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

分析染色质压缩在核力学、结构和功能中的作用

• 批准号: 9452678
• 负责人: Andrew Daniel Stephens
• 金额: $ 9万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9452678
• 关键词: Aging; Biochemical; Biological Assay; Biophysics; Bulla; Cell Differentiation process; Cell Nucleus; Cell model; Cells; Cellular biology; Chromatin; Chromatin Loop; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Coupled; Cytoskeleton; DNA Damage; DNA Repair; Data; Development; Disease; Environment; Euchromatin; Exhibits; Expression Profiling; Fluorescence; Functional disorder; Gene Expression; Gene Structure; Genetic; Genetic Transcription; Genome; Genomics; Heart Diseases; Heterochromatin; Histones; Homeostasis; Image; Imaging Techniques; Intermediate Filaments; Label; Lamin Type A; Lamins; Malignant Neoplasms; Measurement; Measures; Mechanics; Mediating; Microdissection; Micromanipulation; Microscopy; Mitosis; Modeling; Molecular Conformation; Morphology; Muscular Dystrophies; Nanostructures; Nuclear; Nuclear Envelope; Nuclear Inner Membrane; Organelles; Pattern; Phenotype; Physical condensation; Physiological; Property; Proteins; Resistance; Role; Skin; Spectrum Analysis; Stretching; Structure; Symptoms; Techniques; Tissue Differentiation; Tissues; base; cancer biomarkers; career; chromatin modification; chromatin protein; cohesin; density; diagnostic biomarker; experimental study; histone modification; human disease; insight; keratinocyte; live cell imaging; mechanotransduction; nanonewton; non-genetic; novel; nuclear imaging; progenitor; response; segregation; therapeutic target

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.

项目摘要 细胞核是一种必须适当地抵抗或抵抗生物物理力以决定空间结构的细胞器。 基因组的组织和控制机械转导，决定表达的因素 细胞的轮廓。以前的研究表明，核力学的两个主要贡献者是核纤层， 中间丝内衬内核膜，染色质，它填充细胞核。改变 核纤层蛋白和染色质致密化发生在许多主要的人类疾病中， 癌的这些疾病表现为改变的核形态，如称为泡的核突起。对 另一方面，核纤层蛋白和染色质致密化/组织化改变发生在健康细胞， 在分化过程中，细胞核、细胞和组织的力学和形态会发生急剧变化。 目前，基于疾病的核泡和基于健康分化的核泡两者的机制基础都不清楚。 核形态学和力学的变化是未知的。我的博士后研究发现， 其组蛋白介导的压缩状态决定了初始力响应（<30%应变）和形态， 作为第二因素，在较长的变形下，对层压A的作用决定了应变硬化。不 已知如果决定染色体结构域的更高级染色质构象是这种非- 染色质的遗传结构和抗性作用。为了检验这一点，我首先建议使用我的发达国家， 显微切割，显微操作和纳米级力测量方法，以进一步阐明 通过破坏高级染色质的染色质致密化的核力学作用 构象在核拉伸实验中，我将确定染色质如何响应核拉伸， 通过成像单个染色体基因座（CRISPR标记）和整体染色质纳米结构的变形 (PWS显微术）。其次，我将研究增加或减少染色质的机械影响， 通过测定核泡发生率来压实核泡的疾病相关表型， 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， 染色质致密化在核起泡中的新作用，因为单独的染色质去致密化导致 然后我将确定核泡是一种症状还是一种原因， 疾病，通过活细胞成像和生化技术来检测系统性DNA损伤， 转录和基因组内容物在水泡中的忠实分离。最后，我将使用公认的 角质形成细胞的原代细胞模型，以研究细胞核形态变化的基础 分化，祖细胞到终末细胞，以及Ras激活后体内平衡的丧失，以模拟癌症转变。 通过分析染色质致密化对核力学的贡献，我的目标是过渡到一个 独立的职业生涯调查的形态变化的机械基础观察超过70 在疾病和健康细胞分化中的作用。