Nucleus

The spatiotemporal organisation of chromatin in the eukaryotic nucleus is fundamental for genome regulation. Chromatin undergoes rapid remodelling and rearrangements within minutes, altering its diffusion properties. Considering the tight coupling between genome function and nuclear architecture, a key question is how chromatin dynamics adapt to or promote nuclear processes. To elucidate the underlying physical principles, we employed High-resolution Diffusion mapping (Hi-D) to track chromatin motion throughout interphase in live human cells. Our analysis, that considers both diffusive motion and drift generated by active forces, revealed that chromatin dynamics are heterogeneous, with distinct behaviours in different subnuclear zones. Notably, both diffusive and processive contributions to chromatin motion progressively decrease from G1 to G2 phase, with this reduction occurring uniformly across all subzones. This suggests a global mechanism driving the observed decrease in chromatin mobility during cell cycle progression. By combining genetic knockout experiments and polymer modelling, we demonstrate that the doubling of DNA content, rather than cohesin-mediated sister chromatid entrapment, is responsible for the gradual decrease in chromatin motion during the cell cycle in human nuclei. These findings provide new insights into the physical and functional organisation of chromatin and its regulation during cellular proliferation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/712877v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@75c654org.highwire.dtl.DTLVardef@2fbd3dorg.highwire.dtl.DTLVardef@31025aorg.highwire.dtl.DTLVardef@191808e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Existing databases of interphase chromosome conformations typically store three-dimensional coordinates of genomic segments. However, since interphase chromatin is highly dynamic, such databases are dominated by transient configurations and unstructured regions, whose positions vary continuously between cells and over time, unlike folded proteins such as globin, which adopt similar structures in every cell. These drawbacks motivated the inception of a database based on strion (a portmanteau of a string capturing structure and function). A strion concisely describes the structure and activity of all transcription units in one cell, by retaining only functionally relevant positional information. Sets of strions describing structures in different cells sampled at different times are compiled into a super-strion. Then, 46 super-strions summarise the range of structure and activity of a human cell type, including information on all transcription units, how often each co-fires and co-clusters with others in transcription factories/hubs, enhancer interactomes and small-world expression networks. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/724942v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@13a1263org.highwire.dtl.DTLVardef@18d2c78org.highwire.dtl.DTLVardef@162865corg.highwire.dtl.DTLVardef@1631d65_HPS_FORMAT_FIGEXP M_FIG C_FIG

Hutchinson-Gilford Progeria Syndrome (HGPS) is a genetic disease characterized by the accumulation of progerin, a mutant form of lamin A, at the nuclear envelope. Progerin disrupts the stability of the nuclear lamina, leading to genome instability and accelerated aging phenotypes. While structural nuclear defects are well-documented, the impact of progerin on real-time chromatin dynamics and the ability of current therapeutics to rescue these dynamics remains poorly understood. In this work, we employ single-particle tracking to quantify telomere dynamics in HGPS patient fibroblasts. We demonstrate that HGPS cells exhibit significantly increased telomere dynamics, characterized by expanded scan areas, increased diffusion coefficients, and larger jump distances compared to healthy controls. We further evaluated the efficacy of two clinically relevant treatments, the farnesyltransferase inhibitor Lonafarnib and the ICMT inhibitor C75, to determine if emerging treatments can restore chromatin dynamics compared to healthy controls. Our results reveal that Lonafarnib partially rescues telomere dynamics, shifting chromatin motion back towards healthy control levels, and that C75 provides a complete rescue of the dynamics for all parameters quantified. These findings provide a quantitative framework for understanding how nuclear lamina mutations induce aberrant genome dynamics and the efficacy of HGPS therapies on restoring those dynamics.

Nuclear blebs are herniations of the nucleus that occur in many human conditions including aging, heart disease, muscular dystrophy, and many cancers. Nuclear blebbing causes nuclear rupture and cellular dysfunction. However, understanding the formation, stability, and identification of nuclear blebs remains an ongoing challenge. Our previous studies reveal that nuclear blebs are best hallmarked by decreased DNA density. To determine if chromatin decompaction underlies decreased DNA density in nuclear blebs, we investigated the histone composition of nuclear blebs across multiple cell lines. Time lapse and immunofluorescence imaging revealed that global histone H2B and H3 levels are decreased in the nuclear bleb relative to the nuclear body. Next, we imaged histone modification states of euchromatin and heterochromatin, which respectively track decompact and compact states of chromatin. Overall, we find that nuclear blebs display variable histone modification state across cell lines, as euchromatin does not consistently enrich nor is heterochromatin consistently depleted. Nuclear blebs did consistently show active RNA Pol II initiation is enriched relative to elongation. Thus, we find that the local histone modification state is not an essential component of nuclear blebs while transcription initiation enrichment over elongation is reproducible across cell lines and conditions. Summary statementWe measured histones and their modification states in nuclear blebs. We find that chromatin state is variable while transcription initiation is consistently enriched relative to elongation in nuclear blebs.

Intron length is a fascinating example of form without function. The vast majority of intronic space within genomes remains without a provided utility. It often fascinates us to find introns performing any function at all, establishing an attention bias against the vast lacking of utility of the remaining intergenic space. In an attempt to better understand the greater breadth of intronic length, I investigate here what I term The Kinetic Intron Hypothesis. This hypothesis investigates hypothetical dynamics of intron RNA synthesis and degradation. It explores how NTPs stored within intron RNA might function in mitosis and NTP resource management. Preliminary testing of the hypothesis leads to trends that warrant further exploration and validation by the scientific community. SignificanceCurrently no widely acknowledged model exists to characterize the length of introns within genes, yet intron length is massively abundant in eukaryotic genomes. Here I present an attempt to model the length of introns. In doing so, I explore novel hypothesized intron dynamics, presenting preliminary data for previously uncharacterized intron characteristics. The new data and model have the protentional to unveil new avenues of utility for introns at the intracellular level.

BackgroundDynamic chromatin behavior, which is related to chromatin accessibility, plays a critical role in various genome DNA functions such as RNA transcription and DNA replication/repair. Previous studies using highly synchronized cells showed that average local chromatin motion, captured by single-nucleosome imaging and tracking on a second time scale, remained almost constant throughout G1, S, and G2 phases in living human cells, although possible effects of prolonged drug treatments for cell-cycle synchronization could not be excluded. ResultsTo avoid possible effects of prolonged drug treatment, we combined single-nucleosome imaging with Fucci probes to visualize cell-cycle progression through G1, S, and G2. Using HeLa and HCT116 cells expressing H2B-HaloTag and Fucci probes, we found that local nucleosome motion remained similar on average throughout interphase, except for elevated motion in early G1. Transcription inhibition similarly increased nucleosome motion throughout interphase. Local nucleosome motion also increased following replication stress or DNA damage. ConclusionOur findings suggest that near-constant chromatin motion supports housekeeping functions under similar physical conditions during interphase. Our findings also suggest that cells can transiently change chromatin motion to perform ad hoc tasks in response to signals from inside and outside the cell, such as DNA damage.

Chromosome segregation is a tightly-regulated process that normally occurs with high fidelity. Errors in chromosome segregation are associated with aging, cancer, and infertility. Initially erroneously attached chromosomes are corrected over the course of mitosis, with the spindle assembly checkpoint preventing entry into anaphase until this error correction is complete. Despite extensive work on the molecular basis of error correction and the spindle assembly checkpoint, it is still unclear how disruption of these processes contribute to chromosome segregation errors. Here, we develop and experimentally test a coarse-grained model of error correction in the presence of a faulty spindle assembly checkpoint. We use the resulting model to disentangle the impact of various small molecule and genetic perturbations on both error correction and the spindle assembly checkpoint, and to compare chromosomally stable hTERT-RPE-1 cells and chromosomally unstable U2-OS cells. We find that the probability of error-free chromosome segregation is determined by the ratio of the checkpoint failure rate to the error correction rate, and validate a simple heuristic for understanding the source of chromosome segregation errors: perturbations which cause errors by disrupting the spindle assembly checkpoint decrease anaphase times, while those that disrupt error correction increase anaphase times. Taken together, this work provides a quantitative framework for understanding how error correction and the spindle assembly checkpoint contribute to mitotic timing and fidelity.

Centromeric chromatin is defined by the presence of the histone H3 variant CENP-A, which forms a specialized nucleosome required for kinetochore assembly. Compared to canonical H3 nucleosomes, CENP-A nucleosomes exhibit an open DNA conformation that leaves an additional 13 base pairs of DNA accessible at the entry and exit sites. While the CENP-A N-helix has previously been implicated in promoting this enhanced DNA breathing, the contributions of the intrinsically disordered N-terminal tail and adjacent latch regions of CENP-A in nucleosome conformation remain unknown. The intrinsically disordered N-terminal regions of histone H3 are known to facilitate interactions with DNA to stabilize overall nucleosome conformation. Here, we systematically tested the contribution of each N-terminal histone region to maintaining H3 histones by utilizing a combination of MNase digestion assays and coarse-grained molecular dynamics simulations of H3/CENP-A chimera histone nucleosomes containing targeted swaps of the tail, latch, and N-helix regions. Removal or substitution of individual H3 with CENP-A N-terminal regions increased DNA accessibility and nucleosome unwrapping. While any single CENP-A N-terminal region was sufficient to open the canonical nucleosomal DNA conformation, replacement of any single CENP-A N-terminal region with its H3 counterpart was insufficient to restore the wrapped DNA conformation characteristic of canonical H3 nucleosomes. Instead, progressive incorporation of multiple H3-derived regions produced increasingly closed DNA conformations, demonstrating that the H3 tail, latch, and N-helices act cooperatively to stabilize wrapped nucleosomal DNA. Taken together, these findings demonstrate that the more restricted DNA breathing of canonical nucleosomes arises from coordinated contributions across multiple N-terminal regions and suggest that the multi-region redundancy in the conformational flexibility of the centromeric nucleosome could emphasize the importance of retaining flexibility in the centromeric nucleosome, even upon post-translational modification and binding to structural proteins. SIGNIFICANCEThe centromere is marked by nucleosomes containing CENP-A, which adopt a more open and accessible DNA conformation than canonical nucleosomes. However, the molecular determinants underlying this difference remain unclear. Previous structural investigations of the centromeric nucleosome have placed less emphasis on the intrinsically disordered N-terminal regions of CENP-A. Here, we systematically dissect the contributions of the N-terminal tail, latch, and N-helix via MNase digestion assays and molecular dynamics simulations on nucleosomes containing H3/CENP-A chimeras. We demonstrate that no individual H3-derived region is sufficient to impart a closed conformation to the nucleosomal DNA. Instead, multiple regions act together to stabilize DNA, revealing that nucleosome conformation is controlled by concerted histone-DNA interactions.

Citrullination is a charge-modifying post-translational modification whereby proteinogenic arginine is converted to the non-coded amino acid citrulline by calcium-activated protein arginine deiminases (PADs; EC 3.5.3.15). The five known PAD enzymes in humans (PADs 1, 2, 3, 4, and 6) are differentially expressed and have distinct targets, including histones. While some PAD histone citrullination sites are known, a comprehensive investigation of all histone tail arginines targeted by catalytically active PADs 1-4 is lacking. Here, we sought to identify PAD citrullination sites in histone tails, both within histone peptides and in reconstituted nucleosomes. Toward this objective, we utilized a real-time 1H-15N NMR spectroscopy-based assay. By monitoring both arginine and citrulline backbone amide peak intensities over time, we identified sites of citrullination in 15N-labeled histone tails within peptides and reconstituted nucleosome core particles. We found that PADs 1, 2, and 4 citrullinate all directly observable histone tail arginines to varying degrees. This is distinct from PAD3, which only moderately citrullinates H2A and H4 arginine residues and does not modify H3 tail arginines. Together, these data suggest a level of histone arginine specificity by each PAD. Furthermore, histone tail citrullination is altered within nucleosomes compared to isolated peptides, which we interpret to reflect changes in conformation and accessibility. We speculate that citrullination increases nucleosomal histone tail dynamics, with implications for crosstalk between sites of histone citrullination and other important sites of regulation by PTMs (including lysines) within and between tails.

The nature of the chromatin polymer and its properties are tightly coupled to its function. By analyzing recent microscopy data, we show that chromatin segments at [~] 10-100 kb scales exhibit anomalously broad bond-length fluctuations and acute local angles beyond the scope of existing polymer models. Using polymer simulations at nucleosome resolution and systematic coarse-graining, we show that chromatin at these scales requires a non-equilibrium description. We develop a data-constrained non-equilibrium model that explains these anomalous fluctuations, in which coarse-grained beads experience extensile, angular, and stochastic active forces. Our work provides an experimentally guided framework for incorporating active processes and suggests that the nature of activity is scale-dependent. The model quantitatively reproduces three-dimensional distance distributions across scales and enables inference of effective elastic and active parameters, providing a unified framework for active chromatin simulations and the study of its 3D organization.

1Nuclear envelope stretch and rupture are common to cell spreading and migration in a variety of microenvironments, leading to marked changes in nucleocytoplasmic transport. Predicting cell response to different mechanochemical cues that are transmitted to the nucleus remains an open problem in the field of mechanomedicine. We developed a predictive modeling framework to examine how nuclear deformation on substrates with different nanotopographies influences nucleocytoplasmic transport and rearrangement of the nuclear lamina. Using the finite element method, we simulated nuclear compression by the perinuclear actin cap on substrates with arrays of nanopillars, modeling the nuclear envelope as a nonlinear elastic structure and coupling deformations to a biochemical model of lamin remodeling and nucleocytoplasmic transport. These simulations predicted regions of high nuclear envelope stretch adjacent to cell-nanopillar contacts, leading to maximized nuclear envelope tension on small nanopillars spaced by 4-5 microns. We then considered the effects on nuclear transport of YAP and TAZ and found that increased nuclear compression led to YAP/TAZ nuclear localization in agreement with previous experiments. Furthermore, the simulated force load per lamin was maximized on nanopillar substrates with high nuclear stretch. The magnitude of this load was modulated by the rate of actin cap assembly and the overall expression level of lamin A/C - decreasing lamin content in the nuclear envelope led to a higher likelihood of rupture. We validated this prediction in subsequent experiments with lamin-depleted U2OS cells, establishing the central importance of lamin transport and microenvironment nanotopography to nuclear mechanotransduction. 2 SignificanceCell nuclei commonly experience large strains, but existing computational models do not explain the coupling between such deformations and molecular transport. Here, we present a modeling framework that includes the mechanics of nuclear deformations and the reaction-transport of molecules within the cytoplasm, nuclear envelope, and nuclear interior. As a well-controlled setup for comparing experiments and simulations, we consider nuclear indentations exhibited by cells on nanopillar substrates. Our simulations recapitulate measurements of nuclear YAP/TAZ localization from the literature and predict that low-lamin cells experience higher force loads at the nuclear envelope. We validate this prediction experimentally, showing that lamin-depleted cells are more likely to exhibit nuclear rupture. Overall, our framework presents opportunities to predict nuclear mechanoadaptation to different microenvironments.

Abnormalities in nuclear morphology are an important diagnostic tool to determine malignancy in cancer cells and are characterised by nuclear blebbing and deformations. Nuclear shape is mostly maintained by a dense protein meshwork of lamins, consisting of 4 lamin subtypes, of which the individual contribution to nuclear shape maintenance remains elusive. In this study, we decouple the roles of lamin A, C, and B1 across cancer cell lines with varying malignant potential (HeLa, HT1080, and MDA-MB-231). Using single-cell correlation analysis, we directly link reduced lamin A/C, and not lamin B1, expression levels to nuclear deformability. We found that the nuclear shape of the more malignant MDA-MB-231 cells is approximately 4-fold more sensitive to lamin A/C than HeLa and HT1080 cells. Biochemical analyses reveal cell-type-specific variation in lamin A/C interactions and homodimer formation that correlates with nuclear shape deformations. In contrast to healthy mouse embryonic fibroblast cells, malignant cells exhibit reduced dimerisation, which correlates with nuclear deformability. As such, our study links, for the first time, the lamin A/C dimerisation state to nuclear abnormalities, thereby providing new avenues for investigating cancer progression.

Genomic organization within the nucleus is crucial for gene regulation and cell health, as disruptions in this organization are linked to genetic disorders and cancers. Recent studies suggest that molecular-scale organization of chromatin near the nuclear periphery (lamina-associated domains, LADs) affects gene regulation, providing transciptional supression, but the biophysical mechanisms of supression behind remain unclear. LADs are large heterochromatic regions near the nuclear lamina, where transcriptional factors and RNA polymerase are scarce, implying a nonspecific filtering property. Here, we investigate the steric filtering capabilities of LADs by performing coarse-grained polymer simulations. Our results show that LAD thickness can be affected by the interaction between chromatin and nuclear periphery as well as chromatin self-compaction. Regardless, the LAD layer acts as a size-selective steric partitioning environment for protein particles limiting their access to nuclear periphery. Notably, increasing bulk protein levels enhances protein access linearly. These results align with experimental observations and suggest that LADs could control the presence of transcription machinery on the periphery of the nucleus, providing a polymer-physical mechanism for gene regulation in nuclei.

Understanding cis-regulatory elements (CREs) at the single cell level is fundamental to deciphering transcriptional changes during development, cell differentiation, and homeostasis. Recent studies have shown that arbitrary peak-calling thresholds complicate data interpretation and cross-study comparisons. Furthermore, due to the inherent sparsity of single-nuclei ATAC-seq (snATAC-seq) data, distinguishing between truly inaccessible regions and technical dropouts remains challenging. Our analysis of snATAC-seq experiments performed in a well-established cell line suggests that the dichotomy between accessible (open) or inaccessible (close) CREs is misleading. Thousands of accessible regions are present in a very small fraction of cells of the population but they are repeatedly identified, suggesting that they have a low accessibility or are only transiently accessible. However, depending on the detection threshold selected they could be considered as either genuine CREs or noise. To resolve this inconsistency, we propose a model where chromatin accessibility is treated as a continuum, defined by a probability of accessibility (Pa) for each accessible region across cell types and conditions. Through computational simulations, we demonstrate that snATAC-seq results can be explained by a simple "balls into bins" probability model, offering a theoretical framework for calculating Pa distributions from any snATAC-seq dataset. Furthermore, we examine how Pa distributions shift following activation of the TGF{beta} signaling pathway. This probabilistic approach removes the reliance on arbitrary thresholds and supports a more quantitative, and dynamic understanding of accessible regions function.

Nucleoli and centromeres play essential roles in cellular proliferation and homeostasis, and are structurally and functionally interconnected. Centromeres frequently cluster around nucleoli, and some centromere assembly factors are known to reside in the nucleoli. To investigate the spatial and temporal relationships between these nuclear domains, we examined their dynamics in living cells. We imaged HeLa cells stably expressing mCherry-NPM1 and GFP-CENP-A using time-lapse microscopy. The results show that a subset of centromeres exhibits dynamic behavior during interphase, migrating over micrometer-scale distances within two hours. On average, 40-50% of centromeres maintain an association with nucleoli throughout interphase, with some cells displaying nucleolar-centromere association and dissociation within hours. Upon entry into mitosis, nucleoli are disassembled, and NPM1 localizes to the periphery of mitotic chromosomes. Nucleolar-centromere interactions are re-established in early G1, coinciding with the assembly of new centromeres. Treatment with actinomycin D, an inhibitor of RNA polymerase I, significantly reduces nucleolar size, nucleolar-centromere interactions, and centromere dynamics. Furthermore, post-mitotic nucleolar reformation is impaired. These findings highlight the dynamic nature of centromeres in interphase nuclei and their interactions with nucleoli. This behavior is partially dependent on rDNA transcription and nucleolar integrity, underscoring the critical roles of nucleoli, centromeres, and their interaction in 4D genome organization.

Pancreatic ductal adenocarcinoma (PDA) arises from distinct precursor lesions with different clinical outcomes, yet the mechanisms linking epigenetic regulation to invasive cell behavior remain poorly understood. Here, we investigate how the chromatin remodeler Brg1 influences the dynamic properties of cancer cell migration. Using a biomimetic supported membrane system combined with label-free interferometric imaging, we quantitatively analyze the spatiotemporal dynamics of PDA cells derived from pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasms (IPMN). Despite their similar morphology under conventional conditions, PanIN- and IPMN-derived PDA cells exhibit markedly different migration behaviors. PanIN-derived cells migrate faster and display enhanced dynamic remodeling, whereas IPMN-derived cells show persistent elongation with limited displacement. These differences are captured by quantitative analyses of cell trajectories and deformation dynamics. Mechanistically, PanIN-derived PDA cells exhibit elevated Rac1 activity, supporting a model in which a Brg1-Rac1 axis regulates cytoskeletal dynamics and migration behavior. Together, our findings demonstrate that epigenetic regulation is linked to distinct dynamic phenotypes of cancer cells and highlight the importance of quantitative analysis of cell behavior for understanding invasive potential.

The bacterial nucleoid undergoes extensive structural reorganization during growth, influenced by nucleoid-associated proteins (NAPs) whose interactions and effects on nucleoid organization remain unclear. We investigated these interactions by tracking single molecules of the NAP HU-PAmCherry in living Escherichia coli cells in different growth phases, and we further examined how two NAPs, Dps and H-NS, impact HU dynamics. HU mobility varies with growth phase: In exponential phase, HU has two distinct mobility states: a fast-diffusing state and a slower, interacting state. In stationary phase, we observed a third population of very slow molecules, suggesting stable HU binding or confinement within compacted DNA. Deleting dps increases HU mobility in stationary phase, consistent with findings that Dps promotes short-range DNA contacts and nucleoid compaction in deep stationary phase. We measured in exponential phase that hns deletion leads to nucleoid compaction, faster HU diffusion, and a third population of very slow HU molecules in these cells. In stationary phase, deleting hns increases these stably bound HU molecules. Our results show that growth-phase-dependent nucleoid reorganization by Dps and H-NS influences the behavior and function of other NAPs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=55 SRC="FIGDIR/small/695591v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@16d8f9forg.highwire.dtl.DTLVardef@1f03355org.highwire.dtl.DTLVardef@ba1fc7org.highwire.dtl.DTLVardef@17c5de2_HPS_FORMAT_FIGEXP M_FIG C_FIG The nucleoid-associated proteins Dps, H-NS, and HU shape the bacterial chromosome in the deep stationary phase through their interactions with the nucleoid.

Emerging evidence suggest that gene expression is controlled though the modulation of transcriptional bursting across species. However, the underlying regulatory mechanisms remain largely uncertain. Recent live-imaging studies have reported that transcription factors (TFs) form a cluster at enhancers just prior to gene activation, thereby locally concentrating the active transcription machinery. This process is thought to be mediated by multivalent interaction between Mediator recruited by TFs. Conversely, transcription itself is also suggested that to influence the assembly of the TF clustering, implicating the presence of a feedback mechanism. To understand the theoretical framework underlying the interplay between TF clustering and transcription, we here develop a polymer micelle model of transcriptional bursting. With this model, multiple RNA polymerase II (Pol II) molecules loaded to the promoter, together with enhancer-bound Mediator, assemble a micelle-like structure due to their connectivity via DNA when the chromatin fiber connecting enhancer and promoter adopts closed conformation, analogous to polysoap micelle. This assembly further recruits freely diffusing Pol II and Mediator in the nucleoplasm even at low concentration up to the optimal size. Our theoretical framework enables quantitative prediction of how dynamic transitions of enhancer-promoter conformation and the stability of the micelle impact the kinetics of transcriptional bursting.

Genomic DNA is subject to forces and torsion. Some arise mechanically, while others can be entropic, such as those due to crowding within the nuclear environment. Indeed, about 30-40% of the cell is occupied by molecules other than water, and of these, the vast majority are macromolecules. Here, we explore both experimentally and theoretically the interplay between tension, torsion, and macromolecular crowding. Using pharmaceutically relevant crowders of different molecular weights, Dextran 70, and polyethylene glycol (PEG), we observed that macromolecular crowding of unwound, stretched DNA effectively opposed the tension and promoted the formation of plectonemes. A theoretical model representing the equilibrium between B- and L-form DNA fit to the experimental measurements indicates the contractile tension produced by macromolecular crowding of DNA. SIGNIFICANCE STATEMENTDistinct DNA conformers are involved in different cellular processes. Genomic DNA is both stretched and unwound by enzymes in a crowded intracellular medium. This can induce conformational changes between extended, twisted and more compact, plectonemic forms. This study explores the effect of macro-molecular crowding on the conformations of DNA subject to tension and torque. Fitting experimental data to a model for the right-to-left-handed DNA transition, we show that macromolecular crowding induces a contractile force that favors DNA writhe and that such force depends both on the concentration and molecular weight of the crowder.

Essential nuclear processes require pairs of chromosomal loci to find each other in three-dimensional space. Polymer models of chromosome dynamics typically assume that the stochastic forces driving such locus motion are spatially uncorrelated, implying that relative diffusion follows directly from single-locus dynamics. Here we show that this assumption fails in living cells. Using live-cell imaging in fly embryos and mouse embryonic stem cells, we find that pairwise locus distances diffuse markedly slower than predicted for independent fluctuations. Combining stochastic trajectory analysis with polymer simulations, we demonstrate that this slowdown arises from non-equilibrium spatially correlated fluctuations (SCFs) in the nucleoplasm, which cause nearby loci to move coherently. We establish three experimentally testable signatures of SCFs: fluctuation amplitudes plateau at large distances, are independent of genomic separation, and show an anomalous temporal scaling. All three predictions are confirmed experimentally, including for loci on separate chromosomes. ATP depletion and disruption of cohesin-mediated loop extrusion reveal that both active processes and crosslinking contribute to correlation magnitudes. Because SCFs slow relative motion preferentially at short distances, they reduce encounter frequencies while prolonging encounter durations, generating a trade-off with direct implications for gene regulation. Our results identify spatially correlated fluctuations as a fundamental determinant of relative motion in confined active polymers.