Optogenomic mapping of chromatin accessibility in live cells

摘要 已经使用多种染色质可及性简档方法广泛地研究了DNA的物理可及性， 包括DNase-seq、ATAC-seq、MNase-seq和Nome-seq。这些研究揭示了无障碍的 基因组与顺式DNA调控元件如增强子和启动子大部分重叠，突出了一种 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，高阶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及性。目前的技术不适合解决这一问题 因为它们的技术限制改变了高阶生物的固有结构和生物物理特征 染色质。因此，我们的目标是开发一种新的方法来定量测量染色质的可及性 在活细胞中的自然条件下。在这个项目中，我们将利用最近开发的Ilid-SspB光生 模块设计用于测量局部染色质可及性的光控分子探针 活细胞中的构象。在目标1中，我们将开发一对光控染色质可及性探针， 由MNase-mClover3-SspB示踪剂和Ilid-mRuby3-H2B锚组成。在验证和优化之后， 我们将建立光学基因控制与高通量DNA相结合的实验系统 测序以探测天然可及的染色质构象。在目标2中，我们将应用这项名为 OMAC-SEQ(使用测序的可及染色质光学图谱)和开发计算流水线 检测活体胚胎干细胞中细胞分化偶联染色质构象的变化。在AIM 3，我们将开发一种有丝分裂特异的OMAC-seq工具包来量化有丝分裂中可获得的基因组DNA。 染色体。通过实现这些目标，拟议的OMAC-SEQ技术将成为 了解高阶染色质对基因组可及性的影响。

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.