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，整体染色质浓缩和异染色质扩散与细胞死亡和组织变性的易感性增加，重新进入细胞的可能性降低健康再生或恶性转化的循环已被确定。提议通过称为液-液相的过程通过核小体缩合物的扩张来介导分离（LLPS）仍然是导致大量染色质浓缩和分离的分子机制。它的逆转仍然未知，核小体凝聚物的液体或固体性质是有争议的取决于特定的实验条件。我们发现某些表观遗传修饰剂会抑制。异染色质浓缩可以恢复视网膜层并部分逆转小鼠模型的失明视网膜变性疾病、色素性视网膜炎在这里我们建议研究异染色质的机制。使用新方法进行缩合和解缩合：解析核小体缩合体的结构我们的主要假设是核小体。冷凝物包含内部“熔核”和随机定位的液体状核小体和固体 “外壳”，核小体以短距离并列成部分交错的锯齿形组蛋白堆。翻译后电荷修饰（例如乙酰化）在破坏核小体稳定方面发挥着重要作用堆叠使得某些未堆叠的核小体簇展开并打开其核小体界面适应转录激活因子并减少抑制性异染色质区室。为了检验这一假设，我们提出两个具体目标：在 SA1 中，我们将从视网膜杆状光感受器和小脑颗粒细胞中分离染色质对照小鼠和经 HDAC1 和 LSD1 抑制剂治疗的小鼠我们将确定核小体的效率。缩合、控制和修饰组蛋白的相关电荷并研究哪些基因组在视杆细胞发育过程中凝结，以及哪些特定的组蛋白修饰和非组蛋白蛋白质（如果有）区分凝聚相和可溶相。在 SA2 中，我们将使用冷冻 ET。分离的核小体凝聚物和玻璃化视网膜杆光感受器和培养的小脑颗粒原位细胞样本以确定单个核小体位置、中心到中心距离、轴向角度和 3D 空间中的平面角度预计这些实验将为染色质提供机制见解。成熟神经细胞中的凝结及其通过表观遗传修饰剂的抑制可能会导致更有效的治疗视网膜色素变性和其他神经退行性疾病，并开发新的成像工具核小体在细胞核中凝结。