Chromatin packaging in eukaryotic chromosomes has been traditionally viewed as a hierarchical process, in which nucleosome chains fold into helical chromatin fibers. These fibers would then fold into more complex coiled structures. However, recent chromatin microscopic imaging and analyses of DNA contacts within the 3D space of the cell nucleus have necessitated a radical revision of the hierarchical chromatin packing model. To reveal the nanoscale anatomy of condensed human and mouse chromatin, we have combined Cryo-electron tomography with a newly developed deep-learning denoising and quantitative stereological analysis of the denoised cryo-tomograms. Cryo-ET imaging revealed the following features specific to the condensed eukaryotic chromatin: 1) the discontinuous asymmetric nucleosome stacking; 2) the alternating parallel and perpendicular orientations of the juxtaposed nucleosome disks; 3) regressive folding of the nucleosome zigzag chains into discontinuous nanoparticles rather than continuous fibers; 4) great variability of the nucleosome linker length comprising very long, short , and negative-length nucleosome linkers within the same arrays; 5) Histone charge modifications strongly inhibited nucleosome stacking and self-association with a modest effect on chromatin folding of native chromatin, in sharp contrast with a dramatic unfolding regular reconstituted chromatin. The striking linker length heterogeneity is consistent with a discontinuous regressive folding in which the variable linkers destabilize chromatin fibers and confine nucleosome chain folding to localized nanoparticles. We conclude that cryo-ET in combination with 3D nucleosome modeling may further reveal nanoscale chromatin structural transitions vital for understanding the mechanism(s) of gene regulation underlying genetic abnormalities and epigenetic memory.
This work was supported by US National Science Foundation grant 1911940.