BPS2026 – Cryo-electron tomography reveals distinct nanoscale organization of eu- and heterochromatin

Structural and spatial segregation of the eukaryotic genome into the open euchromatin and condensed heterochromatin is critical for establishing cell-specific epigenetic programs. To resolve the nanoscale 3D organization and reveal chromatin higher order structures underlying eu- and heterochromatin formation in human and mouse cells, we apply cryo-electron tomography (cryo-ET) aided by deep-learning image denoising. We find that either nucleosome arrays isolated from immature mouse retina cells enriched with euchromatin or the fractionated human euchromatin, both contain nucleosomes with strikingly heterogeneous linker DNA lengths in which very long linkers alternate with closely apposed and overlapping nucleosomes featuring extremely short or absent nucleosome linkers. By cryo-ET-assisted nucleosome interaction capture, we observe that mouse and human euchromatin samples are enriched with nearest-neighbor i ± 1 interactions. These structural features are consistent with a discontinuous regressive folding in which the variable nucleosome linkers destabilize chromatin fibers and confine nucleosome chain folding to localized nanoparticles rather than extended chromatin fibers. In contrast, in mature retina cells, and in fractionated human heterochromatin, the short-linker nucleosomes are much less frequent, supporting stronger chromatin compaction while both mouse and human heterochromatin are enriched with i ± 2 interactions typical of the two-start zigzag. The unusually short linkers enriched in euchromatin are predicted to inhibit the two-start zigzag and limit chromatin compaction by interfering with formation of linker DNA stems and introducing higher chromatin folding entropy. This mechanism may be broadly utilized for understanding the molecular mechanism(s) underlying epigenomic plasticity and epigenetic changes driving heterochromatin spreading and cell differentiation. This work is supported by National Science Foundation grant 2521597.