page 394
4. Notes......Page 398
References......Page 399
1. Introduction......Page 402
2.1. Nucleosome Isolation......Page 403
2.3.2. Library Preparation......Page 404
3.1. Growth of Cultures and Nucleosome Isolation......Page 406
3.2.1. Sample Labelling......Page 408
3.3.1. Sequencing: Quality Control #1 (Prior to Library Preparation)......Page 410
3.3.2. Library Preparation......Page 411
3.3.4. Sequencing: Quality Control #3 (Library Quantification with Quantitative PCR)......Page 417
4. Notes......Page 421
References......Page 424
1. Introduction......Page 426
2.1. Preparation of Nucleosome DNA Templates......Page 427
3.1. Preparation of Nucleosome DNA Templates......Page 428
3.2. Preparation of DNA for Sequencing......Page 429
3.3. Sequencing and Data Analysis......Page 430
4. Notes......Page 431
References......Page 432
1. Introduction......Page 434
2.2. Chromatin Extraction and DNA Isolation......Page 437
3.1. Preparation of Nuclei and MNase Digestion......Page 438
3.2. Salt Fractionation and DNA Isolation......Page 439
3.3.1. Microarray Analysis......Page 441
4. Notes......Page 442
References......Page 444
1. Introduction......Page 446
2. Data and Sources of Bias......Page 448
3.1. Building a Tag Density Profile for Data Visualization in a Browser......Page 449
3.2.2. Peak Detection......Page 450
3.3. Digital Footprinting Analysis......Page 451
4. Software......Page 452
5. Notes......Page 453
References......Page 454
1. Introduction......Page 456
2.2. Estimating Characteristic DNA Fragment Length......Page 458
2.3. Calculating Nucleosome Occupancy......Page 459
2.4. Estimating Nucleosome Center Positions......Page 460
3. Notes......Page 461
References......Page 462
Index\r......Page 464