Optogenomic mapping of chromatin accessibility in live cells

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.