Locus-specific Imaging of Dynamic Histone Methylations during Reprogramming

重编程过程中动态组蛋白甲基化的位点特异性成像 将成纤维细胞重编程为诱导多能干细胞（iPSC）代表了革命性的进步 在理解特定的基因调控如何指导细胞的生命方面。表观遗传修饰 包括染色质重塑是重编程过程中的早期事件。组蛋白甲基化. 不同的残基可以募集不同的染色质重塑复合物来调节染色质 结构并相应地沉默/激活基因表达。这些组蛋白甲基化及其 在不同基因组基因座的组合可以作为确定总体基因表达谱的代码， 表型结果。然而，在特定位点的组蛋白甲基化是如何动态变化的， 在重编程过程中，细胞经历高度异质性的调节， 单细胞水平。在这项提案中，我们将利用定向进化和高通量筛选的力量 系统开发特异性/灵敏性FRET生物传感器的方法 用于监测单细胞中关键的组蛋白甲基化。我们将进一步开发生物传感器， 和正交FRET对，可以同时监测两个不同的组蛋白甲基化在一个单一的生活 细胞生产的高分辨率图像的多表观遗传景观。这些多重组蛋白 然后分析在重编程期间从单个细胞获得的甲基化并将其整合在一起 构建动态组蛋白甲基化景观。这些表观遗传调节也将被可视化 在特定的位点上分配相应的基因组地址， 甲基化已建立的细胞命运的荧光标记物将进一步应用于确定组蛋白 协调甲基化密码以调节重编程。因此，该项目的成功 应该在单细胞水平的表观遗传学和遗传学领域产生变革性的影响，特别是有关 干细胞重编程因此，提出了三个具体目标：目标1。开发高通量 筛选FRET生物传感器的工程方法，以监测各种组蛋白甲基化;目的2。 设计具有不同颜色的FRET生物传感器，以监测组蛋白不断变化的多重景观 重编程过程中的甲基化;目标3。揭开特定基因座上不断演变的组蛋白甲基化景观 在重新编程过程中。虽然该提案的重点是开发针对组蛋白甲基化的工具， 具体位点和重编程结果，策略和方法可以扩展到监测， 原则上，单细胞中的任何表观遗传修饰，包括但不限于组蛋白乙酰化和 磷酸化所开发的用于表观遗传景观进化的单细胞成像的生物传感器应该提供 生命科学、生物医学研究和组织工程的强大工具。结果是 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加密的 以一种综合的方式来调节生命。

Locus-specific Imaging of Dynamic Histone Methylations during Reprogramming Reprogramming fibroblasts into induced pluripotent stem cells (iPSCs) represents a revolutionary advancement in the understanding of how specific gene regulations can guide the life of a cell. Epigenetic modifications including chromatin remodeling are early events during the reprogramming process. 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 at specific loci are dynamically regulated during the reprogramming processes in which cells undergo a highly 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 biosensors with distinct and orthogonal FRET pairs that can simultaneously monitor two different histone methylations in a single live cell for the production of high-resolution images of multiplex epigenetic landscapes. These multiplex histone methylations obtained from individual cells during reprogramming will then be analyzed and integrated together to construct the dynamic histone methylation landscapes. These epigenetic modulations will also be visualized at specific loci to assign the corresponding genomic addresses on the evolving landscape of histone methylations. Established fluorescence markers of cell fate will further be applied to determine how histone methylation codes are coordinated for the regulation of reprogramming. As such, the success of the project should have transformative impact in the field of epigenetics and genetics at single cell levels, particularly related to stem cell reprogramming. Three specific aims are accordingly proposed: Aim 1. Develop high-throughput screening methods for the engineering of FRET biosensors to monitor various histone methylations; Aim 2. Engineer FRET biosensors with distinct colors to monitor the evolving multiplex landscape of histone methylations during reprogramming; Aim 3. Unravel the evolving histone methylation landscapes at specific loci during reprogramming. While the focus of this proposal is to develop tools targeting histone methylations at specific loci and reprogramming outcomes, the strategies and approaches can be extended to monitor, in principle, any epigenetic modification in single cells, including but not limited to histone acetylation and phosphorylation. The developed biosensors for single cell imaging of epigenetic landscape evolution should offer powerful tools for life science, biomedical research, and tissue engineering in general. 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.