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.

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