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In mammals, interactions between sequences within topologically associating domains enable control of gene expression across large genomic distances. Yet it is unknown how frequently such contacts occur, how long they last and how they depend on the dynamics of chromosome folding and loop extrusion activity of cohesin. By imaging chromosomal locations at high spatial and temporal resolution in living cells, we show that interactions within topologically associating domains are transient and occur frequently during the course of a cell cycle. Interactions become more frequent and longer in the presence of convergent CTCF sites, resulting in suppression of variability in chromosome folding across time. Supported by physical models of chromosome dynamics, our data suggest that CTCF-anchored loops last around 10 min. Our results show that long-range transcriptional regulation might rely on transient physical proximity, and that cohesin and CTCF stabilize highly dynamic chromosome structures, facilitating selected subsets of chromosomal interactions. Live-cell imaging shows that interactions within topologically associating domains are transient and frequent throughout the cell cycle. Convergent CTCF sites regulate the frequency and duration of interactions, which last a few minutes on average.

Gene expression in mammalian cells is controlled by enhancers that are often dispersed across large cis-regulatory landscapes around a promoter. Yet how enhancers determine transcription of their target genes, and how this depends on their relative position inside a cis-regulatory landscape remains unclear. Here we use live-cell imaging to track the activity of a promoter under the control of the same enhancer, but inserted at different positions across a simplified regulatory landscape with minimal complexity. Combined with mathematical modeling, this reveals that RNA production from the promoter occurs in clusters of transcriptional bursts, with enhancer position controlling the frequency at which such clusters appear. This results in bursts being more frequent and occurring more uniformly across cells when the enhancer is genomically close to the promoter than when it is located at large genomic distance. Mathematical modeling further indicates that the enhancer modulates the promoter’s ability to transition from its basal transcriptional state to a regime where clusters of bursts become more frequent. Our results challenge existing models of mammalian promoter operation, and reveal that enhancer position within a cis-regulatory landscape critically controls the timing and variability of transcriptional output in single cells.

The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study on the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rates with certain rhodamine substrates, which are more than two orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rates however are less affected by the structure of the label than those of HaloTag7, which vary over six orders of magnitude for commonly employed substrates. Solving the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics.

Physical proximity between genomic sequences in mammalian chromosomes controls key biological processes such as transcriptional regulation and DNA repair. Yet it is currently unknown if chromosomal contacts are rare and stable or instead frequent and dynamic, and how they depend on the loop extrusion activity of cohesin or barriers such as CTCF. By imaging chromosomal locations at high spatial and temporal resolution over several hours in living cells, we show that sequences within topological associating domains (TADs) frequently come into physical proximity during the course of a cell cycle and remain close to each other only for a few minutes. Such contacts become nonetheless substantially longer and more frequent in the presence of convergent CTCF sites, resulting in a suppression of variability in chromosome folding in single cells across time. Supported by physical models of chromosome dynamics, our data additionally suggests that individual CTCF-anchored loops last around 10 minutes. The estimates of chromosomal contact dynamics in our study provide a novel quantitative framework to link chromosome structure to function and show that cohesin and CTCF stabilize otherwise highly dynamic chromosome structures to facilitate selected subsets of chromosomal interactions. Competing Interest Statement The authors have declared no competing interest.