Methods for Mapping Genetic Regulatory Elements in Single Cells and Single Molecules

绘制单细胞和单分子遗传调控元件的方法

• 批准号: 10657351
• 负责人: Aaron Streets
• 金额: $ 42.92万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10657351
• 关键词: Address; Adenine; Antibodies; Atlases; Base Pairing; Binding; Binding Proteins; Bioinformatics; Biological Models; Cell Line; Cell Nucleus; Cell physiology; Cells; Centromere; ChIP-seq; Chimeric Proteins; Chromosome Mapping; Chromosome Segregation; Complex; DNA; DNA Binding; DNA Methylation; DNA Sequence; DNA sequencing; DNA-Protein Interaction; Dedications; Detection; Development; Developmental Process; Digestion; Disease; Encyclopedia of DNA Elements; Epigenetic Process; Evolution; Exclusion; Genetic; Genetic Transcription; Genome; Genomic DNA; Genomics; Goals; High-Throughput Nucleotide Sequencing; Human; Human Genome; Individual; Investigation; Libraries; Ligation; Maps; Measurement; Measures; Methods; Methylation; Methyltransferase; Microfluidics; Modification; Noise; Nuclear; Nucleic Acid Regulatory Sequences; Pathogenesis; Phenotype; Play; Population; Positioning Attribute; Preparation; Process; Proteins; Protocols documentation; Regulation; Regulatory Element; Repetitive Sequence; Research; Resolution; Ribosomal DNA; Ribosomal RNA; Role; Signal Transduction; Site; Site-Directed Mutagenesis; Stretching; Techniques; Technology; Tissues; Transcriptional Regulation; Translational Regulation; Variant; base; cell type; epigenetic profiling; epigenetic regulation; epigenetic variation; epigenomics; genome-wide; genomic locus; high standard; histone modification; interest; nanopore; novel; prevent; programs; sequencing platform; single cell analysis; single cell sequencing; single molecule; telomere; tool; transcription factor

PROJECT SUMMARY The human genome is regulated through interactions between DNA and proteins in the nucleus that define and maintain the epigenetic state of cells. Therefore, large consortia such as the Encyclopedia of DNA Elements (ENCODE) are dedicated to comprehensively mapping regulatory elements such as transcription factor binding or histone modification so that we may understand regulatory processes that guide development, disease, and the everyday functioning of cells in our body. However, current methods for genome-wide measurement of protein-DNA interactions are unable to map regulatory elements in highly repetitive regions of the genome because they rely on high-throughput, short-read DNA sequencing platforms. This limitation prohibits comprehensive investigation of roughly 8% of the human genome including centromeres and ribosomal DNA arrays, which play essential roles in chromosome segregation and nuclear organization. Furthermore, these methods typically lack the sensitivity to profile the epigenetic landscape of single cells, preventing high-resolution measurements of regulatory variation in complex tissues. The goal of this research program is to expand the toolbox for mapping protein-DNA interactions genome-wide and extend capabilities to long-read sequencing and single-cell sequencing technologies with the development of two methods: (1) Directed methylation and long- read sequencing (DiMeLo-Seq) and (2) single-cell directed methylation and sequencing (scDiMe-Seq). To record the genomic position of protein binding or histone modification, a methyltransferase fused to protein A will be directed to the targeted regulatory element with a primary antibody. Upon activation, the methyltransferase will methylate adenines in proximal DNA sequences. DiMeLo-Seq will implement long-read DNA sequencing technologies such as nanopore sequencing to directly detect the position of these modifications on long molecules of DNA, taking advantage of the differential signal generated by methyl-adenines as they pass through the nanopore. This approach will produce sequencing reads of up to hundreds of kilobases long, providing high- confidence mapping of regulatory elements to regions of the genome that are unmappable with short-read sequencing. To detect these modifications with single-cell sensitivity, scDiMe-Seq will enrich genomic loci containing methyl adenines through targeted digestion, adapter ligation, and PCR amplification. These enriched fragments will then be sequenced using standard high-throughput sequencing. This project aims to develop DiMeLo-Seq and scDiMe-Seq through rigorous protocol optimization of the directed methylation strategy and sequencing library preparation for long and short-read sequencing. The methods will then be characterized and validated by targeting well studied features such as lamina associated domains, and CTCF landscapes, as well as H3K9me3 and CENPA which are both enriched in centromeres. The overall goal of this project is to produce two robust and scalable methods that may shed light on regulatory mechanism in previously unexplored regions of the genome and aid in Human Cell Atlas initiatives by providing epigenetic information for single-cell analysis.

项目摘要 人类基因组是通过定义和 保持细胞的表观遗传状态。因此，大型财团，例如DNA元素的百科全书 （编码）专用于全面绘制调节元素，例如转录因子结合 或组蛋白修饰，以便我们可以理解指导发展，疾病和 细胞在我们体内的日常功能。但是，目前的全基因组测量方法 蛋白-DNA相互作用无法映射基因组高度重复区域的调节元件 因为他们依靠高通量，短阅读的DNA测序平台。这种限制禁止 大约8％的人类基因组（包括centromeres和核糖体DNA）的全面研究 阵列，在染色体分离和核组织中起着重要作用。此外，这些 方法通常缺乏对单个细胞表观遗传景观的敏感性，从而防止高分辨率 测量复杂组织中调节性变异的测量。该研究计划的目的是扩展 用于绘制蛋白质-DNA相互作用基因组的工具箱，并将功能扩展到长阅读测序和 通过开发两种方法的单细胞测序技术：（1）定向甲基化和长期 读取测序（Dimelo-Seq）和（2）单细胞定向甲基化和测序（SCDIME-SEQ）。记录 蛋白结合或组蛋白修饰的基因组位置，融合到蛋白A的甲基转移酶将是 用原代抗体针对靶向调节元件。激活后，甲基转移酶将 近端DNA序列中的甲基腺苷。 Dimelo-Seq将实施长阅读的DNA测序 诸如纳米孔测序之类的技术直接检测这些修饰的位置 DNA的分子，利用甲基腺苷通过时产生的差异信号 纳米孔。这种方法将产生多达数百千碱基的测序读物，从而提供高度 调节元素的置信度映射到基因组区域，这些区域无法与短读 测序。为了通过单细胞灵敏度检测这些修饰，scdime-seq将富含基因组基因座 通过靶向消化，适配器连接和PCR扩增含有甲基腺苷。这些丰富 然后，将使用标准的高通量测序对片段进行测序。该项目旨在发展 通过对定向甲基化策略的严格协议优化Dimelo-Seq和Scdime-Seq， 测序库的准备，以进行长阅读测序。然后将这些方法进行表征，并 通过针对诸如Lamina相关域和CTCF景观等精心研究的特征来验证 作为H3K9me3和CENPA，都富含着丝粒。该项目的总体目标是生产 两种可靠且可扩展的方法，可以阐明以前未开发的区域中的调节机制 通过提供单细胞分析的表观遗传信息，基因组和人类细胞倡议的帮助。