Affiliation:
1. Department of Zoology, University of Geneva, Geneva, Switzerland
Abstract
ABSTRACT
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.
Publisher
American Society for Microbiology
Subject
Cell Biology,Molecular Biology
Reference30 articles.
1. Bollag D. M. M. D. Rozycki and S. J. Edelstein. 1996. Protein methods. Wiley-Liss Inc. New York N.Y.
2. Breiling, A., B. M. Turner, M. E. Bianchi, and V. Orlando. 2001. General transcription factors bind promoters repressed by Polycomb group proteins. Nature 412 : 651-655.
3. Brutlag, D., C. Schlehuber, and J. Bonner. 1969. Properties of formaldehyde-treated nucleohistone. Biochemistry 8 : 3214-3218.
4. Cavalli, G., V. Orlando, and R. Paro. 1999. Mapping DNA target sites of chromatin-associated proteins by formaldehyde cross-linking in Drosophila embryos, p. 20-30. In W. A. Bickmore (ed.), Chromosome structural analysis: a practical approach. Oxford University Press, Oxford, United Kingdom.
5. de Cock, J. G., E. C. Klink, W. Ferro, P. H. Lohman, and J. C. Eeken. 1991. Repair of UV-induced pyrimidine dimers in the individual genes Gart, Notch and white from Drosophila melanogaster cell lines. Nucleic Acids Res. 19 : 3289-3294.
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