Ultra-resolution, multiscale live imaging reveals TADs as domains of cohesin-accelerated search

In the last decade, sequencing methods like Hi-C have made it clear the genome is intricately folded, (e.g. in TADs, stripes and dots), and that this organization contributes significantly to the control of gene expression and thence cell fate and behavior. Single-cell DNA tracing microscopy and polymer physics-based simulations of genome folding, have proposed these population-scale patterns arise from motor-driven, heterogeneous movement, rather than stable 3D genomic architecture, implying that motion, rather than structure, is key to understanding genome function. However tools to directly observe this motion in vivo have been limited in coverage and resolution. Here, we introduce TRansposon Assisted Chromatin Kinetic Imaging Technology (TRACK-IT), which combines a suite of imaging and labeling improvements to achieve ultra-resolution in space and time, with self-mapping transposons to distribute labels across the chromosome. We find, in the presence of cohesin, that elements at submegabase separation exhibit search times as fast as a few to few tens of seconds - faster than the sampling interval of most recent studies. Acute degradation of cohesin leads to longer search times and a stronger dependence of search time on the linear separation at submegabase scales, but minimal change at multimegabase scales. Loci separated by a TAD border exhibited increased search times compared to distance matched controls. Finally, cohesin containing cells exhibited rare, processive movements, not seen in cohesin depleted cells. These processive trajectories exhibit extrusion rates of four kb/s across three distinct genomic intervals, four fold faster than recent in vitro measurements. Taken together, these results reveal a genome in constant motion on the seconds timescale, where motor dependent extrusion produces TADs that manifest as kinetic domains of accelerated local search. Overall design: RNA-seq of T7 RNAP in situ transcripts (isT-seq) originating from T7 promoter positioned within sleeping beauty transposon, which is then used to identify ITR-genome junction to efficiently map transposon insertion sites. 4 bulk samples (ctrl: no transposon, pre: TRACK-IT parent cell line before transposon remobilization, post: TRACK-IT labels remobilized following sleeping beauty expression, rnd: positive control where TRACK-IT labels were remobilized from multi copy transfected plasmids) and 9 single clonal cell line (5kb, 20kb, 55kb, 70kb, 134kb, 260kb, 2Mb, 12Mb, 73Mb) were used as samples.