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

• 批准号: 9922921
• 负责人: SHU CHIEN
• 金额: $ 58.54万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-03 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9922921
• 关键词: 3-Dimensional; Acetylation; Address; Affinity; Atlases; Binding; Biological Sciences; Biomedical Research; Biosensor; Cell physiology; Cells; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Code; Color; Directed Molecular Evolution; Engineering; Epigenetic Process; Event; Evolution; Fibroblasts; Fluorescence; Fluorescence Resonance Energy Transfer; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genetic; Genomics; Guide RNA; Histone Acetylation; Histone Code; Histones; Image; Individual; Lead; Life; Masks; Medicine; Methods; Methylation; Modification; Molecular; Monitor; Nuclear; Oranges; Outcome; Pattern; Phenotype; Phosphorylation; Positioning Attribute; Process; Production; Proteins; Regenerative Medicine; Regulation; Reporter; Role; Sensitivity and Specificity; Signal Transduction; Specificity; Spottings; System; Tissue Engineering; Visualization; Yeasts; base; cellular imaging; chromatin remodeling; design; encryption; endonuclease; genomic locus; high resolution imaging; high throughput screening; histone methylation; histone modification; improved; induced pluripotent stem cell; insight; matrigel; multiplexed imaging; nanobodies; novel; photoactivation; recruit; spatiotemporal; stem cell biology; stem cells; stemness; success; tool

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.

重新编程过程中动态组蛋白甲基化的基因座特异性成像 将成纤维细胞重编程为诱导的多能干细胞（IPSC）代表革命性的进步 在理解特定基因法规如何指导细胞寿命的过程中。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染色质重塑络合物的差异集以调节染色质 结构和沉默/激活基因表达。这些组蛋白甲基及其 不同基因组基因局的组合可以用作确定总体基因表达谱和 表型结果。但是，仍然尚不清楚特定基因座的组蛋白甲基化如何动态 在重编程过程中受到调节，其中细胞经过高度异质的调制 单细胞水平。在此提案中，我们将利用定向进化和高通量筛查的力量 系统地开发特定/敏感的品格（荧光共振能量传递）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。我们将进一步开发具有独特的生物传感器 和正交的晶格对，可以同时监测单个活的两个不同的组蛋白甲基化 用于生产多重表观遗传景观高分辨率图像的细胞。这些多重组蛋白 然后将在重编程过程中从单个细胞获得的甲基化进行分析并集成在一起 构建动态组蛋白甲基化景观。这些表观遗传调制也将被可视化 在特定基因座，分配组蛋白不断发展的景观的相应基因组地址 甲基化。细胞命运的已建立荧光标记将进一步应用以确定组蛋白 甲基化代码是协调的，以调节重编程。因此，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具有变革性影响，尤其是相关的 进行干细胞重编程。因此提出了三个具体目标：目标1。发展高通量 筛选方法的FRET生物传感器来监测各种组蛋白甲基化的方法；目标2。 工程师FRET生物传感器具有不同颜色，以监视组蛋白的不断发展的多重景观 重编程过程中的甲基化；瞄准3。在特定基因座揭示不断发展的组蛋白甲基化景观 在重新编程期间。虽然该提案的重点是开发针对组蛋白甲基化的工具 特定的基因座和重编程结果，可以扩展策略和方法以监视， 原理，单个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。用于表观遗传景观演化的单细胞成像的开发的生物传感器应提供 一般而言，生命科学，生物医学研究和组织工程的强大工具。从中的结果 项目还可以直接导致动态核地图集，以说明特定组蛋白代码的加密方式 以整合生活的方式调节生活。

项目成果

Removing physiological motion from intravital and clinical functional imaging data.

• DOI: 10.7554/elife.35800
• 发表时间: 2018-07-09
• 期刊: eLife
• 影响因子: 7.7
• 作者: Warren SC;Nobis M;Magenau A;Mohammed YH;Herrmann D;Moran I;Vennin C;Conway JR;Mélénec P;Cox TR;Wang Y;Morton JP;Welch HC;Strathdee D;Anderson KI;Phan TG;Roberts MS;Timpson P
• 通讯作者: Timpson P