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。哺乳动物期间 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然不清楚， 这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期间的早期事件。不同残基的组蛋白甲基化可以募集 不同的染色质重塑复合物组来调节染色质结构并沉默/激活基因 相应的表情。这些组蛋白甲基化和它们在不同基因组位点的组合可以 作为确定总体基因表达谱和表型结果的代码。然而， 了解组蛋白甲基化以及特定位点的染色质结构是如何动态调节的 在PED期间，细胞在单细胞水平上经历异质性调节。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， 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。我们将进一步发展和应用RNA-染色质相互作用的作图技术， 细胞（sciMARGI），以确定PED期间的关键RNA-基因组相互作用位点。然后我们将使用 使用核酸内切酶缺陷型Cas9（dCas 9）、小向导RNA（sgRNA）和分裂FP来鉴定和追踪这些基因。 对胚胎细胞分化至关重要的特定位点的位置。最终，我们将应用我们可控的 表观遗传调节剂，以指导特定位点的组蛋白调节，并阐明其在决定细胞增殖中的作用。 在PED期间的命运。鉴于表观遗传修饰在不同基因座的重要性，该项目的成功 应该对理解基因座特异性表观遗传学在决定细胞遗传学中的作用产生变革性的影响。 在PED期间的命运因此，提出了三个目标：目标1。关键组蛋白的时空显像 单个活细胞和PED期间的甲基化;目的2。可视化基因座特异性组蛋白修饰， 目标3.在PED期间重新编程基因座特异性组蛋白修饰。虽然该提案的重点是 开发针对特定基因座和分化的组蛋白甲基化和染色质结构的工具 结果，策略和方法可以扩展到监测，原则上，任何其他表观遗传， 这些修饰包括但不限于组蛋白乙酰化和磷酸化。的结果 该项目还可以直接导致动态核图谱，说明特定的组蛋白编码是如何 以一种整合的方式进行加密，以调节生命。

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.