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

摘要 在成熟的神经元细胞中，整体染色质凝聚和异染色质扩散与神经元细胞的凋亡有关。 增加对细胞死亡和组织变性的敏感性，降低重新进入细胞的可能性 健康再生或恶性转化的循环。异染色质凝聚一直是 提出通过称为液-液相的过程通过核小体凝聚物的膨胀介导 分离（LLPS）。尽管如此，负责大量染色质凝聚和 其逆转仍然未知，核小体凝聚物的液体或固体性质是有争议的， 这取决于具体的实验条件。我们发现，某些表观遗传修饰剂， 异染色质凝聚可以恢复视网膜层，并在小鼠模型中部分逆转失明。 视网膜变性疾病、视网膜色素变性。在此我们提出研究异染色质的机制 使用一种新的方法进行缩合和去缩合：解析核小体缩合物的结构 在异染色质中使用冷冻电子断层扫描（cryo-ET）。我们的主要假设是核小体 凝聚物包含内部的“熔融核心”，具有随机定位的液体样核小体和固体核小体。 “外壳”，核小体在短距离内并置成部分交叉的锯齿形堆叠。组蛋白 翻译后电荷修饰（如乙酰化）在核小体的不稳定中起主要作用 堆叠，使得未堆叠的核小体的某些簇会展开并打开它们的核小体界面 以适应转录激活因子并减少抑制性异染色质区室。 为了验证这一假设，我们提出了两个具体目标： 在SA 1中，我们将从视网膜视杆细胞和小脑颗粒细胞中分离染色质， 对照小鼠和用HDAC 1和LSD 1抑制剂处理的小鼠。我们将确定核小体的效率 凝聚，控制和修饰组蛋白的相关电荷，并研究什么样的基因集是 在视杆细胞发育过程中，有哪些特定的组蛋白修饰和非组蛋白修饰， 蛋白质（如果有的话）区分凝聚相和可溶相。在SA 2中，我们将使用 玻璃化视网膜视杆细胞和培养的小脑颗粒的分离核小体凝聚物 原位细胞样本，以确定单个核小体的位置、中心到中心的距离、轴向角度，以及 3D空间中的平面角。这些实验有望为染色质的机制提供见解 在成熟的神经元细胞中的凝聚和它的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聚。

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.