A marker-free technology for mapping the epigenome of cell types in mammalian tissues

用于绘制哺乳动物组织细胞类型表观基因组图谱的无标记技术

基本信息

批准号：
10341084
负责人：
Siddharth Subhas Dey
金额：
$ 37.43万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA BARBARA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-04 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10341084
关键词：
Address Aging Animal Model Antibodies Bar Codes Binding Biochemical Biological Assay Cell Line Cell Separation Cell surface Cells Chromatin Chromosome Mapping Complex Consumption DNA DNA Methylation DNA-Protein Interaction Data Development Disease Epigenetic Process Eye Fluorescence Frequencies Gene Expression Gene Expression Regulation Generations Genetic Transcription Genome Genome Mappings Genomic DNA Goals Homeostasis Human Investigation Knowledge Malignant Neoplasms Maps Measurement Messenger RNA Methods Nuclear Lamina Nucleic Acids Organism Pattern Phenotype Play Polycomb Proteins Rattus Regulator Genes Reporter Retina Sampling System Techniques Technology Time Tissue Sample Tissues Transgenic Animals Work cell type combinatorial developmental disease epigenome experimental study genome-wide high throughput technology histone modification improved in silico in vivo methylome multiple omics new technology physical separation programs relating to nervous system single cell sequencing single cell technology transcription factor transcriptome transcriptomics

Address Aging Animal Model Antibodies Bar Codes Binding Biochemical Biological Assay Cell Line Cell Separation Cell surface Cells Chromatin Chromosome Mapping Complex Consumption DNA DNA Methylation DNA-Protein Interaction Data Development Disease Epigenetic Process Eye Fluorescence Frequencies Gene Expression Gene Expression Regulation Generations Genetic Transcription Genome Genome Mappings Genomic DNA Goals Homeostasis Human Investigation Knowledge Malignant Neoplasms Maps Measurement Messenger RNA Methods Nuclear Lamina Nucleic Acids Organism Pattern Phenotype Play Polycomb Proteins Rattus Regulator Genes Reporter Retina Sampling System Techniques Technology Time Tissue Sample Tissues Transgenic Animals Work cell type combinatorial developmental disease epigenome experimental study genome-wide high throughput technology histone modification improved in silico in vivo methylome multiple omics new technology physical separation programs relating to nervous system single cell sequencing single cell technology transcription factor transcriptome transcriptomics

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项目摘要

PROJECT SUMMARY The genome within cells of a multicellular organism is identical, yet distinct cell types display varied functions due to differences in their epigenome. Therefore, mapping the genome-wide epigenetic landscape of different cell types within a tissue is critical for understanding cell type-specific gene expression regulation. Techniques to map epigenetic factors currently rely on our ability to isolate the desired cell types at high purity with certain biochemical assays, such as quantifying protein-DNA contacts, also requiring a large number of starting cells. However, cell type-specific markers and antibodies are frequently unknown or unavailable, presenting a major challenge in isolating cell types at high purity. While transgenic animal models that express cell type-specific fluorescent reporters can overcome this limitation in some cases, generation of these animal models is time consuming. Further, tissues frequently contain rare cell types, making it challenging to isolate large numbers of such cells that are required for assays mapping the binding landscape of transcription factors or chromatin modifying proteins. To overcome limitations of these current approaches, the overall goal of this proposal is to establish a marker-free high-throughput technology to map the epigenome of different cell types within a tissue by developing single-cell sequencing methods to simultaneously quantify the transcriptome and epigenome from the same cell. The single-cell transcriptomes will be used for the unbiased identification of cell types in silico, and the corresponding epigenomes of cells belonging to the same cell type will be pooled to generate high- quality cell type-specific epigenetic landscapes. More specifically, in Aim 1 we propose to develop a single-cell multiomics technology to simultaneously quantify mRNA, 5mC and DNA accessibility from the same cell. Unlike a recently developed method that makes these measurements by physically separating mRNA from genomic DNA, our technology does not involve the physical separation of nucleic acids, thereby enabling high-throughput processing of thousands of single cells per day. Preliminary experiments suggest that we can efficiently make these combined measurements from the same cell. In Aim 2, we propose to develop a new single-cell method to simultaneously quantify mRNA and protein-DNA contacts from the same cell. In preliminary experiments, we mapped genome-nuclear lamina interactions or the binding pattern of a chromatin modifying protein together with mRNA from the same cell. Finally, as proof-of-concept that the methods developed in this proposal can be used to map cell type-specific epigenetic profiles from in vivo tissue samples, we will quantify methylome and DNA accessibility patterns for cell types in the rat retina. The retina is well-studied neural tissue with over 50 cell types, including rare ones, and therefore serves as an excellent testbed to validate our technologies. Thus, through the development of these multiomics single-cell methods, we expect to develop a technology that can be applied to map the epigenome of different cell types in a tissue without a priori knowledge of cell type-specific markers, enabling deeper understanding of the mechanisms of gene regulation in heterogeneous tissues.

项目摘要多细胞生物细胞内的基因组是相同的，但不同的细胞类型显示了多样的功能由于其表观基因组的差异。因此，映射不同基因组的表观遗传景观组织内的细胞类型对于理解细胞类型特异性基因表达调节至关重要。技术为了绘制表观遗传因素，目前依赖于我们在纯度高纯度分离所需的细胞类型的能力中生化测定，例如量化蛋白质-DNA接触，还需要大量的起始细胞。但是，细胞类型特异性的标记和抗体通常是未知或不可用的，这是一个专业在高纯度下隔离细胞类型方面的挑战。而表达细胞类型特异性的转基因动物模型在某些情况下，荧光记者可以克服这一局限性，这些动物模型的产生是时间消费。此外，组织经常包含罕见的细胞类型，使大量的细胞类型具有挑战性测定映射转录因子或染色质的结合景观所需的细胞修饰蛋白质。为了克服这些当前方法的局限性，该提案的总体目标是建立无标记的高通量技术，以绘制组织中不同细胞类型的表观基因组通过开发单细胞测序方法以同时量化转录组和表观基因组相同的单元格。单细胞转录组将用于对硅中细胞类型的无偏鉴定，并且属于同一细胞类型的细胞的相应表观基因组将被合并以产生高优质细胞类型的表观遗传景观。更具体地说，在AIM 1中，我们建议开发一个单细胞多组学技术可同时从同一细胞量化mRNA，5MC和DNA可及性。与众不同最近开发的方法，通过将mRNA与基因组进行物理分离来进行这些测量 DNA，我们的技术不涉及核酸的物理分离，从而实现高通量每天处理数千个单元。初步实验表明我们可以有效地进行这些来自同一细胞的合并测量。在AIM 2中，我们建议开发一种新的单细胞方法同时量化来自同一细胞的mRNA和蛋白-DNA接触。在初步实验中，我们映射的基因组 - 核薄片相互作用或染色质修饰蛋白的结合模式一起带有来自同一细胞的mRNA。最后，作为概念证明，本提案中开发的方法可以是用于绘制体内组织样品的细胞类型特异性表观遗传谱，我们将量化甲基甲基和大鼠视网膜中细胞类型的DNA可及性模式。视网膜是研究良好的神经组织，超过50个细胞包括稀有类型的类型，因此是验证我们技术的出色测试。因此，通过开发这些多域单细胞方法，我们希望开发一种可以应用于组织中不同细胞类型的表观基因组，而没有先验的细胞类型特异性知识标记，使能够更深入地了解异质组织基因调节的机制。