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 内部细胞质量（ICM）和滋养剂（TE）是哺乳动物期间指定的前两个细胞类型 发展。 TE细胞支持植入并引起plapeta，而ICM细胞形成胚胎 还有一些胚外组织。因此，它们的分化对于成功怀孕至关重要。 机械调节的河马信号通路在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的命运 但是，植入尚未完全理解。我假设机械力通过 细胞骨架，通过调节河马信号传导和染色质结构来调节TE转录程序。 在此提案中，我将通过定义核张力如何调节河马信号传导和 早期胚胎区分期间的染色质组织。我的最终目标是定义机械链接 将机械和调节途径连接到细胞和染色质状态。这项工作将增强我们的 了解与早期胚胎发生和植入的关系的细胞命运规范以及更多 广泛。我在Giraldez实验室中的博士后工作表明，lamin a/c在TE中被转录上调， 与ICM相比，它调节TE识别； LMNA/C部署导致类似ICM的转录状态 让人联想到河马途径激活。在AIM 1（K99）中，我将调查层lamin a/c对河马的调节， 确定细胞骨架网络在该信号传导调控中的机械感测的作用。在目标2中 （K99/r00），为了确定机械力如何调节染色质，我将应用高级电子显微镜 在体内可视化核能分辨率染色质结构的方法。在培训期间 Giraldez实验室，我将采用一种新颖的标签策略，并结合冷冻em来表征椎板异染色质 互动。在R00期间及以后，我将采用这些方法来确定如何压实 在核冲击染色质结构和ICM/TE上的胚胎和机械力的产生 命运。在AIM 3（R00）中，我将使用嵌合胚和其他发育测定法来检查如何变化 核的机械性能会影响分化潜力。我还将量化核刚度和 发育小鼠胚胎中的染色质结构。这项工作为更深入了解 机械力在调节基因表达和细胞身份中的作用。该提议汇集了培训 以及我在整个教育和新方法（RNA-Seq和Cryo-EM）中获得的概念 我将从耶鲁大学的其他科学家和专家中学到我的心态（Giraldez）实验室，以及我的共同实验室 导师，伊丽莎白·维拉（Elizabeth Villa）和伯纳（Berna Sozen）。该建议将完成我的博士后培训，并为我做好准备 为了我运行竞争性独立研究计划的最终目标。