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Optogenomic mapping of chromatin accessibility in live cells

活细胞染色质可及性的光基因组图谱

基本信息

批准号：
10709895
负责人：
XIAOZHONG ALEC WANG
金额：
$ 32万
依托单位：
NORTHWESTERN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-24 至 2026-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10709895
关键词：
ATAC-seq Architecture Binding Biophysics Cell Differentiation process Cells Characteristics Charge Chromatin Chromosomes Coupled Crowding DNA DNase I hypersensitive sites sequencing Data Set Detergents Digestion Disease Enhancers Environment Epiblast Exhibits Experimental Designs Gene Expression Genetic Genome Genomic DNA Genomics High-Throughput DNA Sequencing High-Throughput Nucleotide Sequencing Histones Image In Situ Interphase Light Liquid substance Maps Measures Methods Mitosis Mitotic Chromosome Modeling Molecular Molecular Conformation Molecular Probes Mus Names Nucleoplasm Nucleosomes Optics Permeability Physical condensation Procedures Protocols documentation Regulatory Element Role Structure System Techniques Technology Testing Tracer Validation biophysical properties cell fixing computational pipelines computerized data processing crosslink design embryonic stem cell enzyme activity epigenomics genome-wide imaging study in vitro Model indexing insight new technology next generation sequencing novel novel strategies nuclease optogenetics promoter prototype recruit sample fixation technology development tool virtual

项目摘要

ABSTRACT Physical access of DNA has been widely studied using multiple chromatin accessibility profiling methods, including DNase-seq, ATAC-seq, MNase-seq and NOMe-seq. These studies have revealed the accessible genome largely overlaps with cis- DNA regulatory elements such as enhancers and promoters, highlighting a prominent role of chromatin-binding factors in establishing chromatin accessibility. However, recent chromatin conformation imaging studies have suggested that higher-order chromatin organization can also impact accessibility of DNA through packing. A pressing question is whether native chromatin packing conformation regulates chromatin accessibility in living cells. The current technology is not suitable to resolve this issue because of their technical limitations that alter the native configuration and biophysical features of higher-order chromatin. Thus, our objective is to develop a new approach to quantitively measure chromatin accessibility under native conditions in live cells. In this project, we will leverage the recently developed iLID-SspB optogenetic module to design a light-controlled molecule probe for measuring the accessibility of local chromatin conformation in live cells. In Aim 1, we will develop a pair of optically controlled chromatin accessibility probes, composed of MNase-mClover3-SspB tracer and iLID-mRuby3-H2B anchor. Upon validation and optimization, we will establish an experimental system that combine the optical genetic control with high throughput DNA sequencing to probe the native accessible chromatin conformation. In Aim 2, we will apply this technology named OMAC-seq (Optical Mapping of Accessible Chromatin using Sequencing) and develop a computational pipeline to measure cell differentiation-coupled changes of chromatin conformation in live embryonic stem cells. In Aim 3, we will develop a mitosis-specific OMAC-seq toolkit to quantify accessible genome DNA in mitotic chromosome. By completing these aims, the proposed OMAC-seq technology will be an enabling toolkit for understanding the impact of higher order chromatin on genome accessibility.

摘要已经使用多种染色质可及性分析方法广泛研究了DNA的物理可及性，包括DNase-seq、ATAC-seq、MNase-seq和NOMe-seq。这些研究揭示了基因组与顺式DNA调控元件如增强子和启动子有很大的重叠，突出了染色质结合因子在建立染色质可及性中的突出作用。然而，最近的染色质构象成像研究表明，高级染色质组织也可以影响通过包装实现DNA的可及性。一个紧迫的问题是天然染色质包装构象调节活细胞中染色质的可接近性。目前的技术不适合解决这个问题由于它们的技术限制，改变了更高阶的天然构型和生物物理特征，染色质因此，我们的目标是发展一种新的方法来定量测量染色质可及性在活细胞中的天然条件下。在这个项目中，我们将利用最近开发的iLID-SspB光遗传学设计一个光控分子探针的模块，用于测量局部染色质的可及性活细胞中的构象。在目标1中，我们将开发一对光学控制的染色质可及性探针，由MNase-mClover 3-SspB示踪剂和iLID-mRuby 3-H2 B锚组成。经过验证和优化，我们将建立一个联合收割机，将光学遗传控制和高通量DNA结合起来的实验系统测序以探测天然可接近的染色质构象。在目标2中，我们将应用这种名为 OMAC-seq（Optical Mapping of Disposable Chromatin using Sequencing），并开发一个计算管道以测量活胚胎干细胞中染色质构象的细胞分化偶联变化。在Aim中 3.我们将开发一个有丝分裂特异性的OMAC-seq工具包，以量化有丝分裂中可获得的基因组DNA。染色体通过完成这些目标，拟议的OMAC-seq技术将成为一个使能工具包，了解高阶染色质对基因组可及性的影响。