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.

间期染色质在染色体区域、活性区室和非活性区室中分层组织，拓扑关联域（TAD）。基因表达由调节染色质元件（增强子）控制和启动子）结合转录因子并在 TAD 边界内相互作用，但不跨越 TAD 边界。最近的进展单细胞生物学揭示了染色质拓扑和蛋白质的巨大细胞间变异性覆盖范围。尽管 TAD 似乎在人口水平上描绘了功能单位，但它们的边界仅在以下情况下出现：来自大型细胞群的平均特性。即使在没有边界的情况下，类似 TAD 的结构仍然存在合奏级别。同样，单细胞 ChIP-seq 揭示了增强子中具有不同表观遗传特征的亚状态。干细胞群体。但目前还没有通过读取蛋白质覆盖率来联系拓扑和功能变异性的分析方法和表观遗传特征同时来自单细胞染色质痕迹。在这里，我们将利用最新的进展多重染色质成像和单分子超分辨率显微镜填补了这一空白。在目标一中，我们将表征多重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以基因组分辨率将超分辨蛋白质信号映射到染色质拓扑。瞄准 II，我们将扩展该测定的能力，以同时检测多个蛋白质特征。这样的组合任何其他单细胞都无法获得基因组分辨率下调控元件的蛋白质特征数据化验。我们将通过稳定地映射两者来进一步表征该测定在具有挑战性的条件下的性能整合的、广泛的组蛋白修饰和瞬时结合序列特异性转录因子未结合染色质的部分。使用计算聚类策略，我们将按染色质拓扑对数据进行分层确定结构变异是否是由特定的蛋白质因子驱动的，例如协调长链的转录因子范围增强剂相互作用。最后，我们将建立协议和技术解决方案来加速收购，对包含 100-1000 个单个等位基因的具有统计意义的数据集进行处理和可视化。由此产生的数据集将为细胞间染色质变异的分子机制提供前所未有的见解拓扑并作为研究单细胞基因组结构功能的强大假设生成器关系。