Physicochemical characterization of self-associated chromatin oligomers

2016 Spring.Includes bibliographical references.The DNA of chromosomes is extensively compacted within the nuclei of eukaryotic nuclei. Chromosomes are composed of chromatin which is a repeating polymer of nucleosomes bound by additional chromatin proteins. Chromatin can be reconstituted in vitro using purified DNA and histone proteins to form nucleosomal arrays. Reconstituted chromatin fibers are structurally dynamic and the structures formed are highly dependent on the buffer conditions, particularly polyvalent cations. The addition of Mg2+ favors nucleosome-nucleosome interactions. At low concentrations nucleosomes on the same fiber interact resulting in folding, while at higher concentrations inter-fiber interactions result in chromatin self-association. Unlike folded chromatin, the oligomeric structure of chromatin is unkown, to address this deficiency, in this dissertation the oligomeric structures formed by 12-mer nucleosomal arrays were characterized by microscopy, sedimentation velocity, and SAXS experiments. The oligomeric chromatin complexes were globular throughout all stages of the cooperative assembly process, and ranged in size from ~50 nm to a diameter of ~1000 nm. The oligomer sedimentation coefficients under these conditions ranged from 5000-350,000S, corresponding to ~1-400 Mb DNA/oligomer. The nucleosomal arrays were packaged within the oligomers as interdigitated 10-nm fibers, rather than folded 30-nm structures. Linker DNA was freely accessible to micrococcal nuclease, although the oligomers remained partially intact after linker DNA digestion. The organization of chromosomal fibers in human nuclei in situ was stabilized by 1 mM MgCl2, but became disrupted in 0 mM MgCl2, conditions that also dissociated the oligomers in vitro. These results indicate that a 10-nm array of nucleosomes has the intrinsic ability to self-assemble into large chromatin globules stabilized by nucleosome-nucleosome interactions, and suggest that the oligomers are good in vitro model for investigating the structure and organization of interphase chromosomes