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

神经元染色质核小体凝聚物的纳米级冷冻电子断层扫描分析

基本信息

批准号：
10669760
负责人：
SERGEI A GRIGORYEV
金额：
$ 20.75万
依托单位：
PENNSYLVANIA STATE UNIV HERSHEY MED CTR
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10669760
关键词：
3-Dimensional Acetylation Affect Architecture Biochemical Blindness Cell Cycle Cell Death Cell Maturation Cell Nucleus Cells Cerebellum Charge Chromatin Chromatin Remodeling Factor Chromatin Structure Cryo-electron tomography DNA DNA sequencing Dependence Development Disease Dissociation Divalent Cations Environment Epigenetic Process Gel Gene Mutation Gene Structure Genes Genetic Diseases Genetic Transcription Genome Genomic DNA HDAC1 gene Heterochromatin Higher Order Chromatin Structure Histone Deacetylase Histone Deacetylase Inhibitor Histone H1 Histones Hydrogels Image Imaging Device Imaging Techniques In Situ In Vitro Individual Ions KDM1A gene Liquid substance Malignant - descriptor Maps Mass Spectrum Analysis Mediating Methyl-CpG-Binding Protein 2 Modeling Modification Molecular Mus Natural regeneration Nature Neurodegenerative Disorders Neurons Nuclear Nucleosomes Phase Photoreceptors Physical condensation Physiological Play Positioning Attribute Predisposition Process Proteins Resistance Retina Retinal Degeneration Retinitis Pigmentosa Role Sampling Shapes Solid Structure Surface Testing Thinness Time Tissues Transcriptional Activation Type 7 Spinocerebellar Ataxia blind epigenetic drug epigenetic therapy experimental study fortification genome editing granule cell histone modification image reconstruction improved inhibitor insight mouse model nanoscale nervous system disorder neuronal cell body non-histone protein novel novel strategies preservation prevent retinal rods sight restoration three-dimensional modeling tissue degeneration tomography

项目摘要

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.

摘要