Chromatin and Chromatin Remodeling Enzymes

30.pdf......Page 0
Considerations......Page 9
Comparison of Search Strategies......Page 13
H2A......Page 16
H3......Page 25
H4......Page 26
Cloning Strategy......Page 27
Large-Scale Plasmid Purification......Page 31
Histone Expression and Purification......Page 35
Inclusion Body Preparation......Page 37
Refolding of Histone Octamer......Page 38
Histone Octamer Refolding......Page 39
Reconstitution of Nucleosome Core Particles......Page 41
Microscale reconstitution......Page 43
Large-Scale Reconstitution......Page 44
High-Resolution Gel Shift and Heat Shifting of NCPs......Page 45
Purification of NCP by Preparative Gel Electrophoresis......Page 46
Acknowledgment......Page 48
Structural Biology and Chromatin Studies......Page 49
Materials and Instrumentation......Page 50
Preparation of Soluble Chromatin......Page 53
Chromatography to Extract Histones and Purify Octamer......Page 55
Production of Recombinant Histones......Page 57
Plasmid Construction......Page 58
Production and Isolation of Half-Palindrome DNA Plasmid......Page 59
Isolation of Half-Palindrome from Plasmid......Page 60
Nucleosome Reconstitution......Page 62
Crystallization of NCP......Page 64
Acknowledgments......Page 67
Introduction......Page 68
Histone H3 Ligation Strategy and Design......Page 69
Protected N-Terminal H3 Peptide Synthesis......Page 72
Cleavage of Side Chain-Protected N-Terminal H3 Peptide......Page 73
Thioesterification of N-Terminal H3 Peptide......Page 74
C-Terminal Histone H3 Fragment Synthesis and Purification......Page 76
C-Terminal Histone H3 Fragment Expression and Purification......Page 77
Factor Xa Cleavage and Purification of C-Terminal Histone H3 Fragment......Page 78
Histone H3 Ligation......Page 79
Concluding Remarks......Page 82
Background......Page 83
Preparation of Histones from Tissues, Cultured Cells, and Yeast......Page 84
Extraction of Histones from Nuclear Suspensions......Page 85
Sample Storage, Concentration, and Preparation for Gel Analysis......Page 86
AUT First-Dimension Gels......Page 88
AUC Second-Dimension Gels......Page 90
Immunoblotting......Page 93
UVA and Laser-Guided DNA Double Strand Break Formation......Page 94
Introduction......Page 96
Reconstitution of Recombinant Histone Octamer......Page 97
Expression of Recombinant Histones and Purification......Page 98
Reconstitution of Histone Octamer......Page 99
Reconstitution of Recombinant Nucleosomes......Page 100
Reconstitution of Nucleosomes by Salt Dialysis......Page 101
Purification of Yeast ISWI Complexes......Page 102
Whole Cell Extract Preparation......Page 103
Ion-Exchange Chromatography......Page 104
Purification of Overexpressed Isw2-Itc1 Complex......Page 105
ATPase Assay for ISWI Complex Activity......Page 107
Assembly of Regularly Spaced Nucleosome Arrays......Page 108
Acknowledgments......Page 110
Introduction......Page 111
b-1,3-Glucanase Production......Page 112
Yeast Core Histone Purification......Page 114
Expression......Page 118
Purification......Page 119
One-Dimensional Topological Analysis......Page 121
Two-Dimensional Topological Assay......Page 122
Micrococcal Nuclease Digestion Analysis of Quality of Reconstituted Chromatin......Page 123
General Comments......Page 124
Acknowledgments......Page 125
Introduction......Page 126
High-Speed Egg Extract Preparation and Immunodepletion......Page 128
In Vitro Nucleosome Assembly Assays......Page 132
Control Experiments for Nucleosome Assembly with Depleted HSE Analyzed by Supercoiling Assays......Page 136
Conclusions and Perspectives......Page 139
Acknowledgments......Page 140
Introduction......Page 141
Assembly of Mono- and Dinucleosome Templates by Salt Gradient Dialysis......Page 143
Protocols......Page 144
Gel Shift Analysis of Protein Binding to Mono- and Dinucleosomes......Page 148
Analysis of Transcription Factor Binding, Using DNase I Footprinting......Page 149
Protocol......Page 150
Protocol......Page 152
Protocol......Page 153
Assembly of Nucleosome Arrays......Page 155
Protocols......Page 156
Gel Shift Analysis of Protein Binding to Nucleosome Arrays......Page 158
Preparation of DNA for Nucleosome Arrays Containing a Unique, Internally Labeled Site......Page 159
Protocol......Page 162
Hypersensitive Site DNase Analysis of Transcription Factor Binding to Nucleosome Arrays......Page 163
Generating Histone H1-Compacted Nucleosome Arrays and Assessing Transcription Factor-Mediated Chromatin Opening......Page 164
Protocol for Chromatosome Assay......Page 165
Protocol for Restriction Enzyme Accessibility Assay......Page 167
Acknowledgments......Page 168
Introduction......Page 169
lac Repressor......Page 171
pALT......Page 173
Yeast Transformation, Culture, and Extract Preparation......Page 174
Minichromosome Purification......Page 177
Analysis of Minichromosomes......Page 178
Acknowledgments......Page 181
Introduction......Page 182
Genetic Manipulations......Page 183
Plasmids and Yeast Strains......Page 185
Cell Growth and Cell Disruption......Page 186
Extraction and Differential Centrifugation......Page 187
Affinity Purification......Page 188
Comments......Page 190
Introduction......Page 191
Construction of Histone Cysteine-Substitution Mutant Coding Sequences......Page 192
Preparation of Recombinant Histones: A New Method for Fast Purification of Recombinant H3/H4 Tetramers......Page 194
Construction of Dinucleosome DNA Template......Page 199
Preparation of Model Dinucleosomes......Page 201
UV-Induced Cross-Linking and Identification of Cross-Linking Bands......Page 203
Introduction......Page 206
Identifying Histone Residues for Cysteine Substitutions Suitable for Protein-DNA Cross-Linking......Page 208
Histone Overexpression and Purification and Octamer Refolding......Page 210
Nucleosome Reconstitutions......Page 211
Modification with Azido Phenacyl Bromide (APB)......Page 212
UV Irradiation and Analysis of Cross-Linking Efficiency......Page 213
Separation of Cross-linked and Un-Cross-Linked DNA......Page 214
Heat- and Alkali-Induced DNA Cleavage and Determination of Cross-Linked Nucleotide......Page 215
Using Site-Directed Histone-DNA Mapping to Characterize Remodeled Nucleosomal State......Page 216
Mapping of Dyad Axis Positions in Remodeled Nucleosomes with H4 C47 Reveals Displacement of Histone Octamer beyond DNA Ends......Page 217
Correlation of Remodeled Nucleosome Dyad Axis and H2A-H2B Dimer-DNA Contacts Reveals an Altered Nucleosomal Conform......Page 218
Histone-DNA Contact Mapping of Gel-Purified Remodeled Nucleosomal Conformations......Page 222
Rapid Kinetic Analysis of Remodeling Reaction Using Site-Directed Histone-DNA Contact Mapping......Page 223
Introduction......Page 224
Background......Page 225
Histone Preparation......Page 226
Preparative PCR Method......Page 227
Background......Page 229
Improved Reaction Conditions......Page 230
Background......Page 231
Nucleosome Reconstitution......Page 233
FRET Strategy......Page 234
Fluorescence Labeling of Recombinant Histone Octamer......Page 235
Thermally Induced Nucleosome Sliding......Page 237
Gel Mobility Shift Assay......Page 238
Dimer Exchange......Page 239
Introduction......Page 242
Method......Page 243
Method......Page 244
Method......Page 246
Method......Page 247
Method......Page 248
Confirmation of Reconstitution......Page 249
Method......Page 251
Introduction......Page 253
Mouse Blastocyst Attachment, Outgrowth, and Differentiation In Vitro......Page 254
Isolation of Total RNA......Page 255
RT-PCR Assay for H2A.Z and H2A......Page 256
Immunostaining of Embryos......Page 258
Notes......Page 259
Xenopus as a Model System for Early Development......Page 260
Synthetic mRNA Preparation......Page 261
Microinjection of H2A.Z-EGFP into Xenopus Oocytes and Embryos......Page 262
Isolation and Fluorescence Analysis of Minichromosomes from Oocytes......Page 264
Acknowledgments......Page 266
Introduction......Page 267
Preparation of 0.6 M NaCl Nuclear Extract and Nuclear Pellet......Page 268
First HPLC......Page 269
SDS-PAGE......Page 270
Preparation of Apparatus for Electroelution......Page 274
Setting Up Electroelution Apparatus......Page 275
Purification of Recombinant CENP-A......Page 276
Purification of His6-CENP-A......Page 277
Renaturation Process......Page 278
Purification of Native CENP-A/His6-H4 Complex......Page 279
Reconstitution of CENP-A Core Histones......Page 280
Nucleosome Reconstitution......Page 281
Conclusions......Page 282
Introduction......Page 284
Protocol......Page 285
Isolation of CEN Complex from Interphase HeLa Nuclei......Page 286
Solubilization of Bulk Chromatin with MNase Digestion of Nuclei in 0.3 M NaCl......Page 287
Immunoprecipitation of centromere chromatin with anti-CENP-A IgG beads......Page 289
Results and Discussion......Page 290
Introduction......Page 292
Cation-Exchange Chromatography......Page 293
Cation-Exchange Hydrophilic Interaction Chromatography......Page 296
Chromatographic Method......Page 299
Comments......Page 300
AU/AUT-PAGE......Page 301
Two-Dimensional Electrophoresis......Page 302
Capillary Electrophoresis......Page 303
Procedure for Capillary Electrophoresis of Chicken H1 Variants......Page 304
Analyses of H1 Variant Function......Page 305
Phosphorylation......Page 307
Poly(ADP-Ribosylation)......Page 309
Concluding Remarks......Page 310
HMGA Proteins Exhibit Unusual Properties......Page 312
Production and Purification of Recombinant HMGA Proteins......Page 313
Isolation of Recombinant HMGA Proteins......Page 315
Chromatographic Purification of Recombinant HMGA Proteins......Page 316
Isolation and Purification of HMGA Proteins from Mammalian Cells and Tissues......Page 317
Purification of HMGA Proteins Isolated from Mammalian Cells......Page 318
In Vivo Biochemical Modifications of HMGA Proteins......Page 320
Determination of In Vivo Biochemical Modifications by Mass Spectrometry......Page 322
MALDI/TOF Mass Spectrometry......Page 323
HMGA Proteins Bind to Nucleosome Core Particles Both In Vitro and In Vivo......Page 324
HMGA1 Binding to Nucleosomes Is Orientation Specific......Page 327
Isolation, Mutagenesis, and Radiolabeling of DNA Fragments......Page 330
Isolation of Nucleosomes, Histone Octamers, and Chromatin Reconstitutions......Page 331
A/T-Hook Peptide Motif Binds Nucleosome Core Particles......Page 332
Construction of Hybrid B-Box:A/T-Hook Plasmid Expression Vectors and Recombinant Protein Production......Page 335
DNA and Nucleosome Binding by Hybrid B-Box:A/T-Hook Proteins......Page 336
A/T Hooks Are Essential Components of Many Chromatin-Remodeling Complexes......Page 337
Preparation and Functional Analysis of HMGN Proteins......Page 338
General Considerations......Page 339
Preparation of HMGN Proteins from Cells Grown in Tissue Culture by Extraction with 5% PCA......Page 340
Preparation of HMGN from Tissues......Page 342
HPLC on Reverse-Phase C4 Column......Page 343
Detection and Initial Isolation of HMGN-Containing Multiprotein Complexes......Page 344
Mobility Shift Assay to Study In Vitro Interactions of HMGN with Chromatin Subunits......Page 348
Assays for the Interaction of HMGN Proteins with Chromatin......Page 349
Fluorescence Recovery After Photobleaching Analysis of the Interaction of HMGN Proteins with Chromatin in Living Cells......Page 351
Analysis of the In Vivo Function of HMGN Proteins......Page 352
Immunoprecipitation of HMGN1-Containing Chromatin (ChIP)......Page 353
Micrococcal Nuclease Digestion......Page 357
Introduction......Page 358
Tagging Genomic Loci......Page 360
Nuclear Detection......Page 363
Cell Preparation......Page 364
Time-Lapse Acquisition......Page 366
Positional Information......Page 368
Quantitative Approaches to Motion Analysis......Page 369
Tracking......Page 370
Controls for Nuclear Rotation......Page 372
Characteristic Parameters of Movement......Page 373
Discussion......Page 377
Acknowledgments......Page 378
Introduction......Page 379
Review of Previous Work......Page 380
Basic Approach......Page 383
Ongoing Methodology Development......Page 385
Directional Cloning in pSP Plasmids......Page 390
Generating Stable Cell Lines......Page 391
Acknowledgments......Page 394
Acknowledgments......Page 395
Some Problems Associated with Use of GFP-Tagged Proteins......Page 397
Choice of End for Attaching GFP-Fusion......Page 399
Overview......Page 400
Microscopy......Page 403
Measurements of Relative Intensity......Page 404
Problems Associated with Analysis over Long Periods......Page 405
Present Results and Future Directions......Page 406
Introduction......Page 408
Photobleaching Microscopy......Page 409
Collecting FRAP Data......Page 411
Double Normalization......Page 414
Single Normalization......Page 415
Qualitative Analysis of FRAP Data......Page 416
Quantitative FRAP Analysis......Page 417
Approximation of Well-Mixed Nucleoplasm......Page 418
Estimation of Number of Binding Site Classes......Page 421
Developing Kinetic Model......Page 422
Modeling Experimental Bleaching Protocol......Page 424
Required Equations......Page 425
Experimental Data Constraints and Parameter Optimization......Page 426
Summary......Page 429
Introduction......Page 430
Topology of Living Cell Nucleus......Page 433
Scalar Relationship between Cells and Molecules......Page 437
Study of Chromatin Dynamics, Using FRAP......Page 439
Biological Principles Underlying FRAP of Nuclear Proteins......Page 441
Designing and Characterizing Fluorescent Reporter for Live Cell-Imaging Experiments......Page 442
Performing the Experiment: Optimizing Data Collection......Page 444
Mathematical Analysis of Molecular Binding Events......Page 448
Models......Page 449
Parameter Estimation Methodology: Application to Type H1 Histone......Page 454
Acknowledgments......Page 457
Designing a FRAP Experiment......Page 458
Cells and Cell Culture Conditions......Page 461
Controlling Laser Illumination Intensity......Page 462
Defining Scan and Bleach Regions......Page 463
Collecting Data......Page 464
Experiments with FRAP......Page 466
Quantitative Analysis of FRAP Data......Page 469
Introduction......Page 471
Immunofluorescence Labeling......Page 473
Identification of Cell of Interest......Page 474
Resin Selection, Coverslip Embedding, and Sample Sectioning......Page 475
Embedding and Relocating Cell of Interest......Page 477
Preparation of Grids and Carbon Films for ESI......Page 478
Analysis of Centromeric Heterochromatin In Situ......Page 479
Ultrastructural Analysis of Chromatin during Mitosis......Page 482
ESI of Chromatin in Vicinity of PML Nuclear Bodies and IGCs......Page 485
Lower Limit of Detection of Nucleic Acid-Based Fibers in Sections by ESI......Page 490
Future of ESI: EM Tomography and Multiplex Detection of Nucleic Acids and Protein In Situ......Page 492
Acknowledgments......Page 493
Introduction......Page 494
Principles......Page 495
In Vitro Transcription Reaction......Page 498
Reverse Transcription......Page 499
Principles of Fixation......Page 500
Prehybridization Treatment......Page 501
Washing after Hybridization......Page 502
Principle of Method......Page 503
Method......Page 504
Method......Page 505
Quantitation of Enrichment......Page 507
Perspectives......Page 508
Introduction......Page 509
Principles of 3C Technology......Page 510
In Vivo Formaldehyde Fixation of Cells......Page 511
DNA Digestion in Intact Nuclei......Page 513
Reversal of Cross-Links and Purification of DNA......Page 515
Quantification of Cross-Linking Frequencies......Page 516
Controls......Page 517
Interpretation of Data Obtained by 3C Technology......Page 520
3C Technology and RNA TRAP......Page 521
Acknowledgments......Page 523