Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity sequence domains (LCDs), but how these LCDs drive transactivation remains unclear. We used live-cell single-molecule imaging to reveal that TF LCDs form local high-concentration interaction hubs at synthetic and endogenous genomic loci. TF LCD hubs stabilize DNA binding, recruit RNA polymerase II (RNA Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. Under physiological conditions, rapid and reversible LCD-LCD interactions occur between TFs and the RNA Pol II machinery without detectable phase separation. Our findings reveal fundamental mechanisms underpinning transcriptional control and suggest a framework for developing single-molecule imaging screens for drugs targeting gene regulatory interactions implicated in disease.

Folding of mammalian genomes into spatial domains is critical for gene regulation. The insulator protein CTCF and cohesin control domain location by folding domains into loop structures, which are widely thought to be stable. Combining genomic and biochemical approaches we show that CTCF and cohesin co-occupy the same sites and physically interact as a biochemically stable complex. However, using single-molecule imaging we find that CTCF binds chromatin much more dynamically than cohesin (~1–2 min vs. ~22 min residence time). Moreover, after unbinding, CTCF quickly rebinds another cognate site unlike cohesin for which the search process is long (~1 min vs. ~33 min). Thus, CTCF and cohesin form a rapidly exchanging 'dynamic complex' rather than a typical stable complex. Since CTCF and cohesin are required for loop domain formation, our results suggest that chromatin loops are dynamic and frequently break and reform throughout the cell cycle.DOI: http://dx.doi.org/10.7554/eLife.25776.001

Materials and MethodsMicro-C protocol for mammals was modified from the original protocol for yeast in (1, 2). The protocol was optimized for the input cell number from 1k to 5M and first applied to the mammalian system in (3). We first briefly summarize the critical steps and concepts in the Micro-C method, and then provide detailed step-by-step instructions. Micro-C experiment 1. Cell culture and crosslinkingHere, we performed a dual crosslinking protocol to fix protein-DNA and protein-protein interactions. In addition to formaldehyde, we used the non-cleavable and membrane-permeable protein-protein crosslinker DSG (disuccinimidyl glutarate, 7.7Å) or EGS (ethylene glycol bis(succinimidyl succinate), 16.1Å) to crosslink the primary amines between proximal proteins. The dual-crosslinking method significantly increases the signal-to-noise ratio of Micro-C data in yeast (2).In brief, 1k -5M cells were resuspended by trypsin and fixed by freshly made 1% formaldehyde at room temperature for 10 minutes. The crosslinking reaction was quenched by adding Tris buffer (pH = 7.5) to final 0.75 M at room temperature. Fixed cells were washed twice with 1X PBS and protein-protein interactions fixed by 3 mM DSG for 45 minutes at room temperature. The DSG solution was freshly made at a 300 mM concentration in DMSO and diluted to 3 mM in 1X PBS before use. The crosslinking reaction was quenched by 0.75 M Tris buffer and washed twice with 1X PBS. Crosslinked cells were snap-frozen in liquid nitrogen and stored at -80°C (pellets are stable for up to a year). Note that freshly made crosslinking solution is critical to producing high-reproducibility Micro-C data, and Tris buffer is a faster and stronger quenching agent than glycine.

Summary Comparative genome analyses reveal that organismal complexity scales not with gene number but with gene regulation. Recent efforts indicate that the human genome likely contains hundreds of thousands of enhancers, with a typical gene embedded in a milieu of tens of enhancers. Proliferation of cis-regulatory DNAs is accompanied by increased complexity and functional diversification of transcriptional machineries recognizing distal enhancers and core promoters, and by the high-order spatial organization of genetic elements. We review progress in unraveling one of the outstanding mysteries of modern biology: the dynamic communication of remote enhancers with target promoters in the specification of cellular identity.