Single Cell Tracking of 3D Epigenetic Landscape Evolution During Embryonic Development

胚胎发育过程中 3D 表观遗传景观演化的单细胞追踪

基本信息

批准号：
10344905
负责人：
Yingxiao Wang
金额：
$ 67.94万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-03-01 至 2027-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10344905
关键词：
3-Dimensional Acetylation Affect Architecture Atlases Biological Sciences Biosensor Cell Cycle Cell Differentiation process Cell physiology Cells Cellular biology Chromatin Chromatin Remodeling Factor Chromatin Structure Code DNA biosynthesis Development Directed Molecular Evolution Embryo Embryonic Development Engineering Epigenetic Process Event Evolution Fluorescence Resonance Energy Transfer Gene Expression Gene Expression Profile Gene Expression Regulation Genetic Transcription Genome Genomics Guide RNA Histone Acetylation Histone Code Histones Image Lead Life Light Mammalian Cell Masks Methods Methylation Mitotic Modeling Modification Molecular Monitor Mus Mutagenesis Nuclear Outcome Pattern Phenotype Phosphorylation Play Population Positioning Attribute Process Proteins RNA Regenerative Medicine Regulation Reporting Reproducibility Resolution Role Signal Transduction Site Technology Testing Transcript Work base blastomere structure cellular imaging chromatin remodeling embryo stage 2 encryption endonuclease genomic locus high throughput screening histone methylation histone modification insight next generation sequencing novel preimplantation recruit repaired spatiotemporal stem cells success tool transcriptome

项目摘要

Single Cell Tracking of 3D Epigenetic Landscape Evolution During Embryonic Development An important question to cell biology is how cells break the symmetry during mitotic divisions. During mammalian pre-implantation embryonic development (PED), how the first cell fate decision is made remains unclear and is crucial for the understanding of how specific gene regulations can guide the life of a cell. Epigenetic modifications including chromatin remodeling are early events during PED. Histone methylation at different residues can recruit differential sets of chromatin remodeling complexes to regulate chromatin structures and silence/activate gene expressions accordingly. These histone methylations and their combinations at different genomic loci can serve as codes to determine the overall gene expression profile and phenotypic outcomes. However, it is still not understood how histone methylations and hence chromatin structures at specific loci are dynamically regulated during PED in which cells undergo a heterogeneous modulation at single cell levels. In this proposal, we will harness the power of directed evolution and high-throughput screening method to systematically develop specific/sensitive FRET (fluorescence resonance energy transfer) biosensors for the monitoring of crucial histone methylations in single cells. We will further develop and apply the mapping RNA-chromatin interactions in single cells (sciMARGI) to identify crucial RNA-genome interaction sites during PED. We will then employ the endonuclease-deficient Cas9 (dCas9), small guide RNAs (sgRNAs) and split FPs to identify and track the positons of specific loci crucial for embryonic cell differentiation. Ultimately, we will apply our controllable epigenetic modulators to guide the histone modulations at specific loci and elucidate their role in determining cell fates during PED. Given the importance of epigenetic modifications at different loci, the success of the project should have transformative impact in understanding the role of locus-specific epigenetics in determining the cell fate during PED. Accordingly, three aims are proposed: Aim 1. Spatiotemporal imaging of crucial histone methylations in single live cells and during PED; Aim 2. Visualize the locus-specific histone modifications during PED; Aim 3. Reprogram the locus-specific histone modifications during PED. While the focus of this proposal is to develop tools targeting histone methylations and chromatin structures at specific loci and differentiation outcomes, the strategies and approaches can be extended to monitor, in principle, any other epigenetic modification in single cells, including but not limited to histone acetylation and phosphorylation. The results from this project can also lead directly to the dynamic nuclear atlas illustrating how specific histone codes are encrypted in an integrative manner for the regulation of life.

胚胎发育过程中 3D 表观遗传景观演化的单细胞追踪细胞生物学的一个重要问题是细胞如何在有丝分裂过程中打破对称性。在哺乳动物时期在植入前胚胎发育（PED）中，第一个细胞命运决定是如何做出的仍不清楚，并且对于理解特定基因调控如何指导细胞的生命至关重要。表观遗传修饰包括染色质重塑是 PED 期间的早期事件。不同残基的组蛋白甲基化可以招募不同组的染色质重塑复合物调节染色质结构和沉默/激活基因相应的表达。这些组蛋白甲基化及其在不同基因组位点的组合可以发挥作用作为确定总体基因表达谱和表型结果的代码。然而，这仍然不是了解组蛋白甲基化以及特定位点的染色质结构如何动态调节在 PED 过程中，细胞在单细胞水平上经历异质调节。在本提案中，我们将利用定向进化和高通量筛选方法的力量系统地开发用于监测关键组蛋白的特异性/灵敏 FRET（荧光共振能量转移）生物传感器单细胞中的甲基化。我们将进一步开发和应用 RNA-染色质相互作用作图细胞 (sciMARGI) 来识别 PED 期间关键的 RNA-基因组相互作用位点。然后我们将采用核酸内切酶缺陷型 Cas9 (dCas9)、小向导 RNA (sgRNA) 和分裂 FP 可识别和追踪对胚胎细胞分化至关重要的特定基因座的位置。最终，我们将运用我们的可控表观遗传调节剂指导特定位点的组蛋白调节并阐明它们在决定细胞中的作用 PED 期间的命运。鉴于不同位点表观遗传修饰的重要性，该项目的成功对于理解位点特异性表观遗传学在确定细胞中的作用应该具有变革性的影响 PED期间的命运。因此，提出了三个目标：目标 1. 关键组蛋白的时空成像单个活细胞和 PED 过程中的甲基化；目标 2. 可视化过程中位点特异性组蛋白修饰 PED；目标 3. 在 PED 期间重新编程位点特异性组蛋白修饰。虽然该提案的重点是开发针对特定位点和分化的组蛋白甲基化和染色质结构的工具结果，策略和方法原则上可以扩展到监测任何其他表观遗传单细胞的修饰，包括但不限于组蛋白乙酰化和磷酸化。结果来自该项目还可以直接生成动态核图谱，说明特定组蛋白代码的作用一体化加密，调节生活。