Bulk chromatin motion has not been analyzed at high resolution. We present HiD , a method to quantitatively map dynamics of chromatin and abundant nuclear proteins for every pixel simultaneously over the entire nucleus from fluorescence image series. HiD combines reconstruction of chromatin motion and classification of local diffusion processes by Bayesian inference. We show that DNA dynamics in the nuclear interior are spatially partitioned into 0.3-3-μm domains in a mosaic-like pattern, uncoupled from chromatin compaction. This pattern was remodeled in response to transcriptional activity. HiD can be applied to any dense and bulk structures opening new perspectives towards understanding motion of nuclear molecules.

Intrinsic dynamics of chromatin contribute to gene regulation. How chromatin mobility responds to genomic processes, and whether this response relies on coordinated chromatin movement is still unclear. Here, we introduce an approach called Dense Flow reConstruction and Correlation (DFCC), to quantify correlation of chromatin motion with sub-pixel sensitivity at the level of the whole nucleus. DFCC reconstructs dense global flow fields of fluorescent images acquired in real-time. We applied our approach to analyze stochastic movements of DNA and histones, based on direction and magnitude at different time lags in human cells. We observe long-range correlations extending over several μm between coherently moving regions over the entire nucleus. Spatial correlation of global chromatin dynamics was reduced by inhibiting elongation by RNA polymerase II, and abolished in quiescent cells. Furthermore, quantification of spatial smoothness over time intervals up to 30 s points to clear-cut boundaries between distinct regions, while smooth transitions in small (<1 μm) neighborhoods dominate for short time intervals. Rough transitions between regions of coherent motion indicate directed squeezing or stretching of chromatin boundaries, suggestive of changes in local concentrations of actors regulating gene expression. The DFCC approach hence allows characterizing stochastically forming domains of nuclear activity.

Intrinsic dynamics of chromatin contribute to gene regulation. How chromatin mobility responds to genomic processes and whether this response relies on coordinated movement is still unclear. Here, CC-BY-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was . http://dx.doi.org/10.1101/230789 doi: bioRxiv preprint first posted online Dec. 7, 2017; Significance StatementControl of gene expression relies on modifications of chromatin structure and activity of the transcription machinery. However, how chromatin responds dynamically to this genomic process and whether this response is coordinated in space is still unclear. We introduce a novel approach called Dense Flow reConstruction and Correlation (DFCC) to characterize spatially correlated dynamics of chromatin in living cells at nanoscale resolution. DFCC allows us to detect chromatin domains in living cells with long range correlations over the entire nucleus. Furthermore, transitions between domains can be quantified by the newly introduced smoothness parameter of local chromatin motion. The DFCC approach permits characterizing stochastically forming domains of other DNA dependent activity in any cell type in real time imaging.

Chromatin conformation regulates gene expression and thus, constant remodeling of chromatin structure is essential to guarantee proper cell function. To gain insight into the spatiotemporal organization of the genome, we use high-density photoactivated localization microscopy and deep learning to obtain temporally resolved super-resolution images of chromatin in living cells. In combination with high-resolution dense motion reconstruction, we find elongated ~45- to 90-nm-wide chromatin “blobs.” A computational chromatin model suggests that these blobs are dynamically associating chromatin fragments in close physical and genomic proximity and adopt topologically associated domain–like interactions in the time-average limit. Experimentally, we found that chromatin exhibits a spatiotemporal correlation over ~4 μm in space and tens of seconds in time, while chromatin dynamics are correlated over ~6 μm and last 40 s. Notably, chromatin structure and dynamics are closely related, which may constitute a mechanism to grant access to regions with high local chromatin concentration.

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19Chromatin conformation regulates gene expression and thus constant remodeling of chromatin 20 structure is essential to guarantee proper cell function. To gain insight into the spatio-temporal 21 organization of the genome, we employ high-density photo-activated localization microscopy and 22 deep learning to obtain temporally resolved super-resolution images of chromatin in vivo. In 23 combination with high-resolution dense motion reconstruction, we confirm the existence of 24 elongated ~ 45 to 90 nm wide chromatin 'blobs', which appear to be dynamically associating 25 chromatin fragments in close physical and genomic proximity and adopt TAD-like interactions in 26 the time-average limit. We found the chromatin structure exhibits a spatio-temporal correlation 27 extending ~ 4 μm in space and tens of seconds in time, while chromatin dynamics are correlated 28 over ~ 6 μm and outlast 40 s. Notably, chromatin structure and dynamics are closely interrelated, 29 which may constitute a mechanism to grant access to regions with high local chromatin 30 concentration. 32 The three-dimensional organization of the eukaryotic genome plays a central role in gene regulation 33 (1-3). Its spatial organization has been prominently characterized by molecular and cellular 34 approaches including high-throughput chromosome conformation capture (Hi-C) (4) and 35 fluorescent in situ hybridization (FISH) (5). Topologically associated domains (TADs), genomic 36 regions that display a high degree of interaction, were revealed and found to be a key architectural 37 feature (6). Direct 3D localization microscopy of the chromatin fiber at the nanoscale (7) confirmed 38 the presence of TADs in single cells but also, among others, revealed great structural variation of 39 chromatin architecture (8, 9). To comprehensively resolve the spatial heterogeneity of chromatin, 40 super-resolution microscopy must be employed. Previous work showed that nucleosomes are 41 distributed as segregated, nanometer-sized accumulations throughout the nucleus (10-13) and that 42 the epigenetic state of a locus has a large impact on its folding (14, 15). However, to resolve the 43 fine structure of chromatin, high labeling densities, long acquisition times and, often, cell fixation 44 are required. This precludes capturing dynamic processes of chromatin in single live cells, yet 45 chromatin moves at different spatial and temporal scales. 46The first efforts to relate chromatin organization and its dynamics were made using a combination 47 of Photo-activated Localization Microscopy (PALM) and tracking of single nucleosomes (16). It 48 could be shown that nucleosomes mostly move coherently with their underlying domains, in 49 accordance with conventional microscopy data (17); however, a quantitative link between the 50 observed dynamics and the surrounding chromatin structure could not yet be established in real-51 time. Although it is becoming increasingly clear that chromatin motion and long-range interactions 52 are key to genome o...

The ring-shaped structural-maintenance-of-chromosomes (SMC) complexes condensin and cohesin extrude loops of DNA as a key motif in chromosome organization. It remains, however, unclear how these SMC motor proteins can extrude DNA loops in chromatin that is bound with proteins. Here, using in vitro single-molecule visualization, we show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to DNA loop extrusion by yeast condensin. Strikingly, we find that even DNA-bound nanoparticles as large as 200 nm, much bigger than the SMC ring size, can be translocated into DNA loops during condensin-driven extrusion. Similarly, human cohesin can pass 200 nm particles during loop extrusion, which even occurs for a single-chain version of cohesin in which the ring-forming subunits are covalently linked and cannot open up to entrap DNA. These findings disqualify all common loop-extrusion models where DNA passes through the SMC rings (pseudo)topologically, and instead point to a nontopological mechanism for DNA loop extrusion.

In eukaryotes, genomic DNA is extruded into loops by cohesin. By restraining this process, the DNA-binding protein CTCF generates topologically associating domains (TADs) that play key roles in gene regulation and recombination during development and disease. How CTCF establishes TAD boundaries and to what extent these are permeable to cohesin is unknown. To address these questions, we visualize interactions of single CTCF and cohesin molecules on DNA in vitro. We show that CTCF is sufficient to block diffusing cohesin, possibly reflecting how cohesive cohesin accumulates at TAD boundaries, as well as to block loop-extruding cohesin, reflecting how CTCF establishes TAD boundaries. CTCF functions asymmetrically, as predicted, but unexpectedly is dependent on DNA tension. Moreover, CTCF regulates cohesin's loop extrusion activity by changing its direction and by inducing loop shrinkage. Our data indicate that CTCF is not, as previously assumed, simply a barrier to cohesin-mediated loop extrusion but is an active regulator of this process, where the permeability of TAD boundaries can be modulated by DNA tension. These results reveal mechanistic principles of how CTCF controls loop extrusion and genome architecture.