Nanoscale cryo-electron tomographic analysis of nucleosome condensates in neuronal chromatin

Abstract In mature neuronal cells, global chromatin condensation and heterochromatin spreading are associated with an increased susceptibility to cell death and tissue degeneration and decreased potential of re-entering the cell cycle for either healthy regeneration or malignant transformation. Heterochromatin condensation has been proposed to be mediated by expansion of nucleosome condensates through a process called liquid–liquid phase separation (LLPS). Still, the molecular mechanism(s) responsible for the massive chromatin condensation and its reversal remain unknown and the liquid or solid nature of the nucleosome condensates is disputable and depends on specific experimental conditions. We found that certain epigenetic modifiers that inhibit heterochromatin condensation can restore retina layers and partially reverse blindness in a mouse model of a retina degeneration disorder, retinitis pigmentosa. Here we propose to study mechanism of heterochromatin condensation and de-condensation using a novel approach: resolving the structure of nucleosome condensates in heterochromatin using cryo-electron tomography (cryo-ET). Our main hypothesis is that the nucleosome condensates contain the inner “molten core” with randomly positioned liquid-like nucleosomes and the solid “outer shell” with nucleosomes juxtaposed at short distances into partially interdigitated zigzag stacks. Histone posttranslational charge modifications (such as acetylation) play a major role in destabilizing the nucleosome stacking so that certain clusters of un-stacked nucleosomes would unfold and open their nucleosome interfaces to accommodate transcriptional activation factors and reduce the repressive heterochromatin compartments. To test this hypothesis, we propose two specific aims: In SA1, we will isolate chromatin from retina rod photoreceptors and cerebellum granule cells from control mice and those treated by HDAC1 and LSD1 inhibitors. We will determine efficiency of nucleosome condensation, the associated charge of control and modified histones and study what set of genes are condensed during rod photoreceptors development, and what particular histone modifications and non-histone proteins (if any) discriminate between the condensed and the soluble phases. In SA2, we will use cryo-ET of the isolated nucleosome condensates and of vitrified retina rod photoreceptors and cultured cerebellum granule cells samples in situ to determine individual nucleosome positions, center-to-center distances, axial angles, and plane angles in 3D space. These experiments are expected to provide mechanistic insights for the chromatin condensation in mature neuronal cells and its inhibition by epigenetic modifiers that may lead to a more efficient treatment of retinitis pigmentosa and other neurodegenerative disorders and develop new tools for imaging of nucleosome condensates in the cell nucleus.