Investigating the role of nuclear mechanics in the regulation of chromatin structure and embryonic cell fate

研究核力学在染色质结构和胚胎细胞命运调节中的作用

• 批准号: 10723483
• 负责人: Alice Louisa Sherrard
• 金额: $ 12.47万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-10 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10723483
• 关键词: Adhesions; Affect; Automobile Driving; Binding; Biological Assay; Cell Differentiation process; Cell Nucleus; Cells; Chromatin; Chromatin Modeling; Chromatin Structure; Clustered Regularly Interspaced Short Palindromic Repeats; Critical Pathways; Cryoelectron Microscopy; Cytoplasm; Cytoskeleton; Detection; Development; Education; Electron Microscopy; Embryo; Embryonic Development; Fertilization; Gene Expression; Generations; Genetic Transcription; Genomics; Germ Layers; Goals; Heterochromatin; Image; Immunofluorescence Immunologic; Infertility; Inner Cell Mass; Label; Lamin Type A; Learning; Light; Link; Maintenance; Mammalian Cell; Mammals; Mechanics; Membrane Proteins; Mentors; Methods; Molecular; Mus; Nuclear; Nuclear Envelope; Nuclear Matrix; Nucleosomes; Pathway interactions; Placenta; Postdoctoral Fellow; Pregnancy; Process; Proteins; Protocols documentation; Regulation; Regulatory Pathway; Research; Resistance; Resolution; Role; Running; Scientist; Signal Pathway; Signal Transduction; Specialist; Specific qualifier value; Supporting Cell; Techniques; Testing; Training; Visualization; Work; blastocyst; blastomere structure; cell fate specification; cell type; early pregnancy; embryo cell; embryo tissue; experimental study; implantation; in vivo; insight; mechanical force; mechanical properties; mechanical signal; novel; novel strategies; pluripotency; post-doctoral training; preimplantation; programs; sensor; tomography; transcriptome sequencing; transmission process

SUMMARY The inner cell mass (ICM) and trophectoderm (TE) are the first two cell types specified during mammalian development. TE cells support implantation and give rise to the placenta, whereas ICM cells forms the embryo and some extraembryonic tissues. Their differentiation is therefore critical for successful pregnancy. The mechanically-regulated Hippo signaling pathway is differentially activated in ICM and TE cells, driving gene expression programs that define these cell states. These programs also depend on cell type-specific chromatin landscapes. How mechanical forces regulate chromatin structure and embryonic cell fates during pre- implantation is however not fully understood. I hypothesize that mechanical forces transmitted though the cytoskeleton, regulate TE transcriptional programs by modulating both Hippo signaling and chromatin structure. In this proposal, I will test this hypothesis by defining how nuclear tension regulates Hippo signaling and chromatin organization during early embryonic differentiations. My ultimate goal is to define the mechanistic links connecting mechanical and regulatory pathways to cell and chromatin states. This work will enhance our understanding of cell fate specification, both in relationship to early embryogenesis and implantation, and more broadly. My postdoctoral work in the Giraldez lab showed that Lamin A/C is transcriptionally up-regulated in TE, compared to ICM, and that it regulates TE identify; LMNA/C depletion leads to an ICM-like transcriptional state reminiscent of Hippo pathway activation. In Aim 1 (K99), I will investigate regulation of Hippo by Lamin A/C and determine the role of mechanical sensing by cytoskeletal networks in the regulation of this signaling. In Aim 2 (K99/R00), to determine how mechanical forces regulate chromatin, I will apply advanced electron microscopy approaches to visualize nucleosome resolution chromatin structure in vivo. During the training period in the Giraldez lab, I will apply a novel labeling strategy, combined with cryo EM to characterize lamina-heterochromatin interactions. During the R00 period and beyond, I will apply these approaches to determine how compaction of the embryo and the generation of mechanical forces on the nucleus impact chromatin structure and ICM/TE fates. In Aim 3 (R00), I will use chimeric embryos and other developmental assays to examine how changes in the mechanical properties of the nucleus affects differentiation potential. I will also quantify nuclear stiffness and chromatin structure in developing mouse embryos. This work paves the way for a deeper understanding of the role of mechanical forces in regulating gene expression and cell identity. This proposal brings together training and concepts that I have acquired throughout my education and new approaches (RNA-seq and cryo-EM) that I will learn in my mentor’s (Giraldez) lab, from other scientists and specialists at Yale, and at the lab of my co- mentors, Elizabeth Villa and Berna Sozen. This proposal will complete my postdoctoral training and prepare me for my ultimate goal of running a competitive independent research program.

概括 内细胞团（ICM）和滋养外胚层（TE）是哺乳动物细胞分化过程中最先确定的两种细胞类型。 发展。 TE 细胞支持着床并形成胎盘，而 ICM 细胞则形成胚胎 以及一些胚胎外组织。因此，它们的分化对于成功怀孕至关重要。这 机械调节的 Hippo 信号通路在 ICM 和 TE 细胞中差异激活，驱动基因 定义这些细胞状态的表达程序。这些程序还取决于细胞类型特异性染色质 风景。机械力如何调节染色质结构和胚胎细胞命运 然而，植入尚不完全清楚。我假设机械力通过 细胞骨架，通过调节 Hippo 信号传导和染色质结构来调节 TE 转录程序。 在这个提案中，我将通过定义核张力如何调节 Hippo 信号传导和 早期胚胎分化过程中的染色质组织。我的最终目标是定义机械联系 将机械和调节途径与细胞和染色质状态连接起来。这项工作将增强我们 了解细胞命运规范，与早期胚胎发生和植入等相关 广泛地说。我在 Giraldez 实验室的博士后工作表明 Lamin A/C 在 TE 中转录上调， 与 ICM 相比，它规范 TE 标识； LMNA/C 耗竭导致 ICM 样转录状态 让人想起 Hippo 通路的激活。在目标 1 (K99) 中，我将研究 Lamin A/C 对 Hippo 的调节以及 确定细胞骨架网络的机械传感在信号传导调节中的作用。目标 2 （K99/R00），为了确定机械力如何调节染色质，我将应用先进的电子显微镜 体内核小体分辨率染色质结构可视化的方法。培训期间在 Giraldez 实验室，我将应用一种新颖的标记策略，结合冷冻电镜来表征层异染色质 互动。在 R00 期间及之后，我将应用这些方法来确定如何压缩 胚胎和细胞核上机械力的产生影响染色质结构和 ICM/TE 命运。在目标 3 (R00) 中，我将使用嵌合胚胎和其他发育分析来检查 细胞核的机械特性影响分化潜能。我还将量化核刚度和 发育中的小鼠胚胎中的染色质结构。这项工作为更深入地了解 机械力在调节基因表达和细胞身份中的作用。该提案汇集了培训 以及我在整个教育过程中获得的概念和新方法（RNA-seq 和冷冻电镜） 我将在我的导师（Giraldez）的实验室、耶鲁大学的其他科学家和专家以及我的同事的实验室学习。 导师伊丽莎白·维拉（Elizabeth Villa）和伯纳·索森（Berna Sozen）。该提案将完成我的博士后培训并为我做好准备 我的最终目标是运行一个有竞争力的独立研究项目。