Dynamic Heterochromatin States in Anisotropic Nuclei of Cells on Aligned Nanofibers

[Image: see text] The cancer cell nucleus deforms as it invades the interstitial spaces in tissues and the tumor microenvironment. While alteration of the chromatin structure in a deformed nucleus is expected and documented, the chromatin structure in the nuclei of cells on aligned matrices has not been elucidated. In this work we elucidate the spatiotemporal organization of heterochromatin in the elongated nuclei of cells on aligned nanofibers with stimulated emission depletion nanoscopy and fluorescence correlation spectroscopy. We show that the anisotropy of nuclei is sufficient to drive H3K9me3-heterochromatin alterations, with enhanced H3K9me3 nanocluster compaction and aggregation states that otherwise are indistinguishable from diffraction-limited microscopy. We interrogated the higher-order heterochromatin structures within major chromatin compartments in anisotropic nuclei and discovered a wider spatial dispersion of nanodomain clusters in the nucleoplasm and condensed larger nanoclusters near the periphery and pericentromeric heterochromatin. Upon examining the spatiotemporal dynamics of heterochromatin in anisotropic nuclei, we observed reduced mobility of the constitutive heterochromatin mark H3K9me3 and the associated heterochromatin protein 1 (HP1α) at the nucleoplasm and periphery regions, correlating with increased viscosity and changes in gene expression. Since heterochromatin remodeling is crucial to genome integrity, our results reveal an unconventional H3K9me3 heterochromatin distribution, providing cues to an altered chromatin state due to perturbations of the nuclei in aligned fiber configurations.