Abstract
In eukaryotic cells, genomic DNA is compacted by nucleosomes, as basic repeating units, into chromatin. The nucleosome arrangement in chromatin fibers could be an important determinant for chromatin folding, by which genomic DNA is regulated in the nucleus. To study the structures of chromatin units in cells, we have established a method for the structural analysis of native mono- and poly-nucleosomes prepared from HeLa cells. In this method, the chromatin in isolated nuclei was crosslinked to preserve the proximity information between nucleosomes, followed by chromatin fragmentation by micrococcal nuclease treatment. The mono- and poly-nucleosomes were then fractionated by sucrose gradient ultracentrifugation, and their structures were analyzed by cryo-electron microscopy. Cryo-electron microscopy single particle analysis and cryo-electron tomography visualized a native nucleosome structure and secondary nucleosome arrangements in cellular chromatin. This method provides a complementary strategy to fill the gap between in vitro and in situ analyses of chromatin structure.