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.

抽象的在成熟的神经元细胞中，全局染色质凝结和异染色质扩散与增加对细胞死亡和组织变性的敏感性以及重新进入细胞的潜力增加健康再生或恶性转化的循环。异染色质凝结已经建议通过一种称为液态相的过程介导的核小体冷凝物介导分离（LLP）。尽管如此，负责大量染色质凝结和它的逆转仍然未知，核小体冷凝物的液体或固体性质是有争议的，并且取决于特定的实验条件。我们发现某些抑制的表观遗传修饰符异染色质冷凝可以恢复视网膜层，并在A的小鼠模型中部分逆转失明视网膜变性障碍，色素性视网膜炎。在这里，我们建议研究异染色质的机理使用一种新方法的凝结和去向敏感：解决核体冷凝水的结构在异染色质中使用冷冻电子层析成像（Cryo-ET）。我们的主要假设是核小体冷凝物包含内部“熔融核”，其随机定位液样核小体和固体 “外壳”与核小体在短距离并列为部分互连的锯齿形堆栈中。组蛋白翻译后电荷修饰（例如乙酰化）在破坏核小体稳定的主要作用堆叠以使某些未堆叠核小体的簇展开并打开其核小体界面为了适应转录激活因子并减少反射性异染色质室。为了检验这一假设，我们提出了两个具体目的：在SA1中，我们将从视网膜杆感光器和小脑颗粒细胞中分离染色质对照小鼠以及由HDAC1和LSD1抑制剂治疗的小鼠。我们将确定核武器的效率凝结，相关的对照和修饰组蛋白的电荷，并研究了哪些基因是在杆感光器发育过程中凝结，以及特定的组蛋白修饰和非固定酮蛋白质（如果有）区分凝结的和固相。在SA2中，我们将使用Cryo-Et 孤立的核绿色蛋白酶冷凝物和玻璃化视网膜杆感光体和培养的小脑颗粒细胞原位样品以确定个体核小体位置，中心到中心的距离，轴向角度和平面角度在3D空间中。这些实验有望为染色质提供机械见解成熟神经元细胞中的凝结及其抑制表观遗传修饰剂的抑制作用，可能导致更有效治疗色素性视网膜炎和其他神经退行性疾病，并开发用于成像的新工具细胞核中的核小体冷凝物。