Characteristic Low Density and Shear Sensitivity of Cross-Linked Chromatin Containing Polycomb Complexes

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-highmolecular-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.

Polycomb Complexes and the Propagation of the Methylation Mark at the Drosophila Ubx Gene

“…Chromatin Cross-linking and Immunoprecipitation-Cells of a derivative of the Schneider L2 cultured cell line, Sg4, were grown and cross-linked as described (25), the embryos were collected overnight and cross-linked as described in Ref. 21.…”

“…5 ml of suspension were subjected to sonication with a Branson 250 sonifier equipped with microtip under conditions described in Ref. 25. The resulted lysate was further adjusted to radioimmunoprecipitation assay buffer (140 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride) and subjected to 5 min of centrifugation at 16,000 ϫ g. For ChIP, 500 l of supernatant were precleared by incubation with protein A-Sepharose (Sigma) and further incubated with appropriate antibody overnight at 4°C.…”

“…5-6 g of anti-PC, PSC, me3K27, me2K27, H2B, 1-3 g of anti-H3, E(Z), Trithorax (TRX), GAGA, 1 l of anti-TBP, and 5 l of anti-me3K9 and anti-RNA polymerase (8WG16) antibodies were used per one reaction. The antibody complexes were precipitated, and DNA recovered as described (25). Immunoprecipitated DNA was dissolved in 150 l of water (V 1 ).…”

“…2) A more serious concern is that the low histone immunoprecipitation of the PRE core is because of its smaller DNA fragment size. We have previously reported that the PRE region is highly sensitive to sonication and is rapidly reduced to fragments of 300 bp while the bulk chromatin is still 1-2 kb on the average (25). Short chromatin fragments, containing one or two nucleosomes, would be poorer targets for an anti-histone antibody and therefore be less well precipitated than longer fragments.…”

Chromosome-wide gene-specific targeting of theDrosophiladosage compensation complex

“…ChIP was performed as described previously, including purification over a CsCl gradient (Kageyama et al 2001). For control purposes, chromatin was also prepared using a CsCl-free protocol (Schwartz et al 2005), but no differences to CsCl-prepared chromatin were seen (data available on request). Embryos were fixed in 4% formaldehyde at 18°C for 15 min.…”