title: Nucleosomes stacked with aligned dyad axes are found in native compact chromatin in vitro.
abstract: In this study, electron tomograms of plunge-frozen isolated chromatin in both open and compacted form were recorded. We have resolved individual nucleosomes in these tomograms in order to provide a 3D view of the arrangement of nucleosomes within chromatin fibers at different compaction states. With an optimized template matching procedure we obtained accurate positions and orientations of nucleosomes in open chromatin in "low-salt" conditions (5 mM NaCl). The mean value of the planar angle between three consecutive nucleosomes is 70°, and the mean center-to-center distance between consecutive nucleosomes is 22.3 nm. Since the template matching approach was not effective in crowded conditions, for nucleosome detection in compact fibers (40 mM NaCl and 1 mM MgCl(2)) we developed the nucleosome detection procedure based on the watershed algorithm, followed by sub-tomogram alignment, averaging, and classification by Principal Components Analysis. We find that in compact chromatin the nucleosomes are arranged with a predominant face-to-face stacking organization, which has not been previously shown for native isolated chromatin. Although the path of the DNA cannot be directly seen in compact conditions, it is evident that the nucleosomes stack with their dyad axis aligned in forming a "double track" conformation which is a consequence of DNA joining adjacent nucleosome stacks. Our data suggests that nucleosome stacking is an important mechanism for generating chromatin compaction in vivo.
authors.affiliation: European Molecular Biology Laboratory, Meyerhofstr. 1, 69117 Heidelberg, Germany. scheffer@biophysik.org
authors.forename: Margot P
authors.initials: MP
authors.lastname: Scheffer
authors.forename: Mikhail
authors.initials: M
authors.lastname: Eltsov
authors.forename: Jan
authors.initials: J
authors.lastname: Bednar
authors.forename: Achilleas S
authors.initials: AS
authors.lastname: Frangakis
mesh_terms: D000818:Animals; D002645:Chickens; D002843:Chromatin / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy; D055032:Electron Microscope Tomography; D004912:Erythrocytes / Q000648:ultrastructure; D009707:Nucleosomes / Q000648:ultrastructure*; D013215:Starfish
chemical_list: D002843:Chromatin; D009707:Nucleosomes