Mapping protein signatures to single allele chromatin topologies at genomic resolution

在基因组分辨率下将蛋白质特征映射到单等位基因染色质拓扑

基本信息

批准号：
10649096
负责人：
Jan-Hendrik Spille
金额：
$ 21.14万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-07 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10649096
关键词：
3-Dimensional Acceleration Alleles Antibody Repertoire Architecture Binding Binding Proteins Binding Sites Biological Assay Boundary Elements Cell Culture Techniques Cell Nucleus Cells Cellular Assay Cellular biology ChIP-seq Chromatin Chromatin Structure Chromosome Territory Chromosomes Complement DNA Sequence Data Data Set Detection Development Disease Elements Enhancers Epigenetic Process Frequencies Gene Expression Genes Genetic Transcription Genome Genomics Health Hi-C Human Image Immune system Individual Interphase Knowledge Length Link Maps Measurement Methods Molecular Molecular Conformation Nature Nuclear Nuclear Proteins Nucleosomes Optics Outcome Performance Population Post-Translational Protein Processing Process Property Proteins Protocols documentation Reading Regulatory Element Resolution Sampling Signaling Protein Stretching Structure Structure-Activity Relationship Technology Time Variant Visualization chromatin immunoprecipitation chromatin protein chromosome conformation capture color detection combinatorial detection limit genomic locus high risk histone modification innovation insight instrumentation interest multidimensional data nanometer novel parallelization promoter reconstruction segregation single molecule stem cell population stem cells superresolution microscopy timeline transcription factor ultra high resolution

项目摘要

Interphase chromatin is hierarchically organized in chromosome territories, active and inactive compartments, and topologically associating domains (TADs). Gene expression is controlled by regulatory chromatin elements (enhancers and promoters) that bind transcription factors and interact within, but not across TAD boundaries. Recent advances in single cell biology have revealed a tremendous amount of cell-to-cell variability in both chromatin topology and protein coverage. Even though TADs appear to delineate functional units at the population level, their boundaries only emerge as average properties from large ensembles of cells. TAD-like structures persist even in the absence of boundaries at the ensemble level. Similarly, single cell ChIP-seq has revealed sub-states with distinct epigenetic profiles at enhancers in a population of stem cells. But no assay exists to link topological and functional variability by reading out protein coverage and epigenetic signatures simultaneously from single cell chromatin traces. Here, we will leverage recent advances in multiplexed chromatin imaging and single molecule super-resolution microscopy to fill in this gap. In Aim I, we will characterize the tradeoff between sequence resolution and spatial precision in multiplexed chromatin imaging. We will then use optimized conditions to map super-resolved protein signal to chromatin topologies at genomic resolution. In Aim II, we will extend the capabilities of the assay to detect multiple protein signatures simultaneously. Such combinatorial data on protein signatures of regulatory elements at genomic resolution is not available through any other single cell assay. We will further characterize the performance of the assay under challenging conditions by mapping both stably integrated, widespread histone modifications and transiently binding sequence-specific transcription factors with a sizable unbound fraction to chromatin. Using computational clustering strategies, we will stratify the data by chromatin topology to determine if structural variation is driven by specific protein factors such as transcription factors that orchestrate long- range enhancer interactions. Finally, we will establish protocols and technological solutions to accelerate acquisition, processing, and visualization of statistically meaningful datasets comprising 100s-1000s of single alleles. The resulting datasets will provide unprecedented insight into the molecular mechanisms underlying cell-to-cell variability in chromatin topology and serve as a powerful hypothesis generator for investigating single cell genome structure-function relationships.

相间染色质在染色体区域，活性和不活跃的室内层次组织，以及拓扑关联域（TADS）。基因表达受调节染色质元素的控制（增强剂和启动子）结合转录因子并在内部相互作用但在TAD边界之间进行相互作用。最近的进步单细胞生物学揭示了染色质拓扑和蛋白质中的细胞对细胞变异性大量变化覆盖范围。即使TAD似乎在人口层面描绘了功能单位，但它们的边界也只会出现为大型细胞集合的平均特性。即使在没有边界的情况下，tad状结构仍然存在合奏水平。同样，单细胞芯片seq揭示了在A 干细胞的种群。但是，不存在通过阅读蛋白质覆盖范围来链接拓扑和功能变异性的测定以及来自单细胞染色质痕迹的同时表观遗传特征。在这里，我们将利用最近的进步多路复用染色质成像和单分子超分辨率显微镜以填补这一空白。在目标一世中，我们会表征多路复用染色质成像中序列分辨率和空间精度之间的权衡。我们将然后使用优化的条件将超级分辨蛋白质信号映射到基因组分辨率下的染色质拓扑。目标 II，我们将扩展测定能力同时检测多个蛋白质特征。这样的组合基因组分辨率调节元件的蛋白质特征的数据无法通过任何其他单一细胞获得测定。我们将通过稳定地绘制在具有挑战性的条件下进一步表征分析的性能具有相当大的综合，广泛的组蛋白修饰和瞬时结合序列特异性转录因子与染色质无结合的分数。使用计算聚类策略，我们将通过染色质拓扑分类数据确定结构变化是否由特定蛋白质因子驱动范围增强器相互作用。最后，我们将建立协议和技术解决方案，以加速获取，处理和可视化统计有意义的数据集，其中包含100s-1000的单个等位基因。结果数据集将对染色质细胞对细胞变异性的分子机制提供前所未有的见解拓扑并用作研究单细胞基因组结构功能的强大假设发生器关系。