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

摘要 在成熟的神经细胞中，整体染色质凝聚和异染色质扩散与 增加对细胞死亡和组织退化的易感性，降低重新进入细胞的可能性 健康再生或恶变的周期。异染色质缩合已经被 被提议通过一种称为液-液相的过程来调节核小体冷凝物的膨胀 分离(LLP)。尽管如此，导致染色质大量缩合和 其逆转尚不清楚，核小体冷凝物的液体或固体性质也存在争议。 取决于特定的实验条件。我们发现某些表观遗传修饰物可以抑制 异染色质凝聚可恢复视网膜各层并部分逆转失明 视网膜变性疾病，视网膜色素变性。在这里，我们建议研究异染色质的机制。 用一种新的方法进行缩合和解凝：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 凝析油含有内部的“熔核”，具有随机定位的类液体核小体和固体 “外壳”，核小体在短距离内并列成部分交错的锯齿状堆叠。组蛋白 翻译后的电荷修饰(如乙酰化)在破坏核小体的稳定中起主要作用。 堆积，以便某些未堆叠的核小体簇将展开并打开它们的核小体界面 调节转录激活因子，减少抑制性异染色质隔间。 为了验证这一假设，我们提出了两个具体目标： 在SA1中，我们将从视网膜视杆感光细胞和小脑颗粒细胞中分离染色质 对照组和HDAC1和LSD1抑制剂处理组。我们将测定核小体的效率 缩合，相关的控制和修饰组蛋白的电荷，并研究哪些基因集 在杆状感光细胞发育过程中的浓缩，以及哪些特殊的组蛋白修饰和非组蛋白 蛋白质(如果有的话)区分凝聚相和可溶相。在SA2中，我们将使用Cryo-et of 玻璃化视网膜杆状感光细胞和培养小脑颗粒的分离核小体凝聚体 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、轴角和 3D空间中的平面角度。这些实验有望为染色质提供机械性的见解。 成熟神经细胞中的凝聚及其表观遗传修饰物的抑制可能导致更有效的 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 核小体凝聚在细胞核内。

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.