Cis-regulatory elements (e.g. enhancers, promoters and insulators) within the 3D genome encode essential genetic information to instruct precise spatiotemporal gene expression programs during animal development. However, it is challenging to visualize the nanometer-scale organization of the cis-regulatory element network and to characterize their spatiotemporal dynamics inside the 3D nucleus of single cells.
To address these challenges, I first characterized a long-range enhancer cluster (~50-70kb) regulating the naïve pluripotency gene Klf4 in mouse embryonic stem cells (mESCs). CRISPR/dCas9 tiling screen and ChIP-exo identified key transcription factors (TFs) OCT4, SOX2, ESRRB and STAT3 bound to cis-regulatory elements within the enhancer cluster, in which OCT4 and SOX2 serve as lead factors maintaining an open chromatin architecture and assisting the binding of other TFs/cofactors. Live-cell single-molecule imaging experiments revealed that STAT3 and ESRRB exhibit highly dynamic binding to cognate sites with much slower target site sampling and shorter temporal occupancy than lead factors and that SOX2 facilitates ESRRB target search in both live mESCs and terminally differentiated cells likely involving a protein tethering mechanism. This study was published at Genes and Development in 2017 (http://genesdev.cshlp.org/content/31/17/1795.full.html)
To further understand the organizational principles of cis-regulatory elements directing gene regulation, I developed a single-molecule localization-based super-resolution imaging method–3D ATAC-PALM to selectively illuminate the cis-regulatory chromatin, in collaboration with James Liu’s group at Janelia Research Campus and Howard Chang’s group at Stanford. I adapted a recently developed hyperactive Tn5 transposase-based imaging technology, Assay for transposase-accessible chromatin with visualization (ATAC-see), to insert photo-activatable Janelia Fluor 549 (PA-JF549) labeled oligo DNA probes into accessible chromatin genome-wide. The Tn5 transposase enables selective and high density labeling of short, non-repetitive accessible chromatin DNA compatible with genome-wide ATAC-seq verification. To achieve whole nucleus 3D visualization by photoactivated localization microscopy (PALM), I took advantage of the ultrathin axial sectioning of the Lattice light sheet microscope to minimize out-of-focus photobleaching and maximize the photon budget for accurate single molecule localizations. This method provides a new revenue to characterize the architecture, mechanism and function of the cis-regulatory genome in single cells.
To dissect the spatiotemporal dynamics of cis-regulatory chromatin directed gene regulation at single cell single allele resolution, I am currently combining MS2-based RNA labeling, TetO array based DNA labeling, and HaloTag based protein labeling to understand gene activation dynamics in living cells.