Cohesin and CTCF control the dynamics of chromosome folding.

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.

Nonlinear control of transcription through enhancer-promoter interactions.

Chromosome structure in mammals is thought to regulate transcription by modulating three-dimensional interactions between enhancers and promoters, notably through CTCF-mediated loops and topologically associating domains (TADs)1-4. However, how chromosome interactions are actually translated into transcriptional outputs remains unclear. Here, to address this question, we use an assay to position an enhancer at large numbers of densely spaced chromosomal locations relative to a fixed promoter, and measure promoter output and interactions within a genomic region with minimal regulatory and structural complexity. A quantitative analysis of hundreds of cell lines reveals that the transcriptional effect of an enhancer depends on its contact probabilities with the promoter through a nonlinear relationship. Mathematical modelling suggests that nonlinearity might arise from transient enhancer-promoter interactions being translated into slower promoter bursting dynamics in individual cells, therefore uncoupling the temporal dynamics of interactions from those of transcription. This uncovers a potential mechanism of how distal enhancers act from large genomic distances, and of how topologically associating domain boundaries block distal enhancers. Finally, we show that enhancer strength also determines absolute transcription levels as well as the sensitivity of a promoter to CTCF-mediated transcriptional insulation. Our measurements establish general principles for the context-dependent role of chromosome structure in long-range transcriptional regulation.

Differential regulation of lineage commitment in human and mouse primed pluripotent stem cells by the nucleosome remodelling and deacetylation complex.

Differentiation of mammalian pluripotent cells involves large-scale changes in transcription and, among the molecules that orchestrate these changes, chromatin remodellers are essential to initiate, establish and maintain a new gene regulatory network. The Nucleosome Remodelling and Deacetylation (NuRD) complex is a highly conserved chromatin remodeller which fine-tunes gene expression in embryonic stem cells. While the function of NuRD in mouse pluripotent cells has been well defined, no study yet has defined NuRD function in human pluripotent cells. Here we find that while NuRD activity is required for lineage commitment from primed pluripotency in both human and mouse cells, the nature of this requirement is surprisingly different. While mouse embryonic stem cells (mESC) and epiblast stem cells (mEpiSC) require NuRD to maintain an appropriate differentiation trajectory as judged by gene expression profiling, human induced pluripotent stem cells (hiPSC) lacking NuRD fail to even initiate these trajectories. Further, while NuRD activity is dispensable for self-renewal of mESCs and mEpiSCs, hiPSCs require NuRD to maintain a stable self-renewing state. These studies reveal that failure to properly fine-tune gene expression and/or to reduce transcriptional noise through the action of a highly conserved chromatin remodeller can have different consequences in human and mouse pluripotent stem cells.

The Nucleosome Remodelling and Deacetylation complex suppresses transcriptional noise during lineage commitment.

Multiprotein chromatin remodelling complexes show remarkable conservation of function amongst metazoans, even though components present in invertebrates are often found as multiple paralogous proteins in vertebrate complexes. In some cases, these paralogues specify distinct biochemical and/or functional activities in vertebrate cells. Here, we set out to define the biochemical and functional diversity encoded by one such group of proteins within the mammalian Nucleosome Remodelling and Deacetylation (NuRD) complex: Mta1, Mta2 and Mta3. We find that, in contrast to what has been described in somatic cells, MTA proteins are not mutually exclusive within embryonic stem (ES) cell NuRD and, despite subtle differences in chromatin binding and biochemical interactions, serve largely redundant functions. ES cells lacking all three MTA proteins exhibit complete NuRD loss of function and are viable, allowing us to identify a previously unreported function for NuRD in reducing transcriptional noise, which is essential for maintaining a proper differentiation trajectory during early stages of lineage commitment.

FRET-enhanced photostability allows improved single-molecule tracking of proteins and protein complexes in live mammalian cells.

A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods of time in the dense environments found in living cells. Here, we introduce the concept of using FRET to enhance the photophysical properties of photo-modulatable (PM) fluorophores commonly used in such studies. By developing novel single-molecule FRET pairs, consisting of a PM donor fluorophore (either mEos3.2 or PA-JF549) next to a photostable acceptor dye JF646, we demonstrate that FRET competes with normal photobleaching kinetic pathways to increase the photostability of both donor fluorophores. This effect was further enhanced using a triplet-state quencher. Our approach allows us to significantly improve single-molecule tracking of chromatin-binding proteins in live mammalian cells. In addition, it provides a novel way to track the localization and dynamics of protein complexes by labeling one protein with the PM donor and its interaction partner with the acceptor dye.

3D structures of individual mammalian genomes studied by single-cell Hi-C.

The folding of genomic DNA from the beads-on-a-string-like structure of nucleosomes into higher-order assemblies is crucially linked to nuclear processes. Here we calculate 3D structures of entire mammalian genomes using data from a new chromosome conformation capture procedure that allows us to first image and then process single cells. The technique enables genome folding to be examined at a scale of less than 100 kb, and chromosome structures to be validated. The structures of individual topological-associated domains and loops vary substantially from cell to cell. By contrast, A and B compartments, lamina-associated domains and active enhancers and promoters are organized in a consistent way on a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. By studying genes regulated by pluripotency factor and nucleosome remodelling deacetylase (NuRD), we illustrate how the determination of single-cell genome structure provides a new approach for investigating biological processes.