Visualizing local and global chromatin architecture, and gene expression in the individual cell by structural (SUSHI) and temporal (3D-SMRT) single molecule imaging
通过结构 (SUSHI) 和时间 (3D-SMRT) 单分子成像可视化局部和整体染色质结构以及单个细胞中的基因表达
基本信息
- 批准号:9749351
- 负责人:
- 金额:$ 10万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2018
- 资助国家:美国
- 起止时间:2018-09-01 至 2019-06-30
- 项目状态:已结题
- 来源:
- 关键词:3&apos Untranslated RegionsAlgorithmic AnalysisArchitectureAtlasesBinding ProteinsBiochemicalCRISPR/Cas technologyCell LineCell NucleusCell modelCellsChromatinChromatin LoopChromosome MappingChronologyCodeCore FacilityCustomDNADataDetectionDevelopmentDimensionsDiseaseERG geneElectron MicroscopyElementsEnhancersFOS ProteinFOS geneFluorescenceGene ActivationGene ExpressionGenesGenetic TranscriptionGenomeGenomicsHeLa S3HourImageImage AnalysisImaging DeviceImaging technologyIndividualLabelLightMammalian CellManuscriptsMapsMessenger RNAMethodologyMethodsMicroscopeMicroscopyMovementNuclearNuclear PoreNuclear StructurePathway interactionsProcessProteinsReactionResolutionRestSpecific qualifier valueStainsStructureSystemTechniquesTechnologyTestingTimeTranslatingUnited States National Institutes of HealthValidationbasecellular developmentcomputerized data processingcryogenicsdesignexperimental studyfallsgene productgenome-widehigh resolution imagingimage processingimage registrationimaging modalityimaging platforminterestlight microscopymRNA ExportmRNA Expressionmammalian genomemembermillisecondmolecular imagingnanometernanometer resolutionnovelnovel imaging techniqueorganizational structurequantitative imagingresponsesingle moleculespatiotemporalstemstructural genomicstemporal measurementtool
项目摘要
Project Summary:
Responding to the NIH’s 4D Nucleome FOA for Imaging Tools, we propose here two novel imaging methods.
When combined, these methods provide nanometer spatial resolution, and millisecond to hour temporal
resolution of dynamic chromatin architecture rearrangement and its relation to cell activation and transcription.
The first novel technique, termed SUSHI (SUb-zeroº-Stochastic-High-resolution-Imaging platform), enables
quantitative, stochastic, single molecule imaging by combining intelligent labeling design with cryogenic
fluorescence and emitter control using polarized excitation and depletion. This results in 1-5 nanometer
isotropic structural resolution of nuclear chromatin, and the ability to discern DNA elements such as enhancers,
suppressors or gene loci that can be mapped and tracked.
The second method, termed 3D-SMRT Microscopy (three-dimensional Single-Molecule Real-Time
microscopy, manuscript submitted), is an expansion on the recently described MFM (multi-focus microscopy).
It provides real-time, simultaneous, multicolor, 30-80 nanometer-resolution tracking in the living cell at a
millisecond to hour timescale. By implementing a well thought out labeling strategy, this method also allows for
the detection of DNA elements and their nuclear movement in time and space.
To be able to analyze hundreds of cells in different activation states, we also describe our streamlined image
processing workflows for both SUSHI and 3D-SMRT, permitting the automated analysis of multiple loci of
hundreds of single cells and many activation states.
We will make the imaging platforms available to all members of the 4D Nucleome consortium by placing the
microscopes in a core facility at UMMS, and will share all data processing techniques via code sharing.
We focus on the FOS gene locus, as this “immediate-early response” gene has low to no expression of c-Fos
mRNA and protein at rest. Upon activation, however, there is a rapid induction of c-Fos, which persists only for
a few hours. Heat maps depicting intrachromosomal interaction matrices around the FOS gene indicate
extensive looping at this locus. By labeling DNA in a living cell, we can resolve chromatin looping changes
around the locus and its surrounding enhancers, determine potential gene locus positional movements toward
the nuclear periphery and nuclear pores, and correlate this with mRNA expression and export, both at rest and
upon activation.
By collaborating with members of the 4D Nucleome consortium, we envision the final stage of this project to
include direct correlation with biochemical, structural and genome-wide mapping derived data, thereby
shedding light on how genomic information specifies proper execution of spatial and temporal gene expression
at rest, upon activation, during cellular development and in diseased states.
项目总结:
为了响应美国国立卫生研究院的四维核基因组成像工具,我们在这里提出了两种新的成像方法。
当结合使用时,这些方法可以提供纳米级的空间分辨率,以及毫秒到小时的时间分辨率
染色质结构动态重排的分辨率及其与细胞激活和转录的关系。
第一种新技术,称为寿司(零下随机高分辨率成像平台),使
智能标记设计与低温相结合的定量、随机、单分子成像
利用偏振激发和耗尽控制荧光和发射体。这导致了1-5纳米
核染色质的各向同性结构分辨率,以及识别DNA元件如增强剂的能力,
可被定位和追踪的抑制子或基因位点。
第二种方法,称为3D-SMRT显微镜(三维单分子实时
显微镜,提交的手稿),是对最近描述的MFM(多焦点显微镜)的扩展。
它在活细胞中提供实时、同步、多色、30-80纳米分辨率的跟踪
毫秒到小时的时间刻度。通过实施经过深思熟虑的标签策略,该方法还允许
对DNA元素及其核在时间和空间上的运动的检测。
为了能够分析数百个处于不同激活状态的细胞,我们还描述了我们的流线型图像
寿司和3D-SMRT的处理工作流程,允许自动分析多个基因座
数以百计的单个细胞和许多激活状态。
我们将把成像平台放置在
在密歇根州立大学的核心设施中使用显微镜,并将通过代码共享共享所有数据处理技术。
我们把重点放在fos基因座上,因为这个“即刻-早期反应”基因低表达或不表达c-fos。
静止时的信使核糖核酸和蛋白质。然而,一旦激活,c-Fos就会迅速诱导,这种诱导只在
几个小时。描绘Fos基因周围染色体内相互作用矩阵的热图表明
在这个轨迹上有大范围的循环。通过在活细胞中标记DNA,我们可以解决染色质环状变化
在该基因座及其周围的增强子周围,确定潜在的基因座位置向
核周和核孔,并与mRNA的表达和输出相关,在静息和
在激活时。
通过与4D基因组联盟的成员合作,我们设想这个项目的最后阶段将
包括与生化、结构和全基因组图谱导出数据直接相关,从而
阐明基因组信息如何指定空间和时间基因表达的正确执行
处于静止状态、处于激活状态、处于细胞发育期间以及处于患病状态。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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David Grunwald其他文献
David Grunwald的其他文献
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{{ truncateString('David Grunwald', 18)}}的其他基金
Visualizing local and global chromatin architecture, and gene expression in the individual cell by structural (SUSHI) and temporal (3D-SMRT) single molecule imaging
通过结构 (SUSHI) 和时间 (3D-SMRT) 单分子成像可视化局部和整体染色质结构以及单个细胞中的基因表达
- 批准号:
9306084 - 财政年份:2015
- 资助金额:
$ 10万 - 项目类别:
Visualizing local and global chromatin architecture, and gene expression in the individual cell by structural (SUSHI) and temporal (3D-SMRT) single molecule imaging
通过结构 (SUSHI) 和时间 (3D-SMRT) 单分子成像可视化局部和整体染色质结构以及单个细胞中的基因表达
- 批准号:
9918495 - 财政年份:2015
- 资助金额:
$ 10万 - 项目类别:
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