Mapping 3D-genome motion with next generation tracking technology to distinguish molecular drivers of cis-regulatory control

Transcriptional regulation and resulting development and health/disease states are shaped by the underlying 3D organization of the genome. Recent advances in single-cell genome-organization techniques (including Optical Reconstruction of Chromatin Architecture (ORCA) developed in the Boettiger lab), in parallel to recent theoretical models, have suggested the 3D genome is both flexible and dynamic. Thus, to understand the 3D-genome and its contribution to transcriptional regulation, we need kinetic data from live microscopy. Despite considerable recent growth of chromatin live imaging, substantial limitations in spatial and temporal resolution and difficulties in inserting required chromatin labels constrain the available kinetic data. Here, we propose a new generation of live-imaging approach to study 3D-genome motion, we call Transposon Accelerated Chromatin Kinetic Imaging Technology (TRACK-IT), which combines innovations in 1) ultrabright, miniaturized DNA labels, 2) efficient transposon-based tiling of target loci, and 3) optimized high-speed live microscopy. Our preliminary TRACK-IT data demonstrates ~10x improvement in spatial resolution, ~100x improvement in temporal resolution in live microscopy and enables rapid construction of >10 cell lines with a pair of optimized fluorescent labels tiling a target locus. We will investigate two model loci, one ‘simple’ and the other ‘complex’, with TRACK-IT, to understand how genomic separation and context shape 3D-genome motion (the frequency, speed, and timescales of genomic interaction). We will further test proposed molecular determinants of 3D-genome motion (cohesin loop extrusion, transcription, topoisomerase, etc) through genetic and pharmacological perturbations of these processes. In Aim 2, we will perform advanced biophysical modeling to simulate previously unexplored high-temporal-resolution 3D-genome motion. In particular, these simulations will determine how different parameters/conditions of the candidate molecular mechanisms can accelerate enhancer-promoter communications, and provide a theoretical framework for interpreting live-microscopy data from Aim 1. In Aim 3, we will expand on our TRACK-IT approach in Aim 1 to investigate the 3D motion of enhancers and promoters with respect to transcription busting, using a variation of TRACK-IT we call TRACK-EP. Critically, this aim will use inducible enhancers from the estrogen response pathway, providing a synchronized “time zero” in which to study how the signal flows from enhancer to nascent RNA, as well as contrast the motion of activated vs. inactive enhancers and promoters. Together, our ultra-spatiotemporal-resolution live-microscopy and biophysical simulations will enhance our understanding of 3D-genome biology and resolve key remaining questions in how the 3D genome regulates transcription.