Analyzing the role of chromatin compaction in nuclear mechanics, structure, and function

项目摘要 核是细胞器，它必须适当地转换或抵抗生物物理力来支配空间 基因组的组织和控制机械转导，这是决定表达的因素 单元的配置文件。先前的研究表明，核力学的两个主要贡献者是板层， 中间丝排列在核内的包膜上，染色质充满细胞核。更改 层粘连蛋白和染色质压缩存在于许多主要的人类疾病中，包括椎板病症和许多 癌症。这些疾病表现为核形态的改变，即核的突起，称为泡状核。在……上面 另一方面，在健康细胞中会发生板层和染色质紧凑/组织的变化 分化，在此过程中，核、细胞和组织的力学和形态会发生巨大的变化。 目前，基于疾病的核泡和基于健康分化的核泡的机制基础 原子核形态和力学的变化是未知的。我的博士后研究发现染色质和 它的组蛋白介导的压实状态决定了初始力响应(30%应变)和形态，同时还 作为层蛋白A的次要因素，决定了应变在较长变形时变硬。它不是 已知决定染色体结构域的高阶染色质构象是否是导致这种非 染色质的遗传、结构和抗性作用。为了检验这一点，我首先建议使用我开发的 进一步阐明显微解剖、微操作和纳米牛顿水平的力测量方法 高阶染色质断裂致染色质紧凑的核力学作用 构象。在核拉伸实验中，我将确定染色质对核的反应 单染色体位点成像变形(CRISPR标记)和整体染色质纳米结构 (PWS显微镜)。第二，我将研究染色质增加或减少的机械性影响 核泡形成与疾病相关表型的紧凑 荧光和非荧光固定细胞和活细胞成像技术。我们的初步数据还显示， 染色质压缩在核泡形成中的新作用，即染色质本身的解缩导致 凝结时发出的呜咽声拯救了它。然后我会确定核泡是症状还是原因 疾病，通过活细胞成像和生化技术来检测系统DNA损伤，适当 转录，以及基因组内容在水泡中的忠实分离。最后，我将使用成熟的 角质形成细胞的原代细胞模型研究核形态变化的基础 分化、祖细胞到终末，以及RAS激活后动态平衡的丧失，以模拟癌症的过渡。 通过分析染色质压缩对核力学的贡献，我的目标是过渡到一个 独立研究观察了70多年的形态变化的力学基础 在疾病和健康细胞分化方面都有多年的研究。

Project Summary The nucleus is the organelle which must properly transduce or resist biophysical forces to dictate the spatial organization of the genome and to control mechanotransduction, factors which determine the expression profile of the cell. Previous studies revealed that the two major contributors to nuclear mechanics are lamins, intermediate filaments lining the inner nuclear envelope, and chromatin, which fills the nucleus. Alteration of lamins and chromatin compaction occur in many major human diseases including laminopathies and many cancers. These diseases present altered nuclear morphology as protrusions of the nucleus termed blebs. On the other hand, alteration of lamins and chromatin compaction/organization occurs in healthy cells during differentiation, during which nuclear, cell, and tissue mechanics and morphology can change drastically. Currently, the mechanistic basis for both disease-based nuclear blebs and healthy differentiation-based changes in nuclear morphology and mechanics is unknown. My postdoctoral studies found that chromatin and its histone-mediated compaction state dictated initial force response (< 30% strain) and morphology while also contributing as a secondary factor to the lamin A dictated strain stiffening at longer deformations. It is not known if higher order chromatin conformation dictating chromosome domains is another contributor to this non- genetic structural and resistive role of chromatin. To examine this, I first propose to use my developed microdissection, micromanipulation, and nanonewton-level force measurement approach to further elucidate the nuclear mechanics role of chromatin compaction through disruption of higher order chromatin conformation. During nuclear stretching experiments I will determine how the chromatin responds to nuclear deformation through imaging single chromosome loci (CRISPR labeling) and overall chromatin nano-structure (PWS microscopy). Second, I will investigate the mechanistic impact of increasing or decreasing chromatin compaction on the disease-relevant phenotype of nuclear blebbing by assaying for bleb occurrence with fluorescence and non-florescence fixed- and live-cell imaging techniques. Our preliminary data also reveal a novel role for chromatin compaction in nuclear blebbing, in that decondensation of chromatin alone leads to blebbing while condensation rescues it. I will then determine if nuclear blebs are a symptom or a cause of disease, via live cell imaging and biochemical techniques to assay for systemic DNA damage, proper transcription, and faithful segregation of genomic content in the bleb. Finally, I will use the well-established primary cell model of keratinocytes to investigate the basis of nuclear morphology changes during differentiation, progenitor to terminal, and loss of homeostasis upon Ras activation to mimic cancer transition. Through analyzing the contribution of chromatin compaction to nuclear mechanics, I aim to transition into an independent career investigating the mechanical basis of morphology changes observed for more than 70 years in both disease and in healthy cell differentiation.