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

胚胎发育过程中三维表观遗传景观演化的单细胞示踪 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 着床前胚胎发育(PED)，第一个细胞命运是如何决定的仍不清楚，也是 对于理解特定的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的早期事件。不同残基上的组蛋白甲基化可以招募 不同染色质重塑复合体调控染色质结构和沉默/激活基因 相应的表达方式。这些组蛋白甲基化和它们在不同基因组位置的组合可以 作为决定整体基因表达谱和表型结果的密码。然而，它仍然不是 了解组蛋白甲基化如何动态调节特定基因座上的染色质结构 在PED期间，细胞在单细胞水平上经历异质调制。在这项提案中，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/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单个作图的RNA-染色质相互作用 在PED过程中识别关键的RNA-基因组相互作用位点。然后，我们将使用 核酸内切酶缺失Cas9(DCas9)、小引导RNA(SgRNAs)和裂解FP识别和跟踪 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性 表观遗传调节剂指导组蛋白在特定位点的调节并阐明它们在细胞决定中的作用 PED期间的命运。鉴于不同基因座表观遗传修饰的重要性，该项目的成功 在理解特定位点的表观遗传学在决定细胞中的作用方面应该有变革性的影响 PED期间的命运。因此，我们提出了三个目标：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：可视化基因位点特异性的组蛋白修饰 PED；目的3.在PED期间重新编程特定位点的组蛋白修饰。虽然这项提案的重点是 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，这些战略和方法原则上可以扩展到监测任何其他表观遗传学 单细胞修饰，包括但不限于组蛋白乙酰化和磷酸化。结果来自于 这个项目还可以直接导致动态核图集，说明组蛋白密码是多么具体 以一种综合的方式加密，以调节生活。

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.