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

项目摘要 人类基因组是通过DNA和细胞核中蛋白质之间的相互作用来调节的， 维持细胞的表观遗传状态。因此，DNA元素百科全书等大型财团 （ENCODE）致力于全面绘制调控元件，如转录因子结合 或组蛋白修饰，以便我们可以了解指导发育，疾病和 我们体内细胞的日常功能。然而，目前的全基因组测量方法， 蛋白质-DNA相互作用不能在基因组的高度重复区域定位调控元件 因为它们依赖于高通量、短读段的DNA测序平台。这一限制禁止 对大约8%的人类基因组进行全面调查，包括着丝粒和核糖体DNA 阵列，在染色体分离和核组织中发挥重要作用。而且这些 这些方法通常缺乏对单细胞表观遗传图谱的敏感性， 测量复杂组织中的调节变化。这项研究计划的目标是扩大 用于绘制全基因组蛋白质-DNA相互作用的工具箱，并将功能扩展到长读测序， 单细胞测序技术的发展有两种方法：（1）定向甲基化和长- 读段测序（DiMeLo-Seq）和（2）单细胞定向甲基化和测序（scDiMe-Seq）。记录 蛋白质结合或组蛋白修饰的基因组位置，与蛋白质A融合的甲基转移酶将被 与一抗一起导向靶向调节元件。一旦激活，甲基转移酶将 在近端DNA序列中甲基化腺嘌呤。DiMeLo-Seq将实现长读段DNA测序 技术，如纳米孔测序，以直接检测这些修饰在长 DNA分子，利用甲基腺嘌呤通过时产生的差异信号， 纳米孔这种方法将产生长达数百个碱基的测序读段，提供高效率的测序。 将调控元件置信度映射到用短读码无法映射的基因组区域 测序为了以单细胞灵敏度检测这些修饰，scDiMe-Seq将富集基因组基因座， 通过靶向消化、衔接子连接和PCR扩增来制备含有甲基腺嘌呤的重组质粒。这些富集 然后使用标准高通量测序对片段进行测序。该项目旨在开发 DiMeLo-Seq和scDiMe-Seq通过定向甲基化策略的严格方案优化， 用于长和短读段测序的测序文库制备。然后将对这些方法进行表征， 通过针对诸如叶片相关结构域和CTCF景观等研究充分的特征进行验证， 如H3 K9 me 3和CENPA，它们都在着丝粒中富集。该项目的总体目标是生产 两种强大且可扩展的方法，可能揭示以前未探索区域的调控机制 基因组和援助人类细胞图谱倡议提供表观遗传信息的单细胞分析。

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.