Characteristic low density and shear sensitivity of cross-linked chromatin containing polycomb complexes.
Chromatin cross-linking is widely used for mapping the distribution of chromosomal proteins by immunoprecipitation, but our knowledge of the physical properties of chromatin complexes remains rudimentary. Density gradients have been long used to separate fragments of cross-linked chromatin with their bound proteins from free protein or free DNA. We find that the association of DNA fragments with very-high-molecular-weight protein complexes shifts their buoyant density to values much lower then that of bulk chromatin. We show that in a CsCl gradient, Polycomb response elements, promoters of active genes, and insulator or boundary elements are found at buoyant densities similar to those of free protein and are depleted from the bulk chromatin fractions. In these regions, the low density is associated with the presence of large protein complexes and with high sensitivity to sonication. Our results suggest that separation of different chromatin regions according to their buoyant density may bias chromatin immunoprecipitation results. Density centrifugation of cross-linked chromatin may provide a simple approach to investigate the properties of large chromatin complexes in vivo.
Schwartz
Yuri B
YB
Rutgers University, Department of Molecular Biology and Biochemistry, Nelson Laboratories, 604 Allison Road, Piscataway, NJ 08854, USA.
Kahn
Tatyana G
TG
Pirrotta
Vincenzo
V
D000818:Animals; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods*; D002460:Cell Line; D002465:Cell Movement; D002499:Centrifugation, Density Gradient; D002586:Cesium / Q000737:chemistry; D002712:Chlorides / Q000737:chemistry; D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D029721:Drosophila Proteins / Q000737:chemistry*; D004586:Electrophoresis; D005821:Genetic Techniques; D047468:Immunoprecipitation; D063150:Polycomb Repressive Complex 1; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002712:Chlorides; D002843:Chromatin; D003432:Cross-Linking Reagents; D029721:Drosophila Proteins; C066770:Pc protein, Drosophila; D002586:Cesium; D004247:DNA; D063150:Polycomb Repressive Complex 1; C028019:cesium chloride